METHOD FOR THE SCREENING OF alpha 2 delta -1 SUBUNIT BINDING LIGANDS

Abstract: A method for the screening of ligands which bind to soluble alpha 2 delta -1 subtype polypeptides. The invention also relates to compositions and kits for implementing such methods, as well as to the ligands selected or identified using the same.

Description: Method for the screening of α 2 δ-l subunit binding ligands FIELD OF THE INVENTION The invention relates to a method for the screening of ligands which bind a soluble secreted cerebral cortical voltage-dependent calcium channel α 2 δ subunit polypeptide, in particular calcium channel α 2 δ-l subunit. BACKGROUND OF THE INVENTION Gabapentin (1-aminoethyl-cyclohexane acetic acid) is currently commercialized for the treatment of epilepsy. The compound has however been recognized as being also useful for the treatment of pain and anxiety. Recent reports have suggested an interaction between gabapentin and the α 2 δ subunit of a voltage-dependent calcium channel (VDCC). But electro-physiological studies have yielded conflicting data on the action of gabapentin at VDCCs, even though the relevance of the interaction of gabapentin at the α 2 δ subunit to the clinical utility of the drug is becoming clearer. However, none of the prototype anticonvulsant drugs displace [ 3 H] gabapentin binding from the α 2 δ-l subunit. The most frequently used assay currently available for the screening of ligands that bind the α 2 δ subunit involves the use of pig membrane extracts as a source of the α 2 δ subunit. Such an assay presents major inconvenients. Firstly, because the assay material is a membrane extract, it is very difficult to accurately determine the protein composition from one assay preparation to another particularly with regard to the subtype. Also, the presence of various impurities in the assay preparation is a problem in small plate assays. Furthermore, as the protein preparation lacks homogeneity, the interaction between the targeted protein and the assay plate is often quite uneven. This renders the streamlining of the assay in a high throughput format almost impossible to achieve. SUMMARY OF THE INVENTION The inventors have found that it was possible to use a soluble secreted form of a voltage- dependant calcium channel α 2 δ-l subunit polypeptide (hereinafter α 2 δ-l subunit polypeptide) in an assay for the screening of ligands which bind the α 2 δ-l subunit. The exact position and configuration of the [ 3 H]gabapentin binding site on the α 2 δ subunit is not currently known. Furthermore, recent deletion experiments on the porcine α 2 δ-l subunit coding sequence have shown that amino-acids close to the C-terminal region are needed in order for the protein to bind [ 3 H]gabapentin. For this very reason, the use of truncated forms of the porcine α 2 δ-l subunit in screening assays has not been disclosed or suggested in the prior art because there was concern as to whether relevant levels of binding capacity would be achieved in an assay environment. The assay of the invention is of considerable interest because it confirms that a recombinant soluble secreted α 2 δ-l subunit polypeptide can be used in high throughput α 2 δ-l ligand screening. It also provides a useful advantage over the pig membrane extract screening assay as it allows the study of α 2 δ-l subtype-specific binding ligands. Proteins can be tagged which makes purifying convenient and possible to use a tagged antibody for recognition. It was not clear whether the addition of the 6His tag to the C-terminus of the protein would affect the [ 3 H]gabapentin binding properties of α 2 δ. It was also unclear whether a C-terminally located 6His tag on α 2 δ would be accessible for interaction with the Ni NTA chromatography matrix (for purification purposes) and SPA bead, or Ni flashplate well surface (for purposes of the assay). The invention concerns a method for the screening of ligands which bind a calcium channel α 2 δ-l subunit. The method comprises the steps of: - contacting a secreted soluble recombinant calcium channel α 2 δ-l subunit polypeptide with: - a ligand of interest; and a labelled compound which binds a α 2 δ-l subunit; and - measuring the level of binding of the labelled compound to the secreted soluble α 2 δ-l subunit. The invention also concerns a method for the screening of biologically active products, in particular products that modulate a nervous system function in a subject, comprising the steps of: - contacting a secreted soluble recombinant calcium channel α 2 δ-l subunit polypeptide with: - a candidate product; and a labelled compound which binds a α 2 δ-l subunit; and - measuring the level of binding of the labelled compound to the secreted soluble α 2 δ-l subunit. The invention also concerns a kit for the screening of ligands which bind a calcium channel α 2 δ-l subunit. The kit comprises: a secreted soluble recombinant calcium channel α 2 δ-l subunit polypeptide; and a labelled compound which binds a calcium channel α 2 δ-l subunit. The invention also concerns a kit or a method for the screening of ligands which bind a calcium channel α 2 δ subunit, a method for the screening of biologically active products, in particular products that modulate a nervous system function in a subject, characterized in that said kits or methods comprise at least one of the following compounds : 1) A calcium channel α 2 δ subunit that is soluble and retain the functional characteristics of the full-length or wild-type α 2 δ subunit from which it derives. 2) A calcium channel α 2 δ subunit according to 1) wherein the full-length or wild-type α 2 δ subunit from which it derives is of mammalian origin. 3) A calcium channel α 2 δ subunit according to 2) wherein the mammalian origin is a human, a porcine, a rat or a mouse origin. 4) A calcium channel α 2 δ subunit according to 3) wherein the mammalian origin is a human origin. 5) A calcium channel α 2 δ subunit according to any one of 1) to 4) above, wherein the full-length or wild-type α 2 δ subunit from which it derives is naturally expressed in the cerebral cortical. 6) A calcium channel α 2 δ subunit according to any one of 1) to 5) above, wherein the full-length or wild-type α 2 δ subunit from which it derives is voltage-dependent. 7) A calcium channel α 2 δ subunit according to any one of 1) to 6) above, wherein the α 2 δ subunit is cleaved. 8) A calcium channel α 2 δ subunit according to any one of 1 ) to 7) above, wherein the α 2 δ subunit is cleaved into separate α 2 and δ peptides. 9) A calcium channel α 2 δ subunit according to 8), wherein the α 2 and δ peptides are disulfide-bridged. 10) A calcium channel α 2 δ subunit according to any one of 1) to 6) above, wherein the α 2 δ subunit is not cleaved. 11) A calcium channel α 2 δ subunit according to any one of 1) to 10) above characterized in that it is purified or isolated. 12) A calcium channel α 2 δ subunit according to any one of 1) to 11) above characterized in that it is processed as the full-length or wild-type α 2 δ subunit from which it derives is naturally processed. 13) A calcium channel α 2 δ subunit according to any one of 1) to 12) above characterized in that it is producable by the baculovirus/insect cells expression system. 14) A calcium channel α 2 δ subunit according to any one of 1) to 13) above characterized in that it is produced by the baculovirus/insect cells expression system. 15) A calcium channel α 2 δ subunit according to any one of 1) to 14) above characterized in that its δ peptide comprises at least the ligand-interacting part(s) of the complete δ peptide from which it originates 16) A calcium channel α 2 δ subunit according to any one of 1) to 15) above characterized in that its δ peptide has a C-terminal truncation with respect to the complete δ peptide from which it originates, said truncation being sufficient to render the truncated δ peptide soluble. 17) A calcium channel α 2 δ subunit according to any one of 1) to 16) above characterized in that its α 2 peptide comprises at least the ligand-interacting part(s) of the complete α 2 peptide from which it originates. 18) A calcium channel α 2 δ subunit according to any one of 15) or 17) above characterized in that ligand is gabapentin, L-Norleucine, L-Allo-Isoleucine, L- Methionine, L-Leucine, L-Isoleucine, L-Naline, Spermine or L-Phenylalanine. 19) A calcium channel α δ subunit according to any one of 1) to 18) above characterized in that its α 2 peptide comprises at least the ligand-interacting part(s) of the complete α 2 peptide from which it originates, its δ peptide comprises at least the ligand- interacting part(s) of the complete δ peptide from which it originates and its δ peptide does not comprise a part of the transmembrane domain of the complete δ peptide from which it originates which renders said calcium channel insoluble. 20) A calcium channel α 2 δ subunit according to any one of 1) to 19) above wherein the full-length or wild-type α δ subunit from which it derives or originates is αr 2 <5J , α 2 - 2, a 2 δ-3 or a. 2 δ-4. 21) A calcium channel α 2 δ subunit according to any one of 1) to 20) above wherein the full-length or wild-type α 2 δ subunit from which it derives or originates has the amino acid sequence of SEQ ID Ν°20. 22) A calcium channel α 2 δ subunit according to 20) or 21) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of SEQ ID N° 4, SEQ ID N° 5 or SEQ ID N° 6. 23) A calcium channel α 2 δ subunit according to any one of 20) to 22) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of the region comprised between amino acid number 340 and amino acid number 1062 of SEQ ID N°20. 24) A calcium channel α 2 δ subunit according to any one of 1) to 20) above wherein the full-length or wild-type α 2 δ subunit from which it derives or originates has the amino acid sequence of SEQ ID N°21. 25) A calcium channel α 2 δ subunit according to 20) or 24) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of SEQ ID N° 10, SEQ ID N° 11 or SEQ ID N° 12. 26) A calcium channel α 2 δ subunit according to any one of 20), 24) or 25) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of the region comprised between amino acid number 306 and amino acid number 1019 of SEQ ID N°20. 27) A calcium channel α 2 δ subunit according to any one of 1) to 20) above wherein the full-length or wild-type α 2 δ subunit from which it derives or originates has the amino acid sequence of SEQ ID N°55. 28) A calcium channel α 2 δ subunit according to 20) or 27) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of SEQ ID N° 53, SEQ ID N° 54 or SEQ ID N° 55. 29) A calcium channel α 2 δ subunit according to any one of 20), 27) or 28) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of the region comprised between amino acid number 302 and amino acid number 1050 of SEQ ID N°55. 