Title of Invention

COMPOSITIONS COMPRISING ANALOGS OF SHK FOR SELECTIVE INHIBITION OF HV1.3 POTASSIUM CHANNELS

Abstract

Analogs of ShK toxin and methods for using such ShK analogs. The ShK analogs generally comprise ShK toxin attached to a chemical entity (e.g. an atom, molecule, group, residue, compound, moiety, etc.) that has an anionic charge. In some embodiments the chemical entity attached to the ShK toxin may comprise an amino acid residue. The ShK analogs may be administered to human or non-human animal subjects to cause inhibition of potassium channels or to otherwise treat diseases or disorders. In some embodiments, the chemical entity to which the ShK toxin is attached may be chosen to provide selective inhibition of certain potassium channels (e.g., KvL3 channels) over other potassium channels (e.g., Kvl.l channels). In some embodiments, the chemical entity to which the ShK toxin is attached may include a fluorophore, thereby providing a fluorophore, thereby providing a fluorophore tagged ShK analog. Such fluorophore tagged ShK analogs may be used in flow cytometry alone, or in conjunction with class II tetramers that can detect auto reactive cells.

Full Text

ANALOGS OF ShK TOXIN AND THEIR USES IN SELECTIVE INHIBITION OF Kv1.3 POTASSIUM CHANNELS RELATED APPLICATION This patent application claims priority to United States Provisional Patent Application No. 60/617,395 filed on October 7, 2004, the entirety of which is expressly incorporated herein by reference. FIELD OF THE INVENTION The present invention provides a) novel compositions of matter, b) methods and kits for in vivo and/or in vitro inhibition of the Kv1.3 channel in T- and B-lyrnphocytes and other cell types and c) methods for treating autoimmune and other disorders in human or animal subjects. BACKGROUND OF THE INVENTION Cell plasma membranes form the outer surfaces of eukaryotic cells. Various ions (e.g., sodium, potassium, calcium, etc.) move in and out of cells by passive diffusion through the cells' plasma membranes. Such diffusion of ions into and out of cells is facilitated by the presence of "ion channels" within the cell membranes. Ion channels are proteins embedded within the cell membrane that control the selective flux of ions across the membrane, thereby allowing for the formation of concentration gradients between the intracellular contents of the cell and the surrounding extracellular fluid. Because ion concentrations are directly involved in the electrical activity of excitable cells (e.g., neurons), the functioning (or malfunctioning) of ion channels can substantially control the electrical properties and behavior of such ceils. Indeed, a variety of disorders, broadly termed "channelopathies," are believed to be linked to ion channel insufficiencies or dysfunctions. Ion channels are referred to as "gated" if they can be opened or closed. The basic types of gated ion channels include a) ligand gated channels, b) mechanically gated channels and c) voltage gated channels. In particular, voltage gated channels are found in neurons, muscle cells and non-excitable cells such as lymphocytes. They open or close in response to changes in the charge across the plasma membrane. Kv1.3 Channels and Autoimmune Diseases. Autoimmune diseases such as multiple sclerosis (MS), type-1 diabetes mellitus (T1DM), rheumatoid arthritis (RA) and psoriasis affect several hundred million people worldwide. In these disorders specific autoreactive T cells - for instance myelin-specific T ceils in MS patients - are believed to undergo repeated autoantigen stimulation during the course of disease and differentiate into chronically activated memory cells that contribute to pathogenesis by migrating to inflamed tissues and secreting cytokines (Viglietta et alM 2002; Vissers et al., 1002; Wulff et at., 2003b). Therapies that preferentially target chronically activated memory T cells would have significant value for autoimmune diseases. Memory T cells are divided into two subsets - central memory (TCM) and effector memory (TEM) - based on the expression of the chemokine receptor CCR7 and the phosphatase CD45RA (Geginat et al., 2001; Sailusto et al., 1999). NaKVe and TCM cells home to the lymph node before they migrate to sites of inflammation, whereas TEM cells home directly to sites of inflammation where they secrete copious amounts of IFN-p and TNF-a and exhibit immediate effector function. It has recently been shown that myelin-specific autoreactive T cells in MS patients are predominantly activated TEM cells (Wulff et al., 2003b), and adoptive transfer of myelin-specific activated rat TEM cells into na'fve recipients induced severe EAE (Beeton et al., 2001a; Beeton et al., 2001b). An exciting new therapeutic target for immunomodulation of TEM cells is the voltage-gated Kv1.3 K* channel. TEM cells up-regulate Kv1.3 channels upon activation and their antigen-driven proliferation is exquisitely sensitive to Kv1.3 blockers (Wulff et alM 2003b). NaTve and TCM cells in contrast are significantly less sensitive to Kv1.3 blockers to begin with and rapidly become resistant to Kv1.3 blockade by up-regulating the calcium-activated \C channel IKCal (Ghanshani et alM 2000; Wulffetal. ,2003b). The dominance of Kv1.3 in TEM cells provides a powerful way to manipulate the activity of this subset with specific Kv1.3 inhibitors. The functionally restricted tissue distribution of the channel and the fact that in vivo Kv1.3 blockade ameliorates TEMHnediated EAE, bone resorption in peridontal disease and delayed type hypersensitivity reactions in animal models without causing obvious side effects has enhanced the attractiveness of Kv1.3 as a therapeutic target (Beeton et al., 2001b; Koo et al., 1997; Valverde et al., 2004). Although Kv1.3 blockers would suppress all activated TEM cells (for example TEM cells specific for vaccine antigens), a Kv1.3-based therapy would be a significant improvement over current therapies that broadly and indiscriminately modulate the entire immune system. An additional advantage of Kv1.3 blockers is that they are reversible. Thus, one could titrate the therapeutic effect of Kv1.3 blockers when needed and stop therapy in the face of infection, unlike chemotherapeutic agents, which take months to subside. Kv1.3 Channels and Obesity The Kv1.3 channel was found to play a role in energy homeostasis and energy balance (Hum Mol Genet 2003 12:551-9). Mice with the Kv1.3 channel genetically knocked out were able to eat fatty diets without gaining weight, while control mice given the same diet became over-weight. Pharmacological blockade of Kv1.3 channels recapitulated the effect of genetic knockout of Kv1.3 channels. Consequently, Kv1.3 blockers are likely to have use in the management of obesity. Kv1.3 Channels and Tvoe-2 Diabetes Mellitus. Kv1.3 channels play a role in regulating insulin-sensitivity in peripheral target organs such as the liver and muscle (Proc Natl Acad Sci USA. 2004 101:3112-7). Genetic knockout of the Kv1.3 channel in mice enhanced the sensitivity of the liver and muscle to insulin. Consequently, Kv1.3 blockers may have use in the treatment of type-2 diabetes mellitus by enhancing insulin's peripheral actions and thereby decreasing blood glucose levels. Naturally Occurring Polypeptides Known to inhibit Kv1.3 Channels The most potent Kvl3 inhibitor is the peptide ShK from the Caribbean sea anemone Stichodactyla helianthus. ShK is a 35-residue polypeptide cross-linked by 3 disulfide bridges. ShK blocks Kv1.3 (Kd = 11 pM) and suppresses proliferation of TEM cells at picomolar concentrations, and ameliorates experimental autoimmune encephalomyelitis (EAE) in rats induced by the adoptive transfer of myelin-specific TEM cells. A potential drawback of ShK is its low picomolar affinity for the neuronal Kv1.1 channel (Kd 28 pM). Although no side effects were observed with ShK in EAE trials, ingress of high concentrations of ShK into the brain, as might happen when the blood-brain-barrier is compromised in MS, could lead to unwanted neurotoxicity. The development of highly specific Kv1.3 inhibitors is therefore necessary. An extensive effort by the pharmaceutical industry and academic groups has yielded several small molecules that inhibit Kv1.3 in the mid-nanomolar range, but these compounds do not have the selectivity or potency to make them viable drug candidates. Several truncated peptidic analogs of ShK have previously been reported. In one of these ShK analogs, the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges). In others, non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. These ShK analogs exhibited varying degrees of Kv1.3 inhibitory activity and specificity. Lanigan, M.D. et al.; Designed Peptide Anaiogues of the Potassium Channei Blocker ShK Toxin; Biochemistry, 25;40(51):15528-37 (December 2001). There remains a need in the art for the development of new analogs of ShK that selectively inhibit Kv1.3 channels in lymphocytes with minimal or no inhibitory effects on Kv1.1 channels or other potassium channels. SUMMARY OF THE INVENTION The present invention provides novel compositions (referred to herein as "ShK analogs") comprising ShK toxin attached (e.g., bound, linked by a linker or otherwise associated with) to an organic or inorganic chemical entity (e.g. an atom, molecule, group, residue, compound, moiety, etc.) that has an anionic charge. Further m accordance with the present invention, there are provided methods for inhibiting potassium channels and/or treating diseases or disorders in human or animal subjects by administering to the subject an effective amount of an ShK analog of the present invention. In some embodiments, the chemical entity to which the ShK toxin is attached may be chosen to provide selective inhibition of certain potassium channels (e.g., Kv1.3 channels) over other potassium channels (e.g., Kv1,1 channels). Still further in accordance with the present invention, ShK analogs of the foregoing character may include a fluorophore tag and such fluorophore tagged ShK analogs of the present invention may be used in flow cytometry alone, or in conjunction with class (I tetramers that can detect autoreactive cells. Further aspects, elements and details of the present invention will be apparent to those of skill in the art upon reading the detailed description and examples set forth herebelow. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the chemical structures of a number of ShK analogs of the present invention. Figure 2A shows a molecular model of ShK based on the published NMR structure wherein the Lys22, critical for channel blockade, is highlighted In one shade of grey. L-pTyr was attached to the cc-amino group of Arg1 of ShK (highlighted in a second shade of grey) through an Aeea linker (right). The structures of the linker and L-pTyr were modeled with AM1 in Hyperchem. Figure 2B shows the effect of ShK (top) and ShK(L5) (bottom) on Kv1.3 and Kv1.1 currents in stably transfected cells. Figure 2C shows dose-dependent inhibition of Kv1.3 (open symbols) and Kv1.1 (closed symbols) by ShK (dark) and ShK(L5) (light). Kds on Kv1.3 =10 ± 1 pM (ShK) and 69 ± 5 pM (ShK(L5)); Kds on Kv1.1 = 28 ± 6 pM (ShK) and 7.4 ± 0.8 nM (ShK(L5)). Figure 2D shows the time course of wash-in and wash-out of ShK(L5) on Kv1.3 wherein cells were held at a holding potential of 80 mV and depolarized for 200 msec to 40 mV every 30 sees. Figure 2E shows Ka values for inhibition of Kv1.3 and Kv1.1 by ShK analogs. K& for ShK-F6CA and ShK-Dap22 based on published sources. Figure 3A is a graph showing staining intensities of CD45RA and CCR7 as determined by flow cytometry in the CD3+-gated population of human PBMCs stained with antibodies against CD3, CD45RA and CCR7. Figure 3B is a graph showing staining intensities of CD45RA and CCR7 as determined by flow cytometry in the CD3+-gated population in cells of a human TEM line stained with antibodies against CD3, CD45RA and CCR7. Figure 3C is a graph showing the inhibitory effects of ShK (dark grey) and ShK(L5) (light grey) of [3H] thymidine incorporation by PBMCs (open symbols, a mixture of naTve/TCM cells) and TEM cells (closed symbols) stimulated for 48 hours with anti-CD3 antibody. Figure 3D is a graphic showing of pre-activated human PBMCs (nalVe/Tcwi cells) that up-regulate KCa3.1 expression become resistant to ShK(L5) inhibition when reactivated with anti-CD3 antibody. These cells have previously been reported to become sensitive to the Kca3.1-specific inhibitor TRAM-34. Figure 4A is a graph showing CD45RC staining of rat splenic T cells (left) and PAS T cells (right) detected by flow cytometry. Figure 4B is a graphic showing of Kv1.3 currents exhibited by quiescent (top) and myelin antigen-activated (bottom) PAS T cells. Figure 4C provides a graphic representation of flow cytometry profiles of ShK-F6CA-staining in quiescent (top) and myelin antigen-activated (bottom) PAS T cells. Unstained cells (black lines) and cells stained with ShK-F6CA (area filled in light grey). Competition of ShK-F6CA staining by unlabeled ShK(L5) is represented by the area filled in dark grey. Figure 4D shows confocal images of Kv1.3 immunostaining in quiescent (top) and myelin antigen-activated (bottom) PAS T cells. Statistical analysis was carried out using the Mann-Whitney U-test. Figure 4E shows dose-dependent inhibition by ShK (dark lines) and ShK(L5) (light lines) of [3H] thymidine incorporation by rat (left) naTve/TCM (open symbols) and TEM (closed symbols) cells activated with Con A (1 pg/ml). Figure 4F shows dose-dependent inhibition by ShK (dark lines) and ShK(L5) (light lines) of IL2 secretion by PAS T cells 7 hours after stimulation with MBP. G, ShK(L5)-induced inhibition of myeiin-antigen triggered [3H] thymidine incorporation by PAS T cells (open symbols) is reversed by the addition of 20 u/ml IL2 (closed symbols). Figure 5A is a graph showing Kv1.3 blocking blocking activity of ShK(L5) as determined on Kv1,3 channels stably expressed in L929 cells. Figure 5B is a graph showing blood levels of ShK(L5) at various times after a single subcutaneous injection of 200 mg/kg of ShK(L5) in four rats. Blood was drawn at the indicated times and serum was tested by patch-clamp to determine the amount of ShK(L5). Figure 5C is a graph of the data of Figure 5B fitted to a single exponential decay indicating a half-life of approximately 50 minutes. Figure 5D is a graph showing blood levels of ShK(L5) in five Lewis rats receiving single daily subcutaneous injections of 10 ^ig/kg/day ShK(L5) for 5 days. Blood was drawn each morning (24 hours after the previous injection) and tested for blocking activity on Kv1.3 channels by patch-clamp. Figure 5E is a graph showing serum levels of ShK(L5) in rats at various times following a single dose of 10 mg/kg ShK(L5) either subcutaneously (open bars; n = 4) or intravenously (closed bars; n = 4). Blood was drawn at the indicated times and serum was tested by patch-clamp to determine the amount of ShK(L5) in blood. ShK(L5) maintained a steady-state level of 300 pM in the blood almost 24 bourse after a single subcutaneous injection. This concentration is sufficient to selctively inhibit the function of TEM cells. Figure 5F is a graph showing the % recovery of ShK(L5) after a half-blocking dose of ShK(L5) was added to rat plasma or PBS containing 2% rat plasma and incubated at 37°C for varying duration. Aliquots were taken at the indicated times and blocking activity determined on Kv1.3 channels. ShK(L5) is extremely stable in plasma. Figure 6A is a graph showing scored prevention of EAE. PAS T ceils were activated in vitro, washed, and injected intraperitoneal^ on day 0. Clinical scoring of EAE: 0= no clinical signs, 0.5 = distal limps tail, 1 » |jmp tail, 2 = mild paraparesis or ataxia, 3 = moderate paraparesis, 4 = complete hind limb paralysis, 5 = 4 + incontinence, 6 = death. Rats (n = 6/group) were injected subcutaneous with vehicle alone (n = 6) or ShK(L5) (n = 6; 10mg/kg/day) from day 0 to day 5. Figure 6B is a graph showing scored treatment of EAE. PAS T cells were activated in vitro, washed, and injected intraperitoneally on day 0. Treatment with ShK(L5) at 10mg/kg/day was started when rats developed clinical signs of EAE and was continued for 3 days. Figure 6C is a graph showing ear thicknes as an indicator of DTH reaction elicited against ovalbumin in rats. Animals (n = 6/group) were treated with ShK(L5) 10 mg/kg/day for 2 days, after which ear swelling was measured. Statistical analysis was carried out using the Mann-Whitney U-test. Figure 7A shows the ShK(L5) structure and a graph showing inhibition of Kv1.3 channels in TEM cells as a function of ShK(L5) concentration. Each data-point represents mean of three determinations. Figure 7B is a diagram of Kv1.3-containing signaling complex. Figure 7C shows co-localization of CD4, Kv1.3, Kvp2, SAP97, ZIP and p56™ at IS. Figure 7D shows CD4 and Kv1.3 staining in absence of visible TEM-APC contact. Figure 7E shows CD4 and Kv1,3 staining in GAD65-specific TEM cells exposed to MBP-loaded APCs. Figure 7F shows that ShK(L5) 100 nM does not prevent IS formation. Figure 7G shows that ShK(L5) 100 nM does not disrupt the IS. Figure 8A is a graphic showing of calcium signaling in GAD-specific TEM cells from three T1DM patients triggered by anti-CD3 + cross-linking secondary antibodies (arrow) in the absence (black) or presence of ShK(L5) 0.1 nM (dark grey), 1 nM (medium grey) or 100 nM (light grey). Figure 8B is a graph showing [3H]-thymidine incorporation by naTve/TCM and TEM cells (left) and naTve/TcM-effectors and TEM-effectors from patients with T1DM and RA (right). TEM cells: GAD65-activated TEM clones from three T1DM patients and anti-CD3 antibody activated SF-TEM cells from three RA patients. NaTve/TcM cells: anti-CD3 antibody-activated PB-naTveAcM cells from the same three RA patients. Figure 8C is a series of bar graphs showing Cytokine production by the TEM and naTve/TCM cells used in Figure 8B. Figure 8D shows the phenotype of disease-relevant and disease-irrelevant autoreactive T cells in MS, T1 DM and RA. Figure 8E is a diagram showing the manner in which ShK(L5) inhibits calcium signaling, lymphocyte proliferation and cytokine production but not IS formation. Figure 9 is a diagram representing a rat model of delayed type hypersensitivity (DTH) caused by effector memory T cells. Figure 10 is a diagram showing a treatment protocol for ShK(L5) in a rat model of delayed type hypersensitivity (DTH) caused by effector memory T cells Figure 11 is a diagram represneting specific suppression of effector memory responses in vivo in rats by ShK(L5) without impairing the function of naive and central memory T cells or B cells. Figure 12A shows Kv1.3 currents (top) and channel number/cell (bottom) in GAD65-, insulin and myelin-specific T cells from patients with new onset type-1 diabetes mellitus (T1DM), health controls and patients with multiple sclerosis.. Figure 12B shows Kvl.3 staining (top) and fluorescence intensities of individual T cells (bottom) from these patients. Figure 12C shows graphs of relative cell number vs. CCR7 staining intensity. Cells expressing high levels of Kv1.3 are CCR7-negative i.e. they are