WO2001068681A2

WO2001068681A2 - Altered ion channel proteins

Info

Publication number: WO2001068681A2
Application number: PCT/GB2001/001108
Authority: WO
Inventors: Alan John Aitken; Mao Xing Chen; Zoe Marie Gladwell; Andrew Jonathan Powell; Simon Nicholas Tate
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2000-03-14
Filing date: 2001-03-14
Publication date: 2001-09-20
Anticipated expiration: 2002-09-14
Also published as: GB0006024D0; WO2001068681A3; AU4082201A

Abstract

Altered ion channel proteins having acquired sensitivity to gating agents are disclosed. The invention is directed towards those ion channels that are refractory to gating agents. The altered ion channel proteins are of use in identifying modulators of the unaltered ion channel. Host cells, vectors and screening assays are also disclosed.

Description

Altered ion channel proteins

The present invention concerns altered ion channel proteins. In particular, the present invention concerns altered ion channel proteins having acquired sensitivity to a gating agent, that is an agent that activates the ion channel protein (or slows or otherwise inhibits the rate of inactivation), thereby permitting the flow of ions via the channel into and/or out of the host cell expressing the altered ion channel protein. Other objects and advantages of the present invention will become apparent from the description below.

Voltage-gated sodium ion channels are responsible for the rising phase of the action potential and as such, play a key role in mediating electrical activity in excitable tissues. The sodium ion channel is activated in response to depolarisation of the membrane. This causes a voltage-dependent conformational change in the ion channel, from a resting, closed conformation to an open, active conformation, the result of which increases the membrane permeability to sodium ions (1, 2). In the case of voltage-gated sodium ion channels the activation of the ion channels is usually transient (10-50ms) since the ion channels rapidly inactivate (i.e. switch to a confirmation which is unable to conduct sodium ions) during a sustained membrane depolarisation. This rapid activation and inactivation of sodium conductance causes the rapid rising phase of the action potential in excitable cells.

Noltage-gated sodium ion channels comprise a multisubunit complex consisting of a large (230-270kDa) highly glycosylated alpha (α) subunit which is usually associated with one or two of the smaller beta (β-subunits (β 1 , β2 and β3) (3, 4). The alpha subunits of voltage-gated sodium ion channels form a large multigene family that has expanded over recent years and at least ten different genes have now been identified in mammals (5-13). This alpha subunit consists of four homologous domains (DI-IV) each containing six potential α-helical transmembrane segments (SI-S6) which make-up the pore forming region. Domains critical for the function of the channel are highly conserved throughout the family of voltage-gated sodium ion channels. These include the S4 voltage sensors, the loop between domains III and IV which is involved in the inactivation of the ion channel and the SSI and SS2 segments of the extracellular loop between transmembrane regions S5 and S6, which are responsible for the channels vestibule and ion selectivity (14-16). β subunits appear to have a role in altering the kinetics of the sodium ion channel during activation and inactivation gating. Expression of the β subunits has been associated with an increase in peak current and a role in trafficking of the α subunit to the membrane (17-20).

The most potent blocker of voltage gated sodium ion channels is the puffer fish toxin, tetrodotoxin, (TTX). While most voltage-gated sodium ion channels are inhibited by low nanomolar concentrations of TTX, there are three ion channels which are only inhibited by micromolar concentrations of TTX. These are the major cardiac ion channel (HI or SKM2) and the sensory neurone specific ion channels, SNS/PN3 (here designated SNS1) and SNS2/NaN (3, 7, 8, 12, 13).

Sensory neurones of mammalian dorsal root ganglion (DRG) transmit sensory information from the periphery to the central nervous system and are known to express at least three distinct kinetic types of voltage-gated sodium currents (21, 22). The small diameter neurones co-express a rapidly inactivating, fast TTX sensitive current and a slowly activating and inactivating TTX resistant sodium current. The larger diameter cells only express TTX sensitive sodium currents that have intermediate activation and inactivation kinetics (23, 24). This electrophysiological analysis has now been supported by molecular distribution studies, which suggest that there is a dynamic expression of voltage-gated sodium ion channels in DRG neurones that can change during development, response to injury and upon exposure to inflammatory mediators (25-28). The small diameter neurones are unmyelinated and are involved in the transmission of pain impulses, these neurons are the c-fibres or nociceptive neurones (29).

Recent experimental evidence has associated and implicated sodium currents with the chronic pain and hypersensitivity pathologies of both inflammatory and neuropathic origin. For example in the small diameter nociceptive neurones, hyperalgesic agents such as prostaglandin E₂ (PGE₂) and serotonin enhance TTX resistant sodium currents and decrease the threshold for inactivation (30, 31). Neuronal injury produces dramatic changes in sodium ion channel expression and distribution, for example accumulation of TTX sensitive sodium ion channels at the neuroma of lesioned axons is thought to be responsible for formation of ectopic discharges (32, 33). Following human peripheral nerve injury there is accumulation of sodium ion channels at the site of injury (34). Following brachial plexus injury in human patients the sensory neuron specific ion channel α-subunits, SNS/PN3 and SNS2/NaN, redistribute in the DRG neurons (35). There is an acute decrease of SNS/PN3 and SNS2/NaN protein in the sensory cell bodies of DRG whose central axons have been avulsed from the spinal cord and an increase in the channel proteins in nerve fibres. Recent studies in the rat have also demonstrated that there is a change in the localisation of SNS/PN3 after nerve injury (36). In each case the neuronal hyperexcitability that results is highly likely to contribute to the induction and maintenance of this sensitised state. It follows that voltage-gated sodium ion channels in sensory neurones may provide a highly tractable and attractive target for the development of novel analgesic and anti-hypersensitivity agents, and thus indicated in diseases such as neuropathic pain, inflammatory pain, migraine and headache, multiple sclerosis and other demyelinating conditions (see ref. 37).

This supposition is supported by the observation that anaesthetic, anticonvulsant and antiarrythmic drugs, each with sodium ion channel blocking activity, can produce analgesia. For example, it has been recognised that sub- anaesthetic doses of lignocaine and bupivocaine elevate pain thresholds in man ( 8, 39). In addition the anticonvulsant agents, phenytoin, carbamazepine and the class la antiarrythmic agent mexilitene are used clinically for neuropathic pain (40-42). The anticonvulsant lamotrigine is also weakly analgesic (43). In order to discover novel voltage-gated Na⁺ ion channel modulating agents it would be desirable to carry out a high-throughput plate-based screen. In order to determine the ability of an agent to inhibit a sodium ion channel it is necessary to determine the ability of the ion channel to open and to conduct Na⁺ ions in the presence of the agent. However, the physiological method of gating these ion channels by membrane depolarisation is difficult to reproduce in a high throughput plate- based assay format.

It is established that α-scorpion toxins (α-ScTx) bind to a common extracellular site on brain Na⁺ ion channels thus inhibiting rapid inactivation, and that domains I and IN of the brain Νa⁺ ion channel α-subunits are implicated in α-ScTx binding (44, 45). The ability of α-ScTx to prevent inactivation of the brain sodium channels provides a convenient method to gate these Na⁺ ion channels in plate-based assays appropriate for high-throughput drug screening (46). Mammalian cells expressing the brain Na⁺ ion channel to be assayed are plated in multi-well plates and pre-incubated in a fluorescent membrane potential-sensitive dye (DiBAC(4)₃). At resting membrane potentials, the ion channels »are believed to be slowly cycling between the closed, open and inactivated configurations (upon the membrane depolarisation that causes physiological gating the rate of transition from closed to open to inactivated is believed to increase). Upon addition of α-ScTx to the multi-well plates the transition from the open to inactivated conformation is inhibited resulting in a slight membrane depolarisation. This initial depolarisation in turn causes more channels to open and not inactivate. Thus a cascade occurs that leads to a pronounced membrane depolarisation which causes a change in the fluorescence of the membrane potential-sensitive dye. This fluorescence change can be detected on a fluorometric imaging plate reader (FLIPR, Molecular Devices, Sunnyvale, CA, USA; 47). If the cells are pre-incubated with agents that inhibit the Na⁺ channel the fluorescence change upon addition of α-ScTx is reduced or completely ablated in a concentration-dependent manner. This method has allowed the activity of the brain Na⁺ ion channels to be assayed in a plate-based system.

However, the present inventors have found that α-ScTx does not inhibit inactivation of the SNS Na⁺ ion channel suggesting that the α-ScTx binding site is not conserved between the brain and SNS Na⁺ ion channels. Thus a similar high throughput plate-based assay using α-ScTx cannot be easily developed to assay the activity of the wild-type SNS Na⁺ ion channel. This presents a barrier to the rapid HTS identification of SNS Na⁺ ion channel inhibitory compounds that would potentially be effective analgesic and anti-hypersensitivity drugs.

In U.S.5,858,713 there is disclosed a voltage-sensitive sodium ion channel having a mutation that renders it permeable to calcium. The purpose of the mutation is to allow detection of calcium flow with readily available calcium sensitive dyes. There is no teaching to mutate an ion channel to make it sensitive to a gating agent. Furthermore, it is believed that the mutation of '713 is introduced into the pore itself which brings with it the risk that the ion flow detected is not an accurate representation of the in vivo situation with the unmodified ion channel.

All references referred to throughout this specification are expressly and entirely incorporated herein by reference.

It is an object of the present invention to provide altered ion channel proteins that may be used in identifying modulators, particularly antagonists, of the unaltered ion channel protein. Such modulators may be used in the preparation of pharmaceutical compositions for the treatment of a range of diseases or disorders, such as those mentioned supra, e.g. pain and hyper sensitivity.

In accordance with the present invention there is provided an altered ion channel protein (e.g. altered voltage sensitive sodium ion channel) having acquired sensitivity to a gating agent. The terms "ion channel protein" and "ion channel" are used interchangeably, unless the context suggests otherwise.

The term "altered ion channel protein" or "altered ion channel" is intended to be understood in a structural sense as opposed to implying that for any given example, a particular process has necessarily been followed in its production. For example, an ion channel protein produced by expression of a mutagenic oligonucleotide of the invention which in turn has been produced by e.g. PCR driven amplification of an ancestry mutagenic oligonucleotide, is an altered ion channel protein according to the present invention, even though the oligonucleotide it is directly produced from has not in itself undergone alteration in a process sense.

By the term "acquired sensitivity" we mean that the altered ion channel protein has substantially increased sensitivity to a gating agent compared to the unaltered ion channel protein. The increased sensitivity is at least two fold, more typically, at least 10 fold, preferably at least 100 fold. The present invention provides means for conferring on an altered ion channel protein, sensitivity to gating agents in circumstances where the unaltered ion channel protein has no or little sensitivity to such agents or a particular gating agent of interest. This enables investigation of the altered ion channel protein, particularly when expressed in a host cell, and identifying modulators of the unaltered ion channel protein for use in the preparation of pharmaceutical compositions for the treatment of diseases or disorders.

This may be achieved by identifying within the primary amino acid sequence (or the polynucleotide e.g. DNA encoding it) of the unaltered, gating agent insensitive ion channel protein that region equivalent to a gating agent sensitive region in a family member. This may be achieved, at least in part, by e.g. sequence alignment studies.

Once identified, the gating agent insensitive region of the unaltered ion channel protein may be changed to have a primary amino acid sequence corresponding (e.g. identical or similar/conservative change) to the gating agent sensitive region of the family member. Polynucleotides encoding the altered ion channel protein may be generated by site-directed mutagenesis with mutagenic oligonucleotides being employed using a single-stranded (e.g. Ml 3 bacteriophage based techniques) or double stranded template (e.g. Chameleon™ or Quikchange™ - Stratagene™). After verifying each mutant by sequencing, the mutated polynucleotide is excised and inserted into a suitable vector so that the altered ion channel protein can be expressed by a host cell (although mechanically based carriers are not excluded). Where a plurality of contiguous polynucleotide bases of the unaltered ion channel protein are changed (i.e. a cassette mutation), splice overlapping extension PCR may be employed.

Thus in a general scheme, an altered ion channel protein according to the invention may be produced as follows: (a) identifying the binding site of a gating agent in the primary amino acid sequence of a gating agent sensitive family member;

(b) aligning the sequence of step (a) with the sequence of the gating agent insensitive ion channel protein;

(c) comparing the sequence of the gating agent binding region of the sensitive family region with its corresponding region in the gating agent insensitive ion channel protein;

(d) producing an oligonucleotide encoding an altered gating agent insensitive ion channel protein having a gating agent binding region identical or similar to the gating agent binding region of the gating sensitive member;

(e) incorporating the oligonucleotide of step (d) into a vector;

(f) introducing the vector of step (e) into a host cell;

(g) permitting the cell of step (f) to express said altered ion channel.

It will be apparent that many variations particularly with respect to the strict order presented here are possible.

Determination of the gating agent binding region can be achieved by a number of routes known or apparent to those skilled in the art. For example, the binding region may be well characterised in the literature. In the event that the binding region is not known or poorly characterised then the skilled person has a number of options available. As one example, synthesise of a radiolabelled gating agent and incubation of this with cells expressing said ion channel protein or alternatively with purified ion channel protein will result in a radiolabelled gating agent-ion channel protein complex which can be proteolytically digested and the fragments probed with e.g. antibodies to isolate the ion channel protein fragment - radiolabelled gating agent complex. Once isolated, the complex can be sequenced to obtain a general picture of the binding region. Further information is then available by site-directed mutagenesis (for example alanine scanning site-directed mutagenesis) of the fragment to identify those bases critical in the binding of the gating agent. From this a comprehensive picture of the binding region can be built up. The term "family" is used herein to indicate a group of proteins which share substantial sequence similarities, either at the level of the primary sequence of the proteins themselves or at the level of the DNA encoding them. The sequence similarities may extend over the entire protein/gene or may be limited to particular regions or domains. Similarities may be based on nucleotide/amino acid sequence identity as well as similarity (for example, those skilled in the art recognise certain amino acids as similar and identify differences based on switches of similar amino acids as conservative changes). Some members of a protein family may be related in the sense that they share a common evolutionary ancestry. The members of a protein family do not necessarily share the same biochemical properties or functions though their similarities are usually reflected in common functional features. The criteria by which protein families are recognised are well known in the art and include computer analysis of large collections of sequences at the level of DNA and protein as well as biochemical techniques such as hybridisation analysis and enzymatic assays (see for example Pearson and Lipman (1988) PNAS USA, 85 :2444). The term "family" embraces "homologue".

The term "homologue" is used herein in two distinct senses. It is used sensu stricto to define the corresponding protein from a different organism (i.e. a species variant or orthologue) in which case there is a direct evolutionary relationship between the protein and its homologue. The term is also used sensu lacto to define a protein which is structurally similar to a given ion channel protein. In this sense, homology is recognised on the basis of purely structural criteria by the presence of amino acid identities and/or conservative amino acid changes (as set forth by Dayhoff et al, Atlas of protein structure, vol. 5, National Biomed Dd'n, Washington D.C., 1979).

Preferably, the unaltered ion channel protein has at least 35% identity with the gating agent sensitive family member or homologue. However preferably the ion channel protein has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, more preferably, at least 90%, 95%, 96%, 97%, 98% or even 99% identity.

The present invention is of particular use in conferring gating agent sensitivity on fast voltage gated ion channel proteins which are insensitive or otherwise refractory to gating agents. Preferred examples are sodium ion channel proteins such as the sensory neurone specific (SNS) family as disclosed in WO 99/47670 (SNS2) WO 97/01577 (here termed SNSl) and purportedly so in Jeong et al (2000; ref. 48). The reader is specifically referred to each reference. Particularly preferred is SNSl as disclosed in WO 97/01577 which is insensitive to α-scorpion toxin. Thus in accordance with the present invention there is provided an altered sodium ion channel protein, particularly SNSl ion channel protein having acquired sensitivity to a gating agent, particularly α-scorpion toxin. The altered SNS ion channel protein comprises a region corresponding to (e.g. identical or similar) to a sodium ion channel protein family member that has sensitivity to α- scorpion toxin such as the brain I, II or III sodium ion channel protein. The alteration takes place in the SNS α subunit.

α-scorpion toxin sensitive and insensitive family members are shown in table 1 below.

Table 1.

In accordance with the present invention there is provided an altered rat SNS 1 ion channel protein wherein positions 1554 to 1569 inclusively are identical to or similar to positions 1607 to 1620 inclusively in rat brain II sodium ion channel protein so as to confer gating agent sensitivity on said altered rat SNSl ion channel protein to α-scorpion toxin. Host cells expressing said altered rat SNSl ion channel protein as hereinbefore described are also provided.

In accordance with the present invention there is provided an altered human SNSl sodium ion channel protein having gating agent sensitivity particularly to scorpion toxin. Positions 1552 to 1566 inclusively of said altered human SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel (e.g. I, II or III). The term "gating agent sensitive region" meaning that region responsible for conferring gating agent sensitivity on the gating agent sensitive family member.

In accordance with the present invention there is provided an altered mouse SNS 1 sodium ion channel protein having gating agent sensitivity particularly to α- scorpion toxin. Positions 1553 to 1567 inclusively of said altered mouse SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel.

In accordance with the present invention there is provided an altered rat SNSl sodium ion channel protein having gating agent sensitivity particularly to scorpion toxin. Positions 1554 to 1569 inclusively of said altered rat SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel or similar thereto.

The term "derived from" is intended to define not only a source in the sense of it being the physical origin for the material but also to define material which has structural and/or functional characteristics which correspond to those of material which does originate from the source. Thus a "plurality of contiguous bases derived from a gating agent sensitive region" need not necessarily have been purified from the gating agent sensitive family member. It is preferred that the unaltered, gating agent insensitive ion channel protein is mammalian, more preferably rat (protein is SEQ.I.D.NO:l, DNA is SEQ.I.D.NO:2), mouse (protein is SEQ.I.D.NO:3, DNA is SEQ.I.D.NO:4) or human (protein is SEQ.I.D.NO:5, DNA is SEQ.I.D.NO:6) of which human is most preferred. Particularly preferred is a human sodium ion channel protein such as SNSl or SNS2. The alteration to the gating agent insensitive amino acid region of the unaltered ion channel protein may take the form of a single base change or a plurality of base changes (which plurality of bases maybe contiguous). Indeed, a single amino acid change may in some circumstances be insufficient for conferring gating agent sensitivity on the altered ion channel protein. For example when the primary amino acid sequences of the D-IVS3 to D-IVS4 region of voltage-gated Na⁺ channel family members are aligned (Fig. 1), the acidic glutamic acid residue at 1613 (brain Ila) is conserved across the rat brain channels whereas in the SNS channels it is neutral (rat SNS) or basic (rat SNS2). The glutamic acid residue at 1616 (brain Ila) is also highly conserved across the brain channels, whereas in rat SNS it is basic. The present inventors hypothesised that increased affinity α-ScTx binding to a recombinantly expressed altered SNSl channel with a α-ScTx binding site similar to that of the brain Na⁺ channels would allow the channel to be gated in a manner similar to the brain Na⁺ channels in a α- ScTx-FLIPR assay. This would afford a previously unavailable opportunity to develop a high throughput screen for SNS channel blockers.

Three altered rat SNS ion channel proteins were constructed in an attempt to confer an α-ScTx binding site with affinity equivalent to that seen in the brain Ila Na⁺ channel protein. The present inventors reasoned that if the high binding affinity of the brain Ila Na⁺ channel for ScTx was attributable to non-identical amino acids of the IVS3-S4 loop together with neighbouring amino acids in IVS3 segment (49), then the most likely candidates for changes in the rat SNS gene product were the amino acids at position 1560 and/or 1563. However, if the tertiary amino acid context of these residues was also important, then the entire non-identical region from amino acid 1554 to 1569 in rSNS would need to be changed so as to correspond to (e.g. identical or similar) with amino acids 1607 to 1620 that form the conserved α-ScTx binding site in the brain Na⁺ channels. The three altered SNS ion channel proteins were tested for α-ScTx sensitivity by whole cell patch-clamp electrophysiology. Only the altered ion channel protein with the entire putative α-ScTx binding site substituted from amino acid 1553 to 1569 inhibited inactivation in the presence of α-ScTx. This altered ion channel enables the development of a plated-based assay for the SNS Na⁺ channel protein and potentially other α-ScTx insensitive Na⁺ channel proteins. Thus those skilled in the art are taught that a single base change in the unaltered gating agent insensitive ion channel protein in a position equivalent to a single base in a family member that is gating agent sensitive may not be sufficient to confer acquired sensitivity on the altered ion channel protein, even if the change corresponds to the single base of the family member. In such circumstances, one should consider a plurality of changes such as a cassette mutation in a polynucleotide sequence encoding the unaltered ion channel protein.

The alteration is achieved through the provision of polynucleotide(s) encoding the altered ion channel protein which is then inserted into a suitable vector as outlined above. It will be apparent to those skilled in the art that due to the degeneracy of the genetic code, many variations of the polynucleotide are possible. Such changes may be by way of addition, substitution, deletion, insertion of one or more polynucleotide bases.

The term "region" includes a single base or a plurality of bases, which plurality may or may not be contiguous. It is preferred that the number of alterations introduced into the primary amino acid sequence to confer acquired gating agent sensitivity is kept to a minimum, i.e. as few changes as are necessary. This ensures that the resulting functional character of the altered ion channel protein reflects as far as possible the unaltered ion channel protein.

Suitable vectors may be a plasmid, phage, minichromosome or transposon of which plasmid is particularly preferred. The vector may include other regulatory elements such as a promoter, operator, activator, repressor and/or enhancer, transcription, translation, initiation and termination sequences. They may also contain sequence encoding any of various tags (e.g. to facilitate subsequent purification of the expressed protein such as affinity (e.g. His) tags. Where the altered ion channel protein is a sodium ion channel protein, particularly SNSl, vectors of the present invention may also comprise polynucleotide sequences encoding the unaltered β subunit of the sodium channel protein. Thus the present invention specifically contemplates expression of an altered α subunit of a sodium ion channel particularly SNSl, either alone or together with one or more β sodium ion channel subunit(s) to form an altered sodium ion channel protein.

Particularly preferred are vectors which comprise a regulatory element or elements operably linked to the mutagenic polynucleotide (e.g. DNA) of the present invention to provide expression thereof at suitable levels (e.g. at a level which facilitates its use in drug screening assays).

Any of a wide variety of regulatory elements may be used and may be regulatable for example being inducible. The term "operably linked" refers to a condition in which portions of a linear polynucleotide sequence are capable of influencing the activity of other portions of the same linear sequence, for example, a ribosome binding site is operably linked to a coding sequence if it positioned so as to permit translation. The vector may be inserted into the host cell according to any conventional method. It is preferred that the altered sodium ion channel has acquired sensitivity to α-scorpion toxin although the acquired sensitivity may be towards one or more of the following gating agents;

Veratridine, batrachotoxin, aconitine, grayanotoxin, sea anemone, β scorpion toxin, brevetoxin, ciguatoxin, deltamethrin or cypermethrin. It is preferred that the altered ion channel protein is amenable to ion flow by its natural ion to better reflect the in vivo situation as opposed to the approach taken in US 5,858,713.

Host cells harbouring the vectors of the present invention (and hence expressing the altered ion channel proteins) are preferably eukaryotic , more preferably mammalian, most preferably human, e.g. a human cell such as a HEK 293 cell. Also preferred are mammalian cell lines such as neuroblastoma. For screening purposes it is preferred to use a human cell line so as to best represent the in vivo situation. Vectors of the present invention maybe inserted into a host cell by way of transfection according to techniques well known or apparent to those skilled in the art. Liposomes and other mechanical carriers are not excluded.

Host cells expressing the altered ion channel protein may then be used in assays for identifying modulators of the unaltered ion channel protein. The modulator may be an agonist, antagonist or mimetic. Preferably the modulator is an antagonist.

By altering the ion channel protein so as to render it sensitive to a gating agent, those skilled in the art will recognise that in drug screening assays, particularly for an antagonist, the ability of a candidate agent to modify activation of the altered ion channel protein when in the presence of the gating agent is indicative of a potential modulator of the unaltered ion channel protein in vivo. Such candidate agents may be identified and further selected and/or modified (i.e. developed) to produce a modulator of the unaltered ion channel protein in vivo (particularly for use in treating a mammalian patient such as a human).

Further selection and/or modification criteria may include one or more of the following considerations; Potency as a ion channel protein modulator, selectivity (towards the target unaltered ion channel protein), pharmacokinetics (e.g. ADME in animal models), in vitro electrophysiology characteristics (e.g. on dorsal sensory neurones or spinal nerve preparations), in vivo activity in appropriate animal models of disease (e.g. chronic constriction injury, model of neuropathic pain, carageenan model of inflammatory pain), chemical synthesis, stability and formulation characteristics of the candidate agent, safety and toxicology (acute and chronic) studies in animals. Development may then proceed to proof of concept studies in man before clinical trials begin. It is particularly preferred that where the goal is to develop a modulator of SNSl or SNS2 ion channel proteins, a candidate modulator which is capable of modulating the altered SNS ion channel protein in vitro does not also modulate activation of the unaltered cardiac sodium ion channel protein in vitro is selected or vice-versa. The aim of which is to minimise potential side effects in vivo as a result of unwanted or inappropriate interaction of a SNS ion channel protein modulator with the cardiac sodium ion channel and vice-versa.

Thus in accordance with the present invention there is provided a method of identifying an ion channel protein modulator, particularly an antagonist of ion channel protein activation, which ion channel protein (which is preferably a sodium ion channel, such as SNS 1 or SNS2) is insensitive or otherwise refractory to known gating agents in vitro which method comprises the steps of;

(a) providing a mutagenic oligonucleotide that encodes an altered ion channel protein having a region identical or similar to a region responsible for conferring gating agent sensitivity on a gating agent sensitive family member of said unaltered ion channel protein such that said altered ion channel protein has acquired sensitivity to said gating agent;

(b) contacting said altered ion channel protein with said candidate modulator in the presence of a gating agent (for example by expressing said altered ion channel protein in a host cell then incubating said host cell with said candidate modulator in the presence of said gating agent) ;

(c) identifying a candidate ion channel modulator which modifies activation of said altered ion channel protein by said gating agent (for example by measuring activation of said altered ion channel protein using membrane potential imaging techniques by said gating agent in both the presence and absence of said candidate ion_. channel modulator.);

(d) and optionally synthesising and/or purifying said ion channel modulator of step (c).

Illustrative of commercially available assays that may be employed in identifying a candidate ion channel modulators include Voltage Imaging Plate Reading (VIPR, Aurora Biosciences Corporation), Fluorometric Imaging Plate Reading (FLIPR, Molecular Devices) or the method involving bioluminescence resonance energy transfer (BRET) as disclosed in WO 99/66324 and to which the reader is specifically referred.

Ion channel protein modulators identified by (or identifiably by) said method are also provided. Medicaments, typically pharmaceutical compositions, comprising said modulator are also provided. Such pharmaceutical compositions generally comprise a therapeutically effective amount of the modulator, usually in unit dosage form together with a pharmaceutically acceptable carrier, excipient and the like as known and called for by accepted pharmaceutical practice. Use of a modulator in the manufacture of a medicament for the treatment of pain and/or a hypersensitivity disease or disorder and methods of treating the same are provided.

In accordance with the present invention there is also provided an altered mammalian cardiac sodium ion channel protein having acquired sensitivity to a gating agent.

In accordance with a further aspect of the present invention there is provided a polynucleotide capable of encoding an altered ion channel protein as hereinbefore described. Also provided are polynucleotides capable of hybridising to said encoding polynucleotides. Preferably such hybridisable polynucleotides hybridise under stringent conditions according to methods well known to those skilled in the art. The exact conditions employed are a matter of choice for the skilled person but stringency may be manipulated by varying temperature and salt conditions. Further guidance may be sought from standard textbooks in the art such as Sambrook J et al, Molecular Cloning: A laboratory manual, 2^nd Edtn, Cold Spring Harbour Labs.

Exemplification

In the figures:

Figure 1: Alignment of SNS, brain, cardiac and neuroendocrine sodium channels across the conserved α-ScTx binding site of the brain channels between domains IVS3 and IVS4. The two conserved amino acids are underlined while the extracellular end of the IVS3 domain is indicated (/). Figure 2: Alignment showing the amino acid sequence substituted into rSNSl from brain II (in bold) to generate the rSNS-bll mutant (SNS mutant 1). The two conserved amino acids are underlined while the extracellular end of the IVS3 domain is indicated (/).

Figure 3: DNA alignments showing the single base pair changes made to generate the single and the double amino acid substitutions (SNS mutants 2 and 3 respectively).

Figure 6: Analysis of α-ScTx sensitivity of wild type rSNSl, human brain IIA and the three novel rSNSl mutants by patch-clamp electrophysiology.

Figure_7: Analysis of α-ScTx sensitive SNS mutant 1 in a FLIPR DiBAC assay.

These Examples are intended to illustrate the invention and not to limit the scope of the appended claims.

Cloning of rat SNS into mammalian expression vector pCIN5:

The rat SNS cDNA (rSNSl) was cloned by screening a rat DRG lambda ZAP express cDNA library (constructed by GW; Clontech vector) using a colony hybridisation protocol, and by RT-PCR performed on rat DRG total RNA. This resulted in 7 fragments spanning the coding region which were then assembled in the vector, pBluescript SK⁺ (Stratagene). This plasmid was then modified by site directed mutagenesis to correct a number of PCR derived mutations and to insert a deleted nucleotide. An upstream ATG was removed before the rSNSl coding region was engineered into the mammalian expression vector pBK-CMV (Stratagene) using Notl and Kpnl restriction sites, to give pBK-CMV-rSNSl (Fig. 4a).

The rat SNSl coding region was then subcloned into a pBR322-based shuttle vector pBRLT7 using the Notl and Kpnl enzyme restriction sites to give pBRLT7- rSNSl (Fig. 4b). The Notl and Mlul sites were then used to transfer the coding region into an IRES mammalian expression vector, pCIN5 (GW) which was digested with Notl and Ascl (Mlul compatible) restriction enzymes, to give pCIN5-rSNSl (Fig. 4c).

Constructing the rSNSl-rBII chimeric α- ScTx binding domain in the rSNS cDNA by splice overlap extension PCR:

98mer reverse-complimenting oligonucleotide primers NCI (5'CGACTTCATAGTGGTGATCCTGTCCATTGTAGGAATGTTTCTCGCGG AGCTGATAGAGAAGTATTTCGTGTCCCCGACACTCTTCCGGGTCATCC GTC3') (SEQ.I.D.NO:7) and NC2

(S'GACGGATGACCCGGAAGAGTGTCGGGGACACGAAATACTTCTCTAT CAGCTCCGCGAGAAACATTCCTACAATGGACAGGATCACCACTATGAA GTCG3') (SEQ.LD.NO:8) were designed as a fusion of the rat SNSl and rat brain II sodium channel (underlined) sequences. PCR reactions were carried out on the rat SNS template using primer pairs NC1-SNS2 (TCACTGAGGTCCAGGGCTGTTTCCTTC) (SEQ.LD.N0.9) and NC2-SNS17 (CCTCATCCTCTTCTTGACTGT) (SEQ.I.D.NO:10), and the eLongase amplification system (Life Technologies) according to manufacturer's instructions. The two PCR fragments were mixed and joined by further PCR using the primer pair SNS2-SNS17. This mutant PCR fragment, designated mutant 1 (Fig.2 shows relevant amino acid changes), was then digested vΛt Xhol and Kpnl restriction enzymes, and replaced the Xhol-Kpnl wild type SNSl fragment of pBK-CMV-rSNSl (Fig. 4a). The entire mutant coding region (rSNSl-BII) was excised by Hindlll and Kpnl restriction enzymes, and inserted into the BstXl-Nsil restriction sites of the IRES mammalian expression vector, pCIN5-PL (GW) by blunt end ligation. The resulting construct was designated pCIN5-rSNSl-rBII and the encoded mutant protein designated rSNSl mutant 1. Constructing the point mutations in the rat SNS cDNA by site directed mutagenesis:

The rat SNSl coding region was sub-cloned into the plasmid pBK-CMV (Stratagene) as described previously, to give pBK-CMV-rSNSl (Fig.4a). Complementary oligonucleotide primer pairs were designed which incorporated the required single and double nucleotide mutations respectively.

The sequence of the primers for mutant 2 were:

SCTX1 5' GAGTCTGCTGTTTTCTGAAATCCTTAAGTCACTGG 3'

(SEQ.I.D.NO:l l)

SCTX2 5' CCAGTGACTTAAGGATTTCAGAAAACAGCAGACTC 3';

(SEQ.I.D.NO:12)

and for mutant 3 were:

SCTX3 5'

GAGTCTGCTGTTTTCTGAAATCCTTGAGTCACTGGAAAACTAC 3'

(SEQ.I.D.N0.13)

SCTX4 5'

GTAGTTTTCCAGTGACTCAAGGATTTCAGAAAACAGCAGACTC 3'

(SEQ.I.D.NO:14)

The mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). For each mutagenesis reaction 50ng of pBK-CMV- rSNSl was combined with 125ng of each complementary primer, lμl of dNTP (25mM of each NTP), 5μl of reaction buffer, ddH₂O to a final volume of 50μl and lμl of Pfu DNA polymerase (2.5U/μl).

Temperature cycling was performed on a PTC-200 DNA engine thermal cycler (GRI) using the following protocol:

Segment Cycles Temperature Time

1 1 95°C 30 seconds

2 16 95°C 30 seconds

55°C 1 minute 68°C 20 minutes

4°C 2 minutes

Following temperature cycling, lμl of Dpnl restriction enzyme (lOU/μl) was added to each amplification reaction and incubated in a water bath at 37°C for 1 hour, then lμl of each reaction was transformed into 50μl E. coli XL2-Blue supercompetent cells.

The transformed bacterial cell suspension plus 450μl of NZY+ broth was pre- incubated for 1 hour at 28°C with shaking at 225rpm, before the entire volume was plated out onto LB agar plates supplemented with 50μg/ml of ampicillin. The plates were then incubated for 18 hours at 28°C. Plasmid DNA was purified from overnight cultures of single colonies picked from these plates using the miniprep spin column protocol (Qiagen). The mutations, designated mutant 2 and 3 for the single and double mutations respectively (Fig.3), were then verified by sequencing the plasmid DNA.

Analysis of the α-ScTx sensitivity of the novel rSNSl mutants.

Mammalian cells (either HΕK293, A, C, D & E or CHO, B) were stably (A, B & C) or transiently (D & E) transfected with expression constructs (Figures 4 & 5) for the wild type or mutant sodium ion channel α-subunits indicated. Whole-cell patch clamp recordings were made from these cells using standard methods (50). Briefly, cells were grown on a glass coverslip, placed into a recording chamber (0.5ml volume) and superfused with an extracellular recording solution at 2 ml min^"1. The extracellular recording solution contained (in mM): 140 NaCl, 4.7 KC1, 1.2 MgCl₂, 1 CaCl₂, 11 glucose, 5 HEPES (pH 7.4). Patch electrodes had resistances of 2 to 6MΩ when filled and the pipette-filling solution contained (in mM): 120 CsF, 15 NaCl, 10 Cs- EGTA (ethylene glycol-bis(β-aminoethyl ester) N,N,N',N-tetra acetic acid Cs salt), 10 HEPES. Currents were recorded at room temperature using an Axopatch 200A amplifier (Axon Instruments Inc.). Currents were elicited by a step depolarisation to +20mV (A), -lOmV (B) or OmV (C, D & E) from a holding potential of-90mV, which in each case yielded the peak Na⁺ current in the representative cells. Current traces were initially recorded prior to the addition of α-ScTx (control) after which lOμg/ml α-ScTx (from Leiurus quinquestriatus from North Africa, Sigma-Aldrich Co. Ltd.) was washed on and the subsequent current traces recorded using the same command protocol as for the control trace (figure 6).

The effect of α-ScTx on the inactivation kinetics of each α-subunit is visible in the representative current traces shown in Figure 6. The inactivation kinetics of wild-type SNS (fig. 6A), mutant 2 (fig. 6D) and mutant 3 (fig. 6E) were unaffected by the addition of lOμg/ml α-ScTx, thus indicating that the substitution of conserved amino acids in the α-ScTx binding site alone is insufficient to confer α-ScTx-sensitivity on the rSNSl ion channel. However, addition of α-ScTx to both the brain II (fig. 6B) and SNS mutant 1 (fig. 6C) gave pronounced inhibition of fast inactivation. These results indicate that substitution of the entire region of rSNS from amino acids 1554 to 1569 with the amino acids that form the conserved α-ScTx binding site in the brain Na⁺ ion channels is necessary to confer α-ScTx-sensitivity on the SNS ion channel.

Assay of rSNSl activity in a plate-based assay using rSNSl mutant 1.

The generation of an SNS mutant that is sensitive to α-ScTx (rSNSl mutant 1) has allowed the development of a plate-based assay similar to those that have been developed for the Na⁺ channels (46). Addition of α-ScTx to cells expressing the SNS mutant 1 ion channel, that have been pre-loaded with the fluorescent dye, DiBAC(4)₃, will cause a depolarisation that can be detected as a change in the fluorescence on FLIPR (46, 47). Representative data from plate based assays carried out on a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices Ltd.) is shown in Figure 7.

The expression plasmids pCIN5-rSNSlwt and pCIN5-rSNSl -mutant 1 were transfected into HEK293 cells by electroporation and stable HEK293 clonal cell lines were isolated following selection in 800μg/ml Geneticin G418 (Life Technologies). Two cell lines that functionally expressed the SNS proteins were identified by electrophysiology (one expressing wild type rSNSl and one expressing the α-ScTx-sensitive rSNSl mutant 1) and these cell lines were used for the FLIPR assays described here. The two cell lines were plated in black, clear bottomed 96 well assay plates (Costar®, Corning Inc.) at a seeding density of 5 x 10⁴ cells per well and incubated for 48 hours prior to assay (37°C). Native HEK293 cells were also plated as a negative control.

The cells were loaded with the membrane potential sensitive dye, DiBAC(4)₃ 5μM, Molecular Probes) either in the presence or absence of 30μg/ml α-ScTx in low sodium buffer (5mM NaCl, 140mM tetramethyl ammonium-Cl, 5mM KC1, 2mM CaCl₂, 0.8mM HEPES, lOmM D-glucose, pH7.4, plus 0.5mM Brilliant Black dye; lOOμl per well) at 37°C for 30 minutes. The plates were transferred to the FLIPR and the cell-loaded DiBAC fluorescence (excited at 488nm) assayed during and following addition of lOOμl of sodium add-back buffer (145mM NaCl, 5mM KC1, 2mM CaCl₂, 0.8mM HEPES, lOmM D-glucose, pH7.4, plus 0.5mM Brilliant Black dye) to each well. An increase in the fluorescence of the membrane potential sensitive dye corresponds to the membrane depolarisation caused by the sodium current passing through the channel (if it has been gated by the α- ScTx) (46, 47).

Figures 7 A and 7B show the representative traces of changes in fluorescence in control HEK293, HEK293 rSNSl wild type (wt) and HEK293 rSNSl mutant 1 ion channel cells following 30 minutes incubation in the absence (A) or presence (B) of 30μg/ml α-ScTx. Similar responses as those in the representative traces were obtained in duplicate wells. In the absence of α- ScTx preincubation, only a small depolarisation occurs in each of the 3 cell types upon addition of sodium. This depolarisation does not correspond to SNS ion channel expression since it occurs in the native HEK293. However, on addition of sodium following preincubation with 30μg/ml α-ScTx the HEK293 rSNSl mutant 1 (the α-ScTx-sensitive rSNSl mutant) gives a significant increase in fluorescence that corresponds to membrane depolarisation indicating that the α-ScTx has gated the mutant ion channel to allowing sodium to flow through. The wild type rSNSl and native HEK cells fail to give this increase in fluorescence indicating that the α-ScTx only gates the ScTx-sensitive mutant ion channel. Thus mutating ofthe rSNSl ion channel in order to change its sensitivity to a gating agent (in this case α- ScTx) has successfully enabled analysis of the channel's activity in a plate- based assay.

In order to determine whether this plate-based assay could be used to identify inhibitors of the ion channel's activity, we preincubated the rSNSl mutant 1 cells with 30μg/ml α-ScTx plus or minus a sodium ion channel inhibitor, tetracaine (lOOμM). Figure 7C, shows that the ion channel inhibitor prevents the change in DiBAC(4)₃ fluorescence that is observed in the absence of inhibitor (control). This demonstrates that rSNSl mutant 1 can be used to screen for compounds/agents that inhibit the ion channel which could be potentially therapeutic. This plate-based assay has the advantage that it could be adapted to high throughput, thus enabling the rapid analysis of many compounds/agents for SNS inhibitor activity.

References:

1. Hille, B. (1984). Ionic Channels of Excitable Membranes. Sinauer, Sunderland, M.A.

2. Kandel, E.R., Schwartz, J.H. & Jassell, T.M. (1991). Principals of Neuroscience. Elseivier Science Publishing Co., Inc, USA.

3. Goldin, A.L. (1994). Ligand and voltage-gated ion channels. (2nd vol.) CRC Press.

4. Morgan, K., Stevens, E.B., Shah, B., Cox, P.J., Dixon, A.K., Lee, K., Pinnock, R.D., Hughes, J., Richardson, P.J., Mizuguchi, K. & Jackson, A.P. (2000). β3: An additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics. Proc. Natl. Acad. Sci. USA 97:2308-2313.

5. Mandel, G. (1992). Tissue-specific expression of the voltage-sensitive sodium channel. J. Membrane Biol 125, 193-205.

6. Catterall, W.A. (1992). Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev. 72, S15-S48.

7. Akopian, A.N., Sivilotti, L & Wood, J. (1996). A tefrodotoxin resistant voltage gated sodium channel expressed by sensory neurones. Nature. 379, 257-262.

8. Sangameswaran, L., Delgado, S.G., Fish, L.M., Koc, B.D., Jakeman, L.B., Stewart, G.R., Sze,P., Hunter, J.C., Eglen, R.M. & Herman, R.C. (1996). Structure and function of a novel voltage-gated, tetrodotoxin- resistant sodium channel specific to sensory neurones. 77ze Journal of Biological Chemistry. 271, 5953-5956. 9. Fish, L.M., Sangameswaran, L., Delgado, S.G., Koch, B.D., Jake-man, L.B., Kwan, J., & Herman, R.C. (1995). Cloning of a sodium channel α- subunit (PN-1) from rat dorsal root ganglia. Soc. Neurosci. Abstr. 21, 1824

10. Klugbauer, N., Lacinova, L., Flockerzi, V. & Hofrnann, F. (1995). Structure and functional expression of a new member of the tetrodotoxin- sensitive voltage-gated sodium channel family from human neuroendocrine cells. EMBO J. 14, 1084-1090.

11. Toledo-Aral, J.J., Moss, B.L., He, Z., Koszowski, A.G., Whisenand, T., Levinson, S.R., Wolf, J.J., Silos-Santiago, I., Halegoua, S. & Mandal, G. Identification of PN-1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurones. Proc. Natl. Acad. Sci. USA. 94, 1527-1532.

12. Tate, S., Benn, S., Hick, C, Trezise, D., John, V., Mannion, R.J., Costigan, M., Plumpton, C, Grose, D., Gladwell, Z., Kendall, G., Dale, K., Bountra, C. & Woolf, C.J. (1998). Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons. Nature Neuroscience. 1, 653-655.

13. Dib-Hajj, S.D., Tyrrell, L., Black, J.A. & Waxman, S.G. (1998). NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy. Proc. Natl. Acad. Sci. USA. 95, 8963-8968.

14. Noda, M, Ikeda, T.,Suzuki, H., Takahashi, T., Kuno, M. & Numa, S. (1986). Expression of functional sodium channels from cloned cDNA. Nature, 322, 826-888. 15. Stuhmer, W., Methfessel, C.,Salmann, B., Noda, M. & Numas,S. (1987). Patch clamp characterization of sodium channels expressed from rat brain cDN A. Ewr. Biophys Journal. 14, 131-138.

16. Fozzard, H.A. & Hankock, D.A. (1996). Structure and function of voltage-dependant sodium channels: comparison of brain II and cardiac isoforms. Physiol. Rev. 76, 887-926.

17.Isom, L.L., De Jongh, K.S. & Catterall, W.A. (1994). Auxilliary subunits of voltage-gated ion channels. Neuron, 12, 1183-1194.

18. Isom, L.L., Scheuer, T., Browstein, A.B.,Ragsdale, D.S., Murphy, B.J. & Catterall, W.A. (1995). Functional co-expression of the β 1 and type II A α subunits of sodium channels in a mammalian cell line. J.Biol. Chem. 270, 3306-3312.

19. Isom, L.L., Ragsdale,D.S., De Jogh, K.S., Westenbroek, R.Ε., Reber, B.F.X.,Scheuer, T. & Catterall, W.A. (1995). Structure and function of the β2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif. Cell. 83, 433-442.

20. Pattόn, D.E., Isom, L.L., Catterall, W.A. & Golden, A.L. (1992). The adult rat brain βl subunit modifies activation and inactivation gating of multiple sodium channel α subunits. J. Biol Chem. 269, 17649-17655.

21. Elliott, A. A. & Elliott, J.R. (1993). Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia. Journal of Physiology. 463, 39-56.

22. Caffery, J.M., Eng, D.L., Black, J.A., Waxman, S.G. & Kocsis, J.D. (1992). Three types of sodium channels in adult rat dorsal root ganglion neurones. Brain Research 592, 283-297. 23. Roy, MX. & Narahashi, T. (1992). Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion. Journal ofNeuroscience. 12, 2104-2111.

24. Black, J.A., Dib-Hajj, S., McNabola, K., Jeste, S., Rizzo, M.A., Kocsis, J.D. & Waxman, S.G. (1996). Spinal sensory neurones express multiple sodium channel α-subunit mRNAs. Mol. Brain Res., 43, 117-131.

25. Black, J.A. & Waxman, S.G. (1996). Sodium channel expression: a dynamic process in neurones and non-neuronal cells. Dev. Neurosci., 18, 139- 152.

26. Waxman, S.G., Kocsis, J.D. & Black, J. A. (1994). Type III sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurones, and is re-expressed following axotomy. J. Neurophysiol., 72, 466- 470.

27. Aguayo, LG. & White G. (1992). Effects of nerve growth factor on TTX- and Capsaicin-sensitivity in adult rat sensory neurones. Brain Res. 570, 61-67

28. Haper, A.A & Lawson, S.N. (1985). Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones. Journal of Physiology. 359, 31-46.

29. Gold, M.S., Dastmalchi, S. & Levine, J.D. (1996a). Co-expression of nociceptor properties in dorsal root ganglion neurones from the adult rat in vitro. Neuroscience, 71, 265-275.

30. England, S., Bevan S. & Docherty, R.J. (1996). PGE₂ modulates the tetrodotoxin-resistant sodium current in neonatal rat dorsal root ganglion neurones via the cyclic AMP-protein kinase A cascade. J. Physiol., 495, 429- 440. 31. Cardenas, C.G., Del Mar, L.P., Cooper, B.Y. & Scroggs, R.S. (1997). 5- HT receptors couple positively to tetrodotoxin-insensitive sodium channels in a subpopulation of capsaicin-sensitive rat sensory neurones. J. Neurosci., 17, 7181-7189.

32. Devor, M., Govrin-Lippman, R. & Angelides, K. (1993). Na⁺ channel immunolocalisation in peripheral mammalian axons and changes following nerve injury and neuroma formation. J. Neuroscience, 13, 1976-1992.

33. Matzner, L. & Devor, M. (1994). Hyperexcitability at sites of nerve injury depends on voltage-sensitive Na⁺ channels. J. Neurophysiol., 11, 349-359.

34. England, J.D., Happel, L.T., Kline, D.G., Gamboni, F., Thouron, C.L., Liu, Z.P. & Levinson, S. (1996). Sodium channel accumulation in humans with painful neuromas. Neurology, 47, 272-276.

35. Coward, K., Plumpton, C, Facer, P., Birch, R., Carlstedt, T., Tate, S., Bountra, C. & Anand, P. (1999). Immunolocalisation of SNS/PN3 and NaN/SNS2 sodium channels in human pain states. Pain, in press.

36. Novakovic, S.D., Tzoumaka, E., McGiven, J., Haraguchi, M., Sangameswaran, L., Gogas, K., Eglen, R. & Hunter, J. (1998). Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions. J. Neuroscience, 18, 2174-2187.

37. Black, J.A., Fjell, J., Dib-Hajj, S., Duncan, I.D., O'Connor, L.T., Fried, K., Gladwell, Z., Tate, S. & Waxman, S.G. (1999). Abnormal expression of SNS/PN3 sodium channel in cerebellar Purkinje cells following loss of myelin in the tiaep rat. NeuroReport 10: 913-918.

38. Boas, R.A., Covino, B.G. & Shahnarian. A. (1982). Analgesic responses to i.v. lignocaine. Br. J. Anaesthesiol. 54, 501-505. 39. Marchettini, P., Lacarenza, M., Marangoni, C, Pellegata, G., Sotgiu, M.L. & Mirne, S. (1992). Lidocaine test in neuralgia. Pain, 48, 377-382.

40. Dejgard, A., Peterson, P. & Kastrup, J. (1988). Mexilitine for treatment of chronic painful diabetic neuropathy. Lancet, 29, 9-11.

41. Tanelian, D.L. & Brose, W.G. (1991). Neuropathic pain can be relieved by drugs that are use-dependent Na⁺ channel blockers: lidocaine, carbamazepine and mexilitine. Anaesthesiology, 74, 949-951.

42. Chabal, C, Jacobsen, L., Mariano, A., Chaney, E. & Britell, C.W. (1992). The use of oral mexilitine for the treatment of pain after peripheral nerve injury. Anaesthesiology, 76, 513-516.

43. Nakamura-Craig, M. & Follenfant, R.L. (1995). Effect of lamotrigine in the acute and chronic hyperalgesia induced by PGE₂ and in the chronic hyperalgesia in rats with streptozotocin-induced diabetes. Pain, 63, 33-37.

44. Catterall, W.A. (1980). Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. Ann. Rev. Pharmacol. Toxicol. 20, 15-43.

45. Catterall, W.A. (1995). Structure and function of voltage-gated ion channels. Ann. Rev. Biochem. 64, 493-531.

46. Denyer, J., Worley, J., Cox, B., Allenby, G. & Banks, M. (1998). HTS approaches to voltage-gated ion channel drug discovery. Drug Dis. Today. 3, 323-332.

47. Schroeder, K.S. & Neagle, B.D. (1996). J. Biomol. Screening. 2, 75-80.

48. Jeong, S-Y., Goto, J., Hashida, H., Suzuki, T., Ogata, K., Masuda, N., Hirai, M., Isahara, K., Uchiyama, Y., & Kanazawa, I. (2000). Biochem. Biophys. Res. Comm. 267: 262-270. 49. Rogers, J.C, Qu, Y., Tanada, T.N., Scheuer, T., & Catterall, W.A. (1996). Molecular determinants of high affinity binding of a-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel α subunit. J. Biol Chem. Ill, 27, 15950-15962.

50. Dupere JRB, Dale T, Starkey S and Xie X (1999). The anticonvulsant BW534U87 depresses epileptiform activity in rat hippocampal slices by an adenosine-dependent mechanism and through inhibition of voltage-gated Na⁺ channels. Br.J Pharmacol. 128:1011-20.

Claims

1. An altered ion channel protein having acquired sensitivity to a gating agent.

2. A protein according to claim 1 which is a voltage sensitive sodium ion channel.

3. A protein according to claim 2 wherein the sodium ion channel is a sensory neurone specific (SNS) ion channel.

4. A protein according to claim 3 wherein the SNS is SNSl or SNS2.

5. A protein according to claim 4 wherein the SNS is SNS 1.

6. A protein according to claim 3,4 or 5 wherein the gating agent is α-scorpion toxin.

7. A protein according to claim 2 wherein the gating agent is selected from the group consisting of;

Veratridine, batrachotoxin, aconitine, grayanotoxin, sea anemone, β -scorpion toxin, brevetoxin, ciguatoxin, deltamethrin, cypermethrin.

8. A protein according to any preceding claim wherein the protein is mammalian.

9. A protein according to claim 8 wherein the protein is rat, mouse or human.

10. A protein according to claim 9 wherein the protein is human.

11. A host cell expressing the protein according to any preceding claim.

12. A cell according to claim 11 which is eukaryotic.

13. A cell according to claim 12 which is mammalian.

14. A cell according to claim 13 which is human.

15. A cell according to claim 14 which is HEK293 or a neuroblastoma.

16. A polynucleotide capable of encoding a protein according to any one of claims 1 to 10.

17. A vector comprising a polynucleotide of claim 16.

18. Use of a protein according to any one of claims 1 to 10 in a screening assay for the identification of modulators capable of modulating an unaltered gating agent insensitive ion channel protein.

19. Use of a modulator identified by the assay of claim 18 in the manufacture of a medicament for the treatment of pain, inflammation or hyper-sensitivity.

20. A method of treating a mammalian patient, particularly human comprising the step of administrating a therapeutically effective amount of a modulator identified by the assay of claim 18.

21. A method for the production of an altered ion channel protein having acquired sensitivity to a gating agent which method comprises the steps of ;

(a) identifying the binding site of a gating agent in the primary amino acid sequence of a gating agent sensitive family member;

(b) aligning the sequence of step (a) with the sequence of the gating agent insensitive ion channel;

(c) comparing the sequence of the gating agent binding region of the sensitive family region with its corresponding region in the gating agent insensitive ion channel;

(d) producing an oligonucleotide encoding an altered gating agent insensitive ion channel having a gating agent binding region identical or similar to the gating agent binding region of the gating sensitive member;

(e) incorporating the oligonucleotide of step (d) into a vector;

(f) introducing the vector of step (e) into a host cell;

(g) causing the cell of step (f) to express said altered ion channel.

22. A method of identifying a modulator of a ion channel protein which has no or little sensitivity to a gating agent which method comprises the steps of;

(a) producing a host cell expressing an altered ion channel protein having acquired sensitivity to said gating agent;

(b) contacting said ion protein of step (a) with a candidate agent;

(c) measuring ion channel protein activation in the presence of said agent;

(d) selecting said agents which modify the activation of step (c);

(e) optionally synthesising and/or purifying said agent of step (d);

(f) optionally chemically modifying said agent of step (e) to improve one or more of the following features; selectivity, pharmacokinetics, potency, toxicological profile, stability; formulation characterisitics.

23. A method of claim 22 wherein the modulator is an antagonist.

24. An altered rat SNSl ion channel protein wherein positions 1554 to 1569 inclusively are identical to or similar to positions 1607 to 1620 inclusively in rat brain II sodium ion channel so as to confer gating sensitivity on said altered rat SNSl ion channel protein to α-scorpion toxin.

25. An altered mouse SNSl sodium ion channel protein having gating agent sensitivity particularly to scorpion toxin wherein positions 1553 to T567 inclusively of said altered mouse SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel or similar thereto.

26. An altered human SNSl sodium ion channel protein having gating agent sensitivity particularly to scorpion toxin wherein positions 1552 to 1566 inclusively of said altered human SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel (e.g. I, II or III) or similar thereto.