USE OF POTASSIUM CHANNELS FOR IDENTIFYING COMPOUNDS THAT HAVE AN EVSECTICIDAL EFFECT

Field of the Invention

The present invention relates to compositions that are useful in agrochemical, veterinary or pharmaceutical fields. In particular, the invention relates to nucleotide sequences that encode polypeptides that are useful in the identification or development of compounds that affect voltage-gated potassium channel including A-type potassium channel activity, indicating that such compounds may be useful as pesticides or as pharmaceuticals.

Background of the Invention The voltage-gated potassium channels play an important role in the proper functioning of the nervous system. Following activation of ion channels that underlie nerve impulse conduction via depolarization of the membrane potential difference, potassium channels are activated and participate in the repolarization of the membrane that resets the cell to its resting level (Keynes, 1951, J. Physiol. (London) 114:119-150; Rudy, 1998, Neurosci., 25:729-749; Hille, 1993, 2001, Ion Channels of Excitable Membranes, Sinauer; Aldrich et al. , 1979, J. Physiol. (London) 291:531 -544; Augustine, 1990, J. Physiol. (London), 431 :343-364; Stuart et al., 1997, Trends Neurosci., 20:125-131; Pallotta and Wagoner 1992, Physiol Rev. 72 (suρpl):s49-67; Robitaille et al., 1993, Neuron 11 :645-655; Kang, et al., 2000 J Neurophysiol vol 80: 70-80; Jan and Jan, 1997, Annu. Rev. Neurosci, 20:91-123; Nichols and Lopatin, 1997, Annu. Rev. Physiol, 59:171-191; Sanguinetti and Spector, 1997, Neuropharmacology, 36: 755-762; Mathie et al.,1998, Gen. Pharmacol. 30:13-24; Coetzee et al., 1999, Ann. N.Y. Acad. ScL, 868:233- 285; Nerbonne. 2000, J. Physiol. (London), 525:285-298). As such, there is compelling logic to target these channels as a means of identifying biologically active compounds, including insecticides Voltage-gated potassium channels have been cloned and expressed from vertebrate and from invertebrate species (Pongs, 1992, Physiol. Rev., 729Suppl 40:s69-88; Butler et

al,. 1989, Science. 243:943-947; Zhong and Wu 1993, J. Neurosci., 13:4669-4679; Chouinard et al., Proc. Natl. Acad. Sci. USA, 92:6763-6767; Chandy and Gutman, 1995, In: Handbook of Receptors and Channels; ligand and voltage-gated channels (North RA, ed) CRC; Jan and Jan, 1997, Annu. Rev. Neurosci, 20:91-123; Bauman et al., 1987, EMBO J., 6:3419-3429; Schwarz, 1994, Curr. Opin. Neurobiol. 4:633-639; Jan and Jan, 1997, Annu Rev Neurosci., 20:91-123, Sheng et al, 1992, Nature, 9:271-84; Rhodes et al., J. Neurosci.; 15:5360-71). The Hyperkinetic subunit of the voltage gated potassium channels has been shown to profoundly affect the function of those channels (Wilson, et al., (1998) JBC vol 273, pages 6389-6394; Chouinard et al., (1995), PNAS vol 93, pages 6763-6767). Coexpression of this subunit with the channel genes can yield more robust currents that resemble those observed in vivo.

Summary of the Invention

One embodiment of the invention relates to nucleotide sequences that encode or may be used to express amino acid sequences that are useful in the identification or development of compounds with (potential) activity as pesticides or as pharmaceuticals. These nucleotide sequences, including mutants and fragments thereof, which will be further described below, will also be referred to herein as "nucleotide sequences of the invention".

Another embodiment of the invention relates to the amino acid sequences - such as proteins or polypeptides - that are encoded by, or that may be obtained by suitable expression of, the nucleotide sequences of the invention. These amino acid sequences, including mutants and fragments thereof, which will be further described below, will also be referred to herein as "amino acid sequences of the invention".

The present invention further relates to the use of the nucleotide sequences of the invention, preferably in the form of a suitable genetic construct as described below, in the transformation of host cells or host organisms, for example for the expression of the amino acid sequences of the invention and to such genetic constructs and host cells.

Another aspect of the invention relates to methods for the identification or development of compounds that can modulate and/or inhibit the biological activity of the amino acid sequences of the invention, in which one or more of the above mentioned nucleotide sequences, amino acid sequences, genetic constructs, host cells or host

organisms are used. Such methods, which will usually be in the form of an assay or screen, will also be further described below.

A further aspect of the invention relates to compounds that can modulate the biological activity of, or that can otherwise interact with, an amino acid sequence of the invention, either in vitro or preferably (also) in vivo. The invention also relates to compositions that contain such compounds, and to the use of such compounds in the preparation of these compositions and the control of pests.

Definitions Collectively, the nucleic acids of the present invention will be referred to herein as "nucleic acids of the invention". Also, where appropriate in the context of the further description of the invention below, the terms "nucleotide sequence of the invention" and "nucleic acid of the invention" may be considered essentially equivalent and essentially interchangeable.

Also, for the purposes of the present invention, a nucleic acid or amino acid sequence is considered to be "(in) essentially isolated (form)" — for example, from its native biological source - when it has been separated from at least one other nucleic acid molecule and/or sequence with which it is usually associated. Similarly, a protein or polypeptide of the invention is considered to be "(in) essentially isolated (form)" - for example, from its native biological source - when it has been effectively separated from other polypeptide molecules with which it is normally associated. In particular, a nucleic acid or polypeptide of the invention is considered "essentially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more.

Detailed Description of the Invention The present invention was established from the finding that the amino acid sequences of the invention can be used as "target(s)" in assays to identify chemical compounds and other factors (with the term "target" having its usual meaning in the art, provide for example the definition given in WO 98/06737) which interact with them in vitro or in vivo. Consequently, compounds or factors that have been identified as interacting with the amino acid sequences of the invention (e.g. by the methods as described herein below) may be useful as active agents in the agrochemical, veterinary or

pharmaceutical fields.

In one embodiment, the invention relates to nucleic acid molecules, preferably in essentially isolated form, which nucleic acid molecules comprise a nucleotide sequence of the invention, and in particular the nucleotide sequence of Aphis Gossypii Shal partial cDNA sequence (SEQ ID NO. 1). The nucleotide sequence of SEQ NO.l was derived or isolated from Aphis gossypii in the manner as further described in the Examples below. This sequence provides an incomplete coding sequence for the Aphis gossypii Kv4 voltage-gated potassium channel also known as the Aphis gossypii Shal gene and mutants and fragments thereof. In another embodiment, the invention relates to a nucleic acid, preferably in essentially isolated form, which nucleic acid comprises a nucleotide sequence of the invention, and in particular the nucleotide sequence of the Heliothis virescens Shaker partial cDNA sequence (SEQ ID NO. 3). The nucleotide sequences of Heliothis virescens Shaker partial cDNA was derived from Heliothis virescens, such as in the manner as further described in the Examples below. This sequence provides complete an incomplete coding sequence for the Heliothis virescens voltage gated potassium channel, KvI . hi a further embodiment, the invention relates to a nucleic acid, preferably in essentially isolated form, which nucleic acid comprises a nucleotide sequence of the invention, and in particular the nucleotide sequence of the Aphis Gossypii Shab partial cDNA (SEQ ID NO. 5) or the Aphis Gossypii Shaw partial cDNA (SEQ ID NO. 7). The nucleotide sequences of the Aphis Gossypii Shab partial cDNA or the Aphis Gossypii Shaw partial cDNA were derived from Aphis gossypii, such as in the manner as further described in the Examples below. These sequences provide partial coding sequences for the Aphis gossypii voltage gated potassium channels Kv2 and Kv3, respectively. In another embodiment, the invention relates to a nucleic acid, preferably in essentially isolated form, which nucleic acid comprises a nucleotide sequence of the invention, and in particular the nucleotide sequence of the Aphis gossypii Hyperkinetic cDNA (SEQ ID NO 9). The nucleotide sequences of the Aphis gossypii Hyperkinetic cDNA was derived from Aphis gossypii, such as in the manner as further described in the Examples below. This sequence provides complete or partial coding sequences for the Aphis gossypii Beta subunit for voltage gated potassium channels.

In one further embodiment, the invention relates to a nucleic acid, preferably in essentially isolated form, which nucleic acid comprises a nucleotide sequence of the invention, and in particular the nucleotide sequence of the Drosophila melanogaster Shal cDNA (SEQ ID NO. 11), the Drosophila melanogaster Shaker cDNA (SEQ ID NO.13), the Drosophila melanogaster Shab cDNA (SEQ ID NO. 15), the Drosophila melanogaster Shaw cDNA (SEQ ID NO.17), or the Drosophila Hyperkinetic cDNA (SEQ ID NO. 19). The nucleotide sequences of Drosophila melanogaster Shal cDNA, Drosophila melanogaster Shaker cDNA , Drosophila melanogaster Shab cDNA, Drosophila melanogaster Shaw cDNA, or Drosophila melanogaster Hyperkinetic cDNA were derived from Drosophila melanogaster,. These sequences provide complete coding sequences for the Drosophila melanogaster voltage gated potassium channels, KvI, Kv2, Kv3, Kv4 and voltage gated potassium channel beta subunit respectively.

In a further embodiment, the invention relates to a nucleic acid, preferably in essentially isolated form, which nucleic acid comprises a nucleotide sequence of the invention, and in particular the nucleotide sequence of a chimeric Shal cDNA. The nucleotide sequence of this chimeric Shal cDNA is a chimeric sequence in which the Aphis gossypii sequence of Aphis Gossypii Shal partial cDNA sequence is provided with a complete amino terminus derived from the Drosophila melanogaster Shal gene (SEQ ID NO. 11). Construction of this sequence is further described in the Examples below. This sequence provides a functional coding sequence.