30) A calcium channel α 2 δ subunit according to any one of 1) to 20) above wherein the full-length or wild-type α 2 δ subunit from which it derives or originates has the amino acid sequence of SEQ ID N°33 or SEQ ID N°44. 31) A calcium channel α 2 δ subunit according to 20) or 30) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of SEQ ID N° 34, SEQ ID N° 35, SEQ ID N° 36, SEQ ID N° 41, SEQ ID N° 42 or SEQ ID N° 43. 32) A calcium channel α 2 δ subunit according to any one of 20), 30) or 31) above characterized in that the amino acid sequence of its unprocessed form comprises or consists of the region comprised between amino acid number 302 and amino acid number 1018 of SEQ ID N°33 or SEQ ID N°44. 33) A calcium channel α δ subunit according to any one of 20), 30) or 31) above characterized in that the amino acid sequence of its unprocessed form comprises the region comprised between amino acid number 302 and amino acid number 1018 of SEQ ID N°33 or SEQ ID N°44. 34) A calcium channel α 2 δ subunit according to any one of 20), 30), 31), 32) or 33) above characterized in that its α 2 peptide comprises the region comprised between amino acid number 302 and amino acid number 946 or 997 of SEQ ID N°33 or of SEQ ID N°44 and its δ peptide comprises the region comprised between amino acid number 984 and amino acid number 1018 of SEQ ID N°33 or of SEQ ID N°44. 35) A calcium channel α 2 δ subunit characterized in that its α 2 peptide and its δ peptide have 99%, 98%, 97%, 96%, or 95% homology or identity with the α 2 peptide and the δ peptide respectively of a calcium channel α 2 δ subunit according to any one of 1) to 34) above. 36) A nucleic acid molecule characterized in that its nucleotide sequence comprises a nucleotide sequence which encodes a calcium channel α δ subunit according to any one of 1) to 35). 37) A nucleic acid molecule characterized in that its nucleotide sequence comprises a nucleotide sequence which encodes the α 2 peptide or the δ peptide of a calcium channel α 2 δ subunit according to any one of 1) to 35). 38) A nucleic acid molecule which hybridizes under stringent conditions with a nucleic acid molecule according to 36) or 37) above or 39) herebelow. 39) A nucleic acid molecule according to any one of 36) to 38) above which comprises SEQ ID N°l. SEQ ID N°2, SEQ ID N°3, SEQ ID N°7, SEQ ID N°8, SEQ ID N°9, SEQ ID N°13, SEQ ID N°14, SEQ ID N°15, SEQ ID N°30, SEQ ID N°31, SEQ ID N°32, SEQ ID N°38, SEQ ID N°39, SEQ ID N°40, SEQ ID N°50, SEQ ID N°51, or SEQ ID N°52. 40) A vector capable of expressing a nucleic acid molecule according to any one of 36) to 39) above. 41) An expression vector comprising a nucleic acid molecule according to any one of 36) to 39) above. 42) A vector according to 40) or 41) above which is a baculovirus vector. 43) A cell comprising a nucleic acid molecule according to any one of 36) to 39) above. 44) A cell comprising a vector according to 40), 41) or 42) above. 45) A cell according to 43) or 44) above which is a mammalian cell or an insect cell. 46) A composition comprising a calcium channel α 2 δ subunit according to any one of 7) to 9) above and a calcium channel α 2 δ subunit according to 10) above. 47) A calcium channel α 2 δ subunit according to any one of 1) to 35) above, characterized in that it is tagged or labelled. 48) A nucleic acid molecule according to any one of 36) to 39) above, characterized in that it is tagged or labelled. 49) A calcium channel α 2 δ subunit according to 47), characterized in that it is tagged at the C-terminal part of the δ peptide. 50) A nucleic acid molecule according to 48) above, characterized in that it is tagged at the end of the region of the nucleic acid molecule encoding the C-terminal part of the δ peptide. 51) A calcium channel α 2 δ subunit according to 47) or 49), characterized in that it is tagged by a 6 histidine residue tagg. 52) A nucleic acid molecule according to 48) or 50), characterized in that it is tagged by a 6 histidine residue tagg. The invention also concerns a screening assay using a compound according to any one of 1) to 52) above. This screening assay is preferably an SPA assay, a Flashplate assay, a Nickel Flasplate assay, a Filter binding assay or a Wheat Germ Lectin flasplate assay. The invention also concerns the use of screening assay using a compound according to any one of 1) to 52) above to detect or measure the binding or interaction of a ligand of a calcium channel α 2 δ subunit and a calcium channel α 2 δ subunit. Specific ligands of interest are gabapentin, L-Norleucine, L-Allo-Isoleucine, L-Methionine, L-Leucine, L- Isoleucine, L- Valine, Spermine or L-Phenylalanine. The above screening assays or uses are preferably an SPA assay, a Flashplate assay, a Nickel Flasplate assay, a Filter binding assay or a Wheat Germ Lectin flasplate assay. The invention also concerns a kit to detect or measure the binding or interaction of a ligand of a calcium channel α 2 δ subunit and a calcium channel α 2 δ subunit characterized in that it comprises a compound according to any one of 1) to 52). Specific ligands of interest are gabapentin, L-Norleucine, L-Allo-Isoleucine, L-Methionine, L-Leucine, L- Isoleucine, L- Valine, Spermine or L-Phenylalanine. The kits of the invention recited above are usable in an SPA assay, a Flashplate assay, a Nickel Flasplate assay, a Filter binding assay or a Wheat Germ Lectin flasplate assay. There is a great advantage to use imidazole in the context of the assays and kits of the invention because it increases the possibilities of interactions between the ligand and the receptor. A preferred range of use of imidazole in an SPA assay of the invention is between about 1 mM and about 50 mM. A preferred range of use of imidazole in a Flasplate assay of the invention is between about 1 mM and about 20 mM. There is a great advantage to use Bovine Serum Albumine (BSA) in the context of the assays and kits of the invention because it renders the compounds used in these assays and kits a lot more stable and therefore allows their in large-scale screening, especially in an high-throughput screening format. A preferred range of use of BSA is between about 0.025% and about 0.05%. A great advantage of the compounds described above which are used or comprised in the assays and kits of the invention is that they can be obtained and therefore used as an homogeneous material. Another great advantage of the compounds described above and used or comprised in the assays and kits of the invention is that they can be obtained in large quantity by the technique of the DNA recombinant. A most preferred system to produce these compounds is the baculovirus/insect cells system which furthermore allows to obtain high yields of production. Precise embodiments of the above kits and assays are recited in the continuation of the text of this application, especially in the examples. The invention also resides in a product or ligand isolated, identified or selected using the above screening methods or kits, for use as a medicament or as a lead for further drug development purposes. As indicated above, the compounds are potentially useful for treating disorders of the nervous system, including epilepsy, pain and anxiety BRIEF DESCRIPTION OF THE FIGURES Figure 1 represents the elution profile of the recombinant polypeptide with the amino acid sequence of SEQ ID No 37 purified by Superdex-200 chromatography, either before or after electron on NI-NTA. Figure 2 illustrates the optimization of imidazole concentrations in an embodiment of the SPA assay of the invention. SPA assay of [ 3 H]gabapentin (18.4nM) binding to the recombinant polypeptide of amino acid sequence SEQ ID No 9 (s-α 2 δ-lb-6His) (20μl). Figure 3 illustrates the optimization of imidazole concentrations in an embodiment of the flashplate assay of the invention. Flashplate assay of [ 3 H]gabapentin (14nM) binding to the recombinant polypeptide of amino acid sequence SEQ ID No 37 (s-α 2 δ-lb-6His) (lOμl). Figure 4 illustrates the flashplate time course of [ 3 H]gabapentin (13 nM) binding to various concentrations of the recombinant polypeptide with the amino acid sequence of SEQ ID No 37. Figure 5 illustrates the capacity of the recombinant polypeptide with the amino acid sequence of SEQ ID No 37 in a flashplate assay after 3 hours of incubation. Figure 6 illustrates the optimum imidazole concentration, assayed after 3 hours of incubation, required to maximize [ 3 H]gabapentin binding using a constant amount of the recombinant polypeptide with the amino acid sequence of SEQ ID No 37. Figure 7 illustrates flashplate assay of [ 3 H] gabapentin saturation binding to the purified recombinant polypeptide with the amino acid sequence of SEQ ID No 37, assayed after 3 hours of incubation. Figure 8 illustrates the flashplate time course optimisation of imidazole concentration required to maximize the [ 3 H]Leucine (10.1 nM) binding window to the purified recombinant polypeptide with the amino acid sequence of SEQ ID No 37, assayed after 3 hours of incubation. Figure 9 illustrates competition curves of three compounds in the flashplate assay format, assayed after 3 hours of incubation (see also Table 2). Figure 10 illustrates the dose response nature of [ 3 H]gabapentin binding to s-α 2 δ-lb-6His in the Wheat Germ lectin flashplate assay. The level of non-specific binding is low (around 50-70cpm) and stable, independent of the volume of protein assayed or the point analysed on the time-course. The window is relatively stable over an extended period of time with just a gradual decline from the 15-hour time point (approximately 10% of the window every 24 hours). Figure 11 illustrates the dose response nature of [ 3 H]gabapentin binding to α 2 δ-l and the maintenance of a constant low-level of non-specific binding (around 30-60cpm) independent of protein volume assayed. Figure 12 illustrates the dose response nature of [ 3 H]gabapentin binding to α 2 δ-l in the Nickel flashplate assay. The level of non-specific binding is low (around 70-100cpm) and stable, independent of the volume of protein assayed or the point analysed on the time-course. A stable window is maintained for a period of at least 50 hours (between ~20 and 70 hours on the time-course). Figure 13 illistrates the stability of α2δ-l/[3H] gabapentin binding assay. Each well was performed in triplicate and contains in 200μl of : 0.1M HEPES pH7.3, 0.5mM Imidazole Various concentrations of BSA fraction V (0, 0.025, 0.05, 0.25, 0.5 and 1%). Figure 14 is and example of amino acid sequence alignments of the α 2 δ-l deletion region in four species variants of α 2 δ-l splice isoform b. DETAILED DESCRIPTION OF THE INVENTION The invention concerns a