CHANNELRHODOPSINS FOR OPTICAL CONTROL OF CELLS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 1 1 (e) of U.S. Provisional application serial number 61/559076 filed November 12, 201 1 , the disclosure of which is incorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under grants NIB

IDP2OD002002, NIH 1RC1 MH088182, NIH 1 01 NS067199, NIH IR01.DA029639, and NIH 1R.0INS075421 from the National Institutes of Health; NSF DIvIS 0848804, NSF ^' CAREER Award CBET 1053233, and NSF EFR1 0835878 from the National Science Foundation; a DARPA Living Foundries Early Career Translational Research Award, Wallace H. Coulter Foundation Department of Defense MR C/CDM P, Post-Traumatic Stress Disorder Concept A ward Human Frontiers Science Program. The United States government has certain rights in the invention,

FIELD OF THE INVENTION

The invention, in some aspects relates to compositions and methods for altering conductance across membranes, cell activity, and cell function, also relates to the use of exogenous light-activated ion channels in cells and subjects.

BACKGROUND OF THE IN VENTION

Altering and controlling cell membrane and subcellular region ion permeability has permitted examination of characteristics of ceils, tissues, and organisms. Light-driven pumps and channels have been used to silence or enhance cell activity and their use has been proposed for drug screening, therapeutic applications, and for exploring cellular and subcell ular functio ,

Molecular-genetic methods for preparing ceils that can be activated (e.g., depolarized) or inactivated (e.g., hyperpolarized) by specific wavelengths of light have been developed (see, for example, Han, X. and E. S. Boyden, 2007, PLoS ONE 2, e299). It has been identified that the light-activated cation channel channelrhodopsin-2 ( ^" ChR2), and the light- activated chloride pump halorhodopsin (Flalo N HR), when trans genie ally expressed in cell such as neurons, make them sensitive to being activated by blue light, and silenced by yellow light; respectively (Han, X. and E. S. Boyden, 2007, PLoS ONE 2(3): e299; Boyden, E. S., et. aL 2005. Nat Neurosci. 2005 Sep;8(9): 1.263-8. Epub 2005 Aug 1.4.). Previously identified light-activated pumps and channels have been restricted to activatio by particular wavelengths of light, thus limiting their usefulness.

5

SUMMARY OF THE I NVENTION

The invention, in part, relates to isolated light-activated ion channel polypeptides and methods for their preparation and use. The invention also includes isolated nucleic acid sequences that encode light-driven ion channels of the invention as well as vectors and

{() constructs that comprise such nucleic acid sequences. In addition, the invention in some aspects includes expression of light-activated ion channel polypeptides in cells, tissues, and subjects as well as methods for using the light-activated ion channels to alter conductance across membranes, to alter cell and tissue function, and for use in diagnosis and treatment of disorders.

15 The invention, in part, also relates to methods for adjusting the voltage potential of ceils, subcellular regions, or extracellular regions. Some aspects of the invention include methods of incorporating at least one nucleic acid sequence encoding a light-driven ion channel into at least one target cell, subcellular region, or extracellular region, the ion channel functioning to change transmembrane passage of ions in response to a specific wavelength of 0 light. Exposing an excitable cell that includes an expressed light-driven io channel of the invention to a wavelength of light that activates the channel, may result in depolarization of the excitable cell. By contacting a cell that includes a light activated ion channel of the invention with particular wavelengths of light, the cell is depolarized. A plurality of light- activated ion channels activated by different wavelengths of light in overlapping or non- 5 overlapping pluralities of cells may be used to achieve multi-color depolarization.

In some embodiments, the invention comprises a method for the expression of newly identified cl asses of genes that encode Sight-driv en ion channel s, in genetically targeted cells, to allow millisecond-timescale generation of depolarizing current in response to pulses of light. Channels of the invention can be genetically expressed in specific cells (e.g., using a 0 virus or other means for delivery) and then used to control cells in intact organisms

(including humans) as well as cells in vitro, in response to pulses of light. Given that these channels have different activation spectra from one another and from the state of the art (e.g., ChR2/VChR.l), they also allow multiple colors of light to be used to depolarize different sets of cells in the same tissue, by expressing channels with different activation spectra

genetically in different ceils, and then il!uniinating the tissue with different colors of Sight.

In some aspects, the invention uses eukaryotic chai elriiodpsins, such as eukaryotic channelrhodpsins, such as Chloromon mibdm (a!so referred to herein as: "ChR87"), Ckkmiydamonas nociigama (also referred to herein as: "Chrimson" or "Chr88"), and

Stigeoclottium heiveiicum (also referred to herein as: "Chronos" or "ChR90") rhodopsin, and derivatives thereof, are used to depolarize excitable cells. These channelrhodpsins, or derivatives thereof, can also be used to modify the pH of cells, or to introduce cations as chemical transmitters.

The ability to optically perturb, modify, or control cellular function offers many advantages over physical manipulation mechanisms, such as speed, non-invasiveness, and the ability to easily span vast spatial scales from the nanoscale to macroscale. One such approach is an opio-genetie approach, in which heterologously expressed light-activated membrane polypeptides such as a light activated ion channel of the invention, are used to move ions with various spectr of light.

According to an aspect of the invention, methods of altering ion conductivity of a membrane are provided. The methods including a) expressing in membrane a light- activated ion channel polypeptide comprising an amino acid sequence of a wild-type or modified light-activated Chl mydomonas nociigama, Stigeoclonium heiveiicum., or

Chloromonm mbdivm ^' a polypeptide and b) contacting the light-activated ion channel polypeptide with a light that activates the light-activated ion channel and alters the ion conductivity of the membrane, hi some embodiments, the light-activated ion channel polypeptide comprises an amino acid sequence of a wild-type or modified light-activated Chlamydomonas nociigama polypeptide and the activating light has a wavelength between 365 nm and 700 nm. In certain embodiments, the activating light has a wavelength from 530 nm to 640 nm, and optionally, the activating light has a wavelength of 590 nm. in some embodiments, contacting the light-activated ion channel polypepti.de with a light having a wavelength greater than 720 nm does not activate the ion channel. In some embodiments, the membrane is not a membrane in which the light-activated ion channel naturally occurs, in some embodiments, tire light-activated ion channel is an isolated ion channel. In some embodiments, the membrane is in cell. In some embodiments, the cell is a neuronal cell and the method further comprises contacting the ion channel polypeptide with a light having a wavelength up to 660 nm under conditions suitable to produce a spike in the neuronal cell. In certain embodiments, the nucleic acid sequence encoding the light-activated ion channel polypeptide comprises the nucleic acid sequence set forth as SEQ ID NO:3. In some

embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises SEQ ID NO:2. In some embodiments, the amino acid sequence of the light- activated ion channel polypeptide comprises a modified Chlamydomonas novtigama light- activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 86-320 of SEQ ID O:2 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO: 2. in certain embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises SEQ ID NO:5. In some embodiments, the amino acid sequence of the light- activated ion channel polypeptide comprises a modified Chlamydomonas nocfigama light- activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 86-320 of SEQ ID NO:5 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO: 5, In some embodiments, the light-activated ion channel polypeptide comprises an amino acid sequence of a wild-type or modified light-activated Sligeoclortium helveticum polypeptide and the light that activates the ion channe! has a wavelength between 365 nm and 630 nm. hi some embodiments, the light that activates the ion channel has a wavelength fr m 430 nm to 550 nm, and optionally, has a wavelength of 500 nm. In certain embodiments, contacting the polypeptide with a light having a wavelength greater than 650 nm does not activate the ion channel, in some embodiments, the cell is a neuronal cell and the method further includes contacting the ion channel polypeptide with a light having a wavelength between 430 nm and 550 nm in. a manner to produce a spike in the neuronal cell, in some embodiments, the nucleic acid sequence encoding the light-activated ion channel polypeptide comprises the nucleic acid sequence set forth as -SEQ ID NQ:8, In certain embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises SEQ ID O:7. In some embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises a modified Stigeocionium elvetieum light-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 61 -295 of SEQ ID NQ:7 and 95%, 96%, 97%, 98%, 99% or J 00% identity to the remaining amino acids in the sequence set forth as SEQ ID NO: 7. In some embodiments, the light-activated ion channel comprises an amino acid sequence of a wild-type or modified light-activated Chloromoms frdivisa polypeptide and the light that activates the ion channel is a light having a wavelength of between 365 nm and 630 nm and a peak activating wavelength of 515 nm. In some embodiments, the light-activated ion channel is encoded by the nucieic acid sequence set forth as SEQ ID NO: 12. In certain embodiments, the amino acid sequence of the light- activated ion channel is set forth as SEQ ID NO: 1 1. In some embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises a modified Chloromonas s bdtvisa light-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 81 -315 of SEQ ID NO: 1 1 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO: 1 1. In. some embodiments, the light-activated ion channel does not activate in response to contact with light having a wavelengt greater than 650 am. In certain embodiments, the membrane is a cell membrane. In some embodiments, the cell is a human cell. In some embodiments, the membrane is a cell membrane of a neuronal cell, a nervous system cell, a cardiac cell, a circulatory system cell, a visual system cell, or an auditory system cell. In certain embodiments, altering the ion conductivity of the membrane depolarizes the cell.

According to another aspect of the invention, an isolated light acti vated ion channel polypeptide is provided. The light-activated ion channel polypeptide includes an amino acid sequence of a wild-type or modified light-activated Chlamydomonas nociigama,

Sligeoc tmm heiveticntn , or Chloromonas suhdrvisa channel polypeptide. In some embodiments, the light-activated ion channel polypeptide comprises an amino acid sequence of a wild-type or modified light-activated Chlamydomonas nociigama polypeptide and activating the ion channel comprises contacting the ion channel polypeptide with a light having a wavelength between 365 nni and 700 lira. In some embodiments, activating the ion channel comprises contacting the ion channel polypeptide with a light having a wavelength from 530 nra to 640 ran, and optionally having a wavelength of 590 nni. In some embodiments, contacting the ion channel polypeptide with a light having a wavelength greater than 720 nm does not activate the ion channel, in certain embodiments, the nucleic acid sequence encoding the light-activated ion channel polypeptide comprises the nucleic acid sequence set forth as SEQ ID NO:3. In some embodiments, the amino acid sequence of the Sight-activated ion channel polypeptide comprises SEQ ID O:2. in some embodiments, tire amino acid sequence of the light-activated ion channel polypeptide comprises a modified Chlamydomonas nociigama light-activated ion channel sequence having at least 70%, 75%. 80%, 85%, 90%, 95%, or 99% identity to amino acids 86-320 of SEQ ID NO:2 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO.2. In some embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises SEQ ID NO: 5. In certain embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises a modified Chlamyd monas noctigama light-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 86-320 of SEQ ID NO: 5 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO:5. In some embodiments, the light-activated ion channel polypeptide comprises an amino acid sequence of a wild-type or modified light-activated Stigeochmum helvetteum polypeptide and activating the ion channel comprises contacting the ion channel polypeptide with a Sight having a wavelength between 365 nm and 630 nm. In some embodiments, activating the ion channel includes contacting the ion channel polypeptide with a light having a wavelength -from 430 nm to 550 nm, and optionally having a wavelength of 500 nra. In some embodiments, contacting the ion channel polypeptide with a light having a wavelength greater than 650 nm does not activate the ion channel. In certain embodiments, the nucleic acid sequence encoding the light-activated ion cha nei polypeptide comprises the nucleic acid sequence set forth as SEQ II ⁾ NO:8. In some embodiments, the amino acid sequence of the light-activated ion channei polypeptide comprises SEQ ID NO:7. In some embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises a modified Stigeochmum hetvetivum light-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 61-295 of SEQ ID NO:7 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO: 7. In certain embodiments, the light-activated ion channel includes an amino acid sequence of a wild-type or modified light-activated Ch romonas suhdivisa polypeptide and the light that activates the io channei is a light having a wavelength of between 365 nm and 630 nm and a peak activating wa velength of 515 nm. In some embodiments, the light-activated ion channel is encoded by the nucleic acid sequence set forth as -SEQ ID NO: 12. In some embodiments, the amino acid sequence of the light- activated ion channel is set forth as SEQ ID NO: Π . In certain embodiments, the amino acid sequence of the light-activated ion channel polypeptide comprises a modified Chhromonas suhdivisa Sight-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99 identity to amino acids 82-315 of SEQ ID NO: 1 1 and 95%, 96%, 97%, 98%, 99% or 00% identity to the remaining amino acids in the sequence set forth as SEQ ID NO: 1 1. In some embodiments, the light-activated ion channel does not activate in response to contact with light having a wavelength greater than 515 nm. In some embodiments, the light-activated ion channel polypeptide is expressed in a membrane. In certain embodiments, the membrane is mammalian cell membrane. In some embodiments, the cell is an excitable cell. In some embodiments, the ceil is in a subject. In. some embodiments, the membrane is a eel! membrane of a ne uronal cell, a nervous system cell, a cardiac cell, a circulatory s ystem cell, a visual system cell, or an auditory system cell. In certain embodiments, altering the ion conductivity of the membrane depolarizes the cell.

According to another aspect of the invention, a vector that includes a nuc leic acid sequence that encodes any of the aforementioned light-activated ion channel polypeptides is provided.

According to another aspect of the invention, a cell that includes any of the aforementioned light-activated ion channel polypeptides is provided and the cell is not a cell in which the light-activated ion channel polypeptide naturally occurs, in some embodiments, the cell is a mammalian cell and in certain embodiments the cell is a human cell.

Accordin g to another aspect of the in v ention, methods of assessi ng the effect of a candidate compound on ion conductivity of a membrane are provided. The methods including (a) contacting a test membrane comprising the isolated light-activated ion channel polypeptide of any one of the aforementioned embodiments with light under conditions suitable for altering ion conductivity of the membrane; (b) contacting the test membrane with a candidate compound; and (c) identifying the presence or absence of a change in ion conductivity of the membrane contacted with, the light and the candidate compound compared to ion conductivity in a control cell contacted with the light and not contacted with the candidate compound; wherein a change in the ion conductivity in the test membrane compared to the control indicates an effect of the candidate compound on the ion

conductivity of the test membrane. In some embodiments, the membrane is in a cell. In certain embodiments, altering the ion conductivity of the membrane depolarizes the cell. In some embodiments, a change is an increase in ion conducti vity of the membrane. In some embodiments, the change is a decrease in ion conductivity of the membrane.

According to another aspect of the invention, methods of treating a disorder in a subject are provided. The methods include (a) administering to a subject in need of such treatment, a therapeutically effective amount of a light-activated ion channel polypeptide of any one of the aforementioned embodiments, to treat the disorder and (b) contacting tire ceil with light and activating the light-activated ion channel in the eel! under conditions sufficient to alter ion conductivity of a cell membrane, wherein altering the conductivity of the cell membrane treats the disorder, in some embodiments, altering the ion conductivity of the membrane depolarizes the cell.