In an additional embodiment, the invention relates to a nucleic acid, preferably in essentially isolated form, which nucleic acid comprises a nucleotide sequence of the invention, and in particular the nucleotide sequence of a chimeric Hyperkinetic cDNA. The nucleotide sequence of this chimeric Hyperkinetic cDNA is a chimeric sequence in which the Aphis gossypii sequence of Aphis Gossypii Hyperkinetic partial cDNA sequence is provided with a complete amino terminus derived from the Drosophila melanogaster Hyperkinetic gene (SEQ ID NO. 19). Construction of this sequence is further described in the Examples below. This sequence provides a functional coding sequence. Generally, the nucleotide sequences of the invention, when in the form of a nucleic acid, may be DNA or RNA, and may be single stranded or double stranded. For example,

the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism, which may for instance be designed using suitable computer programs such as the BackTranslate analysis tool in Vector NTI (InforMax, Inc., Bethesda, MD). Thus, the nucleotide sequences of the invention may contain intron sequences, and also generally comprises different splice variants.

Yet another embodiment relates to a double stranded RNA molecule directed against a nucleotide sequence of the invention (one strand of which will usually comprise at least part of a nucleotide sequence of the invention). Such double stranded RNA molecules have particular utility in RNA interference studies of gene function (Zamore et al, Cell 101 :25-33 (2000)). The invention also relates to genetic constructs that can be used to provide such double stranded RNA molecules (e.g. by suitable expression in a host cell or host organism, or for example in a bacterial strain such as E.coli). For such constructs, reference is made to Maniatis et al., Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989).

In a broader sense, the term "nucleotide sequence of the invention" also comprises:

- parts or fragments of the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO23;

(natural or synthetic) mutants, variants, alleles, analogs, orthologs (herein below collectively referred to as "mutants") of the nucleotide sequence of SEQ ID NO: 1,

SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO23; as further described below as well as any nucleotide sequence that encodes a polypeptide that comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:22, SEQ ID NO. 24 and fragments thereof and DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency; parts or fragments of such (natural or synthetic) mutants;

- nucleotide fusions of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO23; (or a part or fragment thereof) with at least one further nucleotide sequence;

nucleotide fusions of (natural or synthetic) mutants (or a part or fragment thereof) with at least one further nucleotide sequence; in which such mutants, parts, fragments or fusions are preferably as further described below. The invention also comprises different splice variants of the above nucleotide sequences.

Another embodiment of the invention covers the full length coding sequence of the cDNAs for Aphis gossypii Shal, Shab, Shaw and Hyperkinetic and the full length coding sequence of Heliothis virescens Shaker. These may be isolated using the disclosed partial sequences as described in the Examples. In some embodiments, the nucleotide sequence of the invention is a fragment of a nucleic acid molecule that encodes an voltage-gated potassium channel including A-type potassium channel. Preferably, a nucleotide sequence of the invention will have a length of at least 500 nucleotides, preferably at least 1,000 nucleotides, more preferably at least 1,200 nucleotides; and up to a length of at most 3,500 nucleotides, preferably at most 3,000 nucleotides, more preferably at most, 2,240 nucleotides. Examples of parts or fragments of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO. 23, preferably SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 21 or SEQ ID NO. 23 or a part or fragment of a (natural or synthetic) mutant thereof include, but are not limited to, 5' or 3' truncated nucleotide sequences, or sequences with an introduced in frame start codon or stop codon. Also, two or more such parts or fragments of one or more nucleotide sequences of the invention may be suitably combined (e.g. ligated in frame) to provide a further nucleotide sequence of the invention.

In some embodiments, such parts or fragments comprise at least one continuous stretch of at least 100 nucleotides, preferably at least 250 nucleotides, more preferably at least 500 nucleotides, even more preferably more than 1,000 nucleotides, of the nucleotide sequence of the invention such as SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO. 23, preferably SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 21 or SEQ ID NO. 23. Also, it is expected that - based upon the disclosure herein - the skilled person will be able to identify, derive or isolate natural "mutants" (as mentioned above) of the

nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, preferably SEQ ID NO. 1, or SEQ ID NO. 9, from (other individuals of) the same species (for example from an individual of a different strain or line, including but not limited to mutant strains or lines). It is also expected that - based upon the disclosure herein - the skilled person will be able to provide or derive synthetic mutants (as defined hereinabove) of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO. 23, preferably SEQ ID NO. 1, SEQ ID NO. 9. It is also expected that - based upon the disclosure herein - the skilled person will be able to provide or derive polypeptide having polypeptide sequences of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO. 24 by means of protein expression.

In one specific embodiment, the nucleotide sequence of the invention is such that it encodes Aphis Gossypii Shal, such as that set forth in SEQ ID NO:2. A preferred nucleotide sequence of Aphis Gossypii Shal cDNA sequence is SEQ ID NO: 1. The nucleotide sequence of the invention may be one of these nucleic acid sequences, a part or fragment thereof or a DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency.

In another specific embodiment, the nucleotide sequence of the invention is such that it encodes Heliothis virescens Shaker, such as that set forth in SEQ ID NO:4. A preferred nucleotide sequence of Heliothis virescens Shaker cDNA sequence is SEQ ID NO:3. The nucleotide sequence of the invention may be one of these nucleic acid sequences, a part or fragment thereof or a DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency. In another specific embodiment, the nucleotide sequence of the invention is such that it encodes Aphis Gossypii Shab, such as that set forth in SEQ ID NO:6. A preferred nucleotide sequence of Aphis Gossypii Shab cDNA sequence is SEQ ID NO:5. The nucleotide sequence of the invention may be one of these nucleic acid sequences, a part or fragment thereof or a DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency.

In another specific embodiment, the nucleotide sequence of the invention is such that it encodes Aphis Gossypii Shaw, such as that set forth in SEQ ID NO: 8. A preferred nucleotide sequence of Aphis Gossypii Shaw cDNA sequence is SEQ ID NO:7. The nucleotide sequence of the invention may be one of these nucleic acid sequences, a part or fragment thereof or a DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency.

In another specific embodiment, the nucleotide sequence of the invention is such that it encodes Aphis Gossypii Hyperkinetic, such as that set forth in SEQ ID NO: 10. A preferred nucleotide sequence of Aphis Gossypii Hyperkinetic cDNA sequence is SEQ ID NO:9. The nucleotide sequence of the invention may be one of these nucleic acid sequences, a part or fragment thereof or a DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency.

In another specific embodiment, the nucleotide sequence of the invention is such that it encodes Chimeric Shal, such as that set forth in SEQ ID NO: 22. A preferred nucleotide sequence of Chimeric Shal cDNA sequence is SEQ ID NO:21. The nucleotide sequence of the invention maybe one of these nucleic acid sequences, a part or fragment thereof or a DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency. hi another specific embodiment, the nucleotide sequence of the invention is such that it encodes Chimeric Hyperkinetic, such as that set forth in SEQ ID NO:24. A preferred nucleotide sequence of Chimeric Hyperkinetic cDNA sequence is SEQ ID NO:23. The nucleotide sequence of the invention may be one of these nucleic acid sequences, a part or fragment thereof or a DNA sequences that detectably hybridize to such DNA sequences or their complementary strands under conditions of moderate or high stringency.

Preferably, any mutants as described herein will have one or more, and preferably all, of the structural characteristics or conserved features referred to below for the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO. 23

In particular, any mutants, parts or fragments as described herein may be such that they at least encode the K channel pore and selectivity filter of the corresponding amino acid sequence of the invention

Also, any mutants, parts or fragments as described herein will, in some embodiments, preferably have a degree of "sequence identity", at the nucleotide level, with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, of at least 75%, preferably at least 80%, more preferably at least 85%, and in particular more than 90%, and up to 95% or more.

Also, preferably, any mutants, parts or fragments of the nucleotide sequence of the invention will be such that they encode an amino acid sequence which has a degree of

"sequence identity", at the amino acid level, with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ₁ ID NO. 6, SEQ ID NO: 8, SEQ ID NO: 10, preferably SEQ ID NO. 1, SEQ ID NO 4 or SEQ ID NO. 10, of at least 55%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and in particular more than 90% and up to 95% or more, in which the percentage of "sequence identity" is calculated as described below.

For this purpose, the percentage of "sequence identity" between a given nucleotide sequence and the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, may be calculated by dividing the number of nucleotides in the given nucleotide sequence that are identical to the nucleotide at the corresponding position in the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, by the total number of nucleotides in the given nucleotide sequence and multiplying by 100%, in which each deletion, insertion, substitution or addition of a nucleotide - compared to the sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, is considered as a difference at a single nucleotide position.

The preferred computer program for performing global sequence alignments and determining sequence identity is ClustalW (Higgins et al., Nucleic Acids Research 22:4673-4680 (1994)), which is publicly available for a variety of computer platforms. Preferably the parameters used with the ClustalW program for protein sequence

alignments are ktuple = 1, diagonals = 5, windows = 5, gap = 3, score = PERCENTAGE, matrix = BLOSUM, open penalty = 10.0 and extension penalty = 0.05.

Also, in a preferred aspect, any mutants, parts or fragments as described herein will encode proteins or polypeptides having amino acid sequences similar to the biological active sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, i.e. to a degree of at least 55%, preferably at least 75%, and up to 90%.

Any mutants, parts or fragments as described herein are preferably such that they are capable of hybridizing with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, i.e. under conditions of "moderate stringency", and preferably under conditions of "high stringency". Such conditions will be clear to the skilled person, for example from the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as in EP 0 967 284, EP 1 085 089 or WO 00/55318.

It is also within the scope of the invention to use a fusion of a nucleotide sequence of the invention (as described above) with one or more further nucleotide sequence(s), including but not limited to one or more coding sequences, non-coding sequences or regulatory sequences. Preferably, in such fusions, the one or more further nucleotide sequences are operably connected (as described below) to the nucleotide sequence of the invention (for example so that, when the further nucleotide sequence is a coding sequence, the nucleotide fusion encodes a protein fusion as described below).

In another embodiment, the invention relates to an antisense molecule against a nucleotide sequence of the invention.

