Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a methionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg, approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells.

Novel truncated isoform of SK3 potassium channel is a potent dominant-negative regulator of SK currents: implications in schizophrenia.

The small-conductance calcium-activated K(+) channel SK3 (SKCa3/KCNN3) regulates electrical excitability and neurotransmitter release in monoaminergic neurons, and has been implicated in schizophrenia, ataxia and anorexia nervosa. We have identified a novel SK3 transcript, SK3-1B that utilizes an alternative first exon (exon 1B), but is otherwise identical to SK3. SK3-1B, mRNA is widely distributed in human tissues and is present at 20-60% of SK3 in the brain. The SK3-1B protein lacks the N-terminus and first transmembrane segment, and begins eight residues upstream of the second transmembrane segment. When expressed alone, SK3-1B did not produce functional channels, but selectively suppressed endogenous SK3 currents in the pheochromocytoma cell line, PC12, in a dominant-negative fashion. This dominant inhibitory effect extended to other members of the SK subfamily, but not to voltage-gated K(+) channels, and appears to be due to intracellular trapping of endogenous SK channels. The effect of SK3-1B expression is very similar to that produced by expression of the rare SK3 truncation allele, SK3-Delta, found in a patient with schizophrenia. Regulation of SK3 and SK3-1B levels may provide a potent mechanism to titrate neuronal firing rates and neurotransmitter release in monoaminergic neurons, and alterations in the relative abundance of these proteins could contribute to abnormal neuronal excitability, and to the pathogenesis of schizophrenia.

Selective blockade of T lymphocyte K(+) channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis.

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4(+) T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype ("chronically activated") with large numbers of Kv1.3 voltage-gated channels (approximately 1500 per cell) and small numbers of IKCa1 Ca(2+)-activated K(+) channels (approximately 50-120 per cell). In contrast, resting T cells displayed 0-10 Kv1.3 and 10-20 IKCa1 channels per cell ("quiescent" phenotype), whereas T cells stimulated once or twice expressed approximately 200 Kv1.3 and approximately 350 IKCa1 channels per cell ("acutely activated" phenotype). Consistent with their channel phenotype, [(3)H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap(22)) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap(22) and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on these in vitro results, we assessed the efficacy of K(+) channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap(22) plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap(22), but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap(22) plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.

Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SkCa2.

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.

Molecular properties and physiological roles of ion channels in the immune system.

The discovery of a diverse and unique set of ion channels in T lymphocytes has led to a rapidly growing body of knowledge about their functional roles in the immune system. Here we review the biophysical and molecular characterization of K+, Ca2+, and Cl- channels in T lymphocytes. Potent and specific blockers, especially of K+ channels, have provided molecular tools to elucidate the involvement of voltage- and calcium-activated potassium channels in T-cell activation and cell-volume regulation. Their unique and differential expression makes lymphocyte K+ channels excellent pharmaceutical targets for modulating immune system function. This review surveys recent progress at the biophysical, molecular, and functional roles of the ion channels found in T lymphocytes.

Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1.

Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr(250) or Val(275) in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr(250) and Val(275) line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr(250) and Val(275) may lock the triarylmethanes in place via hydrophobic interactions with the pi-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside.

Nuclear localization and dominant-negative suppression by a mutant SKCa3 N-terminal channel fragment identified in a patient with schizophrenia.

The small conductance calcium-activated K+ channel gene SKCa3/KCNN3 maps to 1q21, a region strongly linked to schizophrenia. Recently, a 4-base pair deletion in SKCa3 was reported in a patient with schizophrenia, which truncates the protein at the end of the N-terminal cytoplasmic region (SKCa3Delta). We generated a green fluorescent protein-SKCa3 N-terminal construct (SKCa3-1/285) that is identical to SKCa3Delta except for the last two residues. Using confocal microscopy we demonstrate that SKCa3-1/285 localizes rapidly and exclusively to the nucleus of mammalian cells like several other pathogenic polyglutamine-containing proteins. This nuclear targeting is mediated in part by two polybasic sequences present at the C-terminal end of SKCa3-1/285. In contrast, full-length SKCa3, SKCa2, and IKCa1 polypeptides are all excluded from the nucleus and express as functional channels. When overexpressed in human Jurkat T cells, SKCa3-1/285 can suppress endogenous SKCa2 currents but not voltage-gated K+ currents. This dominant-negative suppression is most likely mediated through the co-assembly of SKCa3-1/285 with native subunits and the formation of non-functional tetramers. The nuclear localization of SKCa3-1/285 may alter neuronal architecture, and its ability to dominantly suppress endogenous small conductance K(Ca) currents may affect patterns of neuronal firing. Together, these two effects may play a part in the pathogenesis of schizophrenia and other neuropsychiatric disorders.

Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels.

To maintain Ca(2+) entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca(2+)-activated K(+) (K(Ca)) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of K(Ca) channels but not K(V) channels reduce Ca(2+) entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native K(Ca) channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca(2+) influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca(2+) entry despite pharmacological inhibition of the native small conductance K(Ca) channel. Thus, K(Ca) channels play a vital role in T cell Ca(2+) signaling.