method for the screening of ligands which bind a soluble secreted α 2 δ subunit polypeptide, in particular a soluble secreted α 2 δ-l subunit polypeptide. The term α 2 δ subunit polypeptide, when used herein, is intended to designate a structure containing two polypeptides (α 2 and δ) attached or associated to one another, in particular by covalent disulfide bridges. More particularly, the targeted α 2 δ subunit binding site is preferably the [ 3 H]gabapentin binding site. The various parameters of the method of the invention are described in further detail below. A - Secreted soluble recombinant α 2 δ-l subunit polypeptide Several nucleotide sequences encoding a secreted soluble form of an α 2 δ-l subunit can be used in the context of the present invention. Preferred soluble secreted α δ-l subunit polypeptides are derived from eukaryotic α 2 δ-l subunits, more preferably from mammal, such as mouse, rat, rabbit, porcine, bovine or others and human α 2 δ-l subunits. Most preferred soluble secreted α 2 δ-l subunit polypeptides are derived from the human or porcine α 2 δ-l subunits. More specifically, the selected nucleotide sequences encode a secreted soluble polypeptide having at least 80%, preferably 90%, more preferably 95%, and most preferably 98 or 99% amino-acid identity with the polypeptide comprising from amino acid 1 to between amino-acids 985 and 1087 of SEQ ID N°33 or SEQ ID N°44, preferably between amino-acids 985 and 1085, more preferably between amino-acids 985 and 1078, most preferably between amino-acids 985 and 1064, 1059 or 1044 of SEQ ID N°33 or SEQ ID N°44. In particular embodiments, selected nucleotide sequences encode a secreted soluble polypeptide having at least 80%, preferably 90%, more preferably 95%, and most preferably 98 or 99% amino-acid identity with the polypeptide comprising from amino acid 1 to between amino-acids 1008 and 1087 of SEQ ID N°33 or SEQ ID N°44, in particular between 1018 and 1078, specifically between 1043 and 1078. More specifically, the selected nucleotide sequences encode a secreted soluble polypeptide having at least 80%, preferably 90%, more preferably 95%, and most preferably 98 or 99% amino-acid identity with the polypeptide comprising from amino acid 1 to between amino-acids 985 and 1054, preferably between amino-acids 985 and 1059, and most preferably between amino-acids 1019 and 1044 of SEQ ID N°33 or SEQ ID N°44. In order to determine the optimal deletions on the α 2 δ-l subunit cDNA that yield a soluble secreted polypeptide devoid of membrane anchorage structures and having a functional [ 3 H]gabapentin binding site, the inventors tested the expression of several human or porcine α 2 δ-l subunit cDNA deletion mutants. The discussion provided below provides detailed comments on possible truncations, giving as an example the porcine α 2 δ-l subunit. However, given the very substantial cross-species homology for α 2 δ-l subunit sequences, the comments below can also be applied to other eukaryotic species, and more particularly other mammalian species such as the rat, the mouse or the rabbit. Their α 2 δ-l subunit sequences, which are available in public databases, share a very substantial homology with the human and porcine α 2 δ-l subunit sequences. Therefore, the inventore believe that this succesefull identification and characterization of a region in α 2 δ-l which can be modified to produce a soluble α 2 δ-l of pig and human whilst maintaining Gabapentin binding, enables those skilled in the art to modify other species α 2 δ-l polypeptides. Exambles of this modification site in other α 2 δ-l from other species is shown in figure 14. The inventors found that by deleting from the porcine α δ-l subunit cDNA a nucleotide sequence encoding as much as amino-acids 967 to 1091 of the native protein, soluble polypeptides could be obtained. On the other hand, the minimal deletion required to achieve solubility appears to be located around nucleotides encoding amino-acids 1064 to 1091 of the sequence of SEQ ID N°33. In this regard, the mutant polypeptide expressed using a cDNA deletion mutant from which a sequence encoding amino-acids 1064 to 1091 is removed is found in both soluble and membrane-associated forms, with [ 3 H]gabapentin and/or other derivatives or compounds such as pregabalin and gabapentoids binding properties similar to that of the wild type protein. Furthermore, a mutant protein expressed using a cDNA deletion mutant from which a nucleotide sequence encoding amino-acids 1085 to 1091 is removed recovers its membrane anchorage properties. Also, mutant proteins expressed using cDNA deletion mutants from which nucleotide sequences encoding either amino-acids 1037 to 1091 or amino- acids 1019 to 1091 of SEQ ID N°33 or 44 are removed are found in soluble form. The inventors believe that the soluble secreted α 2 δ-l subunit polypeptides which are as close as possible to the native sequence and which are therefore more likely to retain their native folding and hence their [ 3 H] gabapentin- binding properties are those corresponding to a protein in which amino-acid stretch 985-1091 to 1074-1091, the amino-acid sequence of SEQ ID N°33 or 44 has been deleted. The skilled scientist can quite easily determine within this amino-acid stretch the optimal mutant protein. The invention therefore particularly concerns a screening assay in which the secreted soluble α 2 δ-l subunit polypeptide is preferably a polypeptide having at least 80% identity with the polypeptide comprising from amino-acid 1 to between amino-acid 985 and 1054, preferably between amino-acids 985 and 1059, and most preferably between amino-acids 1019 and 1064 of SEQ ID N°33 or SEQ ID N°44. Preferred α 2 δ-l subunit polypeptides which can be used in the present invention are those of SEQ ID N°34, 35, 36, 37, 41, 42 and 43, with the polypeptides of SEQ ID N°33 and SEQ ID N°43 being most preferred. In a first and preferred embodiment of the invention, the α 2 δ-l subunit polypeptide is purified before it is used in the assay. The purification step, an example of which is provided further in this specification, can be carried out using several purification techniques well-known to the skilled person. In some instances, it is required to tag the α 2 δ-l subunit polypeptide prior to purification. The tag is then in most instances encoded into the nucleotide sequence that is needed to express the polypeptide. Examples of such tags include, but are not limited to sequences encoding C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST. Most of these tags can be incorporated directly into the sequence, for instance through PCR amplification by incoφorating the appropriate coding sequence in one of the PCR amplification primers. However, the tag can also be introduced by other means such as covalent binding of the appropriate nucleic acid sequence encoding the tag moiety with the 5' or 3' end of the nucleic acid sequence encoding the polypeptide sequence. This is the case for GST. It should be noted that the tag can be located at either end of the polypeptide sequence. Furthermore, in some instances, it can be advantageous to insert a cleavage site between the tag and the polypeptide sequence in order to permit removal of the tag sequence if needed. Therefore a first object of this invention of the invention is the use of C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST taggs for the purification and screening of α 2 δ-l. Preferably, the use of C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST taggs for the purification and screening of α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 or 38. More preferably, the use of a sequence of histidine residues tagg for the purification and screening of α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 or 38, more preferably, the use of a sequence of 6 histidine residues tagg for the purification and screening of α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 or 38 and most preferably the use of C-terminal a 6 histidine residues tagg of α 2 δ-l of SEQ ID N°30, 31 , 32, 33, 34, 35, 36, 37 or 38 for the purification and screening A secont object of the invention is the use of nucleic acids encoding C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST taggs for the purification and screening of expressed α 2 δ-l. Preferably, the use of nucleic acids encoding C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST taggs for the purification and screening of expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 or 38. More preferably, the use of nucleic acids encoding a sequence of histidine residues tagg for the purification and screening of an expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 or 38, more preferably, the use nucleic acid sewuence encoding a sequence of 6 histidine residue tagg for the purification and screening of an expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 or 38 and most preferably the use of C-terminal a nucleic acid sequence encoding 6 histidine residue tagg of expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 or 38 for purification and screening In other cases, providing a tag to the polypeptide is not needed. For instance, the protein can be purified using affinity columns loaded with specific monoclonal antibodies. In a second embodiment of the invention, the α 2 δ-l subunit polypeptide can be only partially purified. For instance, it can be purified along with other contaminating proteins using an appropriate chromatography matrix such as ion-exchange chromatography column. In such instances, it is not required to tag the desired polypeptide of interest. The most preferred embodiment contemplated by the inventors concerns the use of a purified tagged α 2 δ-l subunit polypeptide. A particularly preferred tag is a nucleotide sequence encoding from 2 to 10, and preferably 6 histidine residues as provided in the polypeptide of SEQ ID No 37. The polypeptide may be prepared by expression of the corresponding nucleic acid molecule in any appropriate host cell, using various vector systems known to the skilled person. Typical examples of suitable cells include prokaryotic and eucaryotic cells, such as bacteria, yeasts, mammalian cells (including human cells), insect cells, etc. The vector may be a plasmid, virus, episome, phage, etc. As illustrated in the examples, the polypeptide can be produced for instance by expression of the nucleic acid in insect cells, using a baculovirus vector. A most preferred system of expression of the calcium channel α 2 δ of the invention is the baculovirus/insect cell system. In fact, this system of expression allows to produce only the soluble form, is easy to use because the insect cells can be cultured without adherency and results in