According to yet another aspect of the invention, methods of pe.rfonni.ng a 2, 3, 4. 5 or more-color light ion channel activatio assay in a population of cells are provided. The methods include (a) expressing a blue-Iight-aetrvated ion channel in a first subpopulation of a population of cells; (b) expressing a red-light-aciivated ion channel in a second subpopulation of the population of cells, wherein the first and second subpopulations are non-overlapping subpopulations; (c) contacting the population of ceils with a plurality of blue Sight test doses 5 comprising combinations of blue light wavelength, pulse width, and power; (d) measuring transmembrane voltage deflection in a cell of the second subpopulation of cells contacted with the blue light test doses; (e) determining the test blue light dose comprising a maximum blue light power that activates the blue-light activated ion channel in first subpopulation of ceils and results in a minimum sub-threshold transmembrane voltage deflection in the second

{() subpopulation of cells; (f) contacting the population of cells with a plurality of blue light test doses comprising a lower power than the maximum blue light power of (e); (g) determining the blue light test doses of (f) that activate the blue-light activated ion channel; (h) contacting the population of cells with a plurality of red light test doses comprising combinations of red light wavelength, pulse width, and power, (i) determining a red light test dose comprising a

15 red light powe that activates the second subpopulation of cells; and (j) performing an activity assay comprising contacting the population of ceils with the blue light test dose determined in (g) and the red light test dose determined in (i) . In certain embodiments, the pluralit of blue light test doses comprise wavelengths, pulse widths, and powers independently selected from between 400 ran and 500 nm, I ras and 5 ms. and 10 W/mm ² and 1.0 mW/mnT,

0 respectively. In some embodiments, the red light test dose of (i) is the test dose comprising a minimum red light power that activates the second population of cells. In some

embodiments, measuring the transmembrane voltage deflection in (d) comprises patch clamping a cell of the second population of cells and determining one or more voltage changes in the ceil, in certain embodiments, the determining in (e) comprises altering the 5 blue light dose by increasing the blue light power from 0.5raW/mm ² to 1 OmW/nan ² ; and measuring the sub-threshold transmembrane voltage deflection in the second subpopulation of cells. In some embodiments, the minimum sub-threshold voltage deflection is less than 15 mV, less than 10 mV, or less than 5 mV. In some embodiments, the maximum blue light power in (e) is between 0.4 mW/mm ² and 0,6 mW/ami ² , In some embodiments, the blue 0 light power in (g) is between 50 pW/ m" and 0.4 mW/mm". In certain embodiments, the red-light activated ion channel comprises an amino acid sequence of a wild-type or modified light -activated Chlamydomon s nociig ma polypeptide. In some embodiments, the nucleic acid sequence encoding the red light-activated ion channel polypeptide comprises the nucleic acid sequence set forth as SEQ ID NO:3. In some embodiments, the amino acid sequence of the red ligl -activated ion channel polypeptide comprises SEQ ID NO: 2. in some

embodiments, the amino acid sequence of the red Sight-activated ion channel polypeptide comprises a modified Chlamydomonas noctigama light-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 86-320 of SEQ ID NO:2 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO:2. in certain embodiments, the amino acid sequence of the red light -activated ion channel polypeptide comprises SEQ ID NO:5. in some

embodiments, the amino acid sequence of the red light-activated ion channel polypeptide comprises a modified Chl m domon noctigama light-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to ammo acids 86-320 of SEQ ID NO:5 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO: 5. In some embodiments, the blue-light activated ion channel comprises an amino acid sequence of a wild-type or modified light-activated

Sfigeociomum helveticum polypeptide, in some embodiments, the nucleic acid sequence encoding the blue light-activated ion channel polypeptide comprises the nucleic acid sequence set forth as SEQ ID NO:8. In some embodiments, the amino acid sequence of the blue light-activated ion channel polypeptide comprises SEQ ID NQ:7, in some

embodiments, the amino acid sequence of the blue light-activated ion channel polypeptide comprises a modified Stigeoclomum hehmicum light-activated ion channel sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to amino acids 6.1 -295 of SEQ ID NO;7 and 95%, 96%, 97%, 98%, 99% or 100% identity to the remaining amino acids in the sequence set forth as SEQ ID NO.7. In some embodiments, the plurality of red light test doses comprise wavelengths, pulse widths, and powers independently selected from between 600 nm and 740 nm, .1 ms and 5 ms, and 0.1 mW/rnrn ² and 100 mW/mm ² , respectively,

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a graph of channelrhodopsin photoeiirrents measured in cultured hippocanipal neurons. Fig, 1 A shows results using reel light (660nm) peak photocirrrent at iO mW mm ^'3 for I s illumination. CH.R88 is the only red light sensitive channelrhodopsin with significant photocurrenf at 660nm. Fig. 1 B shows results using blue (4.23 mW mm ^"2 ) or green (3.66 mW mm ^' ) light peak phoiocurrent at equal photon flux for 5ms illumination. ChR87, ChRSS, and ChR90 all have greater or comparable photocurrent than ChR2. Solid bar indicates blue light, horizontal striped bar indicates green light. Figure 2 is a graph showing action spectrum at equal photon dose at all wavelengths recorded in HEK293FT cells. ChR2 (470nni peak) and VChRl (545nm peak) represent the existing channelrhodopsin color sensitivity range. ChR87 (515nm peak) and ChR90 (500nm peak) are blue green light sensitive channelrhodopsins. Whereas ChR88 (590nm peak) is the first red light sensitive natural channelrhodopsin.

Figure 3 provides example traces of optically-driven spikes in cultured hippocampal neurons. Fig. 3 A shows red-light-driven spike trains at low frequency for Ch88. Generally ChR88 can reliably drive spikes up to 5Ez. However at higher frequency such as 20Hz, ChR88 desensitizes and/or causes depolarization block. Fig. 3B shows green-light-driven spike trains at high frequency for Ch90. Due to C R9Q fast iau off and peak photocurrent recovery kinetics, it is able to dri ve temporally precise spikes at the highest frequency a neuron is capable of mediating.

Figure 4 provides graphs showing channelrhodopsin kinetics measured in hippocampal neuron culture voltage clamped at ^« 65mV. Fig, 4A shows single exponential channel turn-off kinetics based on 5 ins pulse. ChR90 has the fastest turn-off kinetics (3.5ms) observed across all natural channelrbodopsins. Fig. 4B shows peak photocurrent recovery ratio based on 1 s illumination. ChR87 and ChR90 both have fast peak photocurrent recovery at around 70%. However ChR88 has slow recovery kinetics similar to ChR2.

Figure 5 provides and graph and traces showing Chrimson blue light crosstalk

characterization in cultured neurons. Fig. 5 A shows action spectrum of Chrimson and the blue light (470nm) wavelength used fo illumination. Wavelength was chosen to minimize crosstalk. Fig. 5B provides representative traces from a single neuron at various illumination conditions. When the blue light power is doubled from 0.1 to 0.2 mW mm ^'2 while the stimulation protocol is fixed as Sins 5Hz, the voltage deflection is also doubled. However when the blue light power is fixed at 0. i raW mm ^" " but the pulse duration is changed from 5ms to 1000ms, the crosstalk, is changed from <5 tnV to full spiking correspondingly. This means blue light crosstalk is a function of both light power and light pulse duration (total photon count).

Figure 6 provides graphs and traces illustrating Chronos and ChR2 blue light sensitivity in cultured hippocampal neurons. Fig. 6A is a spike irradiance curve for individual neurons.

! 0 Fig. 6B shows lowest, light power needed for single-cell 100% spike probability vs GFP fluorescence. Ciironos (circles) is approximately 5 times more light sensitive than ChR2 (triangles) at a given (GFP) expression level. Fig, 6C provides example traces of Chronos spiking at various light powers. Fig. 6D illustrates thai controls shows no significant 5 electrical differences between ChR2 and Chronos expressing neurons.

Figure 7 provides a graph and photomicrographic images illustrating the strategy used for slice characterization of Chronos and Chrimson. Fig. 7A shows illumination wavelength used for slice experiments. Fig. 7B is raicrographic images showing histology for Chronos {() and Chrimson GFP fusion construct singly expressed in layer 2/3 visual cortex in mice.

Figure 8 provides graphs illustrating whole cell patch clamp characterization of Chrimson and Chronos blue and red light sensitivity in slice. Fig. SA illustrates that red light elicits 100% spiking in Chrimson expressing neurons but not Chronos expressing neurons between 15 1-6.5 mW mm ^'2 . Fig. SB shows that blue light at 0.2-0.5 mW mm ^"2 can elicit 100% spiking in Chronos expressing ceils but not Chrimson expressing ceils. However full spiking crosstalk in Chrimson expressing cells can occur at powers higher than 0,6 in W mm" ⁴ . Fig, 8C shows blue l ight crosstalk voltage of Chrimson expressing neurons. 0 Figure 9 provides example traces of current-clamped opsin-expressing neurons in layer 2/3 slice blue light 0.1 raW mm ^" ", red light 1 raW mm ^"2 expressing. No crosstalk was observ ed under red light for Chronos while minimal subthreshold (<5mV) crosstalk was observed under blue light fo Chrimson. 5 Figure 10 provides example traces of voltage-clamped non-opsin-ex pressing neurons in layer 2/3 or 5, post-synaptic to opsin-expressing cells. Zero posi-synaptic crosstalk was observed for both Chronos and Chrimson under red and blue light illumination respectively.

Chronos: blue light 0.13 mW mm ^' ", red light 1.7 mW tim ,

Chrimson: blue light 0,37 mW mm ^"* , red light 1.7 mW mm ^'2 .

Figure 1 1 provides a schematic diagram, photomicrographic image and traces illustrating paired-pulse illumination in slice that differentially express Chrimson and Chronos in separate neurons. Fig. 1 1 A shows a triple p!asmid in utero electroporation scheme to obtain non-overlapping expression of Chrimson and Chronos. Fig. 1 1 B shows opsin expression in ϋ visual cortex no overlap of GFP and m02 is observed ratio of Chronos to Chrirason labeling can be tuned by titrating Cre p!asmid. Fig, 1 1 C shows voltage-clamped non-opsin-expressing neuron in layer 2/3 paired-pulse stimulation to demonstrate different synapses are selectively driven by blue and red light, blue: 0.2 mW mm ^"2 ; red: 5 mW mm ^"* . Arrows represent light application. First trace from top: first arrow indicates bine light, second arrow indicates red light; second trace from top: first arrow indicates red light, second arrow indicates blue light; third trace from top: both arrows represent red light; and fourth trace from top: both arrows represent blue Sight. Figure 12 is a trace illustrating that Chrirason can drive spikes in the tar-red (660 em) using 5 ins pulses at 2.6 mW mm ^" ' in cultured hippocampal neurons.

Figure 13 provides traces illustrating that the ChR88 KJ 76R mutant has improved kinetics f 13ms tau oil) and can mediate high frequency spikes in cultured hippocampal neurons. Exemplar current clamped traces of a single ChR88 K1.76R expressing neuron are shown. Fig, 13A shows that ChR88 176R can reliably drive spikes from 1 to J O mW nmf at

625nm 5Hz stimulation. Fig. I3B shows red light (625nm) driven spike trains at various frequency for ChRSS K.176R. 1 mW mm ^"2 light power is used for ail frequencies. Fig, 13C shows current injection control to show the neuron is capable of spiking at the indicated frequencies.

Figure 14 provides graphs showing channelrhodopsin ion selectivity measured in E 293FT ceils. ChRSS and ChR 0 have comparable ion selectivity as ChR2. However ChR87 has less sodium (Na) current compared to calcium (Ca), proton (FT), and potassium (K) current,

BR IEF DESCR IPTION OF THE SEQUENCES

SEQ II) NO: 1 is amino acid sequence from Chlamydomonas nociigama

MAELiSSATRSLFAAGGiNPWPNPYI iHEDMGCGGMTPTGECFSTEWWCDPSYGLSD

AGYGYCFVEATGGYLVVGVE QAWLHSRGTPGEKIGAOVCQWIAFSIAIALLTFY GFSAWKATCGWEEVYVCCVEVLFVTLE I FKEFSSPAT VYLSTGNHAYCLRYFEWLLS CPVILlKLSNLSGLKNDYSKRT^fGLlVSCVG IVFG AAGLATDWLKWLLYIVSCIY GGY YFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLL KLSPYANS:iGHSICDnA EFWTFLAFfHLRI :iHEH]:OHGDIRKl KME H3EEVEVEEF VEEEDEDTVTHPTSNLANRNSFVI AER RARGIDVRASLDRNGPMiESGRVlLADT DIFVTEMF AQFAQLPAAIELIPALGADNALQLVO^ASVLGGCDFVMX^HPQFL DMS PSGLVARLRMMGORVVAFGPANLREIJESCDVmWfFAPPI LYOLRQVVAQMQLM RRQAAMMGG GGGMKGGMSGMGMGMHAGSMWKQQQMM QODGSAMMMPA

! 2 MQGGAASMRGSGLISAQPGRQASLGGPQSVMMGSAMVGSNPLFGTAPSPLGSAVG

AEAMGH LYGNQAAAGGiPAASAAADGTDVEMMQQLMSEiDRL GELGEQDMPR.

SEQ ID NO:2: ChR88 coding amino acid sequence that includes residues 1-350 of SEQ ID

5 NO: !

MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSD

AGYGYCFVEATGGYLVVGVEK QAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFY GFSAWKATCGWEEVYVCCVEVLFVT EIF EFSSPATVYLSTGNHAYCLRYFEWLES CPVlUKLSNLSGLKNDYS RTMGIJVSCVGMIVFGMAAGLATDWLKWLLYlVSCiY t o GGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLL KLSPYANSiG BS!CDOAKEFWlTEAHeLRIKfflEHiEieGD!R TKME!GGEEVEVEEF VEEEDEDTV.

SEQ ID NG:3 is a raamrnalian-codon optimized DNA sequence encoding ChRSS light-

15 activated ion channel polypeptide

atggctgagctgatcagcagcgccaccagatctcigttigccgccggaggcatcaaccct iggcctaacccct

atgggctgtggaggaatgac cctacaggcgagtgcltcagc^

tacggatattgcticgtggaggccacaggcggctacctggtcgtgggagtggagaagaag caggctt

accaggagaaaagatcggcgcccaggtctgccagtggattgctttcagcatcgccatcgc cctgctgacattctacggcttcagcgcc 0 tggaaggccacttgeggttgggaggaggtctaegtctgttgcgtcgagg

ccccgccacagtgtacctgtciaccggcaaccacgcctattgccigcgctacttc

gctgagcaacctgagcggx tgaagaacgactacagcaagcggaccatgggcctgatcgtgtcttgcgtgggaatgatcg tgttcgg catggccgcaggactggctaccgattggctcaagtggctgctgtatatcgtgtctigcat ctacggcggctacatgtactt

caagtgctacgtggaagccaaccacagcgtgcctaaaggccattgccgcatggtcgtgaa gctgatggcctacgcttacttcgcctctt 5 ggggcagctacccaatcctctgggcagtgggaccagaaggactgctgaagctgagcc

tgcgacatcatcgccaaggagrttiggaccticctggcccaccacctgaggatcaaga ccac

tccggaagaccaccaagatggagatcggaggcgaggaggtggaagtggaagagttcgtgg aggaggaggacgaggacacagtg 30 SEQ ID NO:4 is transmembrane region of ChRSS including residues 86-320 of SEQ ID NO:2

GTPGEKIGAQVCQWIAFSLAIALLTFYGFSAW ATCG EEVYVCCVEVLFVTLEIFKE FSSPAWYLSTGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGM: IVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVP GHCRMVVK LMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTF.LAHHLRIK3 5 FiEHlLIH.

SEQ ID NO:5 is derived from ChR88 and includes 176R substitution

MAELISSATRSLFAAGGI PWPNPYIIHEDMGCGGMTPTGECFSTEWWCDPSYGLSD AGYGYCFVEATGGYLVVGVE QAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFY

0 GFSAWKATCGWEEVYVCCVEVLFVTLEIF EFSSPATVYLSTGNHAYCLRYFEWLLS CPVIL RL·S LSGLKNDYSKRTMGL VSCVGMΓVTGMAAGI.ATDWL WLLYIV ^' SCIY GGY YFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLE KLSPYANS:IGHSICDIIA EFWTFLAHHLRI :IHEH]:OHGDIRKI KMEICH3EEVEVEEF VEEEDEDTV.

5

SEQ ID NO:6 is amino acid sequence -from Sligeoc mum helveiicum

METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFP HINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSF ATTGWEEVWCVIELVKCFIE

I. HEVDSPAT QTNGGAVIWLRYSMWLETCPVILIB LSNLTGLILEEYS RTMTILV 50 TDIGNIVWGITAAFTKGPL ILFFMIGLFYGVTCFFQIA VYIESYHTLP GVCR IC ]

! 3 MAYVFFCSWI FPVMFIAGHEGLGIJTPYTSGIGHIJLDnSK TWGFLGHHLRV IH

EHlLiBGDiR TTTINVAGENMEiETFVDEEEEGGVNHGTADLAHRASFQKMGDRLR AQGVTVIlASLDAeEVPPADEENKFAOKSAAANMPAYNPGKVlLlVPDMSMVDYFR DQF EQLPTRME LLP ALGMDT .

SEQ ID NO:7 is ChR90 coding amino acid sequence that includes residues 1-325 of SEQ I ^' D NO:6

METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFP

HlNFiGTELOFl iAVGLOWFTViVAIVOLIFYG HSFKATTGWEEVYVCViELVKCFiE

LFHEVDSPATVTQTNGGAVIWLRYS WLLTCPVILIHLSNLTGLHEEYSKRTMTILV

TDIGNIVWGlTAAn GPLKlLFF IGLFYGVTCFFQJAKVYIESYHTLPKGVCR ICKl

MAYVFFCSWi.MFPVMFIAGHEGLGlJTPYTSGIGHLILDIJSK rWGFLGHHLR.VKIH

EHILIHGDiRKTT lNVAGENMEiETFVDEEEEGGV.

SEQ ID NO:8 is a manimalian-codon optimized DNA sequence encoding ChR90 light- activated ion channel polypeptide

atggaaacagccgccacaatgacccacgcctttatctcagccgtgcctagcgccgaa gccacaattagaggcctgctgagcgccgc agcagtggtgacaccagcagcagacgctcacggagaaacctct2^cgccacaacagccgg agccgatcacggttgcttcccccac atcaaccacggaaccgagctgcagcacaagatcgcagtgggactccagtggttcaccgtg atcgtggctatcgtgcagctcatcttct acggttggcacagcttcaaggccacaaccggctgggaggaggtctacgtctgcgtgatcg agctcgtcaagtgcttcatcgagctgtt ccacgaggtcgacagcccagccacagtgtaccagaccaacggaggagccgtgatttggct gcggtacagcaigtggctc gccecgtgatecigatecaceigagcaacctgaecggactgcacgaagagtacagcaagc ggaccatgaccatcctggtgaccgac ateggeaacatcgtgtgggggatcaeagccgcetitacaaagg

gacitgcttcttccagatcgccaaggtgtatatcgagagctaccacacccigcccaa aggcgtctgccggaagatttg

cetacgtctrcticigetc^

gaatcggccacctgatcctggatctgatcagcaagaacacttggggcttcctgggcc accacctgagagtgaagatccacgagcaca tcctgatccacggcga.catccggaagacaacc-accat.caacgt.ggccggcgagaac a.tggagatcga.ga.ccttcglcgacgagga ggaggagggaggagtg.