Up-regulation of the IKCa1 potassium channel during T-cell activation. Molecular mechanism and functional consequences.

We used whole cell recording to evaluate functional expression of the intermediate conductance Ca(2+)-activated K(+) channel, IKCa1, in response to various mitogenic stimuli. One to two days following engagement of T-cell receptors to trigger both PKC- and Ca(2+)-dependent events, IKCa1 expression increased from an average of 8 to 300-800 channels/cell. Selective stimulation of the PKC pathway resulted in equivalent up-regulation, whereas a calcium ionophore was relatively ineffective. Enhancement in IKCa1 mRNA levels paralleled the increased channel number. The genomic organization of IKCa1, SKCa2, and SKCa3 were defined, and IK(Ca) and SK(Ca) genes were found to have a remarkably similar intron-exon structure. Mitogens enhanced IKCa1 promoter activity proportional to the increase in IKCa1 mRNA, suggesting that transcriptional mechanisms underlie channel up-regulation. Mutation of motifs for AP1 and Ikaros-2 in the promoter abolished this induction. Selective Kv1.3 inhibitors ShK-Dap(22), margatoxin, and correolide suppressed mitogenesis of resting T-cells but not preactivated T-cells with up-regulated IKCa1 channel expression. Selectively blocking IKCa1 channels with clotrimazole or TRAM-34 suppressed mitogenesis of preactivated lymphocytes, whereas resting T-cells were less sensitive. Thus, Kv1.3 channels are essential for activation of quiescent cells, but signaling through the PKC pathway enhances expression of IKCa1 channels that are required for continued proliferation.

Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant.

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K(+) channel, IKCa1, is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The "IKCa1-pharmacophore" consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar pi-electron-rich heterocycle (pyrazole or tetrazole) or a -C≡N group, whereas cytochrome P450 inhibition absolutely requires the imidazole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synthesized and found to selectively block IKCa1. TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) inhibits the cloned and the native IKCa1 channel in human T lymphocytes with a K(d) of 20-25 nM and is 200- to 1,500-fold selective over other ion channels. Using TRAM-34, we show that blocking IKCa1 in human lymphocytes, in the absence of P450-inhibition, results in suppression of mitogen-stimulated [(3)H]thymidine incorporation of preactivated lymphocytes with EC(50)-values of 100 nM-1 microM depending on the donor. Combinations of TRAM-34 and cyclosporin A are more effective in suppressing lymphocyte mitogenesis than either compound alone. Our studies suggest that TRAM-34 and related compounds may hold therapeutic promise as immunosuppressants.

Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca(2+)-activated over voltage-gated K(+) channels.

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca(2+)-activated K(+) channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3 and the Ca(2+)-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys(32), which lies in close proximity to this cluster. These analogs block IKCa1 with approximately 20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 and Kv1. 6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-K(Ca) channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K(+) channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K(+) channels. The availability of potent and selective inhibitors of IKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.

Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35 residue polypeptide cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. To investigate the role of these disulfides in the structure and channel-blocking activity of ShK toxin, a series of analogues was synthesized by selective replacement of each pair of half-cystines with two alpha-amino-butyrate (Abu) residues. The remaining two disulfide pairs were formed unambiguously using an orthogonal protecting group strategy of Cys(Trt) or Cys(Acm) at the appropriate position. The peptides were tested in vitro for their ability to block Kv1.1 and Kv1.3 potassium channels and their ability to displace [(125)I]dendrotoxin binding to rat brain synaptosomal membranes. The monocyclic peptides showed no activity in these assays. Of the dicyclic peptides, [Abu12,28]ShK(3-35,17)(-)(32) (where the subscript indicates disulfide connectivities) had weak activity on Kv1.3 and Kv1.1. [Abu17,32]ShK(3-35,12)(-)(28) blocked Kv1.3 with low nanomolar potency, but was less effective (being comparable to [Abu12,28]ShK(3-35,17)(-)(32)) against Kv1.1. [Abu3, 35]ShK(12-28,17)(-)(32), retained high picomolar affinity against both channels. Corroborating these results, [Abu3,35]ShK(12-28, 17)(-)(32) had an IC(50) ratio relative to native toxin of 18 in the displacement assay, whereas [Abu17,32]ShK(3-35,12)(-)(28) and [Abu12, 28]ShK(3-35,17)(-)(32) had ratios of 69 and 390, respectively. Thus, the disulfide bond linking the N- and C-terminal regions is less important for activity than the internal disulfides. NMR analysis of the [Abu12,28] and [Abu17,32] analogues indicated that they had little residual structure, consistent with their significantly reduced activities. By contrast, [Abu3,35]ShK(12-28,17)(-)(32) had a moderately well-defined solution structure, with a mean pairwise root-mean-square deviation of 1.33 A over the backbone heavy atoms. This structure nevertheless showed significant differences from that of native ShK toxin. The possible interactions of this analogue with the channel and the distinction between native secondary and tertiary structure on one hand and global topology imposed by the disulfide bridges on the other are discussed.