very high yield of production. Thus, this system allows mass- production of the calcium channel α 2 δ of the invention, provides an homogeneous production and is therefore particularly suitable for the preparation of this target for screening, in particular for high-throughput screening. With regard to the α 2 δ-l subunit polypeptide used subsequently in the screening assay of the invention, several possibilities are also open to the skilled person. In a first and preferred embodiment, the α 2 δ-l subunit polypeptide comprises a tag moiety which can be selected among the tags referred to above. Such tagged polypeptides are particularly useful in SPA or flashplate assays. A preferred tag is the nucleotide sequence encoding histidine residues referred to above. In a second embodiment, the α 2 δ-l subunit polypeptide can be used without a tag. This is the case for instance in SPA or flashplate assays comprising beads or plates coated with wheat germ lectin. In such an embodiment, the tag is not needed as the carbohydrate moieties of the α 2 δ-l subunit polypeptide bind directly to the wheat germ lectin-coated beads or plates. B - Labelled compounds which bind the α 2 δ-l subunit polypeptide In cases where the α 2 δ-l binding site is the [ 3 H] gabapentin binding site, the preferred labelled compound which can be used is of course gabapentin itself. However, gabapentin is not the only labelled compound which can be used in this context. Indeed, it has been previously demonstrated that saturation binding analyses on porcine synaptic plasma cerebral cortex membranes performed in the presence of L-leucine indicate a competitive interaction of the amino acid with the [ H] gabapentin binding site, significantly reducing [ 3 H] gabapentin binding affinity for the site. The inventors believe that this competitive interaction is true across all the amino-acids listed in table 1 below. Table 1 Binding affinities of selected amino acids (IC 5 o <500nM) for the [ 3 H] gabapentin site in porcine cortical membranes COMPOUND IC 50 (NM) ARITHMETIC MEAN (N=3) ± S.E.M. Gabapentin 42.1 + 5.5 L-Norleucine 23.6 ± 6.7 L-Allo-Isoleucine 32.8 ± 6.0 L-Methionine 49.6 ± 10.0 L-Leucine 61.3 + 20.9 L-Isoleucine 68.8 1 1.9 L- Valine 330 + 18 L-Phenylalanine 351 + 89 It is therefore possible to use commercialy available labelled forms of these high affinity ligands in replacement of gabapentin. The utility of [ 3 H]L-leucine has been demonstrated in a filter binding assay and in a flashplate assay format. The inventors believe that labelled amino acids but also other compounds, with affinities preferably below 500 nM in the binding assay can be used as replacements of gabapentin. With regard to the label, several embodiments can be used in the context of the invention. Preferred labels are of course radioactive labels, a list of which is provided further in this specification. An object of this invention is the use of labelled compounds which have an affinity of less than 500nm for the gabapentin binding site of α2δ-l for the screening of ligands that bind to α2δ-l, preferably the use of labelled of Gabapentin, L-Norleucine, L-Allo- Isoleucine, L-Methionine, L-Leucine, L-Isoleucine, L- Valine or L-Phenylalanine, more preferably the use of labelled Gabapentin. C - Assay formats and conditions Several assay formats can be used to carry out the method of the present invention. Preferred assay formats include scintillation assays such as the scintillation proximity assay (SPA) or the flashplate assay. Other assay formats well known to those skilled in the arts such as the filter binding assay and the centrifugation assay are also contemplated in the present invention. SPA and flashplate assays are preferred assay formats for the present invention. Additional details on these assays are provided below. Scintillation assay format Scintillation assays technology either involves the use of scintillant beads (for the SPA assay) or plates (for the flashplate assay). SPA beads are usually made from either cerium-doped yttrium ion silicate (y2SiO5:Ce) or polyvinyltoluene (PVT) containing an organic scintillant such as PPO. Flashplates commonly used are those such as Ni chelate flashplates although other flashplates can also be used. Assays are usually carried out in aqueous buffers using radioisotopes such as 3 H, 125 I, C, S or 33 P that emit low-energy radiation, the energy of which is easily dissipated in an aqueous environment. For example, the electrons emitted by 3 H have an average energy of only 6 keV and have a very short path length (-1 ~tm) in water. If a molecule labelled with one of these isotopes is bound to the bead or flashplate surface, either directly or via interaction with another molecule previously coupled to the bead or flashplate, the emitted radiation will activate the scintillant and produce light. The amount of light produced, which is proportional to the amount of labelled molecules bound to the beads, can be measured conveniently with a liquid scintillation (LS) counter. If the labelled molecule is not attached to the bead or a flashplate surface, its radiation energy is absorbed by the surrounding aqueous solvent before it reaches the bead, and no light is produced. Thus, bound ligands give a scintillation signal, but free ligands do not, and the need for a time- consuming separation step, characteristic of conventional radioligand binding assays, is eliminated. The manipulations required in the assays are reduced to a few simple pipetting steps leading to better precision and reproducibility. The conditions under which SPA and flashplate assays are performed in the context of the present invention are provided below. Scintillation assay conditions 1) SPA assay The SPA assays is first developed to optimize the conditions under which the radioligand binds the α 2 δ-l subunit polypeptide. The parameters which can be varied to optimize radioligand binding in a typical SPA assay using Amersham beads include assay temperature, α 2 δ-l subunit polypeptide interaction with the radioligand and the SPA beads, radioligand concentration as well as pH variations. The temperature at which the assay can be carried out can vary from 1 to 30°C. Preferred temperatures range from 18 to 23°C, with 21°C being the most preferred temperature. The interaction of the α 2 δ-l subunit polypeptide with the SPA beads can be optimized by adjusting the concentration of the polypeptide and by introducing a reagent which will favor this interaction. When 50 mg of Amersham SPA beads are used, the α 2 δ-l subunit polypeptide concentration may vary from 0.1 to 10 pmoles per well, with the optimal concentration being generally around 5 to 6 pmoles per well. As for the reagent favoring the interaction between the α 2 δ-l subunit polypeptide and the radioligand as well as the Amersham SPA beads, the inventors found that imidazole could be efficiently used for that purpose when the α 2 δ-l subunit polypeptide is tagged with an amino acid sequence including 6 histidine residues. Furthermore, and more importantly, it was found that imidazole also enhanced binding of the radioligand to the α 2 δ-l polypeptide. The optimal concentration of imidazole used to enhance radioligand binding varies depending on the concentration of α 2 δ-l subunit polypeptide used in the assay. For instance, when the volume of the α 2 δ-l subunit polypeptide is about 20 μl (α δ-l polypeptide concentration of 0.6 pmol/ul), imidazole concentrations ranging from 10 to 50 mM can be used, with concentrations ranging between 10 and 30 mM being preferred. A most preferred imidazole concentration is 20 mM. It is to be noted that other compounds such as histidine can be used to enhance radioligand binding. Furthermore, pH variations can also influence radioligand binding although pH variations should be closely monitored as they may have an effect on the structural configuration of the of α 2 δ-l subunit polypeptide. Although the use of imidazole is preferred to enhance radioligand binding, the person skilled in the art knows that the use of imidazole is preferred but is absolutely not essential. The concentration of the radioligand is evaluated with respect to the concentration of α 2 δ-l subunit polypeptide present in the assay medium. Generally, the concentration of radioligand varies from 1 nM to 100 nM. A preferred [ 3 H]gabapentin concentration is about 5 to 20 nM, with a most preferred concentration being about 10 nM. A preferred [ 3 H]leucine concentration is also about 5 to 20 nM, with a most preferred concentration being about 10 nM. It is to be noted that the concentration of other radioligands having affinities similar to those of [ 3 H]gabapentin and [ 3 H]leucine should also be in the range ofabout 5 to 20 nM. Once the optimal radioligand binding conditions have been determined, a test ligand can be introduced in the assay medium to evaluate the level of displacement of the radioligand. The concentration of test ligand to be introduced in the assay medium usually varies from 0.1 nM to about 100 μM. A preferred test ligand concentration of about 10 μM is usually a starting point in a high throughput screening assay. Then, depending on the number of hits obtained, it may be lowered or increased. It is to be noted that the parameters set forth above, which have been evaluated for a typical SPA assay using Amersham SPA beads can be adjusted by the skilled person, for example if SPA beads of a different type are used. 2) Flashplate assay Similarly to the SPA assays, the flashplate can first be developed in order to optimize the conditions under which the radioligand binds the α 2 δ-l subunit polypeptide. The parameters which can be varied to optimize radioligand binding in a typical flashplate assay using NEN Ni chelate flashplates or Wheat Germ lectin flashplates also include assay temperature, α 2 δ-l subunit polypeptide interaction with both the radioligand and the flashplates, radioligand concentration as well as pH variations. The temperature at which the assay can be carried out can vary from 1 to 30°C. Preferred temperatures range from 18 to 23 °C, with 21°C being the most preferred temperature. The interaction of the α 2 δ-l subunit polypeptide with the flashplates can be optimized by adjusting the concentration of the polypeptide and by introducing a reagent which will favor this interaction. When a standard NEN Ni chelate flashplate is used, the α 2 δ-l subunit polypeptide volume usually varies between 0.5 and 20 μl for a concentration of α 2 δ-l subunit polypeptide of 0.6 pmol/μl. As the published maximum binding capacity of NEN plates is about 6 pmol per well, the inventors consider that an optimal concentration of α 2 δ-l subunit polypeptide is probably around 5 pmol