SEQ ID MO:9 is transmembrane region of C R90 including residues 61 -295 of SEQ ID NO: 7

GTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFH E

VDSPATVYQT GGAVIWLRYSMWLLTCPVILiHLSNLTGLHEEYSKRTMTILVTDIG

NIVWGrrAAFTKGPLKILFFMIGLFYGVTCFFQlAKVYiESYHTLP iVCRK iCKIMAY

VFFCSWLMFPVMFIAGHEGLGLlTPYTSGiGHLiLDLISK TWGFLGHHLRVKlHEHIL

IH.

SEQ ID NO: 10 is amino acid sequence from Chloromon s subdivisa

MSRLVAASWLLALLLCGITSTTTASSAPAASSTDGTAAAAVSHYAMNGFDELAKGA

WPEDHFVCGPADKCYCSAWLHSHGSKEEKTAFTVMQWIVFAVCHSLLFYAYQTW

RATCGWEEVYVTHELVHVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTCPVi

LrHLS LTGMKMDY KRTMALLVSDVGCI\AVG ^' TTAALSTDFVKiIFFFLGLf..YGFYTF

YAAAKIYIEAYHTVPKGICRQLVRLQAYDFFFTWSMFPILFMVGPEGFGK.1TAYSS GI

AHEVCDLLSK LWGLMGHFIRVKIHEHiLVHGNITKKTKVNVAGDMVELDTYVDQ

DEEHDEGTIDRGTQELANRHSFVVMRENMRAKGVDVRASLGDIDGTEMTKAGNMN

GTLEPGRilLCVPDMSLVDFFREQFSQMPVPFE VPALGPEVALQLVQQALSIGGANY

IDYVM.

SEQ ID NO: 1 1 ChR87 coding amino acid sequence that includes residues 1-346 of SEQ ID NO: 10

!4 MSRLVAASWLLALfJXGITSTTTASSAPA.ASSTDGTAAAAVSHYAMNGFDELAK.GA VVPEDHFVCGPADKCYCSAWLHSHGSKEEKTAFTVMQWIVFAVCIISLLFYAYQTW RATCGWEEVYVTIiELVeVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTCP l IJ.HLSNLTGMK DYNKRTMALLVSDVGCfVWGTTAALSTDFVKI]FFFLGLLYGFYTF YAAAKlYIEAYHTVPKGlCRQLVRLQAYDFFFTWSMFPiLFMVGPEGFGKITAYSSGI AHEVC;DLLSKNLWGLMGHFiRVKlHEHlLV HG rrKKTKV VAGDMV£LDTYVDQ DEEHDEG.

SEQ II) ^' NOT 2 is a mammaiian-codon optimized DNA sequence encoding ChR.87 light- acti ated ion channel polypeptide

atgagcagactggtcgccgcttctt^

cttcttctacagacggaacagccgccgcagc gtgtctcactacgccatgaacggcttcgacgagctggctaaaggagccgtggtgc cagaagaceactHgtctgeggaccagcegacaag

gccticaccgtcatgcagiggaicgtgitcgccgicigcatcatcagcctgcigttc iacgcciacca

gggaggaggtgtacgtgaccatcatcgagctggtccacgtctgcttcggactctggc acgaggtcgatagcccttgtaccctgtacctg agcacaggcaacatggtcctctggctgagaiacgccgagtggctgctgacitgccccgtg atcctgaiccaccigagcaacc gcatgaagaaegacfacaacaagcggaccatggcc^^

ageacegatttcgigaagatcateticitcrt^

cctaccacaccgtgcccaagggcatt^

tgttcatggtcggcccagagggattc-ggcaagatcaccgcciacagcagcggaatc gcccacgaagtgtgcgatctgctgagc-aag aaccfc ggggcctgatgggccacrtca ccgcgtgaagatccacgagcacatcctgg

ggtcaacgiggccggcgacaiggiggaaciggacacctacgtggaccaggacgagga acacgacgaggga,

SEQ ID NO; 13 is transmembrane region of ChR87 including residues 81 -315 of SEQ ID NO: 1 1

GSKEEKTAFTVyQWlVFAVCIiSLLFYAYQTWRATCGWEEVYVTliELVHVCFGLWH EVDSPCTLYLSTG MVLWLRYAEW.LLTCPVILIHLSNLTGMKNDYNKRTM.ALLVSD VGCIVWGTTAALSTDFVKIIFFFLGLLYOFYTFYAAAKIYTEAY ITVPKGICRQLVRLQ AYDFFFTWSMFPlLFMVGPEGFG iTAYSSGIAHEVCDLLS NLWGLMGHFlRVKiHE f ill A i l.

SEQ ID NO: 14 amino acid sequence for Neoch ros rci sp. Rhodopsin. This light- activated ion channel is referred to herein as ChR62.

MADFVWQGAGNGGPSAMVSHYPNGSVLLESSGSCYC.EDWYTSRGNHVEHS.LSNAC DWFAFAISViFLVYYAWAAFNSSVGWEErYVCTVELiKVSlDQFLSSNSPCTLYLSTG NRVLWIRYGEWLLirPVILiHLSNWGLKDNYSKRTMALLVSDIGTIVFGVTSAMCT GYPKVlFFlLGCCYGANTFFNAAKVYLEAHHTLP GSCRTLiRLMAYTYYASWGMFP ILFVLGPESFGHMNMYQSNlAHTVIDLMSKNIWGMLGHFLRHKrREHILIHGDLRTTT TVNVAGEEMQVETMVAAEDADETTV.

SEQ ID ^' NOT 5 is the mammalian codon-optiraixed DNA sequence for the Neochloro rcin rhodopsin. This light-activated ion channel is referred to herein as ChR62.

atggccgacttcgtgtggcagggagctggaaacggaggaccaagcgccatggtgtcc cactaccccaatggcagcgtgctgctgga gagctccggcagctgeiactgtgaagactggtataeitetcggggeaaceacgtggagca ttctctgagtaatgcttgcgattggttcgc ctttgctaicagcgtgattttcctggtgtactaigcctgggccgcttttaactctagtgt gggctggg

agcigatcaaggtgagcatigatcagttccigagctccaactctccttgtaccctgt acctgagtacag

gaiaiggcgaatggctgctgacttgtecagigatcctgaitcaecigtceaacgtga cagggctgaagg

tggetetgctggtgagtgatatcgggaecatcgfgttc^

ggetgetgltatggagctaacaeatt^

etgaicaaaciaataacctataccta^

! 5 acatgtaecagtccaataicgcccataecgtgatt

attcgcgagcacatcctgaiicaiggagatctgcggaccacaactaccg

gtggcegctgaggacgcegatgaaacaactgtg.

SEQ ID NO.16 is the amino acid sequence for Heterochlamydomonas maequaU rhodopsin. This !ig i-activated ion channel is referred to herein as ChR 3.

MGGIGGGGIQPRDYSYGANGTVCVNPDVCFCLDWQQPFGSN ENNVSQGFQLFnA

LSAClL FYAYEWYKATCGWEEIYVCVVE SKlCiELVHEYDTPFCLYLATGSRVLW

L YAEWLMTCPVIIJHLSN!TGLGTDYMKRTMVLLMSDIGCIVFGATAAFANEGYV

CACFLLGMAWG NTFYNAA VYYESYVLVPSGiCKLLVAVMAGLYYVSWSLFPIL

FAIGPEGFGVlSLQASTlGHTlADVLSKN !WGLMGHFLRVQIYKHILLHGNIRi PIKLH

MLGEEVEVM.ALVSEEGEDTV.

SEQ ID NO: 17 is the mammalian eodon-optirnized DNA sequence for the

Heterochlamydomonas inaequalis rhodopsin, this light-activated io channel is referred to herein as ChR93.

atgggaggaattggcggaggcggcattcagcciagagaetacagetacggcgccaac ggaaeagtctgcgtgaaccecgacgtet gcttctgtetggattggcageagcccttcggctctaaeatggagaa

gectgcatectgaigttctacgccta^

agatttgcatcgagctggtgca-cgagtacgacacccccttttgcctgtacctggcc accggcagcagagtcctctggctgagatacgc cgagtggetcatgacitgecccgtgatcctgatccaccigagcaacatcaceggactggg cacegaciaeaacaagcg gctcctgatgagcgacatcggttgcatcgtgttcggcgccacagcagcattcgccaacga gggctacgigaagtgcgcttgtttcctgc tgggcatggcttggggcatgaacaccttctacaacgccgccaaggtgtactacgagagct acgtgctggtgccctccggaatttgcaa gc getggtggccgtgatggecggactgtactacgtgtcttggagc^^

gatcagcctgcaggccageaccattggccacacaatcgccgacgtgctgagcaagaa catgtggggcctgatgggecacttcetgc gggtgcagatctacaagcacatcctgctgcacggcaacatccggaagcctatcaagctgc acatgctgggcgaggaggtggaagtg atggctctggtgtccgaggagggagaggataecgtg.

SEQ ID NO: 18 is the mammalian codon -optimized DNA sequence that encodes the wild- tvpe Chaanelrhodopsin>2, (see: Boyclen, E. et al.. Nature Neuroscience 8, 1263 - 1268 (2005) and NageL G., et al. PNAS ovember 25, 2003 vol. 100 no. 24 13940-13945}, also referred to herein as ChR 2:

atggactatggcggcgctttgtctgcegtcggacgcgaact

aggatcaatgttactgtgc^ggatggattgaatctcgcggcacgaacggcgc^gacc gcgtcaaatgtcc^cagtggcttgeagc aggattcagcaitttgctgctgatgttctatgcctaccaaacctggaaatctacatgcgg ctgggaggagatctatgtgtgcgccati atggttaaggtgaitcicgagttcittiitga

geagagiggctgetcaettgieetgtcatccrt^^

gactceitgtctcagacatcgggaciatcgtglggggggctaccagc^

attgtgctatggc gcgaacaca^^^

cgigaccggcatggcaiggctgttmcgtgagctggggtatgttcccaattctcttca mig

tctatggctecacegiaggtcaeacgat^

agacgaagccgaggecggagccgtg.

SEQ ID NO: 19 is the amino acid sequence of the wild-type Channeirhodopsin-2, , (see: Boyden, E. et aL Nature Neuroscience 8, 1263 - 1268 (2005) and Nagel, G., et al. PNAS November 25, 2003 vol 100 no, 24 13940-13945), also referred to herein as ChR2:

MDYGGALS AVGRELLFVT PVV VNGSVLVPEDQCYC AG W !ESRGTNGAQTASN VE QWLAAGFSiLLf. FYAYQTWKSTCG\VT,EIYVCAIEMVKViLEFFFEFKNPSMLYLA.T GHRVQWLRYAEWLLTCPVIliHLSNLTGLSNDYSRRTIVlGEEVSDIGT!VWGATSA

! 6 ATGYV VfFFCLGLCYGA TFFHAAKAYIEGYHTVPKGRCRO TGMAWLFFVSW

GMFPILFILGPEGFGVLSVYGSTVGBTIIDLMSKNCWGLLGHYLRYLIHEHILIHGD IR KTTKLNIGGTEIEVETLVEDEAEAGAV.

5 SEQ ID NO.20 is the DNA sequence of the 'ss' signal sequence from truncated MHC class Ϊ antigen; gtcccgtgcacgctgctcctgctgttggcagccgccctggctccgactcagacgcgggcc .

SEQ ID NO:21 is the amino acid sequence of the 'ss' signal sequence from truncated MHC class I antigen: MVPCTLLLLLAAALAPTQTRA.

t o

SEQ ID NO:22 is the DNA sequence of the ER export sequence (also referred to herein as ER2": ttctgctacgagaaigaagtg,

SEQ I D NO:23 is the amino acid sequence of the ER export sequence (also referred to herein 15 as "ER2": FCYENEV.

SEQ ID NO:24 is the DN A sequence of KGC, which is a C terminal export sequence from the potassium channel Kir2.1 : aaatccagaattacttctgaaggggagtatatccctctggatcaaatagacatcaatgtt . 0 SEQ ID NO:25 is the amino acid sequence of KGC, which is a C terminal export sequence from the potassium channel Kir2. I : S ITSEGE YIPEDQIDI V.

DETAILED DESCRIPTION

The in vention in some aspects relates to the expression in cells of light-dri ven ion 5 channel polypeptides that can be activated by contact with one or more pulses of light, which resul ts in strong depolarization of the ceil. Light-activated channels of the invention, also referred to herein as light-activated ion channels can he expressed in specific cells, tissues, and/or organisms and used to control cells in vivo, ex vivo, and in vitro in response to pulses of light of a suitable wavelength. Sequences from Chlamydomonas such as Cimmson and

30 derivatives thereof, are strongly activated by contact with red light. In addition, light- activated ion channel polypeptides derived from Stigeockmiwn rhodopsin sequences, have now been identified and characterized as being activated by light having a wavelength between 365 nm and 630 nra.

The light-activated ion channels of the invention are ion channels and may be

35 expressed in a membrane of a cell . An io channel is an integral membrane protei that forms a pore through a membrane and assist in establishing and modulating the small voltage gradient that exists across the plasma membrane of all cells and are also found in subcellular membranes of organelles such as the endoplasmic reticulum (ER), mitochondria, etc, When a light-activated ion channel of the invention is activated by contacting the cell with

0 appropriate light, the pore opens and permits conductance of ions such as sodium, potassium, calcium, etc, through the pore.

! 7 In some embodiments of the invention, light-activated channels may be used to modify the transmembrane potential (and/or ionic composition) of cells (and or their sub-cellular regions, and their local environment). For example, the use of inwardly rectifying cationic channels will, depolarize cells by moving positively charged ions from the

extracellular environment to the cytoplasm. Under certain conditions, their use can decrease the intracellular pH (and/or cation concentration) or increase the extracellular pH (and/or ca tion concentrati on), i some embodiments, the presence of light-activ ated ion channels in one, two, three, or more (e.g. a plurality) of cells in a tissue or organism, can resul t in depolarization of the single cell or the plurality of cells by contacting the light- activated ion channels with light of suitable wavelength.

Chlamydomonas-derived Light-activated Ion Channels

When expressed in a cell, light-activated ion channels of the invention identified having a Chlamydomonas light-activated ion channel sequence or a derivative thereof, can be activated by contacting the cell with light having a wavelength between about 365 nm and 700 nm, between 530 nm and 640 nm, and in some embodiments, a peak activation may occur upon contact with light having a wavelength of 590 nm. Examples of these light- activated ion channels include ChR88 (also referred to herein as Chrimson), 176

substituted Chrimson sequence such as SEQ ID NO: 5; and functional derivatives thereof . in some embodiments of the invention, a Chlamydomonas light-activated ion channel, is a Chlamydomonas noctigama light-activated ion channel.

Contacting an excitable cell that includes a Chlamydomonas-dedvsd light-activated ion channel of the invention with a light in the activating range of wavelengths strongly depolarizes the cell. Exemplary wavelengths of li ht that may be used to depolarize a eel! expressing a Chlamydomonas-deiived light-activated ion channel of the invention, include wavelengths from at least about 365 nm, 385 nm, 405 nm, 425 nm, 445 nm, 465 nm, 485 nm, 505 nm, 525 nm, 545 nm, 565 nm, 585 nm; 605 nm, 625 nm. 645 nm. 665 nm. 6S5 nm; and 700 nm, including all wavelengths therebetweeen. In some embodiments, Chlamydomonas- derived light-activated ion channels of the invention have a peak wavelength sensitiviiy in of 590 nm, and may elicit spikes up to 660 nm.

In some embodiments of the invention, a Chlamydomonas-derived light-activated ion channel, a non-limiting example of which is Chrimson or a K176R substituted Chrimson set forth as SEQ ID NO:5, can drive temporally precise spikes with 1 -5ms pulse width at

0.5m\V/xnm ² to >10mW/xnm ² in neurons at its optimal wavelength m slice and m cell culture; and can stimulate at -frequency up to l Oi !z reliably at its optimal wavelength. In some embodiments of the invention., an optima! wavelength for a Chlamydomonas-de ed Sight- activated ion channel is between 530 nm and 640 nm. in certain embodiments of the invention, the substituted C lamydomonas-denved light-activated ion channel having an 5 amino acid sequence set forth as SEQ ID NO: 5 , has a decreased tau off from 25ms to 13ms, and this K176R mutant can stimulate at frequency up to 60Hz reliably at optimal wavelength, which ma be between 530 nm and 640 nm.