A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies.

Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K+-channel-blocking peptides belonging to family 2 peptides and comprising 30-40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named alpha-KTx1-12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.

Structural conservation of the pores of calcium-activated and voltage-gated potassium channels determined by a sea anemone toxin.

The structurally defined sea anemone peptide toxins ShK and BgK potently block the intermediate conductance, Ca(2+)-activated potassium channel IKCa1, a well recognized therapeutic target present in erythrocytes, human T-lymphocytes, and the colon. The well characterized voltage-gated Kv1.3 channel in human T-lymphocytes is also blocked by both peptides, although ShK has a approximately 1,000-fold greater affinity for Kv1.3 than IKCa1. To gain insight into the architecture of the toxin receptor in IKCa1, we used alanine-scanning in combination with mutant cycle analyses to map the ShK-IKCa1 interface, and compared it with the ShK-Kv1.3 interaction surface. ShK uses the same five core residues, all clustered around the critical Lys(22), to interact with IKCa1 and Kv1.3, although it relies on a larger number of contacts to stabilize its weaker interactions with IKCa1 than with Kv1.3. The toxin binds to IKCa1 in a region corresponding to the external vestibule of Kv1.3, and the turret and outer pore of the structurally defined bacterial potassium channel, KcsA. Based on the NMR structure of ShK, we deduce the toxin receptor in IKCa1 to have x-y dimensions of approximately 22 A, a diameter of approximately 31 A, and a depth of approximately 8 A; we estimate that the ion selectivity lies approximately 13 A below the outer lip of the toxin receptor. These dimensions are in good agreement with those of the KcsA channel determined from its crystal structure, and the inferred structure of Kv1.3 based on mapping with scorpion toxins. Thus, these distantly related channels exhibit architectural similarities in the outer pore region. This information could facilitate development of specific and potent modulators of the therapeutically important IKCa1 channel.

hKCa3/KCNN3 potassium channel gene: association of longer CAG repeats with schizophrenia in Israeli Ashkenazi Jews, expression in human tissues and localization to chromosome 1q21.

We demonstrate a significant association between longer CAG repeats in the hKCa3/KCNN3 calcium-activated potassium channel gene and schizophrenia in Israeli Ashkenazi Jews. We genotyped alleles from 84 Israeli Jewish patients with schizophrenia and from 102 matched controls. The overall allele frequency distribution is significantly different in patients vs controls (P = 0.00017, Wilcoxon Rank Sum test), with patients showing greater lengths of the CAG repeat. Northern blots reveal substantial levels of approximately 9 kb and approximately 13 kb hKCa3/KCNN3transcripts in brain, striated muscle, spleen and lymph nodes. Within the brain, hKCa3/KCNN3transcripts are most abundantly expressed in the substantia nigra, lesser amounts are detected in the basal ganglia, amygdala, hippocampus and subthalamic nuclei, while little is seen in the cerebral cortex, cerebellum and thalamus. In situ hybridization reveals abundant hKCa3/KCNN3 message localized within the substantia nigra and ventral tegmental area, and along the distributions of dopaminergic neurons from these regions into the nigrostriatal and mesolimbic pathways. FISH analysis shows that hKCa3/KCNN3 is located on chromosome 1q21.

Lack of linkage or association between schizophrenia and the polymorphic trinucleotide repeat within the KCNN3 gene on chromosome 1q21.

To determine the importance of a candidate gene KCNN3 (formerly named hSKCa3) in the susceptibility to schizophrenia, we have studied the genotypes of a (CAG)n polymorphism within this gene in the DNAs of the members of 54 multiplex families with this disease. Parametric and nonparametric linkage analysis did not provide evidence for linkage between KCNN3 (that we mapped to chromosome 1q21) and schizophrenia. Furthermore, we observed no difference in the distribution of the (CAG)n alleles between affected and normal individuals. These results do not support the hypothesis that larger KCNN3 alleles are preferentially associated with schizophrenia [Chandy et al. 1998 Mol Psychiatr 3:32-37] in individuals from multiply affected families.

UK-78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage-gated potassium channel and inhibits human T cell activation.

1. UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high-throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of approximately 200 nM and 1:1 stoichiometry. A closely related compound, CP-190,325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is significantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at - 50 mV enhances the channel's sensitivity to UK-78,282. Decreasing the number of inactivated channels by exposure to approximately 160 mM external K+ decreases the sensitivity to UK-78,282. Mutations that alter the rate of C-type inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between tau(h) and IC50 values. 3. Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4. UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage-gated K+ channel, Kv1.4. 5. UK-78,282 effectively suppresses human T-lymphocyte activation.

Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1.

Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail of hSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+ channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1. 3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed with hIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide.

The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys22 in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap22) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap22, at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [3H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of approximately 200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap22 in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap22 or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.