TEM-effectors. Cells expressing low levels of Kv1.3 are CCR&-positive i.e. they are either naive or TCM cells Figure 12D shows Kv1.3 number/cell in autoreactive T cells from a patient having T1DM and MS (left), patients having T1DM for greater than 5 years duration (middle) and patients having non-autoimmune type-2 DM. Figure 12E shows Kv1.3 numbers in CD4+GAD65-tetramer+ T cells from a patient with new-onset T1 DM. Figure 13A shows Kv1.3 channel numbers per cellin peripheral T cells blood and synovial fluid T cellsof RA patients and synovial fluid T cellsof OA patients. Figure 13B shows confocal images of Kv1.3 (light grey) and Kvp2 (darker grey) staining in the cells shown in Figure 13A. Figure 13C shows graphs of relative cell number vs. CCR7 staining intensity. Figure 13D shows micrographs (top) and bar graphs of inflammatory index (bottom) of synovium from RA and OA patients stained with anti-CD3 or anti-Kv1.3 antibodies and counter-stained with hematoxylin/eosin (40X). DETAILED DESCRIPTION The following detailed description and the accompanying drawings are intended to describe some, but not necessarily all, examples or embodiments of the invention only. This detailed description and the accompanying drawings do not limit the scope of the invention in any way. The present invention provides novel analogs of ShK, methods for making such compositions and methods for using such compositions to inhibit Kv1.3 channels (or other ion channels) in human or animal cells and for treatment or prevention of diseases and disorders, such as T cell mediated autoimmune disorders. The compositions of the present invention comprise ShK toxin attached (e.g., bound, linked by a linker or otherwise associated with) to an organic or inorganic, anionic-charged chemical entity (e.g. an atom, molecule, group, residue, compound, moiety, etc.). In at least some embodiments of the invention, the organic or inorganic, anionic-charged chemical entity may be selected to increase or optimize the affinity of the composition for inhibition of Kv1.3 channels over Kv1.1 channels. Examples of organic or inorganic, anionic-charged molecules or groups that may be linked or bound to ShK in accordance with the present invention include but are not necessarily limited to: amino acids; polypeptides; amino acid residues; unnatural amino acid residues; threonine; threonine derivatives; phospho-threonine; serine; serine derivatives; phospho-serine; glutamic acid; glutamic acid derivatives; gammacarboxy-glutamic acid; aspartic acid; aspartic acid derivatives; inorganic compounds or groups; organic compounds or groups; succinic anhydride; and phthalic anhydride. In accordance with the present invention, some non-limiting examples of compositions of the present invention, wherein the anionic-charged chemical entity comprises an amino acid residue, are shown in Figures 1 and 2C and referred to herein by alphaneumeric designations, as shown in Table 1 below: With specific reference to Figure 1, tyrosine or phenylalanine or their charged non-naturai derivatives were conjugated to ShK (top left) through a linker attached on its N terminus (residue Arg1 shown in shaded grey ). The Lys22, required for channel blockade, is shown in a darker shade of grey. The molecular model of ShK is based on the published NMR structure and the structures of the linker and the new residues were modeled. These embodiments of compositions of the present invention generally comprise the ShK toxin, which is a polypeptide, bound to (e.g., chemically bound, linked or otherwise associated with) at least one anionic-charged amino acid residue. In embodiments where the aminio acid residue has a chiral center, the D and/or L enantiomer of such amino acid residue may be used. The anionic-charged amino acid residue may be an unnatural residue and may be attached or linked to an N-terminus of the ShK polypeptide. In some embodiments, the anionic-charged amino acid residue may be linked to an N terminus of ShK through a linker, such as an aminoethyloxyethyloxy-acetyl linker. These analogs of ShK inhibit the Kv1.3 channel more specifically than ShK because they have reduced affinity for other potassium channels (e.g., Kvl.1). The ShK may be isolated from natural sources as known in the art, or it may be synthesized. Synthesis of ShK Toxin ShK Toxin may be synthesized by any suitable method. In one such method, Fmoc-amino acids (Bachem Feinchemikalien) including Arg(Pmc), Asp(OtBu), Cys(Trt), Gln(Trt), His(Trt), Lys(Boc), Ser(tBu) and Thr(tBu) are obtained commercially and assembled to form ShK Toxin. Stepwise assembly of the amino acids may be carried out on an Applied Biosystems 431A peptide synthesizer at the 0.25 mmol scale starting with Fmoc-Cys(Trt)-R. Residues 34 through 22 are single coupled. Thereafter, an aliquot (e.g., half) of the resin is removed to effect better mixing. The remainder of the peptide sequence is then double coupled to the remaining resin aliquot All couplings are mediated by dicyctohexylcarbodiimide in the presence of 2 eq of 1-hydroxybenzotriazole. The final two residues are also coupled via HBTU/DIEA chemistry. These residues are Aeea (Fmoc-aminoethyloxyethyloxyacetic acid) and as the N-terminal residue Fmoc-Tyr (P04) monobenzyl ester. Following final removal of the Fmoc-group, the peptide resin (2.42 g) is cleaved from the resin and simultaneously deprotected using reagent K for 2 h at room temperature. Reagent K is known in the art and has been described in the literature. See, King, D.S., Fields, C.G. and Fields, G.B. (1990) Int. J. Peptide Protein Res. 36, 255-266. Following cleavage, the peptide is filtered to remove the spent resin beads and precipitated with ice cold diethyl ether. The peptide is then collected on a fine filter funnel, washed with ice cold ether and finally extracted with 20% AcOH in H2O. The peptide extract is subsequently diluted into 2 liters of H2O, the pH is adjusted to 8.0 with NH4OH and allowed to air oxidize at room temperature for 36 hours. Following oxidation of the disulfide bonds with a 2:1 ratio of reduced to oxidized glutathione, the peptide solution is acidified to pH 2.5 and pumped onto a Rainin Dynamax Ci8 column (5.0 x 30 cm). The sample is eluted with a linear gradient from 5-30% acetonitrile into H2O containing 0.1% TFA. The resulting fractions are analyzed using two analytical RP-HPLC systems: TFA and TEAP. Pure fractions are pooled and lyophiiized. (See, Pennington, M.W., Byrnes, M.E., Zaydenberg, I., Khaytin, I., de Chastonay, J., Krafte, DM Hill, R., Mahnir, V., Volberg, WA, Gorczyca, W. and Kern, W.R. (1995) Int. J. Peptide Protein Res, 46, 354-358.) Alternatively, solid-phase peptide synthesis employing a Boc-Bzl protecting group strategy may be utilized to assemble the primary structure as well as analogs of the peptide. The peptide could then be cleaved from the solid-phase by anhydrous HF, yielding the linear peptide ready for folding as described above for the Fmoc synthesized peptide. (See, Stewart, J.M. and Young J.D. (1984) Solid Phase Peptide Synthesis. 2nd Edition. Pierce Chemical Company. Rockford, II.) Alternatively, other synthetic methods to assemble the primary structure of ShK or analogs could include chemical ligation technology where the peptide is prepared as a series of designed fragments with C-terminal thioester peptides. The the thioester peptide can react with another peptide containing an N-terminal Cys residue to form a peptide containing a native peptide bond. By using this technology, one could effectively assemble the primary structure of ShK (See, (4) Wilken, J. and Kent S.B.H. (1998) Chemical protein synthesis. Current Opin. Biotech. 9, 412-426.) Alternatively, another synthetic method that may be employed to assemble the primary structure of ShK would utilize a protected peptide fragment convergent approach as described in Albericio, F.( Lloyd-Williams, P., and Giralt, E. (1997) Convergent peptide synthesis; in Methods in Enzymol. Ed G. Fields, Academic Press, New York, NY. pp 313-335. In this method, linear protected fragments are assembled as fully side chain protected fragments. These fragments can then be coupled together in a convergent manner to assemble the primary sequence of ShK or one of its analogs. Assembly of the fragments could also utilize a solid-phase resin to facilitate coupling and wash steps. Alternatively, recombinant methods may be used wherein the cDNA coding sequence for ShK could be generated for expression in either a prokaryotic or eucaryotic expression system. Recombinant ShK analogs containing unnatural amino acids are also possible by utilizing preload tRNA molecules which utilize non-standard condons. The cCNA construct can be engineered to use one of these unused codons to add the phosphotyrosine residue as well as the Aeea residue. Folding of the recombinantty produced ShK analog could then be accomplished in a similar method to that used for the synthetic peptides. (See, Pennington, M.W., Byrnes, M.E., Zaydenberg, I., Khaytin, I., de Chastonay, J., Krafte, D., Hill, RM Mahnir, V., Volberg, W.A., Gorczyca, W. and Kern, W.R. (1995) Int. J. Peptide Protein Res. 46, 354-358.) Attaching Anionic Amino Acid Residues To ShK and Optional Modifications to ShK Anionic amino acid residues may be attached to the N terminus of natural or synthetic ShK Toxin by way of a linker, such as an aminoethyloxyethyloxy-acetyl linker, or bay any other suitable means. In this example, the nine (9) ShK analogs shown in Figure 1 are prepared. Initially, Fmoc-Aeea-OH is coupled to the N-terminus of synthetic ShK Toxin assembled