Li ht-aciivated ion channels of the invention such as ChR88 and derivatives thereof can be used to depolarize excitable cells in which one or more light-activated ion channels of {( ⁾ the invention are expressed. In some embodiments, Chlamydomoms-dedved light-activated ion channels of the invention can be expressed in a sub-population of cells in a cell population that also includes one or more additional subpopulations of cells that express light-activated ion channels that are acti vated by wavelengths of light thai do not activate a Chiam ⁱ dnmonas-denved light-activated ion channel of the invention.

1 5

Sligeoclonium derived J ght~activated Jon Channels

When expressed in a cell, light-activated ion channels of the invention identified having a Stigeoctonium light-activated, ion channel sequence or a derivative thereof, can be activated by contacting the cell with light having a wavelength between about 365 nm and 0 630 nm, between 430 nm and 550 nm; and in some embodiments, a peak activation may- occur upon contact with light having a wavelength of 500 nm. Examples of these light- activated ion channels include ChR90 (also referred to herein as Chronos) and functional derivatives thereof, hi some embodiments of the invention, a Stigeockmium light-activated ion channel is a Stigeockmium helveii um light-activated ion channel.

5 Contacting an excitable cell that includes a Stigeockmium-demed light-activated ion channel of the invention with a light in the activating range of wavelengths strongly depolarizes the cell . Exemplary wa velengths of Sight that may be used to depolarize a cell expressing a Stigeoehnhtm-denved light-activated ion channel of the invention, include wavelengths from at least about 365 nm, 385 nm, 405 nm, 425 nm, 445 nm, 465 nm, 485 nm, 0 505 nm, 525 nm, 545 nm, 565 nm, 585 nm; 605 nm, and 630 nm, including all wavelengths therebefweeen. In some embodiments, Stigeoc nium-denved light-activated ion channels of the invention have a peak wavelength sensiti vity in of 500 nm. In some embodiments of the invention, a Stigeocionium-dexived light-activated ion channel can drive temporally precise spikes with l -5ms pulse width at 50u\¥/mn to >10m\¥/mnr in neurons at "optimal

! wavelength" in slice and cultured cells; and can stimulate at frequency >100Hz at "optima! wavelength" As used herein an optima! wavelength for a Sfigeockmium-detived light- activated ion channel may be a wavelength between 430nm and 550nm.

Light-activated ion channels of the invention such as ChR90 and derivatives thereof can be used to depolarize excitabie cells in which one or more light-activated ion channels of the invention are expressed. n some embodiments, Stigeochnium-derived light-activated ion channels of the in vention can be expressed in a sub-population of cells in a cell population that also includes one or more additional subpopulaiions of cells that express light-activated ion channels that are activated by wavelengths of light that do not activate a Siigeoc ni m- derived light -activated ion channel of the invention.

Chloro onas-derived Lighl-aclivaled Ion Channels

When expressed in a cell, light-activated ion channels of the invention identified having a Chhromonas light-activated ion channel sequence or a derivative thereof, can be activated by contacting the cell with light having a wavelength between about 365 nm and 630 nm, between 450 nm and 570 nm; and in some embodiments, a peak activation may occur upon contact with light having a wavelength of 525 nm. In some embodiments of the invention, a Chhromonas light-activated ion channel, a non-limiting example of which is ChR87, does not exhibit light sensitivity (activation) at wavelengths greater than 650nm, and can drive temporally precise spikes with l.-5ms pulse width at O. lmW/rara'' to greater than !OmW/mnT ⁴ in neurons at its optimal wavelength in both slice and cell culture. In some embodiments of the in vention, a Chhromonas light-activated ion channel, (such as ChR.87) can stimulate at frequency >60Hz at its optimal wavelength, in some aspects of the invention the optimal wavelength for a Chhromonas light-activated ion channel, a non-limiting example of which is ChR87, is between 450 and 570 mn. Examples of Chhromonas light- activaied ion channels include ChR.87 and functional derivatives thereof. In some embodiments of the invention, a Chloromonas light-activated ion channel is a Chhromonas s hdivisa light-activated ion channel, Chhromonas-, CMamydomonas^ and Siigeochnium-denv d light-activated ion channels of the invention permit ion conductance and depolarization when contacted under suitable conditions with an appropriate wavelength of light. As will be understood by those in the art, the term "depolarized" used in the context of cells means an upward change in the cell voltage. For example, in an excitable cell at a baseline voltage of about -65 mV. a positive change m voltage, e.g., up to 5, 10, 1.5, 20, 30, 40, or more millivolts (mV) is a depolarization of that cell. When the change in voltage is sufficient to reach the cell's spike initiation voltage threshold an action potential (e.g. a spike) results. Whe a ceil is depolarized by activating a light-activated ion channel of the invention with an appropriate wavelength of light, the cell voltage becomes more positive than the baseline level, and an incoming signal may more easily raise the cell's voltage sufficiently to reach the threshold and trigger an action potential in the cell, it has been, discovered that by contacting a cell expressing a Ch mydomonas-denved light-activated ion channel of the invention with light in the range between about 365 nm to about 700 ran, the voltage of the cell becomes less negative and may rise by at least about 20, 30, 40, 50, 0, 70, 80, 90, 100, raV (depending on the cell type) thus, depolarizing the ceil. Similarly, it has been discovered that by contacting a cell expressing a Stigeocfonium-denved light-activated ion channel of the invention with light in the range between about 365 ran and 630 nm the voltage of the cell becomes less negative by as much as at least 20, 30, 40, 50, 60, 70. SO, 90, 100, raV, (depending on cell type). Similarly, it has been discovered that by contacting a eel! expressing a Chhromonas- derived light-activated ion channel of the invention with light in the range between about 365 nm and 630 nm, or between 450 nm and 570 nm the voltage of the eel! becomes less negative by as much as at least 20. 30, 40, 50, 60, 70, SO, 90, 100, mV, (depending on ceil type). As used herein, the term "activate" when used in reference to a light-activated ion channel of the invention, means to ope the channel making it permissive to ion conduction through the channel .

Specific ranges of wavelengths of light that in some embodiments of the invention are useful to activate ion channels of the invention are provided and described herein, it will be understood that a light of appropriate wavelength for activation and will have a power and intensity appropriate for activatio . It is well known in the art that light pulse duration, intensity, and power are parameters that can be altered when activating a channel with light. Thus, one skil!ed in the art will be able to adjust power, intensity appropriately when using a wavelength taught herein to activate a light-acti vated ion channel of the invention. A dose l ight that contacts a light-activated ion channel of the in vention may be determined based on the wavelength, pulse Length, and power of the light that contacts the light-activated ion channel. Thus, as a non-limiting example, a dose may have a wavelength of 550 nm, a 4 rns pulse length, and a 0.5 mW/mnV ^* power and another light dose may have a wavelength of 550 nm, a 3 ras pulse length and a 0.5 raW/mm ² power. Those skilled in the art will understand methods to select a dose of light by independently selecting a wavelength, a pulse length, and a power for the light with which a light-activated ion channel of the invention is contacted, in some embodiments, wavelength and pulse length may be held steady; and power incrementally increased to examine activation parameters of a light-activated ion channel of the invention. Similarly, in certain embodiments of the invention may include incremental wavelength increases while pulse length and power are held steady; or incremental pulse length increases while wavelength and power are held steady. In some embodiments of the invention two or more of wa velength, pulse length, and power of a light may he

incrementally altered to examine the effect on activation of a light-activating ion channel of the invention.

A benefit of a light-activated ion channel of the invention is the ability to "tune" the light-activated ion channel's response using appropriate illumination variables (e.g., wavelength, intensity, duration, etc.) (also referred to herein as dose) to activate the channel. Methods of adjusting illumination variables are well-known in the art and representative methods can be found in publications such as: Lin, J., et al.. Biophys. J. 2009 Mar

4;96(5):.l 803-1 ; Wang, H„ et al, 2007 Proc Natl Acad Sci U S A. 2007 May

8; 104(1 ): 8143-8. Epub 2007 May 1 , each of which is incorporated herein by reference. Thus, it is possible to utilize a narrow range of one or more illumination characteristics to activate a light-activated ion channel of the invention. T he expression of light-activated ion channels that are activated by different wavelengths of light in distinct, separate,

subpopulations in a cell population can permit application of different illumination parameters to the population with an effect of differentially activating the different subpopulations through the use specific wa velengths of light. Thus, permitting controlled activation of a mixed population of light-activated channels.

In exemplary implementations, the invention comprises methods for preparing and using genes encoding light- activated ion channels of the invention that have now been identified. The invention, in part, also includes isolated nucleic acids comprising sequences that encode light activated ion channels of the invention as well as vectors and constructs that comprise such nucleic acid sequences. In some embodiments the invention includes expression of polypeptides encoded by the nucleic acid sequences, in cel ls, tissues, and organisms.

Not all cha.onelrhodops.ins can be expressed in cells and utilized in this fashion, because many do not traffic properly and/or function in mammalian cells. Many

channelrhodopsms were screened in order to identify Ch 87, Chrimson, and Chronos as functioning better in mammalian cells than other classes of channelrhodopsms. n addition Chrimson responds strongly to far red light, and therefore, because other channeirhodopsms that depolarize ceils respond strongly to ultraviolet or blue light, Chrimson can be expressed in a separate population of cells from a population of cells expressing one of these other opsins, allowing multiple colors of light to be used to excite these two populations of cells or neuronal projections from one site, at different times.

In some embodiments of the invention, li ht-activated channels are used to modify the transmembrane potential (and/or ionic composition) of cells (and/or their sub-cellular regions, and their local environment). In particular, the use of inwardly rectifying ca tionic channels will depolarize cells by moving positively charged ions from the extracellular environment to the cytoplasm. Under certain conditions, their use can decrease the intracellular pH ( and or cation concentration) or increase the extracellular pH (and/or cation concentration).

Compared to natural gene sequences conventionally used to depolarize neurons, Chronos has demonstrably improved photocurrent generation at all illumination wavelengths except for red wavelength. In addition Chronos can depolarize cells in response to <5ms pulse of 50- 100 uW mm ^"* of blue or green light with sufficient spectral independence from most green or red ehanne hodopsins such as ChRS7 or Chrimson, thus permitting multiple colors of light to be used to depolarize different sets of cells in the same tissue, simply by expressing pumps with different activation spectra genetically in different cells, and then ilhiminating the tissue with different colors of light, hi a non-limiting example of an.

embodiment, one set of cells in a tissue, for example excitatory neurons, express Chrimson. and a second set express Clironos, illuminating the tissue with 630 nra light will

preferentially depolarize the first set, and illuminating the tissue with 470 nm light at low powers (<5 mW mm ^"2 ) will preferenti ll depolarize the second set. Other pairs of targets that could be modulated with two colors of light in the same illumination area include, but are not limited to two projections to/from one site, or combinations of the cell, its projections, and its organelles, given the ability to target the molecule sub-celliilarly.

It has been identified that light-activated ion channels of the invention are, in some embodiments of the invention, activated at different wavelengths than previously identified light-activated ion channels. Thus, light-activated ion channels of the invention can be used in either alone, using a selective light spectrum for activation and depolarization and can also be used in combination with other light-activated ion channels that utilize different wavelength of light for activation and depolarization, thus allowing two, three, four, or more different wavelengths of light to be used to depolarize different sets or subpopulations of cells in a tissue or organism by expressing channels with different activation spectra in different, cells and then iHummatmg the tissue and/or organism with the appropriate wavelengths of light to activate the channeis and depolarize the cells.

According to some aspects of the invention, a light-activated ton channel from

Chlamydomonas nacttgama or a derivative thereof may be used in conjunction with a light- activated ion channel from Stigeoc nium helveticum or a derivative thereof. The two light- activated ion channels are sensitive to and can be activated with different vvaveiengths of light than each other. As described herein, certain Sight-activated ion channels of the invention can depolarize cells in strong response to light with sufficient spectral independence from that of other Sight-activate ion channels of the invention, thus allowing multiple colors of light to be used to depolarize different sets of cells in the same tissue, by expressing channels with different activation spectra genetically in different cells, and then illuminating the tissue with different colors of light in suitable dose to activate one type of light-ac tivated ion channel but not the other type of light-activated ion channel. In a non-limiting example, if a first subset of cells in a tissue (e.g., excitatory neurons) express ChR88, and a second subset express ChR90 light-activated ion channels of the invention, then illuminating the tissue with a dose of light such as 625nin, 2 mWVmm ² will preferentially depolarize/drive spike in the first subset

(ChRSS), and illumina ting the tissue with a dose of light such as 470 nm 0.2 mW/mm ² light will preferentially depolarize/drive spike in the second subset (ChR90). Other pairs of targets that could be modula ted with two colors of light in the same il lumination area include, but are not limited to two projections to/from one site, or combinations of the cell, its projections, and its organelles, given the ability to target the molecule suh-cellutariy.

Taxonomy and sequence sources

In particular, the present invention includes, in part, novel light-activated ion channels and their use to depolarize cells, in some non-limiting embodiments of the invention one or more newly identified light-activated ion channels may be expressed in ceils.

Some light-acti vated ion channels of the invention have amino acid sequences derived from Chlamydomonas rhodopsins that are natural Sy expressed in the genus Chlamydomonas nociigama,. or another member of the Chlamydomonadaceae family. Chlamydomonas noctigama are phytoplankton and can be found in fresh water environmenis. Some embodiments of the invention include isolated wild-type or modified nucleic acid and/or amino acid channelrhodopsin sequences from a member of the chlamydomonadaceae family. for example, from CMamydomonas noctiga a, and m some aspects, the invention also includes methods for their use.

Some light-activated ion channels of the invention have amino acid sequences derived 5 from Stigeochnium rhodopsins that are naturally expressed in the genus Stigeochnium

helveiicum, or another member of the Chaetophoracea family. Stigeochnium helveticum are green, algae and can be found in fresh water environments. Some embodiments of the invention include isolated wild-type or modified nucleic acid and/or amino acid

channelrhodopsin sequences from a member of the Chaetophoracea family, for example, {() from Stigeochnium helveticum, and in some aspects, the invention also includes methods for their use.

Some light-activated ion channels of the invention have amino acid sequences derived from Chhromonas rhodopsins thai are naturally expressed in the genus Chhromonas suhdivisa, or another member of the Chlamydomonadaceae family. Chhromonas suhdivisa 15 are phytoplankton and can be found in fresh water environments. Some embodiments of the invention include isolated wild-type or modified nucleic acid and or amino acid

channelrhodopsin sequences from a member of the Chlamydomonadaceae family, for example, from Chhromonas suhdivisa, and in some aspects, the invention also includes methods for their use.

0 Some light-activated ion channels of the invention have amino acid sequences deri ved from Neochlorosarcma and some light-activated ion channels of the invention have amino acid sequences derived from Hetero hlamydomonas inaequalis rhodopsins. Some embodiments of the invention include isolated wild-type or modified nucleic acid and/or amino acid channelrhodopsin sequences from Neochlorosarcma or from

5 Heterochlamydomonas inaequalis, and in some aspects, the invention also includes methods for their use. Sequences of light-activated ion channels of the invention, may be derived from a Chloromanas sequence, a CMamydomonas sequence, or Stigeochnium sequence and may include a wild-type or modified channelrhodopsin sequence, also referred to as a derivative sequence.

0 A modified light-activated ion channel polypeptide of the invention (also referred to as a derivative of a. light-aciivated ion channel) versus a naturally occurring light activated ion channel may be altered by an substituting 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids, by an insertion and/or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids at one or several positions. Both a wild-type light-activated ion channel poiypepiide and derivatives thereof may have an identity of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, with the sequence of the transmembrane region of the wild-type light-activated ion channel polypeptide, as long as they retain channel functionality. For ChR87, the amino acid sequence of the transmembrane region is set forth as SEQ ID NO: 13, which includes amino acid residues 81-315 of SEQ ID NO: 1 1. For

ChR88, the amino acid sequence of the transmembrane region is set forth as SEQ ID NO:4, which includes amino acid residues 86-320 of SEQ ID NO:2. For ChR.90, the amino acid sequence of the transmembrane region is set forth as SEQ ID NO: 9, which includes amino acid residues 1-295 of SEQ ID NO:7. A derivative or modi fied light-activated ion channel polypeptide of the invention may retain an identity of 20% or more of the transmembrane amino acid sequence from which it was derived.

In contrast, the level of identity between a derivative or modified light-activated ion channel of the mvention and the wild-type from which it is derived may be more constrained to maintain the function of the light-activated ion channel . The amino acid sequence of a non-transmembrane region of a derived or modified li ht-activated ion channel of the invention, may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of the wild-type light-activated ion channel polypeptide from which they are derived.