A nucleic acid, preferably in essentially isolated form, can be used to express an amino acid sequence of the invention, for example, the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO:22, SEQ ID NO. 24

On the basis of the above, and although the invention is not specifically limited to any specific explanation or mechanism, the nucleotide sequences of some embodiments of the invention encode proteins that have biological activity or ion channel properties of voltage-gated potassium channel including A-type potassium channel from insects of the

orders Diptera, Homoptera, Lepidoptera, as well as Arachnids of the order Acari which include Drosophila melanogaster, Anopheles gambiae, Aphis gossypii, Myzus persicae, Bemisia tabaci, Heliothis vires cens, Helicoverpa zea, Spodoptera frugiperda, Tetranychus urticae The nucleic acids of the invention may also be in the form of a genetic construct, again as further described below. Genetic constructs of the invention will generally comprise at least one nucleotide sequence of the invention, optionally linked to one or more elements of genetic constructs known per se, as described below. Such genetic constructs may be DNA or RNA, and are preferably double-stranded DNA. The constructs may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable independent replication, maintenance and inheritance in the intended host organism. For instance, the genetic construct may be in the form of a vector, such as for example a plasmid, cosmid, a yeast artificial chromosome ("YAC"), a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro or in vivo (e.g. in a suitable host cell or host organism as described below). An expression vector comprising a nucleotide sequence of the invention is also referred to herein as a recombinant expression vector. These constructs will also be referred to herein as "genetic constructs of the invention".

In a preferred embodiment, such a construct a recombinant expression vector which will comprise: a) the nucleotide sequence of the invention; operably connected to: b) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also: c) one or more further elements of genetic constructs known per se; in which the terms

"regulatory element', "promoter", "terminator", "further elements" and "operably connected" have the meanings indicated herein below.

As the one or more "further elements" referred to above, the genetic construct(s) of the invention may generally contain one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), or terminator(s)), 3'- or 5 '-untranslated region(s)

("UTR") sequences, leader sequences, selection markers, expression markers or reporter

genes, or elements that may facilitate or increase (the efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to the skilled person, and may for instance depend upon the type of construct used, the intended host cell or host organism; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g. via constitutive, transient or inducible expression); and the transformation technique to be used.

Preferably, in the genetic constructs of the invention, the one or more further elements are "operably linked" to the nucleotide sequence(s) of the invention or to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered "operably linked" to a coding sequence if said promoter is able to initiate or otherwise control or regulate the transcription or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of said promoter)

Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.

Preferably, the optional further elements of the genetic construct(s) used in the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism. For instance, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant that (for example) said promoter should be capable of initiating or otherwise controlling or regulating the transcription or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked (as defined above). Such a promoter may be a constitutive promoter or an inducible promoter, and may also be such that it (only) provides for expression in a specific stage of development of the host cell or host organism, or such that it (only) provides for expression in a specific cell, tissue, organ or part of a multicellular host organism.

Some particularly preferred promoters include, but are not limited to, constitutive promoters, such as cytomegalovirus ("CMV"), Rous sarcoma virus ("RSV"), simian virus- 40 ("SV40"), for example, pSVL SV40 Late Promoter Expression Vector (Pharmacia

Biotech Inc., Piscataway, NJ), or herpes simplex virus ("HSV") for expression in mammalian cells or insect constitutive promoters such as the immediate early baculovirus promoter described by Jarvis et al. (Methods in Molecular Biology Vol. 39 Baculovirus Expression Protocols, ed. C. Richardson., Hamana Press Inc., Totowa, NJ (1995)) available in pIE vectors from Novagen (Novagen, Inc. Madison, WI) or regulated promoters such as the a CMV promoter under the control of the tetracycline repressor (Invitrogen, Corporation, Carlsbad, CA) or insect inducible promoters such as the Drosophila metallothionein promoter described by Bunch et al. (Nucleic Acids Research, Vol. 6, No. 3 1043-106, (1988)) available in vectors from Invitrogen (Invitrogen Corporation, Carlsbad, CA).

A selection marker should be such that it allows - i.e. under appropriate selection conditions - host cells or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells or organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as geneticin or G-418 (GIBCO- BRL, Grand Island, NY), kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds or (food) components in the medium that are essential for survival of the non-transformed cells or organisms. A leader sequence should be such that - in the intended host cell or host organism - it allows for the desired post-translational modifications or such that it directs the transcribed mRNA to a desired part or organelle of a cell such as a signal peptide. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism, including, but not limited to, picornavirus leaders, potyvirus leaders, a human immunoglobulin heavy-chain binding protein ("BiP"), a tobacco mosaic virus leader ("TMV"), and a maize chlorotic mottle virus leader ("MCMV").

An expression marker or reporter gene should be such that - in the host cell or host organism - it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also allow for the localization of the expressed product, e.g. in a specific part or organelle of a cell or in (a)

specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins, such as GFP, antibody recognition proteins, for example, V5 epitope or poly Histidine available in vectors and antibodies supplied by Invitrogen, or purification affinity handles such as polyhistidine which allows for purification on nickel columns or dihydrofolate reductase which allows for purification on methotrexate column, or markers which allow for selection of cells expressing the gene such as the E. coli beta-galactosidase gene.

For some non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present or used in the genetic constructs of the invention - such as terminators, transcriptional or translational enhancers or integration factors - reference is made to the general handbooks such as Sambrook et al. and Ausubel et al. mentioned above, to W.B. Wood et al., "The nematode Caenorhabditis elegans", Cold Spring Harbor Laboratory Press (1988) and D.L. Riddle et al. , "C ELEGANS II", Cold Spring Harbor Laboratory Press ( 1997), as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Patent 6,207,410, U.S. Patent 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Another embodiment of the invention relates to a host cell or host organism that has been transformed with or contains a nucleotide sequence, with a nucleic acid or with a genetic construct of the invention. The invention also relates to a host cell or host organism that expresses, or (at least) is capable of expressing (e.g. under suitable conditions), an amino acid sequence of the invention. Li some embodiments, the host cell may comprises a recombinant vector that expresses Voltage gated Potassium channel chimeric Shal or Drosophila Shal, or an Aphis gossypii Shal with or without co-expression of a chimeric Hyperkinetic or the Aphis gossypii Hyperkinetic beta subunit. to form a functional Voltage gated Potassium channel. Collectively, such host cells or host organisms will also be referred to herein as "host cells or host organisms of the invention". The host cell may be any suitable (fungal, prokaryotic or eukaryotic) cell or cell line, for example:

a bacterial strain, including but not limited to strains of E. coli, Bacillus, Streptomyces or Pseudomonas; a fungal cell, including but not limited to cells from species of Aspergillus or Tήchoderma; - a yeast cell, including but not limited to cells from species of Kluyveromyces or Saccharomyces; an amphibian cell or cell line, such as Xenopus oocytes.

In one specific embodiment, which may particularly useful when the nucleotide sequences of the invention are (to be) used in the discovery and development of insecticidal compounds, the host cell may be an insect-derived cell or cell line, such as: cells or cell lines derived from Lepidoptera, including, but not limited to, Spodoptera SF9 and Sf21 cells and cells or cell lines derived from Heliothis virescens cells or cell lines derived from Drosophila, such as Schneider and Kc cells; and - cells or cell lines derived from a pest species of interest (as mentioned below), such as from Aphis gossypii

The host cell may also be a mammalian cell or cell line, including but not limited to CHO- and BHK-cells and human cells or cell lines such as HεK, HeLa and COS.

The host organism may be any suitable multicellular (vertebrate or invertebrate) organism, including but not limited to: a nematode, including but not limited to nematodes from the genus Caenorhabditis, such as C. elegans, an insect, including but not limited to species of Aphis, Drosophila, Heliothis, or a specific pest species of interest (such as those mentioned above); - other well known model organisms, such as zebrafish; a mammal such as a rat or mouse;

Other suitable host cells or host organisms will be clear to the skilled person, for example from the handbooks and patent applications mentioned above.

It should be noted that when nucleotide sequences of the invention are expressed in a multicellular organism, they may be expressed throughout the entire organism, or only in one or more specific cells, tissues, organs or parts thereof, for example by expression

under the control of a promoter that is specific for said cell(s), tissue(s), organ(s) or part(s).

The nucleotide sequences may also be expressed during only a specific stage of development or life cycle of the host cell or host organism, again for example by expression under the control of a promoter that is specific for said stage of development or life cycle. Also, as already mentioned above, said expression may be constitutive, transient or inducible.

Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions), an amino acid sequence of the invention (and in case of a host org'anism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and offspring of the host cell or host organism of the invention, which may for instance be obtained by cell division or by sexual or asexual reproduction.

In yet another aspect, the invention relates to a nucleic acid, preferably in (essentially) isolated form, which nucleic acid encodes or can be used to express an amino acid sequence of the invention (as defined herein), and in particular the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:6, SEQ ID:8, SEQ ID NO:10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24 .The amino acid sequence SEQ ID NO: 2, SEQ ID NO:6, SEQ ID:8, SEQ ID NO:10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16 and SEQ ID N0:18.may be isolated from the species mentioned above, using any technique(s) for protein isolation and purification known to one skilled in the art. Alternatively, the amino acid sequence SEQ ID NO: 2, SEQ ID NO:6, SEQ ID:8, SEQ ID NO:10, SEQ LD:12, SEQ ID NO: 14, SEQ ID NO:16 and SEQ ID NO:18, SEQ ID NO. 20, SEQ ID NO. 22 and SEQ ID NO. 24 may be obtained by suitable expression of a suitable nucleotide sequence - such as the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO. 23 or a suitable mutant thereof- in an appropriate host cell or host organism, as further described below.

In another aspect, the invention relates to a protein or polypeptide, preferably in (essentially) isolated form, said protein or polypeptide comprising an amino acid sequence of the invention (as defined above), in particular the amino acid sequence of SEQ ID NO:

2, SEQ ID NO:6, SEQ TD:S, SEQ ID NO:10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24, more particularly preferred the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID No. 10, SEQ ID NO. 22, SEQ ID NO. 24. In a broader sense, the term "amino acid sequence of the invention" also comprises:

- parts or fragments of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:6, SEQ E>:8, SEQ ID NO:10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24,i - (natural or synthetic) mutants, variants, alleles, analogs, orthologs or full length cDNAs (herein below collectively referred to as "analogs") of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:6, SEQ ID:8, SEQ ID NO: 10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24 _5ι jparts or fragments of such analogs; - fusions of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:6, SEQ ID:8,

SEQ ID NO:10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24, (or a part or fragment thereof) with at least one further amino acid residue or sequence fusions of the amino acid sequence of an analog (or a part or fragment thereof) with at least one further amino acid residue or sequence; in which such mutants, parts, fragments or fusions are preferably as further described below.