per well at 8μl. Although the use of imidazole is preferred to enhance radioligand binding, the person skilled in the art knows that the use of imidazole is preferred but is absolutely not essential. With regard to the reagent favoring the interaction between the α 2 δ-l subunit polypeptide and the radioligand as well as the flashplates, the inventors found that imidazole could also be efficiently used for that purpose when the α 2 δ-l subunit polypeptide is tagged with an amino acid sequence including 6 histidine residues. It was also found that imidazole concentrations substantially enhanced binding of the radioligand to the α 2 δ-l polypeptide. The optimal concentration of imidazole used to enhance radioligand binding varies depending on the concentration of α 2 δ-l subunit polypeptide used in the assay. For instance, when the volume of the α 2 δ-l subunit polypeptide is about 10 μl (α 2 δ-l polypeptide concentration of 0.6 pmol/μl), the optimal imidazole concentration can vary between 1 and 20 mM, with a concentration of about 10 mM being preferred. As mentioned previously, other compounds such as histidine as well as pH variations may be used to enhance radioligand binding. The concentration of the radioligand is evaluated with respect to the concentration of α 2 δ-l subunit polypeptide present in the assay medium. Generally, the concentration of radioligand varies from 1 nM to 100 nM. A preferred [ 3 H] gabapentin concentration is about 5 to 20 nM, with a most preferred concentration being about 10 nM. A preferred [ 3 H]leucine concentration is also about 5 to 20 nM, with a most preferred concentration being about 10 nM. It is to be noted that the concentration of other radioligands having affinities similar to those of [ 3 H]gabapentin and [ 3 H]leucine should also be in the range of about 5 to 20 nM. Once the optimal radioligand binding conditions have been determined, a test ligand can be introduced in the assay medium to evaluate the level of displacement of the radioligand. The concentration of test ligand to be introduced in the assay medium usually varies from 0.1 nM to about 100 μM. A preferred test ligand concentration of about 10 μM is usually a starting point in a high throughput screening assay. Then, depending on the number of hits obtained, it may be lowered or increased. The inventors have tested the displacement of a particular radioligand, [ 3 H]gabapentin, with (S+)-3-isobutyl gaba, (R-)-3-isobutyl gaba and gabapentin. The data provided in the examples which follow clearly shows that the assay can be used in high throughput competition studies. Example 1 Construction of a nucleotide sequence encoding the putative soluble porcine α 2 δ-lb deletion mutant of SEQ ID N°37 a) Primer design PCR primers were designed to generate the soluble porcine α 2 δ-lb deletion mutant of SEQ ID N° 37 as follows: 5' PCR primer: This was designed to engineer in a KOZAK translation initiation consensus sequence prior to the coding sequence (Kozak JBC 266 19867-19870) 3' PCR primer: This was designed to engineer in six histidine residues followed by a stop-codon at the desired location in the coding sequence. In addition to the stop codon the α 2 δ-l primers also included an Eco Rl restriction site. The bold region in each primer sequence denotes the 'tagged' region; addition of sequences not present in the template. Primers were custom synthesized by Perkin Elmer Applied Biosystems UK to the ABI ready pure grade, supplied lyophilized then resuspended to 15μM in lOmM TE. JB189 and 195 were provided without 5' phosphate groups: 5' primer JB189 (5'-TCGCCACCATGGCTGCTGGCTGCCTGCTG-3\ SEQ ID N°20) 3' primer JB195 (5'- TCGGAATTCCTCAGTGATGGTGATGGTGATGAGAAACACCACCACAGTCG GT-3', SEQ ID N°21) Could you please check the bolded regions? b) PCR protocols for the generation of the α 2 δ-l deletion mutant 1) Generation of the pcDNA3-porcine-α2δ-(+) PCR template An oligo dT-primed λgtlO porcine cerebral cortical cDNA library was screened by ECL (Amersham) using a 2,381 -bp Hindlll fragment (coding sequence 268-2649) of the rabbit skeletal muscle α 2 δ clone (pcDNA3-Rab-α 2 δ-(+) (supplied by Neurex) as the probe. A positive insert was identified and subcloned into pBluescript-SK-(+) to generate pB- PC-α 2 δ-l.l. The clone was sequenced on both strands, except for a 711-bp stretch at one end of the clone, which had a high degree of homology to mitochondrial C oxidase. The α 2 δ coding region was homologous to the 3' region of the human neuronal α 2 δ sequence but lacked 926 bp of 5' coding sequence. The missing sequence was obtained by 5'-RACE using total RNA prepared from porcine cerebral cortex. RACE was performed across a Bgl I site unique in known α 2 δ sequences (rabbit (accession no. M21948)), rat (accession number M86621), human (accession no. M76559) The sequence derived from the 5' RACE product was used to design a primer (JB042, 5'- GGGGATTGATCTTCGATCGCG-3'; SEQ ID N°18) specific for the 5'-untranslated end of the cDNA. PCR was then performed with Pfu DNA polymerase using JB042 and a primer downstream of the Bgl I site (JB040, CTGAGATTTGGGGTTCTTTGG, SEQ ID N°19). The PCR product was ligated to Eco Rl linkers (5'-GGAATTCC-3') and then digested with Eco Rl and Bgl I. The 1,564-bp fragment (5' portion of the α 2 δ cDNA) was gel- purified. Similarly, a 2,303-bp fragment (3' portion of the α 2 δ cDNA) was isolated after digestion of pB-PC-α 2 δ-l .l with Bgl I and Eco Rl. The two fragments of α 2 δ cDNA were then ligated to EcoRI-digested pcDNA3 in a three-way ligation. A clone was picked with the full-length α δ sequence in the positive orientation with respect to the cytomegalovirus promoter (pcDNA3-PC-α 2 δ-(+)). 2) PCR protocol The following reagents were added to obtain two cocktails labelled 'lower' and 'upper' buffers. Lower μl lOx Pfu DNA polymerase buffer 25 lOmM dNTP's 5 100ng/μl pcDNA3-porcine-α 2 δ-(+) 10 15μM JB189 8.5 15μM JB195 8.5 H 2 O 193 Upper μl 1 Ox Pfu DNA polymerase buffer 25 H 2 O 220 2.5units/μl Pfu DNA polymerase 5 50μl aliquots of lower buffer were added to each of four 0.5ml eppendorf tubes. To each was added one PCRgem 100 ampliwax bead (PE biosystems). Tubes were heated to 80°C for 2 minutes then cooled to 4°C. 50μl of upper buffer was then added to each tube. Tubes were then cycled on a Stratagene Robo-Cycler according to the following conditions: 98°C / lmin 30sec, followed by: for 20 cycles 98°C / 45sec, 54°C / 2min, 72°C / 6min, followed by: 72°C / 20min, followed by: hold at 4°C. The 3228bp PCR product was then purified on a QIAquick PCR purification column (Qiagen) and eluted with 61μl of H O. The following reagents were added to the eluted DNA: 0.7μl lOmM ATP, 7μl lOx Polynucleotide Kinase buffer, lμl lunit/μl Polynucleotide Kinase. The above 5' phosphorylation reaction was incubated at 37°C for 1 hour. The reaction was stopped by incubation at 65°C for lOmin. The 3228bp 5' phosphorylated PCR product was then gel purified from a 1% agarose gel using QIAEX (Qiagen) beads and eluted in ~50μl. Example 2 Cloning of the PCR fragments of Example 1 into the Baculovirus transfer vector pFastBacl The PCR products of Example 1 (3228bp JB189/JB195 derived PCR product coding for 6His tagged porcine α 2 δ-lb: SEQ ID No 9) were cloned into Stw I digested, calf intestinal phosphatase dephosphorylated, phenol chloroform extracted and QIAEX gel purified pFastBacl (Life Technologies) using the Rapid DNA ligation kit (Roche Diagnostics) transforming XLl-blue (α 2 δ-lb) E. Coli cells: a) Screening for positive recombinants Given that the PCR product was cloned by blunt-end ligation a screen was required to select a recombinant with the gene ligated in the positive orientation with respect to the polyhedrin promoter in pFastBacl. This was achieved by restriction digest of miniprep DNA (Qiagen miniprep kit) prepared from colony minicultures and analysis on a 1% TAE agarose gel. A positive clone was identified according to the following digest patterns: SEQ ID No 9 in pFastBacl Eco Rl digest performed on miniprep DNA Predicted fragments (bp) PCR product cloned in a positive orientation 4773 and 3230 PCR product cloned in a negative orientation 7989 and 14 b) Sequencing analysis of selected clones One positive was selected for this clone and used to prepare a plasmid DNA stock of the desired construct (QIAGEN maxi kit). Confirmatory sequence reactions were performed using the Big Dye terminator sequencing kit and run on an ABI 310 Prism Genetic Analyzer. Sequence analysis of both coding strands was performed using a selection of sequencing oligonucleotide primers and has yielded the following results: Sequencing of pFBac-Porcine-s-α 2 δ-l-Δ 1064-109 l-6His confirmed that the insert sequence corresponded to the nucleic acid encoding the polypeptide of SEQ ID No 37, except for the deletion of two bases from the 5' end of the 5' PCR primer (JB189). The loss of these two bases did not have any impact on the 5' end of the gene as the KOZAK translation start-site consensus sequence (GCCACC) starts immediately after this deletion. Example 3 Protocol for establishing baculovirus banks for the expression of the α 2 δ-l deletion mutant of SEQ ID N°9 Essentially, the protocol used to generate the baculovirus banks is that outlined in the Life Technologies Bac-to Bac™ baculovirus expression systems manual. a) Transposition of DHlOBac E Coli cells One ng (5μl) of the recombinant pFastBac-1 construct containing the nucleotide sequence encoding the porcine α 2 δ-l deletion mutant of SEQ ID No 37 was added to lOOμl of DHlOBac cells thawed on ice. The cells were then mixed gently by tapping the tube then incubated on ice for 30 minutes before heat shock treatment by incubation in a 42°C water bath for 45 seconds. The mixture was then chilled on ice for 2 minutes before the addition of 900μl of S.O.C. medium. The mixture was then placed in a shaking incubator (200rpm) at 37°C for 4hours. The cells were then serially diluted (10 fold dilutions from IO "1 to IO "3 ) and lOμl of each dilution plated on LB agar plates containing 50μg/ml kanamycin, 7μg/ml gentamicin, lOμg/ml tetracycline, lOOμg/ml Bluo-gal and 40μg/ml IPTG. The plates were incubated at 37°C for between 1 and 3 days until discrete colonies of blue and white colour were discernible. b) Isolation of recombinant DNA White colonies (containing the recombinant bacmid) were picked and grown for 24 hours (to stationary phase) at 37°C with shaking (200 rpm) in 2ml of LB containing 50μg/ ml kanamycin, 7 μg/ml gentamicin and lOμg/ml tetracycline. 1.5ml of culture was then transferred to a microfuge tube and centrifuged at 14,000xg for 1 minute. The supernatant was removed and the cells resuspended gently in 0.3ml of 15mM Tris-HCl (pH8.0), lOmM EDTA, lOOμg/ml RNase A. 0.3ml of 0.2N NaOH, 1% SDS was then added and the mixture mixed gently before incubation at 22°C for 5 minutes. Then 0.3ml of 3M potassium acetate (pH5.5) was added and the sample placed on ice for 10 minutes. After centrifugation at 14,000 x g for 10 minutes the supernatant was transferred to a tube containing 0.8ml of isopropanol, mixed then placed on ice for 10 minutes before centrifugation at 14,000 x g for 10 minutes. The supernatant was then discarded and the pellet rinsed with 0.5ml of 70% ethanol before centrifugation at 14,000 x g for 5 minutes. This 70% ethanol rinse was then repeated before removing all of the supernatant and air drying the pellet for 10 minutes at room temperature. The pellet was finally resuspended in 40μl ofTE. c) Transfection of sf9 cells with the recombinant bacmid DNA A 6-well tissue culture plate was seeded with 0.9x10 6 sf9 cells (cells at log phase having grown from a culture passaged at 0.3xl0 6 cells/ml) per 35mm well in 2ml of Sf-900 II SFM media containing 50units/ ml penicillin and 50μg/ ml streptomycin. Cells were left to attach at 27°C for 1 hour. Bacmid DNA prepared as described above (5μl) was added to 200μl of Sf-900 II SFM media containing 6μl of CELLFECTIN and mixed before incubation at room temperature for 45 minutes. The cells were washed once with 2ml of Sf-900 II SFM media without antibiotics then 0.8ml of Sf-900 II SFM media was added to each tube containing the lipid-DNA complex. The wash buffer was removed from the cells and the 1ml of diluted lipid-DNA complex overlaid on the cells. The cells were incubated for 5hours at 27°C after which time the transfection mixture was removed and 2ml of Sf-900 II SFM media containing 50units/ ml penicillin and 50μg/ ml streptomycin was added. The cells were then incubated for 72 hours. After incubation for 72 hours the media was removed from the cells and centrifuged at 500xg for 5 minutes. The supernatant was then transferred to a fresh tube, this was labelled as the P0 bank and stored at 4°C in the dark. The PI bank was prepared by passaging sf9 cells at approx 5xl0 6 cells/ml to 2xl0 6 cells/ml (100ml in a 250ml Erlenmeyer flask) and adding 0.5ml of the P0 bank harvested above. The cells were then incubated shaking (200rpm) at 27°C for 4 days. Under sterile conditions the culture was centrifuged at 500xg for 10 minutes and the supernatant 0.2μM filtered (PI bank). The P2 bank was prepared by adding 2ml of PI bank per 400ml culture (in IL Erlenmeyer flasks) passaged as above to 2xl0 6 cells/ml. The culture was incubated as before for 4 days and the supernatant harvested and filtered as described for the PI bank. The supernatant was first pooled then aliquoted (10ml) and stored at 4°C. Example 4 Protein expression To sf9 cells passaged from ~5xl 0 6 cells/ml to 2x10 6 cells/ml in Sf-900 II SFM media was added 0.1ml virus per 100 ml of cells of the appropriate viral bank (400ml volumes in IL Erlenmeyer flasks). The cells were then cultured for 4-5 days at 27°C with 110 rpm shaking. Expression of the protein was confirmed by SDS-PAGE and Western blotting using an anti penta-His monoclonal antibody (Qiagen) and was detected in the culture supernatant and cell lysate. Example 5 Purification of α 2 δ-l deletion mutant of SEQ ID N°37 The-α 2 δ-l deletion mutant of SEQ ID N°37 was purified from the cell lysate following the purification strategy outlined below: The culture was centrifuged at 6,000xg for 10 minutes and the supernatant removed. The weight of the cell pellet was determined before re-suspension in 20mM Tris pH8.0, lOOmMKCl, 1% P40-Nonidet (100ml per 20g of wet cells). A protease inhibitor cocktail (Sigma, Cat# P8849), lml/L, was added to the mixture. The solution was then stirred for 10 minutes before centrifugation for lhour at 30,000xg and 4°C. The supernatant was concentrated (30kDa cut off) to approx. ~300ml then centrifuged for lhour at 100,000xg. Supernatant containing the soluble proteins was diluted 1 :3 in lOmM Tris-HCl pH8.0 (equilibration buffer) and loaded onto a pre-equilibrated Q-Sepharose column (2.5cm i.d. x 30cm h.) at a flow rate of 900ml/h. After washing with equilibration buffer until a stable A 280n baseline had been achieved, protein was eluted with 20mM Tris-HCl pH8.0, 0.5M KC1, lOmM Imidazole. The eluate was then loaded onto a Ni-NTA (Qiagen) column (2.5cm i.d. x 6cm h.) pre- equilibrated in 20mM Tris ρH8.0, 0.5M KC1, lOmM Imidazole at a flow rate of 2 ml/min. The column was washed successively with buffer A (20mM Tris pH8.0, 0.5M KC1, 20mM Imidazole), buffer B (lOOmM Tris-HCl pH8.0, IM KC1), and buffer A again. Elution was performed with buffer C (20mM Tris-HCl pH8.0, lOOmM KC1, 0.5M Imidazole). The Ni-NTA eluate (~50ml) was concentrated (30kDa cut-off) to ~2ml and applied at 1 ml/min and in 0.2ml aliquots, to an FPLC Superdex-200 column equilibrated in lOmM HEPES, pH7.4, 150mM NaCl. Fractions containing the polypeptide of SEQ ID No 37 were pooled. As shown in Figure 1, the protein elution profile and associated [ 3 H]gabapentin binding activity is presented together with a silver-stained SDS-PAGE gel (post Ni NTA load of Superdex-200) demonstrating the co-elution of the ~130kDa band (α 2 δ) with the [ 3 H]gabapentin binding activity and A 280nm profile. Example 6 SPA assay of [-HI gabapentin binding to soluble porcine αjδ-lb-6His The assay was carried out at 21°C. Assay components were added in the following order (all reagents were diluted in 1 OmM HEPES (pH 7.4 at 21 °C) to 96- well Optiplates) 25μl imidazole at various concentrations (diluted from a IM stock pH8.0, see assay details) 50μl lOmM HEPES pH 7.4 25μl (50mg) SPA beads (Amersham) lOOμl s-α 2 δ-lb-6His of SEQ ID No 9 (2μl protein diluted to lOOμl) obtained from example 5 25μl radioligand ([ 3 H]gabapentin ) Immediately after adding radioligand, the optiplates were loaded in the Packard Top Count scintillation counter to follow the binding time course. Imidazole was first used in the assay to optimize the specific interaction of the protein's 6His tag with the SPA bead. Imidazole itself (up to 1 OOmM) in the filtration assay has no effect on [ 3 H]gabapentin binding (n=l). As shown in figure 2, specific binding of [ 3 H]gabapentin to the s-α 2 δ-lb-6His was enhanced by imidazole. Of the concentrations, tested the optimal was 50mM. Equilibration was reached after ~2hours. Example 7 Ni Flashplate assay of [ H1 gabapentin binding to soluble porcine ct j δ-lb-όHis (SEQ ID No 37) Assays were carried out at 21 °C in a final volume of 250μl in 96-well NEN Ni chelate flash plates. Assay components were added in the following order (all reagents were diluted in lOmM HEPES (pH 7.4 at 21°C)): 25μl lOmM HEPES pH7.4 25μl imidazole at various concentrations (diluted from a IM stock pH8.0, see assay details) 75μl lOmM HEPES pH 7.4 lOOμl s-α 2 δ-lb-6His (2μl protein diluted to lOOμl) obtained from example 5 25μl radioligand ([ H] gabapentin) Immediately after adding the radioligand, flash plates were loaded in the Packard Top Count scintillation counter to follow the binding time course. The '[ 3 H] flash plate' programme (cpm) was used to monitor activity. Imidazole was first used in the assay to optimize the specific interaction of the protein's 6His tag with the Ni flashplate. Imidazole itself (up to lOOmM) in the filtration assay has no effect on [ 3 H] gabapentin binding (n=l). As shown in figure 3, the specific binding of [ 3 H]gabapentin to the s-α 2 δ-lb-6His was enhanced by imidazole. Initially, from the concentrations tested, the best concentration was found to be 1 OmM. Specific binding was determined at different volumes of s-α 2 δ-lb-6His, in the presence of lOmM imidazole, over a time period of lOh. Results are shown in figure 4 and equilibrium was reached at ~3h. Specific binding increased linearly with increasing amounts of protein, up to 8μl, after which the binding capacity of the Ni chelate in the assay well was probably exceeded (see figure 5). The published maximum binding capacity of NEN plates is 6pmol/well. The concentration of purified s-α 2 δ-lb-6His is estimated at ~0.6pmol/μl, which yields 5pmol/ well at 8μl. Saturation experiments were performed with 12 duplicate data points, [ 3 H]gabapentin concentration ranged from ~1 to 350nM. Data was analyzed using KEL-RADLIG for Windows. The results are presented in Table 2 below. Table 2 Saturation studies Example 8 Ni Flashplate assay of [-HlLeucine binding to soluble porcine α 2 δ-lb-6His The procedure described in example 2 was repeated, except that [ 3 H]gabapentin was replaced by 25 μl (10.1 nM) of [ 3 H]Leucine, as shown in figure 8, [ 3 H]Leucine binds with high affinity to soluble α 2 δ-lb-6His. This demonstrates that it is possible to use commercially available forms of [ 3 H]Leucine in replacement of [ 3 H]gabapentin in the assay. Example 9 Ni Flashplate assay studying competitive binding of HJ gabapentin, (S+)-3-isobutyl GABA and (R-)-3-isobutyl GABA to porcine α 2 δ-lb-6His (SEQ ID No 9) Assays were carried out at 21°C in a final volume of 250μl in 96-well NEN Ni chelate flash plates. Wells were set up for both 'total' and 'non-specific' binding. Specific binding was defined as that remaining after subtraction of the average of the 'non- specific binding' values from the average of the 'total' binding values. Assay components were added in the following order (all reagents were diluted in lOmM HEPES (pH 7.4 at 21°C)): 25μl 1 OmM HEPES pH7.4 or 25 μl of the test compound at the appropriate concentration in HEPES 25μl 200 mM imidazole (diluted from a IM stock pH8.0, see assay details) Total binding 75μl lOmM HEPES pH 7.4 Non-specific binding 50μl lOmM HEPES pH 7.4 and 25 μl lOOμM (S+)-3-isobutyl GABA lOOμl s-α 2 δ-lb-6His (2μl protein* diluted to lOOμl) 25μl radioligand ([ 3 H] gabapentin or [ 3 H]Leucine) * The source of s-α 2 δ- lb-6His was that purified by FPLC Superdex-200 gel filtration (see example 5) Immediately after adding radioligand, flash plates were loaded in the Packard Top Count scintillation counter to follow the binding time course. Incubation time before the assay was 3 hours. The '[ 3 H] flash plate' programme (cpm) was used to monitor activity. Specific binding was ~98% of the 'total' value. Imidazole was used in the assay to optimize the specific interaction of the protein's 6His tag with the Ni flashplate. Imidazole itself (up to lOOmM) in the filtration assay has no effect on [ 3 H]gabapentin binding (n=l). Competition studies were compared across the flash-plate and filter binding methodologies in order to validate the new assay technology with the established filter binding methodology. GraphPad Prism software was used to process competition curve data and determine IC 50 and hill slope values. Twelve point competition curves with half log dilution steps of test compounds were used in the experiments. Results are shown in Table 3 below where IC50 values are presented, and in figure 9 where hill slopes range from -0.9 to 1.3. The [ H]Gabapentin concentration used in assay is in the range of 10-13nM Table 3 Competition studies: GraphPad Prism software was used to process competition curve data and determine IC 50 and hill slope values. Ten point competition curves with half log dilution steps of test compounds were used in the experiments. IC 50 values were converted to Ki values (presented in table) according to the following equation: Ki = IC 50 / (1+[L]/K D ) The K D values used were those mean values presented in table 1. The [ 3 H]Gabapentin concentration in the assay ranged from 10-13nM and was determined for each experiment for the purpose of calculating the Ki value as described above. Hill slopes were all in the range of -0.9 to 1.3 Example 10 Filter binding assay of [-H1 gabapentin binding to the recombinant polypeptide of SEQ ID No 9 Assays were carried out at 21 °C in a final volume of 250μl in 96-deep well plates. Assay components were (all reagents were diluted in lOmM HEPES (pH 7.4 at 21°C)): 25μl compound to test 200μl Polypeptide of SEQ ID No 9 (3μl protein diluted to 200μl) 25μl radioligand ([ H]gabapentin (65Ci/ mmole) Plates were incubated at room temperature for lh prior to filtering on to 96- well GF/B Unifilter plates pre-soaked in 0.3% polyethylenimine. Filters were washed with 3x1 ml 50mM Tris-HCl (pH 7.4 at 4°C), and dried over-night. Scintillant (Microscint O, 50μl) was added and the plates counted using a Packard Top Count scintillation counter. Specific binding was -98% of the 'total' value. In [ 3 H]gabapentin saturation studies, the K D (nM) obtained was about 10.62. Example 11 : Binding of [-H1 gabapentin to the recombinant polypeptide of SEQ ID No 37 using various assay formats. This example further illustrates the screening method of this invention, using various assay formats. a) Preparation of protein stocks Protein was expressed as described in Example 4 except that the cells were infected at lxl 0 6 cells/ml. Additionally the cells were cultured in 20 litre Applikon fermentation vessels (18L culture volume). The culture was maintained at 27°C and 60% dO2 (100% dO 2 equates to [O 2 ] when media - without cells - has been saturated with air at 27°C) with single marine impeller stirring at 125rpm. The protein was expressed in either Sf- 900 II SFM (LTI Inc) or ESF-921 (Expression Systems Inc.) media. b) Purification of s-α. 2 δ-lb-6His protein from cell culture supernatants On the harvest day (day 4-7 post-infection with virus) the cell culture was centrifuged at 9,000xg for 20 minutes to remove the cellular debris, and the supernatant concentrated to approximately 3 litres using a pellicon tangential-flow filtration system employing 30kDa cut-off regenerated-cellulose cassettes. The concentrated sample was re- centrifuged at 9,000xg for 20 minutes then diluted with 2 volumes of lOmM Tris (pH9.0). The diluted sample was then loaded at lOml/min onto a Q-sepharose FF column (5cm i.d. x 50cm h.) which was washed with 20mM Tris-HCl (pH8.0) and eluted with 20mM Tris-HCl (pH8.0), 0.5M KCl, lOmM Imidazole. The eluate was then loaded at lOml/min onto a Ni-superflow (Qiagen) column (2.5cm i.d. x 6cm h.) pre-equilibrated in 20mM Tris (pH8.0), O.IM KCl, lOmM Imidazole. The column was washed successively with buffer A (20mM Tris (pH8.0), 0.5M KCl, 20mM Imidazole), buffer B (20mM Tris-HCl (pH8.0), IM KCl), and buffer A again at lOml/min. Elution was performed with a gradient of buffer C (20mM Tris-HCl (pH8.0), lOOmM KCl, 0.5M Imidazole) against 20mM Tris-HCl (ρH8.0), lOOmM KCl at 2ml/min (over 5 column volumes). Fractions from the gradient elution were assayed for [ 3 H]gabapentin binding activity and the active fractions pooled then dialysed at 4°C four times (each for 24 hours) against lOmM HEPES (pH7.3), 150mM NaCl at a ratio of 1 :60 (sample:dialysate). The dialysed material was then aliquoted and frozen for use in the assays as described below. c) Preparation of protein cocktails for filter, wheat germ lectin and Ni chelate assays (volumes in μl): cocktail xl x23 s-α 2 δ-lb-6His HBS s-θ 2 δ-lb-6His HBS Oμl 0 75 0 1,725 lμl 1 74 23 1,702 2μl 2 73 46 1,679 4μl 4 71 92 1,633 α 2 δ-l protein was sourced from the aliquots generated above. d) Filter and Wheat Germ Lectin flashplate assays The reagents were added in the following order to each well of either a 96-well Wheat Germ Lectin flashplate or a 96-deep well plate. Conditions were prepared in triplicate for both 'total' and 'non-specific' binding (20μl H 2 O added for total binding and 20μl of lOOμM (S+)-3-isobutyl GABA to define non-specific binding) for each of the four volumes of protein tested. Assay set-up per well: 1 OOμM (S+)-3-isobutyl GABA / H 2 O 20μl *100nM [ 3 H]Gabapentin 20μl 235mM HEPES (pH7.3) 85μl α 2 δ- 1 (0, 1 , 2 or 4μl of the x23 cocktail) 75μl * 20μl aliquots of the [ 3 H] gabapentin stock added to each well were counted on a liquid β-scintillation counter (Beckman LS 5000TD) to determine the actual concentration of [ 3 H]gabapentin reached in each well. For these experiments this value was calculated as 10.8nM. The Wheat Germ lectin flashplate was then counted under continuous cycling conditions on a Packard Top Count Microplate scintillation counter. The plate was counted on the '[ 3 H]flashplate' programme with a count delay and count time of 1 minute. Data for the wheat germ lectin assay was plotted as 'specific' binding (i.e. 'total' minus 'non-specific binding'), see figure 10. For the Filter assay, the binding reaction in the deep- well plate was left for 1 hour at 22°C then filtered with three 1ml washes of 4°C 50mM Tris (pH 7.4 at 4°C) onto a 96- well GF/B filter plate pre-soaked for 1 hour in 0.3% Polyethylenemine at 4°C. After leaving at 22°C to dry overnight, 45 μl of Microscint-O (Packard) was added to each filter well and the plate sealed and counted in the Packard Top Count Microplate Scintillation counter on the '[ HJMicroscint' programme with a count delay and count time of 1 minute. The mean of the 'total' and 'non-specific' binding is presented in figure 1 1. e) Nickel flashplate assay 2.35x Nickel flashplate buffer: 4.7ml IM HEPES pH7.3 0.118ml 10% BSA (Sigma A7906, Fraction V (98%), Lot 57H1088) in H 2 O 1.175ml 0.2M Imidazole pH7.3 (NaOH) 14.007ml H 2 O Assay set-up per well: 1 OOμM (S+)-3-isobutyl GABA / H 2 O 20μl *100nM [ 3 H]Gabapentin 20μl 2.35x Nickel Flashplate buffer 85μl s-α 2 δ-lb-6His (0, 1, 2 or 4μl of the x23 cocktail) 75μl * 20μl aliquots of the [ 3 H] gabapentin stock added to each well were counted on a liquid β-scintillation counter (Beckman LS5000TD) to determine the actual concentration of [ 3 H]gabapentin reached in the each well. For these experiments this value was calculated as 10.8nM. The Nickel flashplate was then counted under continuous cycling conditions on the Packard Top Count Microplate scintillation counter. The plate was counted on the '[ 3 H] flashplate' programme with a count delay and count time of 1 minute (Figure 12). f) Discussion This example demonstrates the extended stability of the assay over time (at least 100 hours), which is important if the assay format is to be used for mass-screening purposes. Indeed, such a stability enables the stacking of plates into counters (ideally with appropriate controls on each plate along with test compound wells in order to confirm signal stability across individual plates). The example also shows that it is possible to use the Wheat Germ lectin flashplate assay, as a primary assay or as a secondary screen to further refine ligands identified or selected using the Ni flashplate assay or another assay format of this invention. REFERENCES - Perez-Reyes, E., and Schneider, T. (1994) Drug Dev. Res. 33, 295-318 - Catterall, W. A. (1995) Annu. Rev. Biochem. 64, 493-531 - Birnbaumer, L., Campbell, K. P., Catterall, W. A., Harpold, M. M., Hofmann, F., Home, W. A., Mori, Y., Schwartz, A., Snutch, T. P., Tanabe, T., and Tsien, R. W. (1994) Neuron 13, 505-506 - Brust, P. F., Simerson, S., McCue, A. F., Deal, C. R., Schoonmaker, S., Williams, M. E.Nelicelebi, G., Johnson, E. C, Harpold, M. M., and Ellis, S. B. (1993) Neuropharmacology 32, 1089- 1 102 - Itagaki, K., Koch, W. J., Bodi, L, Klockner, U., Slish, D. F., and Schwartz, A. (1992) FEBS Lett. 297, 221-225 - Mikami, A., Imoto, F_ Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989) Nature 340, 230-233 - Mori, Y., Friedrich, T., Kim, M. S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., Mikoshiba, K., Imoto, K, Tanabe, T., and Numa, S. (1991) Nature 350,398-402 - Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., and Dascal, Ν. (1991) Science 253,1553-1657 - Ramsay, R. E. (1994) Neurology 44, Suppl. 5, 23-30 - Watson, W. P., and Little, H. J. (1995) Br. J. Pharmacol. 116, 33P (abstr.) - Singh, L., Field, M. J., Ferris, P., Hunter, J. C, Oles, R. J., Williams, R. G., and Woodruff, G. Ν. (1996) Psychopharmacology 111, 1-9 - Xiao, W. H., and Bennet, G. L (1995) Soc. Neurosci. 21, 897 (abstr.) - Mellick, G. A., Mellicy, L. B., and Mellick, L. B. (1995) J. Pain Symptom Manage. 10, 265-266 - Shimoyama, Ν., Shimoyama, M., Davis, A. M., Inturrisi, C. E., and Elliott, K. J. (1997) Neurosci. Lett. 222, 65-67 - SegaL A. Z., and Rordorf, G. (1996) Neurology 46, 1175-1176 - Mellick, G. A., and Mellick, L. B. (1996) Sleep 19, 224 -226 - Patel, J., and Νaritoku, D. K (1996) Clin. Neuropharmaco 19,185-188 - Suman Chauhan, Ν., Webdale, L., Hill, D. R., and Woodruff, G. Ν. (1993) Eur, J. Pharmacol. 244, 293-301 - Macdonald, R. L., and Kelly, F_ M. (1993) Epilepsia 34, Suppl. 5, S1-S8 - Taylor, C. P. (1994) Neurology 44, Suppl. 5, 10 -16 - Gotz, E., Feuerstein, T. J., Lais, A., and Meyer, D. K (1993) Arzneimittelforschung 43, 636-638 - Loscher, W., Honack, D., and Taylor, C. P. (1991) Neurosci. Lett. 128,150-154 - Honmou, 0., Knesis, J. D., and Richerson, G. B. (1995) Epilepsy Res. 20, 193-202 - Honmou, 0., Oyelese, A. A., and Kocsis, J. D. (1995) Brain Res. 692,273-277 - Petroff, 0. A. C, Rothman, D. L., Behar, K. L., Lamoureux, D., and Mattson, R. H. (1996) Ann. Neurol 39, 95-99 - Reimann, W. (1983) Eur. J. Pharmacol 94, 341-344 - Dooley, D. J., Bartoszyk, G. D., Hartenstein, J., Reimann, W., Rock, D. M., and Satzinger, G. (1986) Golden Jubilee Conference and Northern European Epilepsy Meeting. Abstracts, University of York, UK, September 1986 (Abstract 8). - Thurlow, R. J., Brown, J. P., Gee, N. S., Hill, D. R., and Woodruff, G. N. (1993) Eur. J. Pharmacol 247,341-345 - Gee, N. S., Brown, J. P., Dissanayake, V. U. I„ Offord, J., Thurlow, R., and Woodruff, G. N. (1996) J Biol Chem. Ill, 5768-5776 - Dissanayake, V. U. I-, Gee, N. S., Brown, J. P., and Woodruff, G. N. (1997) Br. J. Pharmacol. 120, 833-840 - Taylor, C. P., Vartanian, M. G., Yuen, P. W., Bigge, C, Suman Chauhan, N., and Hill, D. R. (1993) Epilepsy Res. 14,11-15 - Rock, D. M., Kelly, K. M., and Macdonald, R. L. (1993) Epilepsy Res. 16, 89 -98 - Wamil, A. W., Mclean, M. J., Nashville, T. N., and Taylor, C. P. (1991) Neurology 41, Suppl. 1, 140 (abstr.) - De Jongh, K S., Warner, C, and Catterall, W. A. (1990) J. Biol. Chem. 265, 14738-14741 - Jay, S . D., Sharp, A. H., Kahl, S. D., Vedvick, T. S., Harpold, M. M., and Campbell, K. P. (1991) J. Biol. Chem. 266, 3287-3293 - Burgess, A. J., and Norman, R. 1. (1988) Eur. J. Biochem. 178, 527-533 - Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W. J., Hai, A., Schwartz, A., and Harpold, M. M. (1988) Science 241, 1661-1664 - Brickley, K., Campbell, V., Berrow, N., Leach, R., Norman, R. I., Wray, D., Dolphin, A. C, and Baldwin, S. A- (1995) FEBS Lett. 364,129-133 - Brice, N. L., Berrow, N. S., Campbell, V., Page, K. M., Brickley, K., Tedder, I., Dolphin, A C. (1997) Eur. J. Neurosci. 9, 749-759 - Wiser, 0., Trus, M., Tobi, D., Halevi, S., Giladi, E., and Atlas, D. (1996) FEBS Lett. 379,15-20 - Xu, X., and Arnason, U. (1994) Gene (Amst.) 148, 357-362 - Williams, M. E., Feldman, D. H., McCue, A. F., Brenner, R., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Neuron 8, 71-84 - Kim, H. L., Kim, H., Lee, P., King, R. G., and Chin, H. (1992) Proc. Natl. Acad Sci. U. S.A. 89,3251-3255 - Brown, J. P., Dissanayake, V. U. K., Briggs, A. R., Milic, M. R., and Gee, N. S. (1998) Anal. Biochem. 255, 236-243 - Higuchi, R. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. eds) pp. 177-183, Academic Press, Ltd., London - Bradford, M. M. (1916) Anal. Biochem. 72, 248-252 - Kyte, J., and Doolittle, F. (1982) J. Mol. Biol 157, 105-132 - Summers, M. F., Henderson, L. E., Chance, M. R., Bess, J. W., Jr., South, T. L., Blake, P. R., Sagi, I., Perez-Alvarado, G., Sowder, R. C, Hare, D. R., and Arthur, L. 0. (1992) Protein Sci. 1, 563-574 - Klug, A. and Rhodes, D. (1987) Trends. Biochem. Sci. 12, 464-469 - Pieler, T., and Bellefroid, E. (1994) Mol. Biol. Rep. 20, 1-8 - Preston, R. A., Manolson, M. F., Becherer, M, Weidenhammer, E., Kirkpatrick, D., Wright, R., and Jones, E. W. (1991) Mol. Cell, Biol. 11, 5801-5812 - Tan, X., Waterham, H. R., Veenhuis, M., and Cregg, J. M. (1995) J. Cell Biol. 128,307-319 - Scotland, P. B., Colledge, M., Melnikova, I., Dai, Z., and Froehner, S. C. (1993) J. Cell Biol. 123, 719-728 - Henderson, L. E., Copeland, T. D., Sowder, R. C, Smythers, G. W., and Oroszlan, S. (1981) J. Biol. Chem. 256, 8400- 8406 - Beaucage et al., Tetrahedron Lett (1981) 22: 1859-1862. - Brown EL, Belagaje R, Ryan MJ, Khorana HG, Methods Enzymol (1979); 68, 109-151. - Feldman and Steg, (1996) Medecine/Sciences, synthese, 12, 47-55. - Houbenweyl, (1974), in Meuthode der Organischen Chemie, E. Wunsch Ed., Volume 15-1 et 15-11, Thieme, Stuttgart. - Koch Y. (1977), Biochem. Biophys. Res. Commun., 74, 488-491. - Kohler G. and Milstein C, (1975) Nature, 256, 495. - Kozbor et al., (1983) Hybridoma, 2(1), 7-16. - Leger OJ, et al. (1997) Hum Antibodies , 8(1), 3-16. - Martineau P, Jones P, Winter G. (1998), J. Mol Biol, 280(1), 117-127. - Merrifield RB, 1965a, Nature, 207(996), 522-523. - Merrifield RB, 1965b, Nature, 201 (996), 22-523. - Narang SA, Hsiung HM, Brousseau R, Methods Enzymol 1979, 68, 90-98. - Ohno et al., (1994), Science, 265, 781-784. - O'Reilly et al., (1992) Baculovirus expression vectors: a Laboratory Manual. W.H. Freeman and Co., New York. - Ridder R. Schmitz R, Legay F, Gram H, (1995) Biotechnology (NY), 13(3), 255-260. - Smith et al., (1983), Mol. Cell. Biol, 3, 2156-2165. - Sternberg N.L. (1992), Trends Genet, 8, 1-16. - Sternberg N.L. (1994) Mamm. Genome, 5, 397-404. - Sambrook, J. Fritsch, E.F. and T. Maniatis (1989). Molecular cloning: a laboratory manual, 2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. - Sanchez-Pescador R., (1988), J. Clin. Microbiol, 26(10), 1934-1938. - Urdea et al., MS (1988) Nucleic Acids Research, 11, 4937-4957. - Urdea et al., MS (1991) Nucleic Acids Symp Ser., 24, 197-200.

Claims: CLAIMS: 1. A method for the screening of ligands which bind a cerebral cortical voltage- dependent calcium channel α 2 δ-l subunit, said method comprising the steps of: - contacting a secreted soluble recombinant calcium channel α δ-l subunit polypeptide with: a ligand of interest; and a labelled compound which binds the α 2 δ-l subunit; and - measuring the level of binding of the labelled compound to the α 2 δ-l subunit. 2. A method for the screening of biologically active products, in particular products that modulate a nervous system function in a subject, comprising the steps of: - contacting a secreted soluble recombinant calcium channel α 2 δ-l subunit polypeptide with: - a candidate product; and a labelled compound which binds a α 2 δ-l subunit; and -measuring the level of binding of the labelled compound to the secreted soluble α 2 δ-l subunit. 3. A method according to claims 1 or 2, wherein said method is an SPA assay. 4. A method according to claim 1 or 2, wherein said method is a flashplate assay. 5 A method according to claims 1 or 2 and 4 where the flashplate assay is a wheat germ lectin flashplate format. 6. A method according to claim lor 2, wherein said method is a filter binding assay. 7. A method according to claim 1 or 2, wherein said secreted soluble recombinant calcium channel α 2 δ-l subunit polypeptide is selected from polypeptides having at least 80%, amino-acid identity with the polypeptide comprising from amino acid 1 to between amino-acids 1008 and 1087, preferably between amino-acids 1018 and 1078, and most preferably between amino-acids 1043 and 1078 of SEQ ID N°33 or SEQ ID N°44. 8. A method according to claim 1 or 2, wherein said secreted soluble recombinant calcium channel α 2 δ-l subunit polypeptide is selected from the group consisting of SEQ ID N°34, 35, 36, 37, 41, 42 and 43 with the polypeptides of SEQ ID N°37 and SEQ ID N°43 being most preferred. 9 A method according to claims 1 or 2 , wherein the α2δ-l subunit polypeptide has at least 80% amino acid sequence identity of any of SEQ ID N°34, 35, 36, 37, 41, 42 and 43. 10. Use of C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST tagg for the purification and screening of α 2 δ-l . 11. Use of C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST tagg for the purification and screening of α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 and 38. 12. Use of a sequence of histidine residues tagg for the purification and screening of α 2 δ- 1 of SEQ ID N°30, 31 , 32, 33, 34, 35, 36, 37 and 38. 13. Use of a sequence of 6 histidine residues tagg for the purification and screening of α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 and 38. 14. Use of C-terminal a 6 histidine residues tagg of α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 and 38 for purification and screening. 15. Use of nucleic acids encoding C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST taggs for the purification and screening of expressed α 2 δ-l. 16. Use of nucleic acids encoding C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST tagg for the purification and screening of expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 and 38. 17. Use of nucleic acids encoding a sequence of histidine residues tagg for the purification and screening of an expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 and 38. 18. Use of a nucleic acid sequence encoding a sequence of 6 histidine residue tagg for the purification and screening of an expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 and 38. 19. Use of the C-terminal part of a nucleic acid sequence encoding 6 histidine residue tagg of expressed α 2 δ-l of SEQ ID N°30, 31, 32, 33, 34, 35, 36, 37 and 38 for purification and screening. 20. Use of labelled compounds which have an affinity of less than 500nm for the gabapentin binding site of α2δ-l for the screening of ligands that bind to α2δ-l. 21. Use according to claim 20, wherein the labelled compound is selected from labelled Gabapentin, L-Norleucine, L-Allo-Isoleucine, L-Methionine, L-Leucine, L-Isoleucine, L- Valine or L-Phenylalanine. 22. A method of screening of claims 1 or 2, wherein, the assay is conducted between 1 and 30°C. 23. A kit for the screening of ligands which bind a cerebral cortical voltage-dependent calcium channel α 2 δ-l subunit, said kit comprising: a secreted soluble recombinant calcium channel α 2 δ-l subunit; and - a labelled compound which binds to the α 2 δ-l subunit. 24. A kit of claim 23, wherein the labelled compound is chosen from any one of the labelled compounds of claims 20 or 21. 25. A method for the screening of ligands which bind a cerebral cortical voltage- dependent calcium channel α 2 δ subunit, said method comprising the steps of: - contacting a secreted soluble recombinant calcium channel α 2 δ subunit polypeptide with: - a ligand of interest; and a labelled compound which binds the α 2 δ subunit; and - measuring the level of binding of the labelled compound to the α 2 δ subunit. 26. A method for the screening of biologically active products, in particular products that modulate a nervous system function in a subject, comprising the steps of: - contacting a secreted soluble recombinant calcium channel α 2 δ subunit polypeptide with: - a candidate product; and a labelled compound which binds a α 2 δ subunit; and - -measuring the level of binding of the labelled compound to the secreted soluble α 2 δ subunit. 27. A method according to claims 25 or 26, wherein said method is an SPA assay. 28. A method according to claim 25 or 26, wherein said method is a flashplate assay. 29. A kit for the screening of ligands which bind a cerebral cortical voltage-dependent calcium channel α 2 δ subunit, said kit comprising: a secreted soluble recombinant calcium channel α 2 δ subunit; and - a labelled compound which binds to the α 2 δ subunit. 30. A kit of claim 29, wherein the labelled compound is chosen from any one of the labelled compounds of claims 20 or 21.

Inventors: BERTELLI FRANCOIS, BROWN JASON PETER, DISSANAYAKE VISAKA, GEE NICOLAS STEVEN, SUMAN-CHAUHAN NIRMALA, BERTELLI, FRANCOIS, BROWN, JASON, PETER, DISSANAYAKE, VISAKA, GEE, NICOLAS, STEVEN, SUMAN-CHAUHAN, NIRMALA
Applicants: BERTELLI FRANCOIS, BROWN JASON PETER, DISSANAYAKE VISAKA, GEE NICOLAS STEVEN, SUMAN CHAUHAN NIRMALA, WARNER LAMBERT CO, BERTELLI, FRANCOIS, BROWN, JASON, PETER, DISSANAYAKE, VISAKA, GEE, NICOLAS, STEVEN, SUMAN-CHAUHAN, NIRMALA, WARNER-LAMBERT COMPANY
Application year: 2000
Main classification: G01N33
IPC codes: A61K31, A61P25, C07K14, C12N15, G01N33