Thus, for example, in some embodiments, a light-activated, ion channel of the invention may be a derivative of Ch 87 and have at least 80%, 85%, 90%, 95%, 96%, 7%, 98%, 99% or 100% identity to the non-transmemhrane regions of the wild-type polypeptide sequence (SEQ ID NO.1 1 ) of the light-activated ion channel polypeptide from which it is derived. In another non-limiting example, in some embodiments, a light-activated ion channel of the invention may be a derivative of ChR90 and have at least 80%, 85%. 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the non-transmembrane regions of the wild- tvpe polypeptide sequence (S EQ ID NO: 7) of the light-ac tivated ion channel polypeptide from which it is derived. Similarly, in another example, in some embodiments, a light- activated ion channel of the invention may be a derivative of ChR88 and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or .100% identity to the non-transmembrane regions of the wild-tvpe polypeptide sequence (SEQ ID NO:2) of the light-activated ion channel polypeptide from which it is derived. In another non-limiting example of a light-activated ion channel derived from ChR88, a polypeptide that includes K176R substitution in the amino acid sequence of SEQ ID NO: 2 functions as a light-activated ion channe! of the invention. As used herein, the term "identity" refers to the degree of tela tecln ess between two or more poiypeptide sequences, which may be determined by the match between the sequences. The percentage is obtained as the percentage of identical amino acids in two or more sequences taking account of gaps and other sequence features. The identity between

polypeptide sequences can be determined by means of known procedures. Algorithms and programs are available and routinely used by those in the art to determine identity between polypeptide sequences. Non-limiting examples of programs and algorithms include

BLASTP, BLASTN and FASTA (Altschul et al. _s NCB ΝΙ.,Μ NIH Bethesda Md. 20894; Altschul et ah. 1990), Onl ine B LAST programs from the National Library of Medicine are available, for example, at blast.tjtcbi.nlm.nih.gov/Blast.cgi.

One skilled in the art will understand that light-activated ion channels of the invention can be identified based on sequence similarity or homology to a light-acti vated ion channel disclosed herein, it will be understood that additional light-activated ion channels may be identified using sequence alignment with one of the light-activated ion channels or derivatives thereof identified herein.

Based on the teaching provided herein regarding the Cholorman s subdivis .

Chlamydomonas noctigama, Stigeocionium heiveiiam channelrhodopsin sequences having light-activated ion channel function and activity, additional rhodopsin sequences with sufficient amino acid sequence homology to a ChR87 _} ChR.88, or ChR90, respectively, can be identified. The presence of functionality, e.g., activation of a channel by contact with suitable light can be determined using methods described herein, and function light-activated ion channels of the invention can be used in methods described herein. It is understood that that the level of sequence identity with a light-activated ion channel of the invention plus functionality with respect to activation by suitable light contact can be characteristics used to identify additional light-activated ion channels using standard procedures for sequence alignment, comparisons, and assays for ion channel activity.

Light-activated ion channels of the invention are transmembrane channel polypeptides that use light energy to open, permitting ion conductance through their pore, thus altering the potential of the membrane in which they are expressed. A non-limiting example of an ion that can be moved through a pore of the invention includes a sodium ion, a potassium ion, a calcium ion, a proton, etc. Routine methods may be used to measure different ion currents for light-activated ion channels of the invention. Light-activated ion channels of the invention can. be activated by sustained light and/or by light pulses and by permitting ion conductance upon activation,, light-activated ion channels of the invention can depolarize cells and alter the voltage in cells and organelles in which they are expressed.

The wild-type and modified (derived) Cholormotms sukdivisa, Chlamydornonas noctigama _i Siigeoc nium helveticum rhodopsin nucleic acid and amino acid sequences used 5 iii aspects and methods of the invention are "isolated" sequences. As used herein, the term "isolated" used in reference to a polynucleotide, nucleic acid sequence, or polypeptide sequence of a rhodopsin, means a polynucleotide, nucleic acid sequence, or polypeptide sequence tha is separate from its native environment and present in sufficient quantity to permit its identification or use. Thus, an isolated polynucleotide, nucleic acid sequence, or

{( ⁾ polypeptide sequence of the invention is a polynucleotide, nucleic acid sequence, or

polypeptide sequence that is not part of, or included in its native, wild-type cell or organism. For example, a nucleic acid or polypeptide sequence may be naturally expressed in a ceil or organism of a member of the Chloromonas genus, but when the sequence is not part of or included in a Chloromonas cell or organism it is considered to be isolated. Similarly, a

15 nucleic acid or poly peptide sequence may be naturally expressed in a cell or organism of a member of the Chlamydomottas genus, but the sequence is not part of or included in a Chlamydornonas cell or organism, it is considered to be isolated. Similarly, a nucleic acid or polypeptide sequence may be naturally expressed in a ceil or organism of a member of the Sdg oclonmm genus, but the sequence is not part of or included in a Sfigeockmmm cell or 0 organism, it is considered to be isolated. Thus, a nucleic acid or polypeptide sequence of a Chloromonas, Chlamydornonas, Stigeoclonium, or other light-activated ion channel nucleic acid or polypeptide that is present in a vector, in a heterologous cell, tissue, or organism, etc., is still considered to be an isolated sequence. As used herein the term "host" used in reference to a membrane or cell in which a light-activated ion channel of the in vention is 5 expressed, means a membrane or cell that is not a ceil or membrane in which the light- activated ion channel is expressed in nature. Thus a host membrane, cell, tissue, or organism for a light-activated ion channel molecule of the invention (such as a light-activated ion channel polypeptide or its encoding nucleic acid), as used herein is a membrane, cell, tissue, or organism in which the light-activated ion channel molecule of the invention does not 0 naturally occur and in which the light-activated ion channel is not naturally expressed.

Examples of a host membrane, cell, tissue, or organism include, but are not limited to mammalian (including but not limited to non-human primate, human, etc.), insect, and avian membranes, cells, and tissues; as well as organisms such as mammals, insects, and birds. The term "heterologous" as used herein, means a membrane, ceil, tissue, or organism that is not the native cell, tissue, or organism, and a light-activated ion channel polypeptide of the invention or its encoding nucleic acid may be present in a heterologous membrane, cell, tissue, or organism. The terms, "protein", "polypeptides", and "peptides" are used

interchangeably herein.

5

Light-activated Ion Channel sequences including modified and derived sequences

A light-activated ion channel of the inventio may comprise a wild-type polypeptide sequence or may be a modified polypeptide sequence. As used herein the ierm "modified" or "modification" in reference to a nucleic acid or polypeptide sequence refers to a change of

{() one, two, three, four, five, six, or more amino acids in the sequence as compared to the wild- type or other sequence from which it was derived. For example, a modified polypeptide sequence may be identical to a wild-type polypeptide sequence except that it has one, two, three, four, five, or more amino acid substitutions, deletions, insertions, or combinations thereof. In some embodiments of the invention a modified sequence may include one, two,

15 three, four, or more amino acid substitutions in a wild-type Sight-acti vated ion channel

polypeptide sequence, or in any other light-activated ion channel polypeptide sequence of the invention.

It will be understood that sequences of light-activated ion channels of the invention may be derived from various members of the Chloromonas genus, Mamyckmrnn s genus, 0 Stlg oclonmm genus. Neoch rosarcina genus, or Heterochlamydomonas genus.

The invention, in some aspects also includes light-activated ion channel polypeptides having one or more substitutions or other modifications from those described herein. For example, sequences of light-activated ion channel polypeptides provided herein can be 5 modified with one or more substitutions, deletions, insertions, or other modifications and such derivative light-activated ion channels can be tested using methods described herein for characteristics including, but not limited to: expression, cell localization, activation and depolarization in response to contact with light using methods disclosed herein. Exemplary modifications include, but are not limited to conservative amino acid substitutions, which will

3 produce molecules having functional characteristics similar to those of the molecule from which such modifications are made. Conservative amino acid substitutions are substitutions that do not result in a significant change in the activi ty or tertiary structure of a selec ted polypeptide or protein. Such substitutions typically involve replacing a selected amino acid residue with a different residue having similar physico-chemical properties. For example. substitution of Giu for Asp is considered a conservative substitution because both are similarly-sized negatively-charged amino acids. Groupings of amino acids by physico- chemical properties are known to those of skill in the art. The following groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Glycine (G ); 5 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginme (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), ^' Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7} Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)}. Light-activated ion channels that include modifications, including but not limited to one, two, three, four, or more conservative amino

{( ⁾ acid substitutions can be identified and tested for characteristics including, but not limited to:

expression, cell localization, activation and depolarization and depolarization-effects in response to contact with light using methods disclosed herein. As described elsewhere herein, in some polypeptide regions such as the transmembrane region of a light-activated ion channel of the invention, may include modifications that result in less than 100%, 95%, 90%,

15 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% identit with the sequence from which its

derived, yet may have at least 90%, 95%, 96%, 97%, 98%, 99%, or J 00% identity in its non- transmembrane regions of the polypeptide.

Sequence identity can be determined using standard techniques known in the art. Light -activated ion channel polypeptides of the invention may be shorter or longer 0 than the light-activated ion channel polypeptide sequences set forth herein. Thus, a light- activated ion channel polypeptide may be a full-length polypeptide or functional fragment thereof in addi tion, nucleic acids of the invention may be used to obtain additional coding regions, and thus additional polypeptide sequences, using techniques known in the art.

In some aspects of the invention, substantially similar light-activated ion channel 5 polypeptide sequences may have at least 80%, 85%, 90%, 9 i%. 92%, 93%, 94%, 95%, 96%.

97%, 98%, or 99 or 100 identity to a light-activated ion channel polypeptide sequence disclosed herein, non-limiting examples of which include as ChR62, ChR.93, ChRS7, ChRSS, ChR90, etc. Art-known alignment methods and tools can be used to align substantially similar sequences permitting positional ideniitication of amino acids thai may be modified as 0 described herein to prepare a light-activated ion channel of the invention. Standard sequence analysis tools and computer programs, such as those used for alignment, etc. can be used to identify light -activated ion channels of the invention that share one or more functional properties with a light-activated ion channel described herein. Sequence modifications can be in one or more of three classes: substitutions, insertions, or deletions. These modified sequences, (which may also be referred to as variants, or derivatives) ordinarily are prepared by site specific mutagenesis of nucleic acids in the DNA encoding a light-activated ion channel polypeptide, using cassette or PGR mutagenesis or other techniques known in the art, to produce DNA encoding the modified light-activated ion channel, and thereafter expressing the DNA in recombinant cell culture. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from natural !y occurring allelic or interspecies variation of the light-activated ion channels of the invention. Modified light-activated ion channels generall exhibit the same qualitative biological activity as the naturally occurring light-activated ion channel ( e.g., wild-type), although variants can also be selected that have modified characteristics.

A site or region for introducing an amino acid sequence modification may be

predetermined, and the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed modified light-activated ion channel screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M l 3 primer mutagenesis and PGR mutagenesis.

Amino acid substitutions are typical ly of single residues; and insertions usual ly will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions may range from about 1 to about 20 residues, although in some cases deletions may he much larger.

Substitutions, deletions, insertions or any combination thereof may be used to arri ve at a final modified light-activated ion channel of the invention. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certai circumstances.

Variants of light-activated ion channels set forth herein, may exhibit tire same qualitative light-activated ion channel activity as one or more of the sequences set forth herein, such as ChR62, CliR 3, ChR87, ChR88, or ChR90 _; but may show some altered characteristics such as altered photocurrent, stability, speed, compatibility, and toxicity, or a combination thereof For example, the polypeptide can be modified such that it has an increased photocurrent and/or has less toxicity than another light-activated ion channel polypeptide. A modified (or derived) light-activated ion channel polypeptide of the invention can incorporate unnatural amino acids as well as natural amino acids. An unnatural amino acid can be included in a light-activated ion channel of the invention to enhance a characteristic such as photocurrent, stability, speed, compatibility, or to lower toxicity, etc.

According to principles of this invention, the performance of light-activated ion channel molecules or classes of molecules can be tuned for optimal use, including in the context of their use in conjunction with other molecules or optical apparatus. For example, in order to achieve optimal contrast for multiple-color stimulation, one may desire to either improve or decrease the performance of one molecule with respect to on another, by the appendage of trafficking enhancing sequences or creation of genetic variants by site-directed mutagenesis, directed evolution, gene shuffling, or altering codon usage, light-activated, ion channel molecules or classes of molecules may have inherently varying spectral sensitivity. This may be used to ad vantage in vivo (where scattering and absorption will vary with respect to wavelength, coherence, and polarization), by tuning the linearity or non-linearity of response to optical illumination with respect to time, power, and illumination history.

^' In some embodiments, the invention includes the use of targeted site-directed mutagenesis at specific amino acid residues of channelrhodopsins including but not limited to residues of channelrhodopsins of the Chloromonas genus, Chlam domonas genus,

Sfigeociomum genus, Neochloros rcina genus, or Het rochlamydomonas genus. Specific locations for single mutations can be identified and alone, or in combination with two or more additional mutations can be placed into a channelrhodopsin sequence and tested with respect to their activation and photocurrent amplitude. Thus, sequences of light-activated ion channels of the invention, and/or similar channelrhodopsin sequences can be modified and the resulting polypeptides tested using methods disclosed herein.

Another aspect of the invention provides nucleic acid sequences that code for a light- activated ion channel of the invention. It would be understood by a person of skil l in the art that light-activated ion channel polypeptides of the present invention can be coded for by various nucleic acids, Each amino acid in the protein is represented by one or more sets of 3 nucleic acids (codons). Because many amino acids are represented by more than one codon. there is not a unique nucleic acid sequence that codes for a given protein, it is well understood by those of skill in the art. how to make a nucleic acid tha t can code for light- activated ion channel polypeptides of the invention by knowing the amino acid sequence of the protein. A nucleic acid sequence that codes for a polypeptide or protein is the "gene" of that polypeptide or protein. A gene can be RNA, DNA, or other nucleic acid than will code for the polypeptide or protein.

It is understood in t e art that the codo systems in different organisms can be slightly different, and thai therefore where the expression of a given protein from a give organism is desired, the nucleic acid sequence can be modified for expression within that organism.

Thus, in some embodiments, a light-activated ion channel polypeptide of the inventio is encoded by a matnmalian-codon-optitnized nucleic acid sequence, which may in some embodiments be a fmman-codon optimized nucleic acid sequence. An aspect of the mveniion provides a nucleic acid sequence that codes for a light-activated ion chamiei that is optimized for expression with a mammalian ceil. In some embodiments of the invention, a nucleic acid that encodes a light-activated ion channel of the invention includes a nucleic acid sequence optimized for expression in a human cell.

Delivery of Lighi- ictivaied Ion Channels

Delivery of light-activated ion channel polypeptide to a cell and/or expression of a light-activated ion channel in a cell can be done using art-known delivery means.

In some embodiments of the invention a light-activated ion channel polypeptide of the invention is included in a fusion protein, it is well known in the art how to prepare and utili ze fusion protei ns that comprise a polypeptide sequence. In c ertai n embodiments of the invention, a fusion protein, can be used to deliver a light-activated ion channel to a cell and can also in some embodiments be used to target a light-activated ion channel of the invention to specific cells or to specific cells, tissues, or regions in a subject. Targeting and suitable targeting sequences for deliver to a desired cell, tissue or region can be performed using art- known procedures .

It is an aspect of the invention to provide a. light-activated ion channel polypeptide of the invention that is non-toxic, or substantially ¹ non-toxic in cells in which it is expressed. In the absence of Sight, a light-activated ion channel of the invention does not significantly alter cell health or ongoing electrical activity in the cell in which it is expressed.

in some embodiments of the invention, a light-activated ion channel of the invention is genetically introduced into a cellular membrane, and reagents and methods are provided for genetically targeted expression of light-activated ion channel polypeptides, including ChR87, ChR88, CKR90, ChR62, ChR93, ChR88 .176R, or a derivative thereof, etc. Genetic targeting can be used to deliver light-activated ion channel polypeptides to specific cell types, to specific cell subtypes, to specific spatial regions within an organism, and to sub-cellular regions within a cell. Genetic targeting also relates to the control of the amount of light- activated ion channel polypeptide expressed, and the timing of the expression.

Some embodiments of the inventio include a reagent for genetically targeted

expression of a l ight-acti vated ion channel polypeptide, wherein the reagent comprises a vector that contains the gene for the light-activated ion channel polypeptide.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operaiiveiy linked. The terra "vector" also refers to a vims or organism thai is capable of transporting the nucleic acid molecule. One type of vector is an episome, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Some useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.

Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Other useful vectors, include, but are not limited to viruses such as lenti viruses, retro viruses, adenoviruses, and phages. Vectors useful in some methods of the invention can geneticall insert light-activated ion channel polypeptides into dividing and non-dividing cells and can insert light-activated ion channel polypeptides to cells that are in vivo, in vitro, or ex vivo cells.

Vectors useful in methods of the invention may include additional sequences including, but not limited to one or more signal sequences and/or promoter sequences, or a combination thereof. Expression, vectors and methods of their use are well known in the art. Non-limiting examples of suitable expression vectors and methods for their use are provided herein.

In certain embodiments of the invention, a vector may be a ientivirus comprising the gene for a light-activated ion channel of the invention, such as ChR.87, ChRSS. ChR 0,

ChR62, ChR93, ChR88 176R, or a derivative or variant thereof. A ientivirus is a non- limiting example of a vector that may be used to create stable cell line. The terra "cell line" as used herein is an established cell culture that will continue to proliferate given the appropriate medium .