The term "amino acid sequence of the invention" also comprises "immature" forms of the above-mentioned amino acid sequences, such as a pre-, pro- or prepro-forms or fusions with suitable leader sequences. Also, the amino acid sequences of the invention may have been subjected to post-translational processing or be suitably glycosylated, depending upon the host cell or host organism used to express or produce said amino acid sequence; or may be otherwise modified (e.g. by chemical techniques known per se in the art). Examples of parts or fragments of the amino acid sequence of SEQ ID NO: 2, SEQ

ID NO. 4, SEQ ID NO. 6, SEQ ID:8, SEQ ID NO: 10, SEQ E>:12, SEQ ID NO: 14, SEQ

ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24,, or a part or fragment of a (natural or synthetic) analog thereof mutant thereof include, but are not limited to, N- and C- truncated amino acid sequence. Also, two or more parts or fragments of one or more amino acid sequences of the invention maybe suitably combined to provide an amino acid sequence of the invention.

Preferably, an amino acid sequence of the invention has a length of at least 100 amino acids, preferably at least 250 amino acids, more preferably at least 300 amino acids; and up to a length of at most 1,000 amino acids, preferably at most 750 amino acids, more preferably at most 600 amino acids. Preferably, any such parts or fragments will be such that they comprise at least one continuous stretch of at least 5 amino acids, preferably at least 10 amino acids, more preferably at least 20 amino acids, even more preferably more than 30 amino acids, of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID:8, SEQ ID NO:10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20 In particular, any parts or fragments as described herein are such that they (at least) encode the K channel pore or selectivity filter of the corresponding amino acid sequence of the invention or a binding domain of the corresponding amino acid sequence of the invention. As will be clear to the skilled person, such parts or fragments may find particular use in assay- and screening techniques (as generally described below) and (when said part or fragment is provided in crystalline form) in X-ray crystallography.

Also, it is expected that - based upon the disclosure herein - the skilled person will be able to identify, derive or isolate natural "analogs" (as mentioned above) of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO:6, SEQ ID:8, SEQ ID NO:10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24,, Such mutants could be derived from (other individuals of) the same species (for example from an individual of a different strain or line, including but not limited to mutant strains or lines); or from (individuals of) other species. For example, such analogs could be derived from the insect species mentioned above.

It is also expected that - based upon the disclosure herein - the skilled person will be able to provide or derive synthetic "analogs" (as mentioned above) of the amino sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO: 10,

SEQ JD:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24, preferably SEQ ID NO: 2, SEQ ID NO:4, SEQ ID: 10, SEQ ID NO. 22 and SEQ ID NO:24. Preferably, any mutants as described herein will have one or more, and preferably all, of the structural characteristics or conserved features referred to below for the sequences of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO:10, SEQ ID: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24, preferably SEQ ID NO: 2, SEQ ID NO:4, SEQ ID: 10, SEQ ID NO. 22 and SEQ ID NO:24. Preferably, any analogs, parts or fragments as described herein will be such that they have a degree of "sequence identity", at the amino acid level, with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO: 10, SEQ JD-λ2, SEQID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, preferably SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID:12, and of at least 55%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and in particular more than 90% and up to 95 % or more.

For this purpose, the percentage of "sequence identity" between a given amino acid sequence and the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO:6, SEQ K>:8, SEQ ID NO:10, SEQ ID:12, SEQID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, may be calculated by dividing the number of amino acid residues in the given amino acid sequence that are identical to the amino acid residue at the corresponding position in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO:6, SEQ ID:8, SEQ ID NO: 10, SEQ ID: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, by the total number of amino acid residues in the given amino acid sequence and multiplying by 100%, in which each deletion, insertion, substitution or addition of an amino acid residue - compared to the sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO:6, SEQ ID:8, SEQ ID NO:10, SEQ ID:12, SEQID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, is considered as a difference at

a single amino acid (position). As mentioned above, the preferred method of performing pairwise global sequence alignments fur such calculations is with the program ClustalW. Also, preferably, any analogs, parts or fragments as described herein will have a biological activity that is essentially similar to the biological activity described above for the sequences of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO: 10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24, preferably SEQ ID NO: 2, SEQ ID NO:4, SEQ ID: 10, SEQ ID NO. 22 and SEQ ID NO:24..

It is also within the scope of the invention to use a fusion of an amino acid sequence of the invention (as described above) with one or more further amino acid sequences, for example to provide a protein fusion. Generally, such fusions may be obtained by suitable expression of a suitable nucleotide sequence of the invention - such as a suitable fusion of a nucleotide sequence of the invention with one or more further coding sequences - in an appropriate host cell or host organism, as further described below. One particular embodiment, such fusions may comprise an amino acid sequence of the invention fused with a reporter protein such as glutathione S-transferase ("GST"), green fluorescent protein ("GFP"), luciferase or another fluorescent protein moiety. As will be clear to the skilled person, such fusions may find particular use in expression analysis and similar methodologies. In another embodiment, the fusion partner may be an amino acid sequence or residue that may be used in purification of the expressed amino acid sequence, for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g. by chemical or enzymatical cleavage) to provide the nucleotide sequence of the invention (for this purpose, the sequence or residue may optionally be linked to the amino acid sequence of the invention via a cleavable linker sequence). Some preferred, but non-limiting examples of such residues are multiple histidine residues and glutatione residues.

In one preferred, but non-limiting aspect, any such fusion will have a biological activity that is essentially similar to the biological activity described above for the sequences of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO: 10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO.

22 and SEQ ID NO:24, preferably SEQ ID NO: 2, SEQ ID NO:4, SEQ ID: 10, SEQ ID NO. 22 and SEQ ID NO:24, preferably SEQ ID NO: 2, SEQ ID NO:4, SEQ ID: 10, SEQ ID NO. 22 and SEQ ID NO:24..

The nucleotide sequences and amino acid sequences of the invention may generally be characterized by the presence of one or more of the following structural characteristics or conserved features:

For the Aphis gossypii Kv ₄ (Shal) potassium channel gene: SEQ IS NO.l is a cDNA sequence encompassing part of the open reading frame; SEQ ID NO. 2 is the protein encoded by SEQ ID NO. 1. The Aphis gossypii Kv ₄ (Shal) potassium channel protein sequence is related to other - voltage-gated potassium channels including A-type potassium channel - as set forth in the table below.

For the Aphis gossypii Hyperkinetic beta subunit gene: SEQ IS NO. 9 is a cDNA sequence encompassing part of the open reading frame; SEQ ID NO. 10 is the protein encoded by SEQ ID NO. 9. The Aphis gossypii Hyperkinetic beta subunit protein sequence is related to other - beta subunits of voltage-gated potassium channels including A-type potassium channel - as set forth in the table below.

Comprehensive pair- wise sequence alignments were made of the following sets of sequences. For the Aphis gossypii Kv ₄ (Shal) potassium channel gene: Drosophila melanogaster Shal (SwissProfcP 17971 SEQ. ID NO 12), Human Shal (UniProt TREMBL:Q5T0M0), and Aphis gossypii Shal partial cDNA (SEQ. ID NO 2). For the Aphis gossypii Hyperkinetic beta subunit gene: Drosophila melanogaster Hyperkinetic (UniProt TREMBL: Q9W2W9 SEQ. ID NO 20), Human potassium channel beta subunit (UniProt TREMBL: Q5TG82), and Aphis gossypii Hyperkinetic partial cDNA (SEQ. ID NO 10). Pairwise alignments were made by the method of Needleman and Wunsch (J MoI Biol 48 :443-453 (1970)) as distributed in the Accelrys GCG (Wisconsin package) program suite version 10.3. Alignment parameters specified the BLOSUM62 scoring matrix for similarity calculations, a gap introduction penalty of 8 and a gap extension penalty of 2.

Scoring Table Shal Alignments.

Species Drosophila Aphid

% Identity % Similarity % Identity % Similarity

Human 74 82 52 60 Drosophila 96 98

Alignment spans:

Scoring Table Hyperkinetic Alignments.

Species Drosophila Aphid % Identity % Similarity % Identity % Similarity

Human 51 63 47 57 Drosophila 57 68

Alignment spans:

By analogy to other voltage-gated potassium channel including A-type potassium channel, it is likely that the functional protein is composed of 4 alpha subunits surrounding a central pore which interact with accessory (beta) subunits. (See, e.g., Hannan and Hall, In Comparative Molecular Neurobiology, Y. Pichon, 1993, Birkhuaser Verlag Basel Switzerland).

On the basis of the above, and although the invention is not specifically limited to any specific explanation or mechanism, the nucleotide sequences and amino acid sequences have (biological) activity as a voltage-gated ion channel. In particular, the present invention has shown activity as a - voltage-gated potassium channel including A- type potassium channel - from insects of the order Lepidoptera, for example, borers, cutworms, armyworms, tobacco budworms, fruit worms, cabbage worms, moths, and loppers, preferably tobacco budworms, Hemiptera, which are aphids, leafhoppers,

whiteflies, scales and true bugs that have mouthparts adapted to piercing and sucking.

As is known in the art, function of the channel may be determined by expressing the putative channel RNA in either a Xenopus oocyte expression system or in a cell line. A functional channel would exhibit voltage gated potassium currents that would be evident using techniques known to those skilled in the arts (Methods in Enzymology, Vol. 207, eds: Rudy, B. and Iverson, L.E., section II, A. Expression of Ion channels in Xenopus oocytes, pp. 225-390; Hamill et al., 1981, Pfleugers Arch., 391:85-100; Fenwick et al., 1982, J. Physol (London), 331 :577-87). In addition high throughput methods such as the Rubidium flux assay may be used (Auro Biomed, Vancouver, BC; Sikander et al., (2003), Drug Discovery. ASSAY and Drug Development Technologies vol. 1, pages 709-717; Terstappen, (1999), Analytical Biochemistry vol. 272, pages 149-155).

In addition, control assays may be performed using other known voltage-gated potassium channel including A-type potassium channel from other species. By performing these types of assays, it may be established that the compound found to be active in the test assay is not active in assays using voltage-gated potassium channel including A-type potassium channel from other species. This result would indicate that the compound has some degree of specificity. Kits can be provided to allow for such assays to be performed. Kits can include containers containing some or all of the reagents needed to perform the assay, samples of a polypeptide of the invention, a nucleic acid of the invention, a genetic construct of the invention or a host cell of the invention.