as described above. The resin is then divided into 9 aliquots. Either Fmoc-Tyr(P04Bzl)-OH, Fmoc-d-Tyr(P04Bzl)-OH, Fmoc-Tyr(P04Me2)-OH, Fmoc-Pmp-OH, Fmoc-d-Pmp-OH, Fmoc-Pmp(Et)-OH, Fmoc-Pmp(Et)2-OH, Fmoc-Tyr(tBu)-OH, or Fmoc-Amp(Boc)-OH is then coupled using DIG and HOBT to one of the resin aliquots. The deblocked peptide resin is then cleaved and deprotected with reagent K (King et al.t 1990) containing 5% triisopropylsilane for 2 h at RT. Met(0) is reduced by addition of solid NH4l to the cleavage cocktail at t-15 min. (Nicolas et al., 1995). For the peptide containing Tyr(P04Me2)-OH, a cleavage cocktail containing 1 M TMSBr in TFA containing thioanisoie as a scavenger for 18 hr at 4° C was used (Tian et ai., 1993). Incomplete removal of the methyl protecting groups is common when using this method and two of the species (Tyr(P04) and Tyr(P04Me)) are easily purified by RP-HPLC The Tyr(P04Me2) containing analog is cleaved via standard Reagent K cleavage keeping both Me groups intact In each case, the cleavage mixture is filtered and the crude peptide is precipitated into ice-cold diethyl ether. The precipitate is collected, yielding approximately 75 mg of peptide from 200 mg of resin. The crude product is dissolved in 20 ml of 50% aqueous AcOH and diluted into 0.75 I of H20. The pH of the solution is adjusted with NH4OH to 8.2, and it was allowed to fold overnight with the addition of glutathione (2mM:1mM) (reduced:oxidized). All analogs are purified using RP-HPLC as described previously (Pennington et alM 1995; Pennington et al., 1996a; Pennington et al., 1996b). Pure fractions are pooled and lyophilized. Each sample is confirmed by RP-HPLC, AAA and MALDI-TOF MS and adjusted to account for peptide content prior to bioassay. In some embodiments of the invention, to improve the PK/PD properties of the ShK structure, residues which are sensitive to degradation properties may be replaced or substituted. Thus, substitution of the Met residue at position 21 may be carried out to impart a stabilizing effect to oxidation. Additionally, substitution of the C-temrinal acid function with an amide will impart stability to C-terminal corboxypeptidase enzymes. These two substitions to the primary structure of ShK combined with the anionic moiety at the N-terminus have been synthesized to generate the most stable and selective Kv1.3 blocker. Nonhydrolyzable phosphate substitutions will also impart a stabitzing effect versus acid and basic hydrolysis of the phosphate as well as stability against phosphatase enzymes. The substitutions are summarized below. The acronyms used are defined as follows: Pmp=p-phosphonomethyl-Phenyialanine; Ppa=p-Phosphatityi-Phenylalanine and Nle=Norleucine. Substitutions: p-phospho-Tyr-Aeea-ShK-Nle21-Cys35-amide p-Phosphono-methyI-Phenylalanine-Aeea-ShK-Nle21-Cys35amide (Pmp) p-Phosphatityl-Phe-Aeea-ShK-Nle21 -Cys35-amide (Ppa) p-phospho-Tyr-Aeea-ShK-Nle2i-Cys35-acid p-Phosphono-methyl-Phenylalanine»Aeea-ShK-Nle21-Cys35acid (Pmp) p-Phosphatityi-Phe-Aeea-ShK-Nle21-Cys35-acid (Ppa) In addition to the nonhydrolyzable Pmp and Ppa, substitution of p-Phosphono(difluoro-methyl)-Phenylalanine (Pfp)and p-Phosphono-methylketo-Phenylalanine (Pkp) are also anionic substituions, providing the following: Pfp-Aeea-Shk-Nle21 Cys35 amide Pkp-Aeea-ShK-Nle21-Cys35 amide Pfp-Aeea-Shk-Nle21 Cys35 acid Pkp-Aeea-ShK-Nle21-Cys35 acid. Structures of the N-terminal substitutions are set forth in Appendix B. Other structures that are within the scope of the present invention are published in Beeton, C. et a!., Targeting Effector Memory T Ceils with a Selective Peptide Inhibitor of Kv1.3 Channels for Therapy of Autoimmune Diseases, Molecular Pharmacology, Vol. 67, No.4,1369- (2005), the entirety of which is expressly incorporated herein by reference and a complete copy of which is appended hereto as Appendix C. Therapeutic Uses of ShK Analogs of the Present Invention The present invention provides methods for treating or preventing certain disorders or diseases, such as T cell mediated disorders (e.g., autoimmune disorders, graft vs. host disease, prevention of rejection of organ transplants etc.), other inflammatory disorders, obesity and Type 2 diabetes, in human or animal subjects by administering to the subject a therapeutically effective (e.g., preventative or effective to reduce or eliminate symptoms or disease progression) amount of a pharmaceutical^ acceptable preparation consisting or comprising an ShK analog of the present invention (e.g., including but not limited to those listed in Table 1 hereabove). Any suitable route of administration (e.g., oral, rectal, intravenous, intramuscular, subcutaneous, intradermal, intranasal, topical, transmucosal, transdermal, by drug delivery implant, etc.) may be used. When used to prevent or treat a T cell mediated disorder, the dosage(s) will be sufficient to inhibit Kv1.3 channels on T cell membranes. In this regard, the ShK analogs of the present invention have the potential to be used to prevent or treat a wide variety of a T cell mediated autoimmune disorders. The following are some examples of some T cell mediated autoimmune diseases that may be prevented or treated by the methods of the present invention, categorized with respect to the target organ that is principally affected by each such disease: Blood: Autoimmune hemolytic anemia Pernicious anemia Autoimmune Thrombocytopenia Vascular Temporal arteritis Anti-phospholipid syndrome Vasculitides such as Wegener's granulomatosis Behcet's disease Skin: Psoriasis Dermatitis herpetiformis Pemphigus vulgaris Vitiligo Endocrine: Type 1 diabetes mellitus Addison's Disease Grave's Disease Hashimoto's thyroiditis Autoimmune oophoritis and orchitis Multiple Organs and/or Musculoskeletal System: Rheumatoid arthritis (RA) Osteoarthritis (OA) Systemic lupus erythematosus Scleroderma Polymyositis, dermatomyositis Spondyloarthropathies such as ankylosing spondylitis Sjogren's syndrome Irrespective of the particular organ(s) affected, T-lymphocytes are believed to contribute to the development of autoimmune diseases. The currently available therapies for these diseases are largely unsatisfactory and typically involve the use of glucocorticoids (e.g. methylprednisolone, prednisone), non-steroidal anti-inflammatory agents, gold salts, methotrexate, antimalarials, and other immunosuppressants such as cyclosporin and FK-506. Also, another T cell mediated disorder that may be prevented or treated by the methods of the present invention is graft vs. host disease and/or rejection of transplanted organs. Indeed, the outcomes of organ transplant procedures have progressively improved with the development of refinements in tissue typing, surgical techniques, and more effective immunosuppressive treatments. However, rejection of transplanted organs remains a major problem. T-lymphocytes play a central role in the immune response and they are responsible, In large measure, for the rejection of many transplanted organs. They are also responsible for the so-called graft-versus host disease in which transplanted bone marrow cells recognize and destroy MHC-mismatched host tissues. Accordingly, drugs such as cyclosporin and FK506 that suppress T-cell immunity are used to prevent transplant rejection and graft-versus-host disease. Unfortunately, these T cell inhibiting drugs are toxic, with liver and renal toxicities limiting their use. Thus, the methods of the present invention may provide les toxic alternatives for the treatment or prevention of graft vs. host disease or transplant rejection. Also, inhibitors of the voltage gated Kv1.3 potassium channel have been shown to be especially effective in suppressing effector memory T cells and, thus, the methods of present invention may be particularly effective in preventing or treating diseases that are associated with effector memory T cells, such as; bone resorption and periodontal disease, psoriasis, rheumatoid arthritis, diabetes mellitus and multiple sclerosis. In addition to T cell mediated diseases, the Kv1.3 channel has been determined to regulate energy homeostasis, body weight and peripheral insulin sensitivity. Thus, the methods of the present invention may be used to treat other diseases and disorders that involve abnormal homeostasis, body weight and peripheral insulin sensitivity by inhibiting Kv1.3 channels on cell membranes, such other diseases and disorders include but are not necessarily limited to bone resorption in periodontal disease, Type 2 diabetes, metabolic syndrome and obesity. Use ofShK Analogs of the Present Invention in Flow Cytometry Further in accordance with the present invention there are provided methods for diagnosing T cell mediated disorders or otherwise sorting or distinguishing between various cell types in vitro using ftuorophore tagged versions of ShK(L5) for use in flow cytometry alone, or in conjunction with class II tetramers that can detect autoreactive cells. Flow Cytometry is a flexible method for characterizing cells in suspension wherein fluorescence activated cell sorting is used to select living cells on the basis of characteristics measured by flow cytometry. The types of cellular features and functions that may be detected by flow cytometry include the expression of proteins outside and within cells, type of DNA content, viability and apoptosis, multiple drug resistance pump activity, enzyme activity, T-cell activation, T-cell receptor specificity, cytokine expression, phagocytosis and oxidative burst activity. Thus, in this method of the present invention, the amino acid residue attached to the ShK may