Promoters that may be used in methods and vectors of the invention include, but are not limited to, cell-specific promoters or general promoters. Methods for selecting and using cell-specific promoters and general promoters are well known in the art. A non-limiting example of a general purpose promoter that allows expression of a light-activated ion channel polypeptide in a w ide variety of cell types - thus a promoter for a. gene that is widely expressed in a variety of cell types, for example a "housekeeping gene" can be used to express a light-activated ion channel polypeptide in a variety of cell types. Non-limiting examples of general promoters are provided elsewhere herein and suitable alternative promoters are well known in the ait.

In certain embodiments of the invention, a promoter may be an inducible promoter, examples of which include, but are not limited to tetraeycline-on or tetracycime-off, or tamoxifen-inducible Cre-ER,

Methods of Use of Light Activated Ion Channels of the Invention

Light activated ion channels of the invention are well suited for targeting cells and specifically altering voltage-associated cell activities. In some embedments of the invention, light-activated ion channels of the invention can utilized to introduce cations into ceils, thus activating endogenous signaling pathways (such as calcium dependent signaling), and then drags are applied that modulate the response of the cell (using a calcium or voltage-sensitive dye). This allows new kinds of drug screening using just light to activate the channels of interest, and using just light to read out the effects of a drug on the channels of interest.

Chrimson is far-red-activatable, and thus allows excitation of ceils with a color of light heretofore not used in biotechnology for excitation of cells. By using for example, Chrimson and Chronos together, excitation of two different populations of cells in the same tissue or in the same culture dish becomes possible. This simultaneous, two-color excitation is particularly promising for complex tissues such as the brain.

The performance of the above said molecules or classes of molecules can be tuned for optimal use, particularly in. context of their use in. conj unction with other molecules or optical apparatus. Such tuning can be done using standard methods known in the art. For example, in order to achieve optimal contrast for multiple-color stimulation, one may desire to either improve or decrease the performance of one molecule with respect to one another, by the appendage of trafficking enhancing sequences or creation of genetic variants by site-directed mutagenesis, directed evolution, gene shuffling, or altering codon usage. Molecules or classes of molecules may have inherently varying spectral sensitivity that may be functionally advantageous in vivo { where scattering and absorption will vary with respect to wavelength, coherence, and polarization), by tuning the linearity or non-linearity of response to optical illumination with respect to time, power, and illumination history.

According to certain principles of this invention, cations may be introduced into cells, thus activating endogenous signaling pathways (such as calcium dependent signaling), and drugs may be applied that modulate the response of the cell (using a calcium or voltage- sensitive dye). This enables new kinds of drug screening using just light to activate the channels of interest, and using j ust Sight to read out the effects of a drug on the channels of interest.

Another aspect of the invention is the use of light-activated channel to decrease the pH of the cell. Such a technique may be used to treat alkalosis.

Another aspect of the invention may involve the use of light -activated proton pumps for the coupled effect of hyperpolarization and intracellular alkalinization. For example, both phenomena can induce spontaneous spiking in neurons by triggering hyperpolarteation- induced cation currents or pH-dependent hyper-excitability.

Another aspect of the invention is to generate sub-cellular voltage or pH gradients, particularly at synapses and in synaptic vesicles to alter synaptic transmission, and

mitochondria to improve ATP synthesis.

Another aspect of the in vention is the use of far-red (660ra«) light to perform noninvasive transcranial and/or transdural stimulation to modulate neural circuits.

Another aspect of the invention is the various compositions of matter that have now been reduced to practice, for example: plasmids encoding for the above genes have been prepared; lenti viruses carrying payloads encoding for the above genes have been prepared; adeno-associated viruses carrying payloads encoding for the above genes have been prepared- cells expressing the above genes have been prepared; and animals expressing those genes have been prepared. (See for example: US Patent Publication 201 10165681 , incorporated herein by reference in its entirety).

Working operation of a prototype of this invention was demonstrated by genetically expressing light-activated ion channel molecules of the invention in excitable cells,

illuminating the cells with suitable wavelengths of light, and demonstrating rapid

depolarization of the ceils in response to the light, as well as rapid release from depolarization upon cessa tion of light. Depending on the particular implementation, methods of the invention allo light control of cellular functions in vivo, ex viva, and in vitro.

In non-limiting examples of methods of the invention, channelrhodopsins of the invention and derivatives thereof are used in mammalian cells without need for any kind of chemical supplement, and in normal cellular environmental conditions and ionic

concentrations. For example, genes encoding channelrhodopsins of Chl mydomonos and Stigeoclonium have been used in exemplary implementa tions of the invention. These sequences in humanized or mouse-optimized form allow depolarization at wavelengths described herein. As used herein, the term "ion channel" means a transmembrane polypeptide that forms a pore, which when activated opens, permitting ion conductance through the pore across the membrane. Many ion channels do not express well in a ceil and/or their expression may be toxic to the cell and reduce eel! health. Thus it was necessary to prepare and screen numerous ehannelrhodopsin light-activated ion channel polypeptides to identify light-activated ion channels of the invention that can be expressed in ceils without

significantly reducing ceil health and viability.

Li ht-aciivated ion channels of the invention have been found to be suitable for expression and use in mammal ian cells w ithout need for any kind of chemical supplement, and in normal cellular environmental conditions and ionic concentrations. light-activated ion channels of the invention have been found to differ from previously identified channels in that the Chronos light-activated ion channels activate at wavelengths of light ranging from 365 tun to 630 nm, with an optimal activation from light ranging from 430 nm to 550 nm, and a peak optimal activation at a wavelength of 500 nm. Chrimson light-activated ion channels activate at wavelengths of light in a range of 365 nm to 700 nm, with an optimal acti vation from light ranging from 530 nm to 640 nm, and a peak optimal activation at a wavelength of 590 nm.

Cells and Subjects

A cell used in methods and with sequences of the invention may be an excitable cell or a non-excitable cell. A ceil in which a light-activated ion channel of the invention may be expressed and may be used in methods of the invention include prokaryotic and eukaryotic ceils. Useful cells include but are not limited to mammalian ceils. Examples of cells in which a light-activated ion channel of the invention may be expressed are excitable cells, which include cells able to produce and respond to electrical signals. Examples of excitable cell types include, but are not limited to neurons, muscles, cardiac cells, and secretory cells (such as pancreatic cells, adrenal medulla cells, pituitary cells, etc.).

Non-limiting examples of cells that may be used in methods of the invention include: neuronal cells, nervous system cells, cardiac cells, circulatory system cells, visual system cells, auditory system cells, secretory cells, endocrine cells, or muscle cells. In some

embodiments, a cell used in conjunction with the invention may be a healthy normal cell, which is not known to have a disease, disorder or abnormal condition. In some embodiments, a cell used in conjunction with methods and channels of the invention may be an abnormal cell, for example, a cell that has been diagnosed as having a disorder, disease, or condition. including, but not limited to a degenerative cell, a neurological, disease-bearing cell, a cell model of a disease or condition, an injured cell, etc. In some embodiments of the invention, a cell may be a control cell.

Light-activated ion channels of the invention may be expressed in cells from culture, cells in solution, cells obtained from subjects, and/or ceils in a subject (in vivo cells). Light- activated ion channels may be expressed and activated in cultured ceils, cultured tissues (e.g., brain slice preparations, etc.), and in living subjects, etc. As used herein, a the term "subject" may refer to a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, rodent, fl or any other vertebrate or invertebrate organism.

Controls and candidate compound testing

Light-activated ion channels of the invention and methods using light-activated ion channels of the invention can be utilized to assess changes in cells, tissues, and subjects in which they are expressed. Some embodiments of the invention include use of light-activated ion channels of the invention to identity effects of candidate compounds on cells, tissues, and subjects. Results of testing a light -activated ion channel of the invention can be

ad vantageously compared to a control. In some embodiments of the invention one or more light-activated ion channels of the invention, non-limiting examples of which are OiR87, ChR88, ChR90 ₅ ChR93, ChR62, ChRSS 176R, or a derivative thereof, may be expressed in a cell population and used to test the effect of candidate compounds on the cells.

As used herein a control may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean, it can be established based upon comparative groups, such as cells or tissues that include the light-activated ion channel and are contacted with light, but are not contacted with the candidate compound and the same type of cells or tissues that under the same testing condition are contacted with the candidate compound. Another example of compara tive groups may include cells or tissues that have a disorder or condition and groups without the disorder or condition. Another comparative group may be cells from a group with a family history of a disease or condition and cells from a group without such a family history. A predetermined value can be arranged, for example, where a tested populatio is divided equally (or unequally) into groups based on results of testing. Those skilled in the art are able to select appropriate contr ol groups and values for use in comparative methods of the invention.

As a non-l imiting example of use of a light-activated ion channel to identify a candida te therapeutic agent or compound, a li ght-activated ion channel of the in ven ti on may be expressed in an excitable cell in culture or in a subject and the excitable ceil may be contacted with a light thai activates the light-activated ion channel and with a candidate therapeutic compound. In one embodiment, a test ceil that includes a light-activated ion channel of the invention can be contacted with a Sight thai depolarizes the ceil and also contacted with a candidate compound. The cell, tissue, and/or subject that include the cell can be monitored for the presence or absence of a change that occurs in the test conditions versus the control conditions. For example, in a cell, a change may be a change in the depolarization or m a depolarization-mediated cell characteristic in the test cell versus a control cell, and a change in depolarization or the depolarization-mediated cell characteristic in the test cell compared to the control may indicate that the candidate compound has an effect on the test cell or tissue that includes the cell, in some embodiments of the invention, a depolarization- mediated cell characteristic may be a an action potential, pH change in a cell, release of a neurotransmitter, etc. and may in come embodiments, include a downstream effect on one or more additional cells, which occurs due to the depolarization of the cell that includes the light-activated ion channel. Aft-known methods can be sued to assess depolarization and depolarization-mediated cell characteristics and changes to the

depolarization or depolarization-mediated cell characteristics upon activation of a light- activated ion channel of the invention, with or without additional contact with a candidate compound.

Candidate-compound identification methods of the invention thai are performed in a subject, may include expressing a light-activated ion channel in a subject, contacting the subject with a light under suitable conditions to activate the light-activated ion channel and depolarize the ceil, and administering to the subject a candidate compound. The subject is then monitored to determine whether any change occurs that differs from a control effect in a subject. Thus, for example, a ceil in cultur can be contacted with a light appropriate to activate a light-activated ion channel of the invention in the presence of a candidate compound. A result of such contact with the candidate compound can be measured and compared to a control value as a determination of the presence or absence of an effect of the candidate compound.

Methods of identifying effects of candidate compounds using light-activated ion channels of the invention may also include additional steps and assays to further

characterizing an identified change in the cell, tissue, or subject when the cell is contacted with the candidate compound, in some embodiments, testing in a. cell, tissue, or subject can also include one or more cells that has a light-acti v ated ion channel of the in vention, and that also has one, two, three, or more additional different light-activated, ion channels, wherein at least one, two, three, four, or more of the additional light-activated ion channels is activated by contact with light having a different wavelength than used to activate the Chronos,

Chrimson, ChR87, or derivative thereof, light-activated ion channel of the invention.

hi a non-Limiting example of a candidate drug identification method of the invention, ceils that include a light-activated ion channel of the invention are depolarized, thus triggering release of a neurotransmitter from the cell, and then drugs are applied that modulate the response of the cell to depolarization (determined for example using patch clamping methods or other suitable art-known means). Such methods enabie new kinds of drag screening using just Light to activate the channels of interest, and using just light to read out the effects of a drag on the channels and channel-containing cells of interest.

In some embodiments, light-activated ion channel pol peptides of the invention can be used in test systems and assays for assessing membrane protein trafficking and

physiological function in heterologous!}' expressed systems and the use of use of light- activated channels to depolarize a cell.

in some aspects of the invention, two-color assays can be performed. For example, Chronos (for blue light activation) and Chrimson (for red light activation) can be expressed in separate sets cells that represent non-overlapping neuronal populations. Following expression, the eel! population can be exposed to light and the wavelength and timing and "dose" of light can be optimized. As used herein the term "dose" in reference to Light, may take into account of wavelength, pulse length, intensity , of the light with which a l ight- activated ion channel of the invention is contacted.

A. non-limiting example of a procedure for optimizing the use of two-color activated populations of cells is provided as follows. A population that has Chronos and Chrimson expressed in different sub-populations is contacted with blue light having a wavelength between 400 am and 500 am, or between 450 rim to 500 rim, and having a pulse width of between 1 and 5 ins for activation. A pulse width of 5 ms provides for minimum subthreshold crosstalk in tire blue light, which is defined as < 15 mV, <10 mV, and optimally as <5 mV, The maximum blue Sight power that can be used is determined using by patch clamping Chrimson expressing cells, illuminating with blue light and measuring voltage deflection. Optimally using blue light power such thai maximum voltage deflection is <10mV, which in some embodiments may be 0.4 to 0.6m W/msrf . The optimal blue light power that can be used to drive Chronos is determined using the same conditions as above, except using lower light power, suc as 50μΨ/ηιηι ² to 0.4m W/mm ² , which in some embodiments may be 0.2mW/mra ² . Power depends on expression system and cell type used to prepare the population. The population can be contacted with red light having a wavelength between 600 nm and 700 nm, or 620 nm and 640nm, and with a pulse width of between 1 and 5 ins for activation, which in some embodiments may be optimized at 5ms, In certain embodiments of the invention, the optimal light power to drive Chrimson in die red may be determined by ramping light powers from for example, 0.1 mW/mm ² to 100 tftW/mirf ₅ or from 0.5mW/mm ² to I0mW/nmr ⁱ . The method may be optimized such that a minimum red light power is used to achieve 100% spiking for Chrimson.

It will be understood that other sets of 2, 3, 4, or more light-activated ion channels may be expressed in separate subpopulations of a population of cells and then exposed to doses of Sight in a manner as described here to optimize their use in assays and treatments of the invention. A non-limiting example of a process to prepare and use a multi-light activated population of cells is as follows. A first light-activated ion channel is expressed in a first subpopulation of a population of cells: a second light-activated ion channel is expressed in a second subpopulation of the population of cells, wherein the first and second subpopulations are non-overlapping subpopulations, and the first light-activated ion channel and second light activated ion channel are have ranges of activating light wavelengths that do not entirely over lap. The population of cells is contacted with a plurality of first light test doses comprising combinations of wavelength, pulse width, and power that activate the first subpopulation, and the transmembrane voltage deflection is measured in. a cell of the second subpopulation of cells contacted with the first l ight test doses. The first light test dose that includes a maximum light power that activates the light activated ion. channel, in first subpopulation of cells and results in a minimum sub-threshold transmembrane voltage deflection in the second subpopulation of cells is determined . The popula tion of cells is then contacted with a plurality of first light test doses comprising a lower power than the maximum first light power that was determined, and a first light test doses that activate the first light activated ion channel (a t the lower powers) are determined. The population of cells is then contacted with a plurality of second light test doses that include combinations of light wavelength, pulse width, and power thai activate the second subpopulation, and a second light test dose comprising a second light power that activates the second subpopulation of cells is determined. Assays can be performed using such a population of cells, that includes contacting the population of cells wi th the first l ight test dose and the second l ight test dose determined using the steps above. The above-described process of optimizing light dose parameters for multi-light activated ion channels can. be used to design and implement assays that include light-activated ion channels of the invention, as well as other light-activated ion channels that are known in the art.

Methods of Treating

5 Some aspects of the invention include methods of treating a disorder or condition in a cell, tissue, or subject using light-activated ion channels of the invention. Treatment methods of the invention may include administering to a subject in need of such treatment, a

therapeutically effective amount of a light-activated ion channel of the invention to treat the disorder. It will be understood that a treatment may be a prophylactic treatment or may be a

{() treatment administered following the diagnosis of a disease or condition. A treatment of the invention may reduce or eliminate a symptom or characteristic of a disorder, disease, or condition or may eliminate the disorder, disease, or condition itself, it will be imderstood thai a treatment of the invention may reduce or eliminate progression of a disease, disorder or condition and may in some instances result in the regression of the disease, disorder, or

15 condition. A. treatment need to entirely eliminate the disease, disorder, or condition to be effective. In some embodiments of the invention one or more light -activated ion channels of the invention, non-limiting examples of which are ChRS7, ChR88 _e ChR90, ChR93, Cli .62 may be expressed in a ceil population and used in methods to treat a disorder or condition.

Administration of a light-activated ion channel of the invention may include

0 administration phai'maceuticai composition that includes a cell, wherein the eel! expresses the light-activated ion channel . Administration of a l ight-activated ion channel of the invention may include adm inistration of a pharmaceutical composition tha includes a vector, wherein the vector comprises a nucleic acid sequence encoding the light-activated ion channel and the administration of the vector results in expression of the light-activated ion channel in a cell in 5 the subject.