Another embodiment of the invention relates to a nucleic acid probe that is capable of hybridizing with a nucleotide sequence of the invention under conditions of moderate stringency, preferably under conditions of high stringency, and in particular under stringent conditions (all as described above). Such nucleotide probes may for instance be used for detecting or isolating a nucleotide sequence of the invention or as a primer for amplifying a nucleotide sequence of the invention; all using techniques known per se, for which reference is again made to the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.

Preferably, when to be used for detecting or isolating another nucleotide sequence of the invention, such a nucleotide probe will usually have a length of between 15 and 100 nucleotides, and preferably between 20 and 80 nucleotides. When used as a primer for

amplification, such a nucleotide probe will have a length of between 25 and 75 nucleotides, and preferably between 20 and 40 nucleotides.

Generally, such probes can be designed by the skilled person starting from a nucleotide sequence or amino acid sequence of the invention - and in particular the sequence of SEQ ID NO: 1, SEQ ID NO. 2, SEQ ID NO: 3, SEQ ID: 4, SEQ ID NO:5, SEQ ID:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO. 9, SEQ ID. 10, optionally using a suitable computer algorithm. Also, as will be clear to the skilled person, such probes may be degenerate probes. Probes and primers preferably have sequences that include unique sequences. A unique sequence is a sequence that is not found on other DNA molecules. The presence of unique sequences ensures that the probe or primer will not cross hybridize to identical sequences found on other genes. One skilled in the art can readily determine if a probe or primer contains unique sequences by first designing the probe or primer and then comparing the sequences thereof with sequences in databases of known nucleic acid sequences. Such comparisons are routinely performed by those skilled in the art. In a further aspect, the invention relates to methods for preparing mutants and genetic constructs of the nucleotide sequences of the present invention.

Natural mutants of the nucleotide sequences of the present invention may be obtained in a manner essentially analogous to the method described in the Examples, or alternatively by: - construction of a DNA library from the species of interest in an appropriate expression vector system, followed by direct expression of the mutant sequence; construction of a DNA library from the species of interest in an appropriate expression vector system, followed by screening of said library with a probe of the invention (as described below) or with a nucleotide sequence of the invention; - isolation of mRNA that encodes the mutant sequence from the species of interest, followed by cDNA synthesis using reverse transcriptase; or by any other suitable method(s) or technique(s) known per se, for which reference is for instance made to the standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd.ed.), VoIs. 1-3, Cold Spring Harbor Laboratory Press (1989) and F. Ausubel et al., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987).

Techniques for generating such synthetic sequences of the nucleotide sequences of the present invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more parts of one or more naturally occurring sequences, introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes or regions that may easily be digested or ligated using suitable restriction enzymes), and the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers, using for example a sequence of a naturally occurring voltage-gated potassium channel as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above.

The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors include: vectors for expression in mammalian cells: pSVL SV40 (Pharmacia), pMAMneo (Clontech), pcDNA3 (Invitrogen), pMClneo (Stratagene), ρSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565); vectors for expression in bacterials cells: pET vectors (Novagen), pGEX vectors (Pharmacia) and pQE vectors (Qiagen); vectors for expression in yeast or other fungal cells: pYES2 (Invitrogen) and Pichia expression vectors (Invitrogen); vectors for expression in insect cells: pBlueBacII (Invitrogen), pEIl (Novagen), pMT/V5His (Invitrogen); - vectors for expression in Xenopus oocytes: pT7Blue2 (Novagen) or a modified version pT7Blue4 (SEQ ID NO.25)

In a further aspect, the invention relates to methods for transforming a host cell or a host organism with a nucleotide sequence, with a nucleic acid or with a genetic construct of the invention. The invention also relates to the use of a nucleotide sequence, of a nucleic acid or of a genetic construct of the invention transforming a host cell or a host organism.

According to one specific embodiment, the expression of a nucleotide sequence of the invention in a host cell or host organism may be reduced, compared to the original (e.g. native) host cell or host organism. This may for instance be achieved in a transient manner using antisense or RNA-interference techniques well known in the art, or in a constitutive manner using random, site specific or chemical mutagenesis of the nucleotide sequence of the invention.

Suitable transformation techniques will be clear to the skilled person and may depend on the intended host cell or host organism and the genetic construct to be used. Some preferred, but non-limiting examples of suitable techniques include ballistic transformation, (micro-)injection, transfection (e.g. using suitable transposons), electroporation and lipofection. For these and other suitable techniques, reference is again made to the handbooks and patent applications mentioned above.

After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence or genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g. using specific antibodies.

The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Li yet another aspect, the invention relates to methods for producing an amino acid sequence of the invention.

To produce or obtain expression of the amino acid sequences of the invention, a transformed host cell or transformed host organism may generally be kept, maintained or cultured under conditions such that the (desired) amino acid sequence of the invention is

expressed or produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell or host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention.

Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium, the presence of a suitable source of food or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the amino acid sequences of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequence of the invention may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell or host organism used. Also, the amino acid sequence of the invention may be glycosylated, again depending on the host cell or host organism used.

The amino acid sequences of the invention may then be isolated from the host cell or host organism or from the medium in which said host cell or host organism was cultivated, using protein isolation and purification techniques known per se, such as

(preparative) chromatography and electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using a specific, cleavable amino acid sequence fused with the amino acid sequence of the invention) and preparative immunological techniques

(i.e. using antibodies against the amino acid sequence to be isolated). In one embodiment, the amino acid sequence thus obtained may also be used to generate antibodies specifically against said sequence or an antigenic part or epitope thereof.

In one embodiment, the present invention relates to antibodies, for example monoclonal and polyclonal antibodies, that are generated specifically against amino acid sequences of the present invention, preferably SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID

NO: 6, SEQ ID: 8, SEQ ID NO: 10, , SEQ ID NO. 22 and SEQ ID NO: 24, or an analog,

variant, allele, ortholog, part, fragment or epitope thereof. In preferred embodiments, the antibody does not cross reactive with other SEQ ID: 12, SEQ ED NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20.

Such antibodies, which form a further aspect of the invention, may be generated in a manner known per se, for example as described in GB-A-2 357 768, USA 5,693,492, WO 95/32734, WO 96/23882, WO 98/02456, WO 98/41633 and WO 98/49306. Often, but not exclusively, such methods will involve as immunizing a immunocompetent host with the pertinent amino acid sequence of the invention or an immungenic part thereof (such as a specific epitope), in amount(s) and according to a regimen such that antibodies against said amino acid sequence are raised, and than harvesting the antibodies thus generated, e.g. from blood or serum derived from said host.

For instance, polyclonal antibodies can be obtained by immunizing a suitable host such as a goat, rabbit, sheep, rat, pig or mouse with (an epitope of) an amino acid sequence of the invention, optionally with the use of an immunogenic carrier (such as bovine serum albumin or keyhole limpet hemocyanin) or an adjuvant such as Freund's, saponin, aluminium hydroxide or a similar mineral gel, or keyhole limpet hemocyanin or a similar surface active substance. After a suitable immune response has been raised (usually within 1-7 days), the antibodies can be isolated from blood or serum taken from the immunized animal in a manner known per se, which optionally may involve a step of screening for an antibody with desired properties (i.e. specificity) using known immunoassay techniques, for which reference is again made to for instance WO 96/23882.

Monoclonal antibodies may for example be produced using continuous cell lines in culture, including hybridoma-based and similar techniques, again essentially as described in the above cited references. Accordingly, cells and cell lines that produce monoclonal antibodies against an amino acid sequence of the invention form a further aspect of the invention, as do methods for producing antibodies against amino acid sequences of the invention, which methods may generally involve cultivating such a cell and isolating the antibodies from the culture or medium, again using techniques known per se.

Also, Fab-fragments against the amino acid sequences of the invention (such as F (ab) ₂ , Fab' and Fab fragments) may be obtained by digestion of an antibody with pepsin or another protease, reducing disulfide-linkages and treatment with papain and a reducing agent,

respectively. Fab-expression libraries may for instance be obtained by the method of Huse et al., 1989, Science 245:1275-1281.

In another embodiment, the amino acid sequence of the invention, or a host cell or host organism that expresses such an amino acid sequence, may also be used to identify or develop compounds or other factors that can modulate the (biological) activity of, or that can otherwise interact with, the amino acid sequences of the invention, and such uses form further aspects of the invention. As will be clear to the skilled person, in this context, the amino acid sequence of the invention will serve as a target for interaction with such a compound or factor. In this context, the terms "modulate", "modulation, "modulator" and "target' will have their usual meaning in the art, for which reference is inter alia made to the definitions given in WO 98/06737. Generally, a modulator is a compound or factor that can enhance, inhibit or reduce or otherwise alter, influence or affect (collectively referred to as "modulation") a functional property of a biological activity or process (for example, the biological activity of an amino acid sequence of the invention). As noted above, it is known in the art that voltage-gated potassium channel including A-type potassium channel activity can be measured using standard assay techniques. Assays based on expression of the channel RNA in either a Xenopus oocyte expression system or in a cell line are known to those skilled in the art. A functional channel would exhibit voltage gated potassium currents that would be evident using known techniques (Methods in Enzymology, Vol. 207, eds: Rudy, B. and Iverson, L.E., section II, A. Expression of Ion channels in Xenopus oocytes, pp. 225-390; Hamill et al., 1981, Pfleugers Arch., 391:85-100; Fenwick et al., 1982, J. Physol (London), 331 :577-87). Such technology, as well as other well-known technology, can be adapted to methods of finding biologically active compounds that specifically effect the compositions of the present invention, particularly, the polypeptide of the invention. In preferred embodiments, the biological activity is the inhibition of voltage-gated potassium channel including A-type potassium channel activity. In addition control assays may be performed using other known voltage-gated potassium channel including A-type potassium channel from other species. By performing these types of assays, it may be established that the compound found to be active in the test assay is not active in assays using voltage-gated potassium channel including A-type potassium

channel from other species. This result would indicate that the compound has some degree of specificity. Kits can be provided to allow for such assays to be performed. Kits can include containers containing some or all of the reagents needed to perform the assay, samples of a polypeptide of the invention, a nucleic acid of the invention, a genetic construct of the invention or a host cell of the invention.

In this context, the amino acid sequence of the invention may serve as a target for modulation in vitro (e.g. as part of an assay or screen) or for modulation in vivo (e.g. for modulation by a compound or factor that is known to modulate the target, which compound or factor may for example be used as an active compound for agrochemical, veterinary or pharmaceutical use).

For example, the amino acid sequences, host cells or host organisms of the invention may be used as part of an assay or screen that may be used to identify or develop modulators of the amino acid sequence of the invention, such as a primary screen (e.g. a screen used to identify modulators of the target from a set or library of test chemicals with unknown activity with respect to the target) or a secondary assay (e.g. an assay used for validating hits from a primary screen or used in optimizing hit molecules, e.g. as part of hits-to-leads chemistry).

For instance, such an assay or screen may be configured as an in vitro assay or screen. Suitable techniques for such in vitro screening will be clear to the skilled person, and are for example described in Tang et al., (2001), J Biomol Screen. Vol. 6, pages 325- 31.

Such an assay or screen may also be configured as a cell-based assay or screen, in which a host cell of the invention is contacted with or exposed to a test chemical, upon which at least one biological response by the host cell is measured. Also, such an assay or screen may also be configured as an whole animal screen, in which a host organism of the invention is contacted with or exposed to a test chemical, upon which at least one biological response (such as a phenotypical, behavioral or physiological change, including but not limited to paralysis or death) by the host organism is measured. Thus, generally, the assays and screens described above will comprise at least one step in which the test chemical is contacted with the target (or with a host cell or host

organism that expresses the target), and in particular in such a way that a signal is generated that is representative for the modulation of the target by the test chemical. In a further step, said signal may then be detected.

Accordingly, in one aspect, the invention relates to a method for generating a signal that is representative for the interaction of an amino acid sequence of the invention with a test chemical, said method at least comprising the steps of: a) contacting the amino acid sequence of the invention, or a host cell or host organism containing or expressing an amino acid sequence, with said test chemical, in such a way that a signal may be generated that is representative for the interaction between said test chemical and said amino acid sequence; and optionally b) detecting the signal that may thus be generated.

In another aspect, the invention relates to a method for identifying modulators and/or inhibitors of an amino acid sequence of the invention (e.g. from a set or library of test chemicals), said method at least comprising the steps of: a) contacting the amino acid sequence of the invention, or a host cell or host organism containing or expressing an amino acid sequence, with a test chemical, in such a way that a signal may be generated that is representative for the interaction between said test chemical and said the target; and optionally b) detecting the signal that may thus be generated, said signal identifying the modulator and/or inhibitor of said amino acid sequence.

Accordingly, the present invention provides methods of identifying a modulator and/or inhibitor of- voltage-gated potassium channel including A-type potassium channel - activity. In preferred embodiments, the A-type potassium channel protein used in the methods has an amino acid sequence containing sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO: 10, SEQ ID: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO:24, a mutant thereof, a fragment thereof, SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO: 10, SEQ ID: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, In some embodiments, the nucleic acid sequence that encodes the - voltage-gated potassium channel including A-type potassium channel - contains SEQ ID NO: 1, SEQ ID NO. 3,

SEQ ID NO: 5, SEQ ID:7, SEQ ID NO:9, SEQ ID:11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO. 17, SEQ ID. 19, SEQ ID NO. 21 and SEQ ID NO: 23, a fragment thereof, SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO: 5, SEQ ID:7, SEQ ID NO:9, SEQ ID:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO. 17, SEQ ID. 19, SEQ ID NO. 21 and SEQ ID NO: 23.

A test chemical may be part of a set or library of compounds, which may be a diverse set or library or a focussed set or library, as will be clear to the skilled person. The libraries that may be used for such screening can be prepared using combinatorial chemical processes known in the art or conventional means for chemical synthesis. The assays and screens of the invention may be carried out at medium throughput to high throughput, for example in an automated fashion using suitable robotics. In particular, in this embodiment, the method of the invention may be carried out by contacting the target with the test compound in a well of a multi-well plate, such as a standard 24, 96, 384, 1536 or 3456 well plate. Usually, in a screen or assay of the invention, for each measurement, the target or host cell or host organism will be contacted with only a single test compound. However, it is also within the scope of the invention to contact the target with two or more test compounds - either simultaneously or sequentially - for example to determine whether said combination provides a synergistic effect. Once a test chemical has been identified as a modulator and/or inhibitor for an amino acid sequence of the invention (e.g. by means of a screen or assay as described hereinabove), it may be used per se as a modulator and/or inhibitor of a protein containing the relevant amino acid sequence of the invention, preferably, an amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID:8, SEQ ID NO: 10, SEQ ID: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, a mutant thereof, a fragment thereof, SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID: 8, SEQ ID NO: 10, SEQ ID: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, (e.g. as an active substance for agrochemical, veterinary or pharmaceutical use), or it may optionally be further optimized for final use, e.g. to improve properties such as solubility, adsorption, bioavailability, toxicity, stability, persistence, environmental impact, etc.. It will be clear to

the skilled person that the nucleotide sequences, preferably SEQ ID NO: 1, SEQ ID NO. 3, SEQ ID NO: 5, SEQ ID:7, SEQ ID NO:9, SEQ ID:11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO. 17, SEQ ID. 19, SEQ ID NO. 21 and SEQ ID NO: 23, more preferably SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO 9, SEQ ID NO. 21 and SEQ ID NO: 23, amino acid sequences, host cells or host organisms and methods of the invention may find further use in such optimization methodology, for example as (part of) secondary assays.

The invention is not particularly limited to any specific manner or mechanism in or via which the modulator and/or inhibitor (e.g. the test chemical, compound or factor) modulates, inhibits, or interacts with, the target (in vivo or in vitro). For example, the modulator and/or inhibitor may be an agonist, an antagonist, an inverse agonist, a partial agonist, a competitive inhibitor, a non-competitive inhibitor, a cofactor, an allosteric inhibitor or other allosteric factor for the target, or may be a compound or factor that enhances or reduces binding of target to another biological component associated with its (biological) activity, such as another protein or polypeptide, a receptor, or a part of organelle of a cell. As such, the modulator and/or inhibitor may bind with the target (at the active site, at an allosteric site, at a binding domain or at another site' on the target, e.g. covalently or via hydrogen bonding), block and/or inhibit the active site of the target (in a reversible, irreversible or competitive manner), block and/or inhibit a binding domain of the target (in a reversible, irreversible or competitive manner), or influence or change the conformation of the target.

As such, the test chemical, modulator and/or inhibitor may for instance be: an analog of a known substrate of the target; an oligopeptide, e.g. comprising between 2 and 20, preferably between 3 and 15 amino acid residues; - an antisense or double stranded RNA molecule; a protein, polypeptide; a cofactor or an analog of a cofactor.

The test chemical, modulator and/or inhibitor may also be a reference compound or factor, which may be a compound that is known to modulate, inhibit or otherwise interact with the target (e.g. a known substrate or inhibitor for the target) or a compound or factor that is generally known to modulate, inhibit or otherwise interact with other members from

the general class to which the target belongs (e.g. a known substrate or inhibitor of said class).

Preferably, however, the test chemical, modulator and/or inhibitor is a small molecule, by which is meant a molecular entity with a molecular weight of less than 1500, preferably less than 1000. This may for example be an organic, inorganic or organometallic molecule, which may also be in the form or a suitable salt, such as a water- soluble salt. The term "small molecule" also covers complexes, chelates and similar molecular entities, as long as their (total) molecular weight is in the range indicated above. As already mentioned above, the compounds or factors that have been identified or developed as modulators and/or inhibitors of voltage gates potassium channels containing the amino acid sequences of the invention, preferably, an amino acid sequence of SEQ ID NO: 2, SEQ ID NO.4, SEQ ID NO: 6, SEQ ID:8 ₅ SEQ ID NO: 10, SEQ ID: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID.20, SEQ ID NO. 22 and SEQ ID NO: 24, a mutant thereof, a fragment thereof, SEQ ID NO: 2, SEQ ID NO. 4, SEQ ID NO: 6, SEQ ID: 8, SEQ ID NO: 10, SEQ ID:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO. 18, SEQ ID. 20, SEQ ID NO. 22 and SEQ ID NO: 24, (and precursors for such compounds) may be useful as active substances in the agrochemical, veterinary or pharmaceutical fields, for example in the preparation of agrochemical, veterinary or pharmaceutical compositions, and both such modulators as well as compositions containing them further aspects of the invention. For example, in the agrochemical field, the modulators and/or inhibitors of the invention may be used as an insecticide, nematicide, molluscide, helminticide, acaricide or other types of pesticides or biocides, e.g. to prevent or control (infestations with) harmful organisms, both as contact agents and as systemic agents. As such, the modulators and/or inhibitors may for example be used as a crop protection agent, as a pesticide for household use, or as an agent to prevent or treat damage caused by harmful organisms (e.g. for the protection of seed, wood or stored crops or fruits). Preferably, the modulators and/or inhibitors of the invention are used as insecticides. For any such application, one or more modulators and/or inhibitors of the invention may be suitably combined with one or more agronomically acceptable carriers, adjuvants or diluents - and optionally also with one or more further compounds known per se with activity as (for example) a plant protection agent (to broaden the spectrum of action and optionally to provide a synergistic effect),

herbicide, fertilizer or plant growth regulator - to provide a formulation suitable for the intended final use. Such a formulation may for example be in the form of a solution, emulsion, dispersion, concentrate, aerosol, spray, powder, flowable, dust, granule, pellet, fumigation candle, bait or other suitable solid, semi-solid or liquid formulation, and may optionally also contain suitable solvents, emulsifiers, stabilizers, surfactants, antifoam agents, wetting agents, spreading agents, sticking agents, attractants or (for a bait) food components. Reference is made to the standard manuals, such as "Pesticidal Formulation Research", ACS-publications (1969) and "Pesticide Formulations", Wade van Valkenburg Ed, Marcel Dekker publications (1973). Such compositions may generally contain one or more modulators and/or inhibitors of the invention in a suitable amount, which generally may be between 0.1 and 99 %, and in particular between 10 and 50 %, by weight of the total composition.

The modulators and/or inhibitors and compositions of the invention may be particularly useful as insecticides, for example to combat or control undesired or harmful insects (both adult and immature forms, such as larvae) from following orders:

Coleoptera, such as Pissodes strobi, Diabrotica undecimpunctata howardi, and Leptinotarsa decemlineata;

Diptera, such as Rhagoletis pomonella, Mayetiola destructor, and Liriomyza huidobrensis; - Hymenoptera, such as Neodiprion taedae tsugae, Camponotus pennsylvanicus, and Solenopsis wagneri;

Hemiptera, such as Pseudatomoscelis seriatus, Lygus lineolaris (Palisot de Beauvois), Acrosternum hilare, and Aphis gossypii Homoptera; and - Lepidoptera such as Heliothis virescens.

When used to control harmful or undesired organisms, these organisms may be directly contacted with the modulators, inhibitors, or compositions of the invention in an amount suitable to control (e.g. kill or paralyze) the organism. This amount may be readily determined by the skilled person (e.g. by testing the compound on the species to be controlled) and will usually be in the region of between particular between 10 and 500 g/ha, in particular between 100 and 250 g/ha.

The modulators, inhibitors, or compositions of the invention may also be applied systemically (e.g. to the habitat of the organism to be controlled or to the soil), and may also be applied to the plant, seed, fruit etc. to be protected, again in suitable amounts, which can be determined by the skilled person. The modulators and/or inhibitors of the invention may also be incorporated - e.g. as additives - in other compositions known per se, for example to replace other pesticidal compounds normally used in such compositions.

In one specific embodiment, the modulators and/or inhibitors and compositions of the invention may be used in the fields of agrochemical, veterinary or human health to prevent or treat infection or damage or discomfort caused by parasitic organisms, and in particular by parasitic arthropods, nematodes and helminths such as: ectoparasitic arthropods such as ticks, mites, fleas, lice, stable flies, horn flies, blowflies and other biting or sucking ectoparasites; - endoparasites organisms such as helminths; and also to prevent or treat diseases that are caused or transferred by such parasites. For such purposes, the modulators and/or inhibitors of the invention may for example be formulated as a tablet, an oral solution or emulsion, an injectable solution or emulsion, a lotion, an aerosol, a spray, a powder, a dip or a concentrate. In the fields of animal and human health, the modulators, inhibitors, and compositions of the invention may also be used for the prevention or treatment of diseases or disorders in which the amino acid sequence of the invention may be involved as a target. For this purpose, the modulators and/or inhibitors of the invention may be formulated with one or more additives, carriers or diluents acceptable for pharmaceutical or veterinary use, which will be clear to the skilled person.

Thus, in a further aspect, the invention relates to the use of a modulator and/or inhibitor of the invention in the preparation of a composition for agrochemical, veterinary or pharmaceutical use, as described hereinabove. The invention relates to the use of the modulators, inhibitors and compositions of the invention in controlling harmful organisms and in preventing infestation or damage caused by harmful organisms, again as described above.

The invention will now be further illustrated by means of the following non- limiting Examples.

Examples Example 1 - Evaluating the Effect of Test Compounds on Isolated Potassium Cannel Currents in Cultured Neurons.

Neurons are studied in a culture system developed by Hayashi and Hildebrand (1990, J. Neurosci.,10:848-59) and Hayashi and Levine (1992, J. Neurosci., J. Exp. Biol. 171:15-42). Central nervous system tissue is dissected free of the experimental animal and transferred to sterile saline (see below). The perineural sheath is removed with fine forceps and the neuronal cell body packets and accompanying neuropil are placed in an enzymatic solution of Liberase Blendzyme (Roche, 11 988 409 001) at a dilution of 2 uL Liberase stock solution (manufacturer's recommendation) to 1 mL divalent free saline in a 1.5 mL sterile, snap-cap centrifuge tube. Exposure time is 2.5 minutes at 37 degrees C. The tissue is then dissociated by trituration with a fire-polished Pasteur pipette. The cells are pelleted by a 7 minute centrifugation at an RCF value of 2. The action of the enzyme is terminated by two centrifugations (7 minutes at RCF 2) of the cells through 1 mL of L-Ll 5 culture media to which O'Dowd additives (see below) are added. After the final centrifugation, the cell pellet is resuspended in sterile culture media and the cells are dispersed onto glass bottomed 1 cm wells cut out of a 35 mm plastic petri dish (MatTeck, P35G-1.5-10C) by aliquoting 150 uL of cell suspension per well. Cells adhered to the coated surface (see below) within 1.5 hours, after which time the dishes were flooded with 1.5 mL of I-L15 plus O'Dowd additives. Cultures are grown at room temperature.

Cells are grown on glass surfaces that are coated by incubation with Concanavalin A (200 ug/mL) and Laminin (2 ug/mL) for 2 hours at 37 degrees C. Excess solution is rinsed off with sterile water and, following vacuum aspiration, the dishes are air dried.

Voltage-gated potassium currents are observed using standard techniques of whole-cell patch clamping (Hamill et al., 1981, Pfleugers Arch., 391:85-100; Fenwick et al., 1982, J. Physol (London), 331:577-87). Recordings are made with an AxoPatch 200 B amplifier (Axon Instruments) and a Digidata 1200 analog to digital converter (Axon Instruments) controlled by pClamp software (Axon Instruments) run on a standard

Pentium class computer. The electrode is positioned with a motor driven translation stage (Newport Electronics) controlled by an ESP 300 motion control driver (Newport Electronics). Cells are visualized for patching with a Hoffman optics 40X lens on an Olympus 1X51 microscope. Superfusing saline is delivered via a Pl peristaltic pump (Invitrogen) and evacuated with vacuum aspiration. Patch-clamp recordings are made with glass pipettes fashioned from 1.7 mm outside diameter (OD) borosilicate 100 uL capillary glass (Drummond,2-000-100). For recordings from smaller, more fragile cells, 1.5 mm OD Corning 7056 glass capillary tubing (Warner, 64-0812) is used. All pipettes are made with a gravity driven two stage pipette puller (Narishige, pp830). The composition of saline solutions used is as follows: Sterile saline solution: Saline solution (after Su and O'Dowd, 2004, J. Neurosci., 23:9246-53) used in the dissociation of cells consisted of (in mM) 126 NaCl, 4 KCl, 33.3 glucose, 43.3 sucrose, 9.9 HEPES. The pH was adjusted to 7 and the osmolarity was 336 mOsm. I-L15 culture medium plus O'Dowd additives: Insect L- 15 medium consisted of L- 15 medium with L-glutamine (GIBCO, 11415-064) to which is added per liter, 379 mg alpha-ketoglutaric acid, 400 mg D-(-)-fructose, 700 mg D- glucose, 670 mg D-malic acid, 60 mg succinic acid, 1 gm TC yeastolate, 1 gm lactalbumin hydrolysate, 0.02 mg niacin, 60 mg imidazole, 10 mL enicillin/streptomycin, 1 mg 20- hydroxyecdysone, 5 mL stable vitamin mix (SVM) and 100 mL heat inactivated fetal bovine serum (Hyclone). The pH is adjusted to 7 and the osmolarity to 350 mOsm. The following additives were added on the week of use, insulin at 50 ug/mL, transferrin at 100 ug/mL, putrescence at 100 uM, selenium at 30 nM, and progesterone at 20 ng/mL (Su and O'Dowd, 2003, J. Neurosci., 23:9246-53). SVM: Stable vitamin mix (Mains and Patterson, 1973) is made by adding the following ingredients to make 100 mL of stock solution: 300 mg L-aspartic acid, 300 mg Sistine, 100 mg beta alanine, 0.4 mg biotin, 40 mg vitamin B12, 200 mg inositol, 200 mg choline chloride, 10 mg lipoic acid, 100 mg p- aminobenzoic acid, 500 mg fumaric acid, 8 mg coenzyme A, 300 mg L-glutamic acid.

Artificial Insect Solution (AIS): Patch-clamp recordings were made in AIS that contained, in mM, 150 NaCl, 6 KCl, 6 CaC12, 10 HEPES, 5 glucose. The pH is set at 7 and the osmolarity at 360 mOsm for lepidopteran cells and 330 for dipteran and cockroach cells. AIS for potassium current patch-clamp recording: Potassium currents are isolated by blockade of sodium currents with the addition 10-6 M tetrodotoxin and by blockade of calcium currents with the addition of 500 uM Cd. The composition of patch pipette solution is as follows: Pipette solution for whole-cell patch-clamp recording: Potassium currents are recorded with a pipette solution that consisted of, in mM, 150 KAspartate, 2 MgC12, 1 CaC12, 11 EGTA, 5 HEPES. The pH was set at 7 and the osmolarity set at 330 mOsm.

All media were sterilized by 0.22 uM filtration.

Potassium currents in this system would be exhibited as a voltage sensitive outward current from a holding potential of -80 mV. During a continuous recording session in voltage clamp, the test compound of interest would be added to the continuously flowing supervising saline. An active agent would result in a change in the voltage sensitive currents observed. An active agent with desirable properties may either increase or decrease the amplitude of the observed voltage-sensitive current because either situation would lead to a disruption of the normal physiologic state of the in situ neuron.

The commercial insecticide Flonicamid (N-cyanomethyl nicotinamide, CAS 158062-67-0) blocks an A-type potassium current in cultured H. virescens neurons (see figure 1). We conclude that the blockade this current causes a disruption in synaptic transmission and that this mechanism underlies the efficacy of this compound against insect pest species. The differing efficacy of Flonicamid against different species, and the inactivity of Flonicamid against vertebrates, is likely due to species specific differences in the sequence of the A-type potassium channel.

Example 2 - Construction of cDNA libraries and sequence databases cDNA libraries enriched for full-length clones were constructed by Invitrogen Corporation (Carlsbad, CA) from poly(A)-containing RNA purified from (a) mixed life stage Aphis gossypii (CA cDNA library), and (b) dissected heads from late instar Heliothis

virescens larvae (TBW head cDNA library). Both libraries were constructed in plasmid vector pCMV-SPORT6.1 and transformed into E. coli strain DHlOB (TonA). The libraries were stored without further amplification as glycerol cultures at -8O ⁰ C. A portion of each cDNA library was amplified, normalized and arrayed in 384 well plates by Invitrogen Corporation. The arrayed clones were subjected to automated sequence analysis using vector primers that flanked the 5' and 3' termini of the cloned cDNA (Genome Therapeutics, Waltham, MA). The sequence reads were trimmed for quality and vector contamination, assembled into contigs and installed into a BLAST-compatible database. Access to the Basic Local Alignment Sequence Tool (BLAST) suite of search tools and instructions for the use of these tools and the construction of BLAST-compatible databases can be obtained from the National Center for Biotechnology Information at the National Institutes for Health (Bethesda, MD).

An additional Heliothis virescens cDNA library was constructed from poly(A)- containing RNA isolated from ventral nerve cords and brains of late instar Heliothis virescens larvae (TBW nerve cord library) as described in detail in WO/04044553, hi a like manner an additional Aphis gossypii cDNA library was constructed from 0.7 grams of cotton aphids collected from cotton plants and frozen at -8O ⁰ C prior to niRNA isolation.

Example 3 Construction of Chimeric Shal and Hyperkinetic

The full-length clone for a functional Shal K channel was constructed by joining a 5' fragment from the Drosophila Shal cDNA to the 3' portion of the Aphis gossypii

Hyperkinetic cDNA. Specifically the 5' Hind III to Nco I fragment from the Drosophila Shal cDNA (SEQ ID NO 11) was ligated to the Nco I to Xho I fragment of the Aphis gossypii Shal cDNA (SEQ ID NO 1) in a reaction also containing either a Hind III to Xho I cut pT7Blue vector or a Hind III to Xho I digested pcDNA 3.1 (Invitrogen Corp., Carlsbad, California). These two ligations will result in an oocyte expression vector and a mammalian cell expression vector respectively containing a full-length chimeric Shal cDNA (SEQ ID NO. 21) with the deduced amino acid sequence for Hyperkinetic Chimeric Hyperkinetic cDNA (SEQ ID NO. 22).

The full-length clone for a functional Hyperkinetic Beta subunit was constructed by joining a 5' fragment from the Drosophila Hyperkinetic cDNA to the 3' portion of the

Aphis gossypii Hyperkinetic cDNA. Specifically, PCR using oligos DmOOl and Dm002 and the cloned Drosophila Hyperkinetic cDNA (SEQ ID NO. 19) is used to generate a fragment encoding the 5' region of the Hyperkinetic cDNA. Use of DmOOl in the PCR reaction will generate an EcoRI site and a Kozak sequence upstream of the start codon of the Hk cDNA. The remainder of the Hyperkinetic gene is isolated from the Aphis gossypii cDNA (SEQ ID NO. 10) as a Pvu II to Xho I fragment. These two fragments are then ligated together in a reaction containing either pT7Blue4 digested with Eco RI and Xho I or alternatively pCMV-SPORT6.1 digested with Eco RI and Xho I. These two ligations will result in an oocyte expression vector and a mammalian cell expression vector respectively containing a full-length chimeric Hyperkinetic cDNA (SEQ ID NO. 23) with the deduced amino acid sequence for Hyperkinetic Chimeric Hyperkinetic cDNA (SEQ ID NO. 24).

DmOOl TAG CGT GAATTC GCCACCATGTGC CGG GCT CCCATT GCG

Dm002 CTC CGT CTC CGAGTGCGC CTC CGAGAT GTC

Example 4 - RecA cloning of the full length genes

Full-length cDNAs corresponding to Aphis Gossypii Shal partial cDNA sequence, Heliothis virescens Shaker partial cDNA sequence, Aphis Gossypii Shab partial cDNA sequence, Aphis Gossypii Shaw partial cDNA sequence and Aphis Gossypii Hyperkinetic partial cDNA sequence were isolated from the cotton aphid cDNA library by RecA- mediated gene enrichment using the RecActive™ Gene Enrichment Kit and protocol from Active Motif (Carlsbad, CA). Briefly, a biotinylated probe was synthesized from the cDNA fragment by a PCR reaction containing the appropriate primers B and C (see Table X) and biotin-dNTP mix from RecActive™. The biotinylated probe was then gel purified and used to clone full length Cotton Aphid cDNAs from the non-normalized Cotton Aphid cDNA library (Example 1) by recA-mediated gene selection, as implemented in the RecActive™ Gene Enrichment Kit. PCR using the appropriate primers A and D were used to screen isolated clones. Resulting clones of the correct size for a full-length insert for each of the K channel genes plus Hyperkinetic were sequenced to confirm

completeness. As these cDNAs were initially cloned in expression vector pCMV- SPORT6.1 (Invitrogen Corp.), no additional subcloning into an expression vector was required.

Example 5 — Functional expression of Insect Voltage-gated Potassium Channels in Xenopus oocytes

Generation of capped mRNA with poly (A) tail: The protein coding regions of the Drosophila Shal channel, the chimera Aphis gossypii Shal channel and the native Aphis gossypii Shal gene were subcloned into the oocyte expression vector pT7Blue4 using PCR with appropriate primers. The newly constructed oocyte expression vectors were

linearized. The linearized DNA was then purified by phenol-chloroform extraction and ethanol precipitation. The DNA was used for making capped mRNA following the manual of mMESSAGE mMACHINE™ high yield capped RNA transcription kit (Ambion, Austin, TX). After transcription reaction, the samples were treated with DNase I to remove the template DNA. The mRNA was phenol-chloroform extracted and ethanol precipitated. The concentration of capped mRNA with poly(A) tail was measured by UV spectrometry. The mRNA solution was stored at -20 ⁰ C for future use.

Evaluation of test compounds on potassium channels in the Xenopus oocyte gene expression system: Oocytes used for gene expression are surgically harvested from Xenopus laevis frogs following anesthesia with 0.75% 3-amino benzoic acid ethyl ester (Tricaine) for 5 minutes. The harvested eggs are rinsed in calcium free OR2 solution. Eggs are defolliculated following incubation in collagenase (crude typel, Sigma, St. Louis, MO) at 4 mg/mL in calcium free OR2 for 2 hours at 17 degreed C under rotary agitation. Eggs are stripped free of the oviduct with forceps and mechanically defolliculated.

Calcium free OR2 solution for Xenopus laevis oocytes: This solution consists of, in mM, 82.5 NaCl, 2 KCl, 1 MgC12, 5 HEPES and is set to a pH of 7.5. ND96 solution for oocyte incubation: This solution consists of, in mM, 96 NaCl, 2 KCl, 1 MgC12, 1.8 CaC12, 5 HEPES and is adjusted to pH 7.4. Defolliculated oocytes are injected with a Nanoject microinjector (Drummond,

Broomhall, PA) with between 3 and 30ng of RNA (4.6 to 92 nL), as appropriate for proper expression. Standard Drummond 1.2 mm OD, 3.5 inch capillaries (Drummond, 3-000- 203-G/X). Pipettes are fashioned on a pp830 pipette puller (Narishige, East Meadow, NY). Injection pipettes are backfilled with mineral oil and then tip filled with mRNA maintained at low temperature. Oocytes are individually injected and maintained in ND96 at 17 degrees C on a rotary shaker until expression levels are favorable for recording, typically 3 to 10 days.

Current flow through ion channels that are expressed in Xenopus laevis oocytes injected with sample mRNA is recorded using the two-electrode voltage-clamp technique. Recordings are made using an AxoClamp 2B amplifier and a Digidata 1322A analog to digital converter controlled by pClamp software (all from Axon Instruments, Molecular

Devices, Sunnyvale, CA) ran on a Pentium class computer. Electrodes are fabricated using a pp830 pipette puller (Narishige, East Meadow, NY) from 100 cm long, 1.5 mm OD, standard wall, borosilicate capillaries with internal filament (Warner, 64-0793). Electrodes are positioned with a MN-153 micromanipulators (Narishige, East Meadow, NY). One electrode injects current and the other is used to measure the electrical potential difference across the membrane and standard recording techniques are used (Stumer, 1992, In: Methods in Enzymology, 207:319-345). Recordings are made in ND96 saline. The recording bath is superfused with fresh ND96 via a Pl peristaltic pump (Invitrogen, Carlsbad, CA) and solution in continuously evacuated via vacuum aspiration. Methods used in the Xenopus oocyte voltage-clamping are well established in the field (Methods in Enzymology, Vol. 207, eds: Rudy, B. and Iverson, L.E., section II, A. Expression of Ion channels in Xenopus oocytes, pp. 225-390).

Potassium currents in this system are exhibited as a voltage sensitive outward current from a holding current of -80 mV. During a continuous recording session in voltage clamp, the test compound of interest is added to the continuously flowing superfusing saline. An active agent results in a change in the voltage sensitive currents observed. An active agent with desirable properties may either increase or decrease the amplitude of the observed voltage-sensitive current because either situation would lead to a disruption of the normal physiologic state of the in situ neuron.

Example 6 - Generation of Stable Mammalian clones expressing functional K channels and evaluation of test compounds on potassium channels expressed in a transfected cell line.

The potassium channel expression vector encoding the deduced amino acid sequence for Drosophila melanogaster Shal (SEQ. ID NO 11) AAF49144 or the deduced amino acid sequence for chimeric Shal (SEQ ID NO 21) was transformed into HEK293 or CHO cells obtained from American Type Tissue Culture ("ATTC", Manassa, VA) using the Lipofectamine 2000 (Invitrogen, Carlsbad CA) according to manufacturer's instruction. Cells are co-transfected with either the potassium channel gene and a plasmid such as pTracer(Invitrogen) encoding a marker gene such as the fluorescent Green

Fluorescent Protein (GFP) or the potassium channel gene plus the hyperkinetic beta subunit gene as well as the marker gene. Successful transfection is signaled by cells that exhibit a green fluorescence under Fluorescine channel illumination. Stable cell lines were generated by selection with the appropriate antibiotic, in the case of cotransfection with pTracer this would be Zeocin™. In addition, each potassium channel expression vector was cotransfected with an expression vector encoding the deduced amino acid sequence for Drosophila melanogaster Hyperkinetic (SEQ ID NO 20) or the deduced amino acid sequence for chimeric Hyperkinetic (SEQ ID NO 24). The cells exhibiting the most intense green fluorescence would be expected to be the cells that also contained the most transfected channel protein and these cells were examined by whole-cell patch clamping. A successful transfection of a potassium channel is indicated by the observation of a voltage-sensitive outward current using standard whole-cell patch clamp technique, see Example 1. Compounds of interest were assessed for activity by inclusion of the compound in the superfusing saline while continuously recording from the cell containing the transfected channel. An active agent with desirable properties may either increase or decrease the amplitude of the observed voltage-sensitive current because either situation would lead to a disruption of the normal physiologic state of the in situ neuron.