incorporate a fluorophore tag for use in flow cytometry alone, or in conjunction with class II tetramers loaded with specific autoantigens that can detect autoreactive cells. Specific descriptions of the methods by which such flow cytometry may be carried out are described in Beeton, C, et al., A Novel Fluorescent Toxin to Detect and Investigate Kv1.3 Channel Up-Regulation in Chronically Activated T Lymhocytes; J.Biol.Chem., Vol. 278, No. 11, 9928-9937 (March 2003). In general, a flow cytometer uses focused laser light to illuminate cells as they pass the laser beam in a fluid stream. Light scattered by the cells and light emitted by fluorescent dyes attached to cells of interest are analyzed by several detectors and processed by a computer. Cells may be distinguished and selected on the basis of size and shape as well as by the presence of many different molecules inside and on the surface of the cells. Examples of Potassium Channel Inhibiting Effects and Therapeutic Utility of ShK Analogs of the Present Invention ShK blocks the neuronal Kv1.1 channel and the Kv1.3 channel with roughly equivalent potency. Neurotoxicity is therefore a concern under circumstances that compromise the blood-brain-barrier and allow the entry of sufficient amounts of ShK to block Kv1.1 channels. Our strategy to design a Kv1.3-specific inhibitor was guided by our finding that ShK-F6CA containing fluorescein-6-carboxylate (F6CA) attached through a 20 A-long Aeea linker to the N-terminus of ShK exhibited 80-fo!d selectivity for Kv1.3 over Kv1.1 (Beeton et al., 2003). Since F6CA can exist as a restricted carboxylate or also as a cyclized lactone, it was not clear whether ShK-F6CA's Kv1.3 specificity was due to the negative charge of F6CA, the hydrophobiclty created by this large bulky fluorescein nucleus, potential planar -p electronic stacking or a combination of all of these potential contributions. To distinguish between these possibilities and with the intention of developing a non-fluorescent Kv1.3-selective inhibitor, we generated a series of 12 novel N-terminally-substituted ShK analogs to probe some of these interactions. By attaching tyrosine, phenylalanine or their derivatives (varying in charge, size and hydrophobiclty) through an Aeea linker to the N-terminus of ShK, we could probe the effects of charge and hydrophobicity to gain insight into our selectivity enhancement seen with F6CA substitution. Selective KV\v1.3 Inhibition overKvl.1 Inhibition: In the example shown in Figures 2A-2D, L-phosphotyrosine (L-pTyr) a negatively charged (net charge 2) post-translationally modified aromatic amino acid, was attached via the AEEA linker to ShK-Arg1 to generate the novel analog ShK(L5). The ShK toxin and ShK(L5) were tested on Kv1.3 and Kv1.1 channels stably expressed in L929 cells. Figure 2B shows the effects of ShK and ShK(L5) on Kv1.3 and Kv1.1 currents elicited by 200 ms depolarizing pulses from a holding potential of 80 mV to 40 mV. Both peptides reversibiy blocked Kvl3 and Kv1.1 in a dose-dependent manner with Hill coefficients of 1. KdS were determined from the dose-response curves shown using Microcal Origin software. ShK blocked Kv1.3 (Kd = 10 ± 1 pM) and Kv1.1 (Kd = 28 ± 6 pM) with roughly equivalent potency as expected (Fig. 1C). In contrast, ShK(L5) was 100-fold selective for Kv1.3 (Ka = 69 ± 5 pM) over Kv1.1 (Kd = 7.4 ± 0.8 nM) (Figs. 1B, 1C). The time course of Kvl3 current block by ShK(L5) and its washout is shown in Figure 1D. The time constant (TON) of ShK(L5) wash-in was 131 ±21 sec (n = 7) while the time constant (TOFF) for peptide wash-out was 150 ± 28 sec (n = 4). The Kd (57 ± 7 pM) calculated from the KON (15 x 106 ± 0.5 x 106 M*1sec*1) and KOFF (0.0059 ± 0.0013 sec'1) values is consistent with the Kd (69 ± 5 pM) determined with Microcal Origin software. Other ShK analogs were also tested on Kv1.3 and Kv1.1 channels. ShK(D5) containing D-phosphotyrosine (D-pTyr) was 35-fold selective for Kv1.3 over Kv1.1 but was an order of magnitude less potent than ShK(L5). ShK(L6) containing L-pTyr-monomethyl showed modest (11-fold) Kvl3 specificity, while ShK analogs containing L-pTyr-dimethyl or L-Tyr were not selective for Kv1.3 over Kv1.1. Analogs that contained phenylalanine or its derivatives (varying in bulk, p electron density and charge) were modestly specific or not specific for Kv1.3 over Kvll ShK(L5)'s 100-fold specificity for Kvl3 over Kvll is greater than that of ShK-F6CA (80-fold), ShK(D5) (35-fold), ShK-Dap22 (33-fold) or any other ShK analog tested. As may be appreciated from the data of Table 2 above, ShK(L5) blocked the Kv1.3 channel in T cells with a Kd (76 pM) equivalent to its Kd on the cloned channel (69 pM). It was 100-fold selective for Kv1.3 over Kv1.1, 260-fold selective over Kv1,6, 280-fold selective over Kv3.2, 680-fold selective over Kv1.2 and >1000-fold selective over all other channels tested. Importantly, it was 1600-fold Kv1.3-selective over KCa3.1, the calcium-activated K* channel (hat regulates activation of human nafve and TCM cells (Wulff et al., 2003). Native ShK was less selective than ShK(L5), ShK was 2.8-fold selective for Kv1.3 (Kd = 10 ± 1 pM) over Kv1.1 (Kd 28 ± 6 pM), 20-fold selective over Kv1.6 (200 ± 20 pM), 500-fold selective over Kv3.2 (Kd = 5,000 ± 1,000 pM), and >1000-fbld selective-over Kv1,2 (10 ± 1 nM) and KCa3.1 (Kd = 28 ± 3 nM). Margatoxin, a peptide from scorpion venom that has been touted as a specific Kv1.3 inhibitor (Koo et al., 1997; Lin et al., 1993; Middleton et al., 2003) was also not specific. It was 5-fold selective for Kv1.3 (110 ± 12 pM) over Kv1.2 (Kd =520 ± 1 pM), 9-fold selective over Kv1.1 (10 ± 1 nM) and > 1000-fold selective over Kvl6 and Kv3.2 (Kd > 100 nM). Luteolin, a . nutriceutical sold for autoimmune diseases tw^jljJtiinax.comJqnL*hA!???'?-°L -' ' it being a Kv1.3 inhibitor (Lahey and Rajadhyaksha, 2004), blocked Kv1.3 weakly (Kd = 65 ± 5 mM) and exhibited no selectivity over Kv1.1 (Kd=77± 5mM)t Kv1.2 {Kd = 63 ± 4 mM) or Kvl5 (Kd = 41 ± 3 mM). ShK(L5)'s exquisite specificity for Kv1.3 together with its picomolar affinity for the channel makes it a potentially attractive immunosuppressant. Preferential Suppression of Human TFM cell Prolifferation With reference to Figures 3A-3D, in order to assess ShK(L5)'s in vitro immunosuppressive activity, Applicants compared its ability to suppress anti-CD3 antibody-stimulated proliferation of human TEM cell lines versus human PBMCs that contain a mixture of naTve and TCM cells. Flow cytometry confirmed the cell surface phenotypes of the two populations studied. As seen in Figure 3A, the TEM lines were >90% CCR7"CD45RAt while as shown in Figure 3B the PBMCs contained 65 % CCR7+CD45RA+ (naYve) and 18% CCR7+CD45RA" (TCM) cells. Figure 3C shows that ShK(L5) and ShK were 60-fbld more effective in suppressing the proliferation of TEM cells (IC50 = -80 pM) compared with PBMCs (IC50 = 5 nM, p Preferential Suppression of Rat TFM Cells Proliferation As a preamble to evaluating ShK(L5)'s therapeutic effectiveness we examined its ability to suppress proliferation of a memory T cell line, PAS, that causes an MS-like disease in rats. As a control, Applicants used rat splenic T cells. To confirm the differentiation status of the two cell populations we assessed the expression of CD45RC, a marker of naive T cells (Bunce and Bell, 1997). Rat splenic T cells were 76% CD45RC* (i.e. mainly naive cells) whereas PAS cells were CD45RC" suggesting that they are memory ceils, as shown in Figure 4A. To determine whether PAS cells are in the TEM- or the TcM-state we examined Kv1.3 expression before and 48 hours after activation. TEM but not TCM ceils are expected to significantly up-regulate Kv1.3 levels upon stimulation. With reference to Figure 4B, patch-clamp experiments revealed a striking increase in Kv1.3 current amplitude after MBP-stimulation of PAS cells consistent with their being TEM cells. As an independent measure of the number of Kvl3 channels on PAS cells, we used ShK-F6CA, a fluorescently labeled ShK analog that has previously been reported to bind specifically to Kv1.3. The intensity of ShK-F6CA staining determined by flow cytometry reflects the number of Kv1.3 tetramers expressed on the cell surface. As seen in Figure 4C, ShK-F6CA (10nM) staining intensity increased with MBP-activation of PAS cells and an excess of unlabeled ShK(L5) (100 nM) competitively inhibited ShK-F6CA staining. As a final test, Applicants performed confocal microscopy on quiescent and MBP-stimuiated PAS cells that had been fixed and stained with a Kv1.3-specific antibody. In keeping with data in Figures 4B and 4C, resting PAS T cells had a Kv1.3 staining intensity of 4.4 ± 0.6 and this value increased to 10.6 ± 2.3 (p MBP-triggered proliferation of PAS cells was suppressed -1000-fold more effectively by ShK(L5) and ShK (IC5o - -80 pM) than mitogen-induced proliferation of rat splenic T cells (See Figure 4E, IC50 "100 nM; p cells, and may therefore have therapeutic use in autoimmune diseases by preferentially targeting TEM cells that contribute to the pathogenesis of these disorders. Circulating Half-Life and Stability A patch-clamp bioassay was used to ascertain whether circulating levels of ShK(L5) following subcutaneous injection were sufficient to inhibit TEM cells. The results of these experiments are shown in Figures 5A-5F. Serum samples from ShK(L5)-treated and control rats were tested for blocking activity on Kv1.3 channels. Control serum did not exhibit detectable blocking activity indicating an absence of endogenous channel blockers. To standardize the assay, known amounts of ShK(L5) were added to rat serum and these samples were tested on Kv1.3 channels. The spiked serum samples blocked Kv1.3 currents in a dose-dependent fashion (Kd 77 ± 9 pM) that was indistinguishable from ShK(L5)'s effect in the absence of serum (Fig. 4A). Levels of ShK(L5) in treated animals were determined by comparison with the standard curve. ShK(L5) was detectable in serum 5 minutes after a single subcutaneous injection of 200 mg/kg. Peak levels (12 nM) were reached in 30 minutes and the level then fell to a baseline of about 300 pM over 420 minutes. The disappearance of ShK(L5) from the blood could be fitted by a single exponential. The circulating half-life was estimated to be -50 min. Since the peak serum level after 200 mg/kg (12 nM) significantly exceeds the requirement for selective blockade of Kv1.3 channels and TEM cell function, we tested lower doses. After a single injection of 10 mg/kg the peak serum concentration of ShK(L5) reached " 500 pM within 30 min (data not shown), a concentration sufficient to block >90% Kv1.3 but not affect Kv1.1. Repeated daily administration of this dose (10mg/kg/day) resulted in steady-state levels of -300 pM (measured 24 hours after injection, Figure 5D), which is sufficient to cause 60-70% suppression of TEM cells with little effect on na'fve/TcM cells. The "steady-state" level is unexpected given the estimated circulating half-life of -50 min and indicates that ShK(L5) "accumulates" on repeated administration. To determine whether the "depot" was in the skin or elsewhere in the body, we measured blood levels of ShK(L5) 10 hours after rats received single intravenous or subcutaneous injections of 10 mg/kg ShK(L5). The peptide disappeared with the same time course following administration by either route (Figure 5E) indicating that the skin is not responsible for the steady-state level of 300 pM ShK(L5) reached after a single 10mg/kg daily injection (Figure 5D), and the depot(s) resides elsewhere. The successful achievement of a steady-state level of 300 pM ShK(L5) following daily single injections of 10mg/kg/day suggests that the peptide may be stable in vivo. To directly examine its stability we incubated ShK(L5) in rat plasma or in PBS containing 2 % rat plasma at 37°C for varying durations and then measured Kv1.3 blocking activity. In both sets of spiked samples (plasma and PBS) we observed a 50% reduction in Kv1.3-blocking activity in about 5 hours, presumably due to peptide binding to the plastic surface of the tube, and the level then remained steady for the next 2-days (Figure 5F). As an added test of stability we compared the Kv1.3- versus Kv1.1-blocking activities of sera from ShK(L5)-treated rats. If ShK(L5) is modified in vivo, either by dephosphorylatlon of pTyr or cleavage of the Aeea-pTyr side-chain, it would yield ShK(L4) and ShK respectively, neither of which is selective for Kv1.3 over Kv1.1. Serum samples from ShK(L5)-treated animals exhibited the same selectivity for Kv1.3 over Kv1.1 as ShK(L5), indicating that the peptide does not undergo the modifications stated above. Taken together, these results indicate that ShK(L5) is remarkably stable in plasma and attains pharmacologically relevant serum concentrations after single daily subcutaneous injections of 10 mg/kg. Nontoxicftv Applicants conducted several in vitro and in vivo tests to determine if ShK(L5) exhibits any toxicity. The results of these studies are summarized in Appendix A. Human and rat lymphoid cells incubated for 48 hours with a concentration (100 nM) of ShK(L5) >1200 times greater than the Kv1.3 half-blocking dose or the IC50 for TEM suppression (70-80 pM), exhibited minimal cytotoxicity. The same high concentration of ShK(L5) was negative in the Ames test on tester strain TA97A suggesting that it is not a mutagen. Both in vitro tests failed to detect any significant toxicity. Drug-induced blockade of Kv11.1 (HERG) channels has contributed to major cardiac toxicity and the withdrawal of several medications from the market ShK(L5) has no effect on Kv11.1 channels at 100 nM (>1430-fold the Kd for Kv1.3), and Applicants' chosen therapeutic regimen (10 mg/kg/day, 300 pM steady-state circulating level) should therefore not cause cardiotoxicity. As a further test, Applicants performed heart rate variability analysis in conscious rats administered vehicle (PBS + 2% rat serum) on day-1, followed by 10 mg/kg/day ShK(L5) on day-2. ShK(L5) had no effect on heart rate or the standard HRV (heart rate variability) parameters in both time and frequency domains (Task force of the European Society of Cardiology and the North American Society of Pacing Electrophysiology, 1996). Encouraged by the acute toxicity experiments, Applicants performed a sub-chronic toxicity study in which rats were administered daily subcutaneous injections of 10mg/kg ShK(L5) or vehicle for 2 weeks (n = 6 in each group). ShK(L5)-treated animals gained weight to the same degree as rats receiving vehicle (Appendix A). Hematological and blood chemistry analysis showed no difference between ShK(L5)- and vehicle-treated rats, and flow cytometric analysis revealed no differences in the proportions of thymocyte or lymphocyte subsets (Appendix A). Collectively, these studies suggest that ShK(L5) is safe. To determine the therapeutic safety index, we administered a 60-fold higher dose (600 mg/kg/day) of ShK(L5) to healthy rats for 5 days and observed no clinical signs of toxicity, and no toxicity was seen when healthy rats received a single injection of 1000 mg/kg ShK(L5). The situation is less sanguine when the blood-brain-barrier is compromised as happens in EAE and MS. Rats with EAE that received ShK(L5) 10 mg/kg/day for 10 days showed no signs of toxicity. In contrast, forty percent of rats (5/12) administered 600 mg/kg/day for five days died on the fifth day when they developed clinical signs of EAE (extrapolated LD50 = 750 mg/kg/day). Since the peak concentration of ShK(L5) in the serum (12 nM) after administration of a single injection of 200 mg/kg is sufficient to block >50% of Kvl.1 channels, toxicity observed in EAE rats administered 600 mg/kg/day ShK(L5) is likely due to the ingress into the brain of sufficient amounts of ShK(L5) to block Kv1.1. Thus, the effective therapeutic safety index of ShK(L5) is well in excess of 100 in situations where the blood-brain barrier is not compromised (as seen in autoimmune diseases that do NOT affect the central nervous system (CNS)), whereas the therapeutic safety index is 75 when the blood-brain barrier is breached. Prevention ofDTH and Acute Adoptive EAE With reference to Figures 6A-6C, ShK(L5) was evaluated for immunosuppressive activity in vivo in two animal models. Applicants tested its ability to prevent and treat acute EAE induced by the transfer of MBP-activated PAS TEM cells into Lewis rats, as welt as to suppress the DTH reaction mediated by TEM cells. PAS cells were activated with MBP for 48 hours in vitro and then adoptively transferred (6-8 x 106 viable cells) into Lewis rats. For the prevention trial, rats then received subcutaneous injections of saline (controls) or ShK(L5) (10 |jg/kg/day) for 5 days. In the first prevention trial control rats developed mild EAE (mean maximum clinical score 2.0 ± 1.2) with an average onset of 5.6 ± 0.6 days (not shown). ShK(L5) reduced disease severity (mean maximum clinical score 0.7 ± 0.6, p In the treatment trial (Figure 6B) rats were injected with MBP-activated PAS cells, administered saline or 10 pg/kg/day ShK(L5) when they initially developed signs of EAE (limp tail, hunched posture and loss of 6% or more of their weight over 24 hours) and therapy was continued for three days. Clinical signs of EAE peaked on day 6 in the control group (score = 3.9 ± 0.7) and on day 7 in the treated group (score = 1.9 ± 0.9; p As an independent assessment of ShK(L5)'s immunosuppressive activity in vivo, Applicants also examined its effectiveness in inhibiting the DTH reaction that is mediated predominantly by skin-homing TEM cells. Lewis rats immunized with ovalbumin and adjuvant were challenged 7 days later with ovalbumin in one ear and saline in the other ear. Rats then received injections of saline (controls) or ShK(L5) (10 pg/kg/day) and ear thickness was measured as an indication of DTH. All control rats developed ear swelling 24 and 48 hours after ovalbumin challenge while the DTH reaction was substantially milder in ShK(L5)-treated animals (Figure 6C). Thus, ShK(L5) inhibits the TgM-mediated DTH response, and prevents and ameliorates severe adoptive EAE induced by myelin-activated TEM cells. Kv1.3 Clusters At The IS Purina Antigen Presentation But K* Efflux Through Kv1.3 Is Not Required For IS Formation Or Stability Referring to Figures 7A-7G, ShK(L5), a highly selective Kv1.3 inhibitor (21), blocked Kv1.3 currents in GAD65-specific TEM cells with a Kd of 72 ± 3 pM. We used ShK(L5) as a pharmacological probe to define those steps in TEM cell activation that require Kv1.3 function. Biochemical studies have shown that Kv1.3 and Kvb2 belong to a signaling complex that includes SAP97 (Synapse-Associated-Protein-97), ZIP (PKC-zeta-interacting-protein, p56h*-associated-p62-protein, A170), pSS** and CD4 (Figure 7B). The existence of this complex in human TEM cells is supported by Applicants' results showing co-capping of Kv1.3, Kvb2, SAP97, ZIP and pSe"* with CD4. Furthermore, FRET (fluorescence energy transfer) studies show Kv1.3 in close proximity to CD3 in Kv1.3-transfected human T cells, and the channel preferentially localizes at the point of contact between Kv1.3-transfected human cytotoxic T cells and their targets. Since CD4 traffics to the IS, the zone of contact between T cells and antigen presenting cells (APC), it is possible that Kv1.3 and other proteins in the signaling complex also localize at the IS during antigen-presentation. To test this idea, GAD65-specific Kv1.3hlgh TEM clones from a T1 DM patient were incubated with HLA-matched APCs that had been loaded with GAD65 557! peptide and stained with DAPI to aid visualization. After 20 min, APC-TEM conjugates were immunostained for proteins in the signaling complex. CD4 co-localized at the IS with Kv1.3, Kvb2, SAP97, ZIP and p56 . in the absence of APC-TEM contact, CD4 and Kv1.3 were distributed throughout the cell. Furthermore, CD4 and Kvl3 failed to localize at points of contact when GAD65-specific TEM cells were exposed to APCs loaded with MBP (an irrelevant antigen), verifying that IS-clustering is antigen-specific. Thus in GAD65-specific TEM cells, a Kv1.3-containing signaling complex traffics together with CD4 to the IS during antigen-presentation, suggesting that Kv1.3 is an integral component of the machinery that transduces signals in TEM cells. Based on these studies, ShK(L5) at a concentration that blocks approximately 99% of Kv1.3 channels (100 nM) did not prevent IS-clustering and did not disrupt the IS once formed, indicating that K* efflux through Kv1.3 channels is unnecessary for IS formation or stability. Suppression of Human T$» Cells With reference to Figures 8A-8E, ShK(L5) inhibited calcium signaling in TEM cells, an early and essential step in T cell activation. GAD65-specific TEM clones from T1DM patients were loaded with the calcium indicator dye Fluo3, pre-incubated in medium alone or with increasing concentrations of ShK(L5) and imaged by flow cytometry before and after the addition of an activating anti-CD3 antibody and a cross-linking secondary antibody. Peak calcium rise occurred in 242±35 seconds after stimulation and was blocked by ShK(L5) with an IC5o of -200 pM (Figure 8A). ShK(L5) was 10-foid more effective in suppressing [3H]-thymidine-incorporation by autoreactive TEM cells from T1DM and RA patients compared with naTve/Tcw cells from these patients (Figure 8B, left). In a second set of experiments (Figure 8B, right), RA-SF and RA-PB T cells were activated with anti-CD3 antibody for 48 hours to generate "TEM-effectors" and HnaTVe/TcM-effectorsn respectively. Cells were rested overnight in medium, re-stimulated with anti-CD3 antibody in the presence or absence of ShK(L5) for a further 48 hours and [3H]-thymidine incorporation was measured. RA-SF-TeM-effectors retained their sensitivity to ShK(L5) inhibition, whereas RA-PB-naTve/TCM-effectors were resistant to Kv1.3 blockade (Figure 8B, right), most likely because they up-regulate the calcium-activated KCa3.1/IKCa1 channel, which substitutes for Kv1.3 in promoting calcium entry. ShK(L5) profoundly suppressed the production of interleukin 2 (IL2) and interferon-g (IFN-g) by TEM cells from T1DM and RA patients, whereas IL2 and IFN-g production by naTve/TCM cells from these patients was less affected (Figure 8C). The production of tumor necrosis factor-a and interleukin 4 by both TEM cells and naTve/TcM cells was less sensitive to ShK(L5) (Figure 8C). Verification of Rat Model of Delayed Type Hypersensitivity (DTH) Caused by Effector Memory T Cells. As shown in Figure 9, rats were immunized with ovalbumin (OVA) in adjuvant. They were challenged in one ear 7 days later with OVA and in the other ear with saline. Ear swelling was measured 24 h later as a sign of delayed type hypersensitivity (DTH). The FACS histograms shown in Figure 9 indicate that T cells in the ears challenged with OVA are CD45RC-negative memory cells while T cells in the blood and spleen of the same rats are mostly naive T cells. Treatment Protocol For Shk(L5) In A Rat Model Of Delayed Type Hypersensitivity (DTH) Caused By Effector Memory T Cells As shown in Figure 10, rats received ShK(L5) 10 pg/kg/day as a subcutaneous injection either from day 0 to day 7 (during the priming phase) to prevent the differentiation of naiv cells to effector memory TEM cells, or during the effector phase after challenge to the ear with ovalbumin to prevent the function of the TEM cells. Shk(L5) Specifically Suppresses Effector Memory Responses In Vivo In Rats Without Impairing The Function Of Naive And Central Memory T Cells Or B Cells As shown in Figure 11, control rats developed ear swelling i.e. a positive DTH response. ShK(L5) was NOT effective in suppressing DTH when administered during the priming phase, indicating that it did not suppress the differentiation of naive and central memory T cells into effector memory cells. ShK(L5) suppressed DTH when administered during the effector phase indicating that it either prevented the ability of effector memory T cells to reach the ear and/or suppressed the activation of effector memory T cells. The first possibility was excluded because the number of T cells in the ears of ShK(L5Hreated rats was the same as in the ears of rats given the vehicle. ShK(L5) suppressed effector memory T cell activation in the ear because these T cells were Kv1.3-negative, while the memory T cells in the ears of vehicle-treated animals were Kv1.3 positive. igM and IgG B-cell responses in these animals was also not affected. Kyi.3 Expression In T Cells Specific For GAD65/555-567. Insulin/9-23- And Myelin Antigens From Patients With T1DM Or MS And Healthy Controls Figure 12A shows KV1.3 currents (top) and channel number/cell (bottom) from antigen-specific T cells from patients with new onset type-1 diabetes mellitus, health controls and patients with mulitple sclerosis. Each data-point represents the mean ± SEM from 20-50 cells from 2-4 T cell lines from a single donor measured 48 hours after the third antigen stimulation. Due to the low frequency of T cells specific for insulin and GAD65 in the blood of T1DM patients and controls, we amplified these populations by generating short-term autoantigen-specific CD4+ T cell lines using the split-well method. As controls, we generated T cell lines specific for the irrelevant autoantigen myelin basic protein (MBP) that is implicated in MS but not T1DM. Following the third antigenic stimulation, Kv1.3 currents were measured by whole-cell patch-clamp in activated cells with a membrane capacitance greater than 4 pF (cell diameter > 11 ^im). Representative Kv1.3 currents and Kv1.3 channel-numbersfl" cell are shown in Figure 12A. The currents displayed biophysical and pharmacological properties characteristic of Kv1.3. T cells specific for insulin (9-23) or GAD65 (555-567) from patients with new onset T1DM displayed large Kv1.3 currents and expressed high numbers of Kv1.3 channels, whereas disease-irrelevant MBP-specific T cells from these patients were Kvl.310* (p = 0.001). For comparison we have plotted our published Kv1.3 data on MS patients In whom the opposite pattern was observed. In MS patients, T cells specific for MBP or myelin oligodendrocyte glycoprotein (peptide 35-55) or proteolipid protein (peptide 139-151) were Kv1.3h,flh, while insulin- and GAD65-specific T cells were Kvl.310" (p = 0.0001). Autoreactive T cells isolated from healthy controls were Kv1.3low regardless of specificity. In one individual with both MS and T1DM, T cells specific for all three autoantigens were Kv1.3hlQh. GAD65-specific and insulin-specific T cells from patients with longstanding T1 DM were Kv1.3WDh reflecting the persistence of autoreactive TEM cells, whereas a Kv1.3,ow pattern was found in GAD65- and insulin-specific T cells from patients with non-autoimmune type-2 DM. As seen in Figure 12B, Kv1.3 staining (top) and fluorescence intensities of individual cells (bottom). Applicants confirmed the patch-clamp data by immunostaining for Kv1.3. Insulin- and GAD65-specific T cells from T1DM patients and MBP-specific T cells from MS patients stained brightly whereas cells specific for irrelevant autoantigens stained dimly. Figure 12C shows CCR7 expression. Flow cytometry revealed that Kv1.3hiflh T cells were CCR7* TEM cells, while Kvl.310* cells were CCR7+ naTve or TCM cells. Figure 12D shows Kv1.3 number/cell in autoreactive T cells from a patient with both T1DM and MS, and from patients having T1DM or type-2 DM for greater than 5 years and 2 years, respectively. Figure 12E shows Kv1.3 numbers in CD4*GAD65-tetramer+ T cells from patient with new-onset T1DM. As a further control, we used fluorescent MHC class II tetramers containing the GAD65 5571 peptide, to isolate GAD65-specific CD4+ T cells from a DR-0401-positive patient with new onset T1DM. Tetramer-sorted GAD65-activated T cells displayed the same Kv1.3hich pattern observed in GAD65-specific T cell lines from T1DM patients. In summary, disease-relevant, autoantigen-acBvated T cells in both T1DM and MS are Kv1.3hl8hCCRr TEM-effectors, while disease-irrelevant autoreactive cells in these patients are Kvt.3,0WCCR7+ nalve/TcM cells. Kv1.3 expression in Rheumatoid Arthritis and Osteoarthritis In RA, disease-relevant T cells can be isolated from affected joints. Applicants patch-clamped T cells from the synovial fluid (SF) of 7 RA patients 48 hours after stimulation with anti-CD3 antibody. As seen in Figure 13A, as controls Applicants analyzed SF-T cells from 7 patients with degenerative, non-autoimmune osteoarthritis (OA) (which had been activated with the same protocol. RA-SF T ceils were Kv1.3h,Qh whereas OA-SF T cells were Kv13*™ (p for Kv1.3 and its associated Kv|J2 subunit corroborated the patch-clamp data. Figure 13B shows confocal images of Kv1.3 (light grey as seen in the figure) and Kv(52 (darker grey as seen in the figure) staining. RA-SF T cells stained brightly for both Kv1.3 and Kv|S2t while OA-SF and RA-PB T cells displayed weak staining. Figure 13C illustrates CCR7 expression. Flow cytometry verified that Kv1.3hi9h RA-SF T cells were CCR7" TEM cells, while Kv1.3,ow OA-SF and RA-PB T cells were CCR7+ naKve/TcM cells. Figure 13D (top) shows micrographs of synovium from RA and OA patients stained with anti-CD3 or antf-Kv1.3 antibodies and counter-stained with hematoxyiin/eosin (40X). As a further test, we immunostained paraffin-embedded synovial tissues (ST) from 5 RA and 5 OA patients for CD3, Kv1.3 and CCR7. We have previously shown that our staining method does not detect Kv1.3 in naTve/TcM cells because of their low numbers of Kv1.3 channels. In RA-ST, a preponderance of CD3+Kv1.3+CCR7~ TEM cells was seen, whereas CD3+ cells were sparse in OA-synovium and these were mainly Kv1.3'CCR7+ naIve/TCM cells. Degree of infiltration by CD3+, Kv1.3+ and CCR7* cells assessed by grading system in Figure S2A. CD3+-inflammatory-index: RA = 3.2±0.1; OA = 1.1 ±0.2 (p It is to be appreciated that the invention has been described herein with reference to certain examples or embodiments of the invention but that various additions, deletions, alterations and modifications may be made to those examples and embodiments without departing from the intended spirit and scope of the invention. For example, any element or attribute of one embodiment or example may be incorporated into or used with another embodiment or example, unless to do so would render the embodiment or example unsuitable for its intended use. Also, where the steps of a method or procedure are listed or stated in a particular order, the order of those steps may be changed unless otherwise specified or unless such change in the order of the steps would render the invention unpatentable or unsuitable for its intended use. All reasonable additions, deletions, modifications and alterations are to be considered equivalents of the described examples and embodiments and are to be included within the scope of the following claims. CLAIMS What is claimed is: 1. A composition of matter comprising ShK attached to an organic or inorganic chemical entity that has an anionic charge. 2. A composition according to claim 1 wherein the chemical entity is selected from the group consisting of: amino acids; polypeptides; amino acid residues; unnatural amino acid residues; threonine; threonine derivatives; phospho-threonine; serine; serine derivatives; phospho-serine; glutamic acid; glutamic acid derivatives; gammacarboxy-glutamic acid; aspartic acid; aspartic acid derivatives; inorganic compounds or groups; organic compounds or groups; succinic anhydride; and phthalic anhydride. 3. A composition according to claim 1 wherein the ShK toxin is obtained from a natural source. 4. A composition according to claim 1 wherein the ShK toxin is synthetic. 5. A composition according to claim 1 wherein the chemical entity is attached to an N terminus of the ShK polypeptide. 6. A composition according to claim 5 wherein the chemical entity is attached to an N-terminus of ShK through a linking molecule or linking group. 7. A composition according to claim 5 wherein the chemical entity is attached to an N-terminus of ShK by an aminoethyloxyethyloxy-acetyl linker. 8. A composition according to claim 1 wherein the chemical entity includes a fluorophore tag. 9. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Pmp(OH2). 10. A composition according to claim 1 wherein the chemical entity is AEEAc-D^Pmp(OH2). 11. A composition according to claim 1 wherein the chemical entity is AEEAc-D-Pmp(OH. Et). 12. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Pmp(Et2). 13. A composition according to claim 1 wherein the chemical entity is AEEAc-D-Pmp(Et2). 14. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Tyr. 15. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Tyr(P03H2). 16. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Phe(p-NH2). 17. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Phe(p-C02H). 18. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Aspartate. 19. A composition according to claim 1 wherein the chemical entity is AEEAc-D-Aspartate. 20. A composition according to claim 1 wherein the chemical entity is AEEAc-L-Glutamate. 21. A composition according to claim 1 wherein the chemical entity is AEEAo-D-Glutamate. 22. A method for causing inhibition of Kvl3 potassium channels in a human or animal subject, said method comprising the step of: (A) administering to the subject a composition that comprises ShK toxin attached to an organic or inorganic chemical entity that has an anionic charge, in a form and amount that is effective to inhibit of Kv1.3 potassium channels. 23. A method according to claim 22 wherein the method is carried out to prevent or treat an autoimmune disorder. 24. A method according to claim 23 wherein the autoimmune disorder is selected from the group consisting of: Multiple sclerosis Myasthenia gravis Autoimmune neuropathies such as Guiilain-Barr§ Autoimmune uveitis Crohn's Disease Ulcerative colitis Primary biliary cirrhosis Autoimmune hepatitis Autoimmune thrombocytopenia Type 1 diabetes mellitus Addison's Disease Grave's Disease Hashimoto's thyroiditis Autoimmune oophoritis and orchitis Behcet's disease Rheumatoid arthritis bone resorption associated with periodontal disease Systemic lupus erythematosus Scleroderma Polymyositis, dermatomyositis Pemphigus vulgaris Spondyloarthropathies such as ankylosing spondylitis Sjogren's syndrome 25. A method according to claim 22 wherein the method is carried out to prevent or treat graft vs. host disease. 26. A method according to claim 22 wherein the method is carried out to treat or prevent rejection of a transplanted tissue or organ. 27. A method according to claim 22 wherein the method is carried out to prevent or treat metabolic syndrome. 28. A method according to claim 22 wherein the method is carried out to treat or prevent Type 2 diabetes. 29. A method according to claim 22 wherein the method is carried out to treat or prevent obesity. 30. A method according to claim 22 wherein the method is carried out to treat or prevent bone resorption associated with periodontal disease. 31. A method according to claim 22 wherein the composition comprises ShK toxin attached to a chemical entity selected from the group consisting of. amino acids; polypeptides; amino acid residues; unnatural amino acid residues; threonine; threonine derivatives; phospho-threonine; serine; serine derivatives; phospho-serine; glutamic acid; glutamic acid derivatives; gammacarboxy-glutamic acid; asparticacid; aspartic acid derivatives; inorganic compounds or groups; organic compounds or groups; succinic anhydride; and phthalic anhydride. 32. A method according to claim 22 wherein the chemical entity is AEEAc-L-Pmp(OH2). 33. A method according to claim 22 wherein the chemical entity is AEEAc-D-Pmp(OH2). 34. A method according to claim 22 wherein the chemical entity Is AEEAo D-Pmp(OH, Et). 35. A method according to claim 22 wherein the chemical entity is AEEAc-L-Pmp(Et2). 36. A method according to claim 22 wherein the chemical entity is AEEAc-D-Pmp(Et2). 37. A method according to claim 22 wherein the chemical entity is AEEAc-L-Tyr. 38. A method according to claim 22 wherein the chemical entity is AEEAc-L-Tyr(P03H2). 39. A method according to claim 22 wherein the chemical entity is AEEAc-L-Phe(p-NH2). 40. A method according to claim 22 wherein the chemical entity is AEEAc-L-Phe(p-C02H). 41. A method according to claim 22 wherein the chemical entity is AEEAc-L-Aspartate. 42. A method according to claim 22 wherein the chemical entity is AEEAc-D-Aspartate. 43. A method according to claim 22 wherein the chemical entity is AEEAc-L-Glutamate. 44. A method according to claim 22 wherein the chemical entity is AEEAc-D-Glutamate. 45. A method for performing flow cytometry, said method comprising the steps of: (A) providing a composition that comprises ShK attached to an organic or inorganic chemical entity that nas an anionic cnarge and a fluorophore tag; (B) combining the composition provided in Step A with cells; and (C) using a flow cytometer to count, isolate, or distinguish cells that have affinity for the composition provided in Step A. 46. A method according to claim 45 wherein Step C comprises using the flow cytometer to count, isolate or distinguish T lymphocytes. 47. A composition according to claim 1 wherein the ShK is modified by substitution of the Met residue at position 21. 48. A composition according to claim 47 wherein the substitution at Met residue 21 deters oxidation. 49. A composition according to claim 1 wherein the ShK is modified by substitution of the of the C-temrinal acid function with an amide. 50. A composition according to claim 49 wherein the substitution of the of the C-terminal acid function with an amide imparts stability to C-terminal corboxypeptidase enzymes. 51. A method according to claim 22 or 45 wherein the ShK is modified by substitution of the Met residue at position 21. 52. A method according to claim 51 wherein the substitution at Met residue 21 deters oxidation. 53. A method according to claim 22 or 45 wherein the ShK is modified by substitution of the of the C-temrinal acid function with an amide. 54. A method according to claim 53 wherein the substitution of the of the C-terminal acid function with an amide imparts stability to C-terminal corboxypeptidase enzymes.

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