An effective amount of a light-activated ion channel is an amount that increases the level of the light-activated ion channel in a cell, tissue or subject to a level that is beneficial for the subject. An effective amount may also be determined by assessing physiological effects of administration on a cell or subject, such as a decrease in symptoms following

0 administration. Other assays will be known to one of ordinary skill in the art and can be

employed for measuring the level of the response to a treatment. The amount of a treatment may be varied for example by increasing or decreasing the amount of the light-activated ion channel administered, by changing the therapeutic composition in which the light-activated ion channel is administered, by changing the route of administration, by changing the dosage timing, by changing the activation amounts and parameters of a light-ac tivated ion channel of the invention, and so on. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated; the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any ), the specific route of administration, and the ^' like factors within the knowledge and expertise of the health practitioner. For example, an effective amount may depend upon the location and number of cells in the subject in which the light-activated ion channel is to be expressed. An effecti v e amoun t may also depend on the location of the tissue to be treated.

Effective amounts will also depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment., the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of a composition to increase the level of light-activated ion channel, and/or to alter the length or timing of activation of a light-activated ion channel in a subject (alone or in combination with, other therapeutic agents) be used, that is. the highest safe dose or amount according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

A light-activated ion channel of the invention (for example. ChR87, ChR88, ChR 0, ChR93, ChR62, or ChRSS K I 76R, or a derivative thereof) ma be administered using art- known methods. In some embodiments a nucleic acid that encodes a light-activated ion channel polypeptide of the invention is administered to a subject and in certain embodiments a light-activated ion channel polypeptide is administered to a subject. The manner and dosage administered, may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. The absolute amount administered will depend upon a variety of factors, including the material selected for administration, whether the

adni.inistrati.on is in single or multiple doses, and individual subject parameters including age, physical condition, size, weight, and the stage of the disease or condition. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation .

Pharmaceutical compositions thai deliver light-activated ion channels of the invention may be administered alone, in combination with each other, and/or in combination with other drag therapies, or other treatment regimens that are administered to subjects. A pharmaceutical composition used in the foregoing methods preferably contain an effective amount of a therapeutic compound that will increase the level of a light-acti vated ion channel polypeptide to a level that produces the desired response in a unit of weight or volume suitable for administration to a subject.

The dose of a pharmaceutical composition that is administered to a subject to increase the level of light-activated ion channel in ceils of the subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the stat of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. The amount and timing of activation of a light- activated ion channel of the invention (e.g., light wavelength, length of light contact, etc.) that has been administered to a subject can also be adjusted based on efficacy of the treatment in a particular subject. Parameters for illumination and activation of Sight-activated ion channels that have been administered to a subject can be determined using art-known methods and without requiring undue experimentation.

Various modes of administration will be known to one of ordinary skill in the art that can be used to effectively deliver a pharmaceutical composition to increase the level of a light-activated ion channel of the invention in a desired ceil, tissue or body region of a subject. Methods for administering such a composition or other pharmaceutical compound of the invention may be topical, intravenous, oral, intraeavity, intrathecal, intrasynovial, buccal, sublingual, intranasal, transdermal, inrravitreal, subcutaneous, intramuscular and intradermal administration. The invention is not limited by the particular modes of administration disclosed herein. Standard references in the art (e.g.. Remington's Pharmaceutical Sciences, 18th edition, 1990) provide modes of administration and formulations for delivery of various pharmaceutical preparations and formulations in pharmaceutical carriers. Other protocols which are useful for the administration of a therapeutic compound of the invention will be known to one of ordinary skill in the art, in which the dose amount, schedule of

administration, sites of administration, mode of administration (e.g., intra-organ) and the like vary from those presented herein.

Administration of a ceil or vector to increase l ight-acti vated ion channel levels in a mammal other than a human; and administration and use of Sight-activated ion channels of the invention, e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above. It will be -understood by one of ordmary skill in the art thai this invention is applicable to both human and animals. Thus this invention is intended to be used in husbandry and veterinary medicine as well as in human therapeutics.

I some aspects of she invention, methods of treatment, using a light-activated ion channel of the invention are applied to cel ls including but not limited to a neuronal cell, a nervous system cell, a neuron, a cardiac cell, a circulatory system cell, a visual system cell, an auditory system cell, a muscle cell, or an endocrine cell, etc. Disorders and conditions thai may be treated using methods of the invention include, injury, brain damage, degenerative neurological conditions (e.g., Parkinson's disease, Alzheimer's disease, seizure, vision loss, hearing loss, etc,

Disorders, Diseases and Conditions

Light-activated ion channels of the invention may be used to target cells and membranes, and to alter voltage-associated cell activities, in some aspects of the invention, a light-activated ion channel of the invention may be used to decrease the pH of a cell in which it is expressed. Such a technique may be used to treat alkalosis.

Another aspect of the invention includes methods of using light-activated proton pumps in conjunction with the use of light-activated ion channels of the invention for the coupled effect of hypeTpoSarization and intracellular aSkali zation. For example, both phenomena can induce spontaneous spiking in neurons by triggering hyperpolarization-induced cation currents or pl l -dependent hyper-excitability.

Another aspect of the invention is to express light-activated ion channels of the invention into cell membranes and then to acti vate the light-acti vated ion channels and generate sub-cellular voltage or pH gradients, particularly at synapses and in synaptic vesicles to alter synaptic transmission, and mitochondria to improve ATP synthesis.

n some embodiments, methods and light-acti vated ion channels of the invention may be used for the treatment of visual system disorders, for example to treat vision reduction or loss. A light -activated ion channel of the invention may be administered to a subject who has a vision reduction or loss and the expressed light-activated ion channel can function as light- sensitive cells in the visual system, thereby permitting a gain of visual function in the subject.

The present invention in some aspects, includes preparing nucleic acid sequences and polynucleotide sequences; expressing in cells and membranes polypeptides encoded by the prepared nucleic acid and polynucleotide sequences; illuminating the cells and/or membranes with suitable light, and demonstrating rapid depolarization of the cells and/or a change in conductance across the membrane in response to light; as well as rapid release from depolarization upon cessation of light. The ability to controliabiy alter voltage across membranes and cell depolarization with light has been demonstrated. The present invention enables light-control of cellular functions in vivo, ex vivo, and in vitro, and the light activated ion channels of the invention and their use, have broad-ranging applications for drug screening, treatments, and research applications, some of wh ich are describe herein.

In illustrative implementations of this invention, the ability to optically perturb, modify, or control cellular function offers many advantages over physical manipulation mechanisms. These advantages comprise speed, non-invasiveness, and the ability to easily span vast spatial scales from the nanoseaie to macroseaie.

The reagents use in the present invention (and the class of molecules that they represent), allow, at least: currents activated by light wavelengths not useful in previous light- activated ion channels, light activated ion channels that when activated, permit effectively zero calcium conductance, and different spectra from older molecules (opening up multicolor control of cells).

Examples

Example 1

Studies were performed to prepare sequences and to express light-activated ion channels in cells, tissues, and subjects, identifications and amino acid sequences of some of the light-activated ion channels in the examples are ChRSS (SEQ ID NO:2); ChR90 (SEQ ID NO:7); ChR8? (SEQ ID NO; 1 1); ChR62 (SEQ D NO: 14), ChR93 (SEQ ID NO: 16) and ChR2 (SEQ ID NO: 19), ChR88 K.176R (SEQ ID NO:5). Non-limiting exemplary methods are set forth Example 1 . General methods also applicable to light-activated channel molecules and methods for their use are disclosed in publications such as US Published

Application No. 2010/0234273, US Published Application No. 201 10165681, Cho BY, er. ai. Methods Enz moi 2011;497:425-43; Chow, BY, et al. Nature 2010 Jan 7;463(7277):9S- 102, the content of each of which is incorporated by reference herein.

Studies were performed to prepare sequences and to express light-activated ion channels in cells, tissues, and subjects. Non-limiting exemplary methods are set forth below.

(a) In ui ro eiectroporation All procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Massachusetts institute of

Technology Committee on Animal Care. C57BL/6J E16~timed pregnant mice were used for electroporation. Surgery was done under ketamine-xyiazine anesthesia and buprenorphine 5 analgesia, DN A solution containing piasmids of interest were injected into laterai ventricle of each embryo using a pulled capillary tube. Five square pulses (50ms width, 1 Hz, 35V) were applied using tweezer electrode for electroporation.

(b) Slice preparation

{() P20-P30 mice were used for slice preparation. In younger animals it was difficult to elicit synaptic responses by phoiostimulating eallosal axons. Mice were anesthetized with isoflttorane and transcardialy perfused with artificial cerebrospinal fluid (ACSF). The brain was removed and placed in an ice-cold cutting solution containing 1 10 mM choline chloride, 25 mM NaHCOs, 25 mM D-glucose, 1 1.6 mM sodium ascorbate, 7 mM MgCl _2s 3.1 mM

15 sodium pyruvate, 2.5 mM KCL 1.25 mM Naf PC^ and 0.5 mM CaCl ₂ . 300~mm~thick

coronal slices of the visual cortex were cut with a vibrating s!icer and incubated in oxygenated ACSF for 45 min at 37"C before the recordings,

(c) Slice ElectrophysioJog

0 Recordings were performed at room temperature (22-24° C) under constant perfusion of oxygenated ACSF. Neurons were visualized using infrared differential interference optics and patched with borosilieate pipettes (resistance 4-6 MO). The intracellular solution contained 120 mM potassium gluconate. 5 mM NaCL 2 mM M.gCI ₂ , 0.1 mM CaCl ₂ . 10 mM HEPES, 1.1 mM EGTA, 4 mM magnesium ATP, 0.4 mM disodium GTP, (pH 7.25; 290 5 mOsml Cells were recorded at a depth of 30-120 urn in the brain slice. Photostiraulation was done using a blue LED (470 urn; Thorlabs, Newton, NJ) and a red LED (625nm with 632/22nm filter; Thorlabs).

(d) Neuron culture, tmnsfetiion, infection, and imaging

0 Ail procedures involving animals were in accordance with the National Institutes of

Health Guide for the care and use of laboratory animals and approved by the Massachusetts Institute of Technology Animal Care and Use Committee. Swiss Webster or C57 mice

[Taconic (Hudson, NY) or Jackson Labs ( Bar Harbor, ME)] were used . For hippocampal cultures, hippocampal regions of postnatal day 0 or day 1 mice were isolated and digested with trypsin (1 rag/ml) for -1.2 min, and then treated with Hanks solution supplemented with 10-20% fetal bovine serum and trypsin inhibitor (Sigma Aldrich, St Louis, MO). Tissue was then mechanically dissociated with Pasteur pipettes, and centrifuged at 1000 rpm at 4°C for 10 min. Dissociated neurons were plated at a density of approximately four hippocampi per 20 glass coversiips, coatecl with Matrigei (BD Biosciences, San Jose, CA). For cortical cultures, dissociated mouse cortical neurons (postnatal day 0 or 1 ) were prepared as previously described, and plated at a density of 100-200R per glass coverslip coated with Matrigei (BD Biosciences). Cultures were maintained in Neurobasal Medium supplemented with B27 (Invitrogen [Life Technologies, Grand Isle, NY]) and glutamine. Fiippocanxpal and cortical cultures were used interchangeably; no differences in reagent performance were noted.

Neurons were transfected at 3-5 days in vitro using calcium phosphate (Invitrogen). GFP fluorescence was used to identify successfully transfected neurons. Alternatively, neurons were infected with 0.1-3 μΐ of lent! virus or adeno-associated virus (AAV) per well at 3-5 days in vitro.

(e) NEK 293 FT ^' cell culture and transfection

HEf 293 FT ceils (Invitrogen) were maintained between 10-70% confluence in D10 medium (Cellgro [Mediatech/Coraing, Manassas, VAJ) supplemented with 10% fetal bovine serum (Invitrogen), 1 % penicillin/streptomycin (Cellgro). and 1 % sodium pyruvate (Biowhittaker, Walkersville, MD). For recording, cells were piated at 5-20% confluence on glass coverslips coated with Matrigei (BD Biosciences). Adherent cells were transfected approximately 24 hours post-plating either with TransLT 293 lipofectamine transfection kits (Minis Bio, LLC, Madison, WI) or with calcium phosphate transfection kits (Invitrogen), and recorded via whole-cell patch clamp between 36-72 hours post-transfection.

(f) hi vitro whole cell patch clamp recording ά optical stimulation

Whole cell patch clamp recordings were made using a Multiclamp 700B amplifier, a Digidata 1440 digitizer, and a PC running pClamp (Molecular Devices). Neurons were bathed in room temperature Tyrode containing 125 mM NaCl, 2 mM KCL 3 mM CaCU, 1 raM MgC , 10 mM HEPES, 30 mM glucose, 0.01 mM NBQX and 0. 1 mM GABAzme. The Tyrode pH was adjusted to 7.3 with NaOH and the osmolarity was adjusted to 300 mOsm with sucrose. HEK cells were bathed in a Tyrode bath solution identical to that for neurons, but lacking GABAxine and NBQX. Borosilicate glass pipettes (Warner Instruments, Hamden, CT) with an outer diameter of 1.2 mm and a wall thickness of 0.255 mm were pulled to a resistance of 3-9 MQ, with a P-97 Flaming/Brown micropipeite puller (Sutter Instruments, Novato, CA ) and filled with a solution containing 125 wM K-gluconate, 8 mM NaCl, 0.1 ni CaCI _2s 0.6 inM MgCi ₂ , 1 iiiM EGTA, 10 mM HEPES, 4 mM Mg-ATP, and 0.4 mM Na-GTP. The pipette solution pH was adjusted to 7.3 with KOH and the osmolality was adjusted to 298 mOsm with sucrose. Access resistance was 5-30 ΜΩ, monitored throughout the voltage-clamp recording. Resting membrane potential was—60 raV for neurons and— 30 raV for HEK 293 FT cells in current-clamp recording.

Photocurrents were measured with 500 ms light pulses in neurons voltage-clamped at -60 mV, and in HEK 293 FT cells voltage-damped, at -30 raV. Light-induced, membrane hyperpolarizations were measured with 500 ms light pulses in cells current-clamped at their resting membrane potential. Li ht pulses for all wavelengths except 660 nra and action spectrum characterization experiments were delivered with a DG-4 optica! switch with 300 W xenon lamp (Sutter Instruments), controlled via TTL pulses generated through a Digidata signal generator. Green light was delivered with, a 575 ± 25 nm bandpass filter (Chroma) and a 575 ± 7.5 nm bandpass filter (Chroma Technology Group, Bellows Falls, VT). Action spectra were taken with a Till Photonics Polychrome V, 150 W Xenon lamp, 15 nm monochfomator bandwidth.

Data was analyzed using Clampfit (Molecular Devices) and ATLAB (Mathworks,

Inc.)

(g) km Conductance Recording

Whole-cell patch clamp recordings were performed in isolated HEK293FT cells to accurately measure parameters from single cells. All. recordings were performed using an Axopatch 200B amplifier and Digidata 1440 digitizer (Molecular Devices) at room temperature. In order to allow isolated cell recording, cells were plated at a lower density of 15,000 ceils per well in 24-well plates that contained round glass coversHps (0, 15 mm thick, 25 mm in diameter, coated with 2 % Growth Factor Reduced Matrigel in DMEM for 1 h at 37 °C). For most recordings, Tyrode was used as the extracellular solution, and the intracellular solution consisted of (in mM) 125 ~Gluconate, 8 NaCl, 0.1 CaCla, 0.6 MgClj, 1 EGTA, 10 HEPES, 4 MgATP, 0.4 NaGTP, pH 7.3 (KOH adjusted), with 295-300 mOsm (sucrose adjusted). Extracellular and intracellular solutions used for testing ion permeability are listed in Table 1. Table 1. Compositions of solutions used in ion permeability experiments

Liquid junction potentials were measured using standard procedures to be 5,8 mV for the 90 mM Ca¾ and 4.9 mV for the 5 mM NaCl extracellular solutions, which were corrected during recording; the others were < 1 mV injunction potential.

In ail patch clamp recordings, a stringent cutoff of access resistance less than 25 ΜΩ and holding current less than ± 50 pA was applied in order to ensure accurate measurement. Typical, membrane resistance was between 500 ΜΩ - 2 GQ and pipette resistance was between 4 - 10 ΜΩ,

Photostimu!ation of patch clamped cells was conduc ted by a 470 nm LED (Thorlabs) at 10 mW/mm ² unless otherwise stated. For most experiments, i s illumination was delivered to measure transient and steady-state photocurrents.

( ) Plasmid construction and site directed mutagenesis.

Opsins were mammalian codon -optimized, and synthesized by Genscript (Gensctipt Corp., NJ). Opsins were fused in frame, without stop codons, ahead of GFP (using Barn HI and Age!) in a lentiviral vector containing the CaMKO promoter, enabling direct neuron transfection, HEK cell transfection (expression in HEK. ceils is enabled by a ubiquitous promoter upstream of the lentiviral cassette), and lentivirus production and transfection. Amino add sequences of some opsins that were tested were as follows: ChRSS (SEQ ID NO:2); CliR.90 (SEQ ID NO:7); ChR87 (SEQ ID NO: I 1 ); C 6 ^" 2 (SEQ ID NO: 1.4), ChR93 (SEQ ID NO: 16) and ChR2 (SEQ ID NO: 19), ChR88 176R (SEQ ID NQ;5).

The 'ss' signal sequence from truncated MHC class I antigen corresponded to amino acid sequence (M)VPCTLLLLLAAALAPTQTRA (SEQ ID NO:21), DNA sequence gtcccgtgcacgctgctcctgctgttggcagccgccctggctccgactcagacgcgggcc (SEQ ID Q:20). The 'ER2 ^* ER. export sequence corresponded to amino acid sequence FCYENEV (SEQ ID

NO:23), DN A sequence ttctgctaegagaatgaagtg (SEQ ID NO:22). The 'KGC' signal sequence corresponded to amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:25), DNA sequence of KGC signal sequence :

aaatccagaat.tact.tctgaa.ggggagt.a.tatccctctggaicaaataga.c atcaatgtt. (SEQ ID NO:24).

Point mutants for I I EK cell testing were generated using the QuikChange kit

[Stratagene, (Agilent Technologies, Santa Clara, CA)] on the opsin-GFP fusion gene inserted between BamHI and Age! sites in a modified version of the pEGF P-N3 backbone [invitrogen, (Life Technologies Corporation, Carlsbad. CA)]. All constructs were verified by sequencing.

(i) Lentivirm preparation

HEK293FF cells [Invitrogen, (Life Technologies Corporation, Carlsbad, CA)] were transfected with the lentiviral plasmid. the viral helper plasmid ρΔ8.74, and the pseudotyping plasmid pMD2.G. The supernatant of transfected HEK cells containing virus was then col lected 48 hours after transfection, purified, and then pelleted through ukracentrirugation. Lenti virus pellet was resuspended in phosphate buffered saline (PBS) and stored at - S0°C until further usage in vitro or in vivo. The estimated final titer is approximately 10 ⁹ infectious units/mL.

Example 2

Light-activated ion channels VChR.1 , Ch l , ChR2, ChR87, ChR90, and ChRSS were expressed in cultured hippocampa ^' I neurons using neuron culture, transfection, infection, and imaging methods described in Example 1. In vitro whole cell patch claim recording and optical stimulation were conducted on the neurons using methods described in Example 1. Figure 1 shows chatmelrhodopsin photocurrenis measured in the cultured hippocampal neurons. Fig. 1 A shows results rising red light (660nm) peak photocurrent at 10 mW ranC for Is illumination. ChRSS is the only red light sensitive channelrhodopsin with significant photocurrent at 660nm. Fig. I B shows results using blue (4.23 raW mm ^"2 ) or green (3.66 raW mm ^'* ) Sight peak photocurrent at equal photon flux for 5ms illumination. ChR87,

ChR88, and ChR90 all have greater or comparable photocurrent than ChR2. Solid bar indicates blue light, horizontal striped bar indicates green light.

Example 3

HEK 293FT cells were transfected to express ChR2, ChR90, VChRI, ChR88, and ChR87 light-activated ion channels using methods of Example 1. hi vitro whole cell patch claim recording and optica! stimulation were conducted on the transfected, cultured cells using methods described in Example 1. Figure 2 show action spectrum at equal photon dose at all wavelengths recorded in HE 293FT cells. ChR2 (470mn peak) and VChRI (545nm peak) represent the existing channelrhodopsin color sensitivity range. ChR87 ( 515nm peak) and ChR90 (500nm peak) are blue green light sensitive channe!rhodopsins. Whereas ChR88 (590nm peak) is the first red light sensitive natural channelrhodopsin.

Example 4

Light-activated ion channels ChR90 and ChR88 were expressed in cultured hippocampal neurons using neuron culture, transfection. infection, and imaging methods described in Example 1 , in vitro whole cell patch claim recording and optical stimulation were conducted on the neurons using methods described in Example 1. Figure 3 shows optically-driven spikes in the cultured hippocampal neurons. Fig. 3A shows red-light-drive spike trains at low frequency for Ch88. Generally ChR88 could reliably drive spikes up to 5Hz. However at higher frequency such as 20Hz, ChR88 desensitizes and/or causes depolarization block. Fig. 3B shows green-light-driven spike trains at high frequency for Ch. 0. Due to ChR.90 fast tau off and peak photocurrent recovery kinetics, it was able to drive temporally precise spikes at the highest frequency a neuron is capabl e of mediating.

Example 5

Light-activated ion channels ChR88, Chr2, ChR87 _; and ChR90 were expressed in cultured hippocampal neurons using neuron culture, transfection, infection, and imaging methods described in Example 1. In vitro whole cell patch claim recording and optical stimulatio were conducted on the neurons using methods described i Example 1. Figure 4 illustrates the results and shows channelrhodopsin kinetics measured in the hippocampal neuron culture voltage clamped at -65mV. Fig. 4A shows single exponential channel turn-off kinetics based on 5 ras pulse. CliR90 had the fastest turn-off kinetics (3.5ms) observed across all natural channelrhodopsms. Fig, 4B showed peak photocurrent recovery ratio based on 1 s

illumination. Ch 87 and C R90 both had fast peak photocurrent recovery at around 70%. However CliR.88 had slow recovery kinetics similar' to ChR2.

5 Example 6

Light-activated ion channels Chrimson (ChR88) were expressed in cultured hippocampal neurons using neuron culture, transfeetion, infection, and imaging methods described in

Example 1. In vitro whole eel! patch claim recording and optical stimulation were conducted on the neurons using methods described in Example 1. Figure 5 shows Chrimson blue light

{() crosstalk characterization in cultured neurons. Fig. 5 A shows action spectrum of Chrimson and the blue light (470nm) wavelength used for illumination. Wavelength was chosen to minimize crosstalk. Fig. 5B shows representative traces from a single neuron at various illumination conditions. When the blue light power was doubled from 0.1 to 0.2 raW mm ^" ' while the stimulation protocol was fixed as 5ms 5 Hz, the voltage deflection was also doubied.

15 However when the blue light power was fixed at 0.1 mW mm ^" " but the pulse duration was changed from 5ms to 1000ms, the crosstalk was changed from <5 mV to full spiking

correspondingly. This means blue light crosstalk was a function of both light power and li ht pulse duration (total photon count). 0 Example 7

Light-activated ion channels Chronos (ChR 0) and ChR2 were expressed in cultured

hippocampal neurons using neuron culture, transfection, infection, and imaging methods described in Example 1 , In vitro whole cell patch claim recording and optical stimulation were conducted on the neurons using methods described in Example 1. Figure 6 ill strates 5 Chronos and ChR2 blue light sensitivity in cultured hippocampal neurons. Fig. 6A is a spike irradiance curve for individual neurons. Fig. 6B shows lowest light power needed for single- cell 100% spike probability vs GFP fluorescence. Chronos (circles) was approximately 5 times more light sensitive than ChR2 (triangles) at a given (GFP) expression level. Fig. 6C show's example traces of Chronos spiking at various light powers. Fig. 6D illustrates that 0 controls showed no significant electrical differences between ChR2 and Chronos expressing neurons.

Example 8 In iero electroporation and slice preparation methods as described in Exampk 1 were used to examine Clironos (ChR90) and Chrimson (ChR88) activation. Figure 7 illustrates the strategy used for slice characterization of Clironos and Chrimson . Fig. 7 A shows

illumination wavelength used for slice experiments. Fig. 7B provides micrographic images showing histology for Chronos and Chrimson GFP fusion construct singly expressed in layer 2/3 visual cortex in mice.

Example 9

In uiero electroporation, slice preparation, and slice electrophysioiogy methods as described in Example 1 were used to characterize Chrimson (Cii SS) and Chronos (ChR90) blue and red light sensitivity in slice preparations. Figure 8 illustrates results obtained using whole cell patch clamp methods. Fig. 8 A shows that red light elicited 100% spiking in Chrimson expressing neurons but not Chronos expressing neurons between 1-6.5 raW mm ^"2 . Fig. 8B shows that blue light at 0.2-0.5 mW mnf could elicit 100% spiking in Chronos expressing cells but not Chrimson expressing cells. However full spiking crosstalk in Chrimson expressing cells can occur at powers higher than 0.6 mW mn . Fig, 8C shows blue light crosstalk voltage of Chrimson expressing neurons.

Example 10

i uiero electroporation, slice preparation, and slice electrophysioiogy methods as described in Example 1 were used to characterize Chrimson (ChR88) and Chronos (ChR90). Figure 9 il lustrates results with example traces of current-clamped opsin-expressing neurons in layer 2/3 slice blue light 0,1 in W mm ", red light I mW mm ^"2 expressing. No crosstalk, was observed under red light for Chronos while minimal subthreshold (<5mV) crosstalk was observed under blue light for Chrimson.

Figure 10 illustrates results with example traces of voltage-clamped non-opsin- expressin neurons in layer 2/3 or 5, post-synaptie to opsin-expressing cells. Zero postsynaptic crosstalk was observed for both Chronos and Chrimson under reel and blue light illumination respectively, Chronos: blue light 0.13 mW mnf ² , red light 1.7 mW mnf, Chrimson: blue light 0.37 mW mm ^' ", red light 1.7 mW mnf.

Figure 1 1 illustr ates results of studies of paired-pulse illumination in slice that differentially express Chrimson and Chronos in separate neurons. Fig. 1 1 A shows a triple plasmid in uiero electroporation scheme used to obtain non-overlapping expression of Chrimson and Chronos. Fig. 1 I B shows opsin expression in visual, cortex no overlap of GFP and m02 was observed ratio of Chronos to Chrirason labeling could be tuned, by titrating Cre plasmid. Fig. 1 1C shows voltage-clamped non-ops in-ex ressing neuron h layer 2/3 paired- poise stimulation to demonstrate different synapses were selectively driven by bine and red light, blue: 0.2 mW mm ^'2 ; red: 5 mW mm ^"2 .

5

Example 1 1

Chrimson light-activated ion. channels (ChR88) were expressed in cultured hippocampal neurons using neuron col tare, transfection, infection, and imaging methods described in

Example I . In vitro whole cell patch claim recording and optical stimulation were conducted {() on the neurons using methods described in Example 1. Figure 12 illustrated results thai showed that Chrimson could drive spikes in the far-red ( 660 am) using 5 ms pulses at 2.6 mW aim ^' " in cultured liippocampal neurons.

Example J 2

15 Substituted Chrimson (ChR88) light activated ion channels, referred to as "ChR.88 K.l 76R", having an amino acid sequence set forth as SEQ ID O:5, were expressed in cultured hippocampal neurons using neuron culture, transfection, infection, and imaging methods described in Example 1. In vitro whole cell patch claim recording and optical stimulation were conducted on the neurons using methods described in Example 1. Figure 33 show 0 results indicating that the C R88 K1.76R mutant had improved kinetics (1.3ms tan off) and couid mediate hi gh frequency spikes in cultured hippocampal neurons. Examples of current clamped traces of a single ChR88 K I 76R expressing neuron are shown. Fig. 13 A shows that ChRSS K 176R could reliably drive spikes from 1 to 10 mW mm ^"2 at 625nm 5 Hz stimulation. Fig, 1.3B show s red light (625mn) driven spike trains at various frequencies for ChRSS 5 K 176R. 1 mW mm ^* " light power is used for all frequencies. Fig. 13C shows current injection control thai demonstrated that the neuron was capable of spiking ai the indicated frequencies.

Example 13

ion conductance recording methods set forth in Example I , were used to examine channel. 30 closing kinetics for ChRSS, ChR 0, ChR87, and ChR2 expressed in HEK293 cells. The closing kinetics were examined and compared. 2ms light pulse was used to activate channelrhodopsin and all measurements were voitage clamped to -65 mV. Chronos had the fastest channel closing kinetics and is independent of voltage. Example 14

Genes described under (a), ( i and (ci were expressed in cells using methods provided below. Genes

a) The Chloromonas subdivisa gene referred to herein as Ch 8? and having the amino acid sequence set forth herein as SEQ ID NO: 5 and a mammalian coclo -optimized DNA sequence set forth herein as SEQ ID O:6;

b) The gene for Chlamydomonas noctigama referred to herein as ChR88 or Chrimson, and having the amino acid sequence set forth herein as SEQ ID NO: 1 and a mammalian codon- optinxized DNA sequence set forth herein as SEQ ID NO:2; and

c) The gene for Sligeoc nmm keiveticum, referred to herein as ChR90 or Chronos and having the amino acid sequence set forth herein as SEQ ID NO:3 and having a mammalian codon-optimized DNA sequence set forth herein as SEQ ID NO:4 are expressed in cells as follows.

Methods

(1) The opsin gene was cloned into a lenti viral or adeno-associated virus (AAV)

pa ckagi ng plasmid, or another desired expression plasmid. and then clone GFP downstream of the preferred gene, eliminating the stop codon of the opsin gene, thus creating a fusion protein.

(2) The viral or expression plasmid contained either a strong general promoter, a cell- specific promoter, or a strong general promoter followed by one more logical elements (such as a lox-stop-lox sequence, which will lie removed by Cre recombinase selectively expressed in cells in a transgenic animal, or in a second virus, thus enabling the strong general promoter to then dri ve the gene.

(3) If using a viral plasmid, synthesize the viral vector using the viral plasmid.

(4) If using a virus, as appropriate for gene therapy (over 600 people have been trea ted w th AAV carrying various genetic payloads to date, in 48 separate clinical trials, without a single adverse event), inject the virus using a small needle or cannula into the area of interest, thus delivering the gene encoding the opsin fusion protein into the cel ls of interest If using another expression vector, directly electroporate or inject that vector into the cell or organism (for acutely expressing the opsin, or making a cell line, or a transgenic mouse or other animal).

(5) Illuminate with Sight. For Chronos, peak illumination wavelengths are 500 ran 4·/- 15 ran. For Chrimson, peak iltimiinaiion wavelengths are 590 nm ÷/- 15 nm. (6) To illuminate two different populations of cells (e.g., in a single tissue) with two different colors of light, first target one population with Chrimson, and the other population with Chronos, using two different viruses (e.g., with different coat proteins or promoters) or two different pSasinkls (e.g. , with two different promoters). Then, after the molecule expresses, illuminate the tissue with 470 ±10 urn or 406 ± 10 nm light to preferentially depolarize the Chfonos-expressing cells, and illuminate the tissue with 406 ± 10 nm or 660 ± 10 nm light, to preferentially depolarize the Chrimson- expressing cells.

(7) The above wavelengths illustrate typical modes of operation, but are not meant to constrain the protocols that can be used. Either narrower or broader wavelengths, or differently-centered illumination spectra, can be used. For prosthetic uses, the devices used to deiiver light .may be implanted. For drug screening, a xenon lamp or TED can be used to deliver the light.

Aspects of the invention include compositions of matter that have been reduced to practice, as described below;

Piasmids encoding for the above genes, have been prepared and used to deliver genes into cells, where the genes have been expressed. As an exemplary vector, lenti viruses carrying pay!oads encoding for the above genes have been prepared and used to deliver genes into cells resulting in expression of the light activated ion channels in the ceils. In addition, adeno-associated. viruses carrying payioads encoding for the above genes have been prepared and used to deliver genes into cells, resulting i the expression of the light activated ion channels in the cells. Cells have been prepared that express the light activated ion channels genes set forth in Example 2. Animals have been prepared that include cells that express the light activated ion channels genes disclosed herein. Example 15

Two-color assays are performed. Chronos (for blue light activation) and Chrimson (for red light activation) are expressed in separate sets cells that represent non-overlapping neuronal populations. Following expression, the cell population is exposed to light and the wavelength and ti ting and "dose" of light is optimized using the following parameters.

The population is contacted with blue light having a wavelength between 450 nm to

500 nm, and with a pulse width of between 1 and 5 ms for activation. A pulse widt of 5 ms provides for minimum sub-threshold crosstalk in the blue light, which is defined as <! 5 mV, <10 mV, and optimally as <5 mV. (!) The maximum blue light power that can be used is detennined using by patch clamping Chrimsoii expressing cells, illuminating with blue light and measuring voltage deflection. Optimally using blue light power such that maximum voltage deflection is < lOmV, usually 0.4 to 0,6mW/mm

(2) The optimal bine light power that can be used to drive Chronos is determined using the same conditions as above in (1 ), except using lower light power, such as to 0.4mW/i»m _} and optimally 0.2mW/rnm\ Power depends on expression system and cell type used in the study.

The population is contacted with red light having a wavelength between 620 ran to 640nm, and with a pulse width of between 1 and 5 ms for activation, which may be optimized at 5ms. The optimal light power to drive chrimson in the red is detennined by ramping light powers from 0.5mW/mrrf to l mW/mm ² . The method is optimized such that a minimum red light power is used to achieve 100% spiking for chrimson. It is to be understood that the methods, compositions, and apparatus which have been described above are merely illustrative applications of the principles of the invention.

Numerous modifications may be made b those skilled in the art without departing from the scope of the invention.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for tha purpose and variations ca be made by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety,

We claim: