Mutating a Critical Lysine in ShK Toxin Alters Its Binding Configuration in the Pore-vestibule Region of the Voltage-gated Potassium Channel, Kv1.3

Biochemistry. Oct, 2002  |  Pubmed ID: 12356296

The voltage-gated potassium channel in T lymphocytes, Kv1.3, an important target for immunosuppressants, is blocked by picomolar concentrations of the polypeptide ShK toxin and its analogue ShK-Dap22. ShK-Dap22 shows increased selectivity for Kv1.3, and our goal was to determine the molecular basis for this selectivity by probing the interactions of ShK and ShK-Dap22 with the pore and vestibule of Kv1.3. The free energies of interactions between toxin and channel residues were measured using mutant cycle analyses. These data, interpreted as approximate distance restraints, guided molecular dynamics simulations in which the toxins were docked with a model of Kv1.3 based on the crystal structure of the bacterial K(+)-channel KcsA. Despite the similar tertiary structures of the two ligands, the mutant cycle data imply that they make different contacts with Kv1.3, and they can be docked with the channel in configurations that are consistent with the mutant cycle data for each toxin but quite distinct from one another. ShK binds to Kv1.3 with Lys22 occupying the negatively charged pore of the channel, whereas the equivalent residue in ShK-Dap22 interacts with residues further out in the vestibule, producing a significant change in toxin orientation. The increased selectivity of ShK-Dap22 is achieved by strong interactions of Dap22 with His404 and Asp386 on Kv1.3, with only weak interactions between the channel pore and the toxin. Potent and specific blockade of Kv1.3 apparently occurs without insertion of a positively charged residue into the channel pore. Moreover, the finding that a single residue substitution alters the binding configuration emphasizes the need to obtain consistent data from multiple mutant cycle experiments in attempts to define protein interaction surfaces using these data.

Modulation of Mouse Paneth Cell Alpha-defensin Secretion by MIKCa1, a Ca2+-activated, Intermediate Conductance Potassium Channel

The Journal of Biological Chemistry. Feb, 2002  |  Pubmed ID: 11724775

Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.

Characterisation of the Human Voltage-gated Potassium Channel Gene, KCNA7, a Candidate Gene for Inherited Cardiac Disorders, and Its Exclusion As Cause of Progressive Familial Heart Block I (PFHBI)

European Journal of Human Genetics : EJHG. Jan, 2002  |  Pubmed ID: 11896454

Mutations in genes encoding cardiac ion channels and their subunits are responsible for several genetic cardiac disorders. We characterised the human gene KCNA7, encoding the voltage-gated potassium channel Kv1.7 and compared its coding sequence with that of the mouse orthologue, kcna7. Both genes are encoded by two exons separated by a conserved intron, unlike all the other Kv1-family genes that contain intronless coding regions. KCNA7 and kcna7 encode proteins of 456 amino acid residues that share >95% sequence identity, and the mouse channel has biophysical and pharmacological properties closely resembling the ultra-rapidly activating delayed rectifier (I(Kur)) in cardiac tissue. Using reverse transcriptase-PCR, KCNA7 mRNA was detected in adult human heart. We determined that KCNA7 resides on chromosome 19q13.3 in a region that also contains the progressive familial heart block I (PFHBI) locus. Direct sequencing of KCNA7's coding sequence in PFHB1-affected individuals revealed no pathogenic sequence changes, but two single nucleotide polymorphisms detected in exon 2 result in amino acid substitutions. These results provide evidence for the exclusion of this candidate as the PFHB1-causative gene, although mutations in regulatory and non-coding regions cannot be excluded. As ion channel-encoding genes have been implicated in a growing number of genetic conditions, the data presented may facilitate further analysis of the role of KCNA7 and its product in the heart.

Blockade of the Intermediate-conductance Calcium-activated Potassium Channel As a New Therapeutic Strategy for Restenosis

Circulation. Sep, 2003  |  Pubmed ID: 12939222

Angioplasty stimulates proliferation and migration of vascular smooth muscle cells (VSMC), leading to neointimal thickening and vascular restenosis. In a rat model of balloon catheter injury (BCI), we investigated whether alterations in expression of Ca2+-activated K+ channels (KCa) contribute to intimal hyperplasia and vascular restenosis.

International Union of Pharmacology. XLI. Compendium of Voltage-gated Ion Channels: Potassium Channels

Pharmacological Reviews. Dec, 2003  |  Pubmed ID: 14657415

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

A Novel Fluorescent Toxin to Detect and Investigate Kv1.3 Channel Up-regulation in Chronically Activated T Lymphocytes

The Journal of Biological Chemistry. Mar, 2003  |  Pubmed ID: 12511563

T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited >80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (>600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation, peaking at 15-20 h and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.

The Voltage-gated Kv1.3 K(+) Channel in Effector Memory T Cells As New Target for MS

The Journal of Clinical Investigation. Jun, 2003  |  Pubmed ID: 12782673

Through a combination of fluorescence microscopy and patch-clamp analysis we have identified a striking alteration in K(+) channel expression in terminally differentiated human CCR7(-)CD45RA(-) effector memory T lymphocytes (T(EM)). Following activation, T(EM) cells expressed significantly higher levels of the voltage-gated K(+) channel Kv1.3 and lower levels of the calcium-activated K(+) channel IKCa1 than naive and central memory T cells (T(CM)). Upon repeated in vitro antigenic stimulation, naive cells differentiated into Kv1.3(high)IKCa1(low) T(EM) cells, and the potent Kv1.3-blocking sea anemone Stichodactyla helianthus peptide (ShK) suppressed proliferation of T(EM) cells without affecting naive or T(CM) lymphocytes. Thus, the Kv1.3(high)IKCa1(low) phenotype is a functional marker of activated T(EM) lymphocytes. Activated myelin-reactive T cells from patients with MS exhibited the Kv1.3(high)IKCa1(low) T(EM) phenotype, suggesting that they have undergone repeated stimulation during the course of disease; these cells may contribute to disease pathogenesis due to their ability to home to inflamed tissues and exhibit immediate effector function. The Kv1.3(high)IKCa1(low) phenotype was not seen in glutamic acid decarboxylase, insulin-peptide or ovalbumin-specific and mitogen-activated T cells from MS patients, or in myelin-specific T cells from healthy controls. Selective targeting of Kv1.3 in T(EM) cells may therefore hold therapeutic promise for MS and other T cell-mediated autoimmune diseases.

Potassium Channels As Therapeutic Targets for Autoimmune Disorders

Current Opinion in Drug Discovery & Development. Sep, 2003  |  Pubmed ID: 14579513

In human T-lymphocytes, the voltage-gated Kv1.3 channel and the Ca(2+)-activated IKCa1 channel play an important role in Ca2+ signaling, activation, adhesion and migration and have, therefore, long been regarded as attractive targets for immunotherapy. Here, we review the pharmacology of the two channels, their expression pattern in naïve and memory T-cells and their functional roles during T-cell activation, and explain the rationale for the use of Kv1.3 blockers for the therapy of T-cell mediated autoimmune diseases.

SK3-1C, a Dominant-negative Suppressor of SKCa and IKCa Channels

The Journal of Biological Chemistry. Feb, 2004  |  Pubmed ID: 14638680

Small conductance Ca2+-activated K+ channels, products of the SK1-SK3 genes, regulate membrane excitability both within and outside the nervous system. We report the characterization of a SK3 variant (SK3-1C) that differs from SK3 by utilizing an alternative first exon (exon 1C) in place of exon 1A used by SK3, but is otherwise identical to SK3. Quantitative RT-PCR detected abundant expression of SK3-1C transcripts in human lymphoid tissues, skeletal muscle, trachea, and salivary gland but not the nervous system. SK3-1C did not produce functional channels when expressed alone in mammalian cells, but suppressed SK1, SK2, SK3, and IKCa1 channels, but not BKCa or KV channels. Confocal microscopy revealed that SK3-1C sequestered SK3 protein intracellularly. Dominant-inhibitory activity of SK3-1C was not due to a nonspecific calmodulin sponge effect since overexpression of calmodulin did not reverse SK3-1C-mediated intracellular trapping of SK3 protein, and calmodulin-Ca2+-dependent inactivation of CaV channels was not affected by SK3-1C overexpression. Deletion analysis identified a dominant-inhibitory segment in the SK3-1C C terminus that resembles tetramerization-coiled-coiled domains reported to enhance tetramer stability and selectivity of multimerization of many K+ channels. SK3-1C may therefore suppress calmodulin-gated SKCa/IKCa channels by trapping these channel proteins intracellularly via subunit interactions mediated by the dominant-inhibitory segment and thereby reduce functional channel expression on the cell surface. Such family-wide dominant-negative suppression by SK3-1C provides a powerful mechanism to titrate membrane excitability and is a useful approach to define the functional in vivo role of these channels in diverse tissues by their targeted silencing.

K+ Channel Expression During B Cell Differentiation: Implications for Immunomodulation and Autoimmunity

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2004  |  Pubmed ID: 15240664

Using whole-cell patch-clamp, fluorescence microscopy and flow cytometry, we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD(+)CD27(+) memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca(2+)-activated intermediate-conductance IKCa1 channel when quiescent, and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast, quiescent class-switched memory B cells express high levels of Kv1.3 ( approximately 2000 channels/cell) and maintain their Kv1.3(high) expression after activation. Consistent with their channel phenotypes, proliferation of naive and IgD(+)CD27(+) memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin, whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore, specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders.

K+ Channels As Targets for Specific Immunomodulation

Trends in Pharmacological Sciences. May, 2004  |  Pubmed ID: 15120495

The voltage-gated Kv1.3 channel and the Ca(2+)-activated IKCa1 K(+) channel are expressed in T cells in a distinct pattern that depends on the state of lymphocyte activation and differentiation. The channel phenotype changes during the progression from the resting to the activated cell state and from naïve to effector memory cells, affording promise for specific immunomodulatory actions of K(+) channel blockers. In this article, we review the functional roles of these channels in both naïve cells and memory cells, describe the development of selective inhibitors of Kv1.3 and IKCa1 channels, and provide a rationale for the potential therapeutic use of these inhibitors in immunological disorders.

Kv1.3-blocking 5-phenylalkoxypsoralens: a New Class of Immunomodulators

Molecular Pharmacology. Jun, 2004  |  Pubmed ID: 15155830

The lymphocyte potassium channel Kv1.3 is widely regarded as a promising new target for immunosuppression. To identify a potent small-molecule Kv1.3 blocker, we synthesized a series of 5-phenylalkoxypsoralens and tested them by whole-cell patch clamp. The most potent compound of this series, 5-(4-phenylbutoxy)psoralen (Psora-4), blocked Kv1.3 in a use-dependent manner, with a Hill coefficient of 2 and an EC50 value of 3 nM, by preferentially binding to the C-type inactivated state of the channel. Psora-4 is the most potent small-molecule Kv1.3 blocker known. It exhibited 17- to 70-fold selectivity for Kv1.3 over closely related Kv1-family channels (Kv1.1, Kv1.2, Kv1.4, and Kv1.7) with the exception of Kv1.5 (EC50, 7.7 nM) and showed no effect on human ether-a-go-go-related channel, Kv3.1, the calcium-activated K+ channels (IKCa1, SK1-SK3, and BKCa), or the neuronal NaV1.2 channel. In a test of in vivo toxicity in rats, Psora-4 did not display any signs of acute toxicity after five daily subcutaneous injections at 33 mg/kg body weight. Psora-4 selectively suppressed the proliferation of human and rat myelin-specific effector memory T cells with EC50 values of 25 and 60 nM, respectively, without persistently suppressing peripheral blood naive and central memory T cells. Because autoantigen-specific effector memory T cells contribute to the pathogenesis of T cell-mediated autoimmune diseases such as multiple sclerosis, Psora-4 and other Kv1.3 blockers may be useful as immunomodulators for the therapy of autoimmune disorders.

Enhanced Neuronal Excitability in the Absence of Neurodegeneration Induces Cerebellar Ataxia

The Journal of Clinical Investigation. Feb, 2004  |  Pubmed ID: 14966567

Cerebellar ataxia, a devastating neurological disease, may be initiated by hyperexcitability of deep cerebellar nuclei (DCN) secondary to loss of inhibitory input from Purkinje neurons that frequently degenerate in this disease. This mechanism predicts that intrinsic DCN hyperexcitability would cause ataxia in the absence of upstream Purkinje degeneration. We report the generation of a transgenic (Tg) model that supports this mechanism of disease initiation. Small-conductance calcium-activated potassium (SK) channels, regulators of firing frequency, were silenced in the CNS of Tg mice with the dominant-inhibitory construct SK3-1B-GFP. Transgene expression was restricted to the DCN within the cerebellum and was detectable beginning on postnatal day 10, concomitant with the onset of cerebellar ataxia. Neurodegeneration was not evident up to the sixth month of age. Recordings from Tg DCN neurons revealed loss of the apamin-sensitive after-hyperpolarization current (IAHP) and increased spontaneous firing through SK channel suppression, indicative of DCN hyperexcitability. Spike duration and other electrogenic conductance were unaffected. Thus, a purely electrical alteration is sufficient to cause cerebellar ataxia, and SK openers such as the neuroprotective agent riluzole may reduce neuronal hyperexcitability and have therapeutic value. This dominant-inhibitory strategy may help define the in vivo role of SK channels in other neuronal pathways.

SKCa Channels Mediate the Medium but Not the Slow Calcium-activated Afterhyperpolarization in Cortical Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2004  |  Pubmed ID: 15071101

Many neurons, including pyramidal cells of the cortex, express a slow afterhyperpolarization (sAHP) that regulates their firing. Although initial findings suggested that the current underlying the sAHP could be carried through SK(Ca) channels, recent work has uncovered anomalies that are not congruent with this idea. Here, we used overexpression and dominant-negative strategies to assess the involvement of SK(Ca) channels in mediating the current underlying the sAHP in pyramidal cells of the cerebral cortex. Pyramidal cells of layer V exhibit robust AHP currents composed of two kinetically and pharmacologically distinguishable currents known as the medium AHP current (I(mAHP)) and the slow AHP current (I(sAHP)). I(mAHP) is blocked by the SK(Ca) channel blockers apamin and bicuculline, whereas I(sAHP) is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SK(Ca) channels, we overexpressed K(Ca)2.1 (SK1) and K(Ca)2.2 (SK2), the predominant SK(Ca) subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of K(Ca)2.1 and K(Ca)2.2 resulted in a fourfold to fivefold increase in the amplitude of I(mAHP) but had no detectable effect on I(sAHP). As an additional test, we examined I(sAHP) in a transgenic mouse expressing a truncated SK(Ca) subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SK(Ca)-IK(Ca) channels. Expression of SK3-1B profoundly inhibited I(mAHP) but again had no discernable effect on I(sAHP). These results are inconsistent with the proposal that SK(Ca) channels mediate I(sAHP) in pyramidal cells and indicate that a different potassium channel mediates this current.

International Union of Pharmacology. LII. Nomenclature and Molecular Relationships of Calcium-activated Potassium Channels

Pharmacological Reviews. Dec, 2005  |  Pubmed ID: 16382103

International Union of Pharmacology. LIII. Nomenclature and Molecular Relationships of Voltage-gated Potassium Channels

Pharmacological Reviews. Dec, 2005  |  Pubmed ID: 16382104

Potassium Channels, Memory T Cells, and Multiple Sclerosis

The Neuroscientist : a Review Journal Bringing Neurobiology, Neurology and Psychiatry. Dec, 2005  |  Pubmed ID: 16282596

Multiple sclerosis is a chronic inflammatory autoimmune disease of the central nervous system characterized by demyelination and axonal damage that result in disabling neurological deficits. Here the authors explain the rationale for the use of inhibitors of the Kv1.3 K+ channel in immune cells as a therapy for multiple sclerosis and other autoimmune disorders.

Targeting Effector Memory T Cells with a Selective Peptide Inhibitor of Kv1.3 Channels for Therapy of Autoimmune Diseases

Molecular Pharmacology. Apr, 2005  |  Pubmed ID: 15665253

The voltage-gated Kv1.3 K(+) channel is a novel target for immunomodulation of autoreactive effector memory T (T(EM)) cells that play a major role in the pathogenesis of autoimmune diseases. We describe the characterization of the novel peptide ShK(L5) that contains l-phosphotyrosine linked via a nine-atom hydrophilic linker to the N terminus of the ShK peptide from the sea anemone Stichodactyla helianthus. ShK(L5) is a highly specific Kv1.3 blocker that exhibits 100-fold selectivity for Kv1.3 (K(d) = 69 pM) over Kv1.1 and greater than 250-fold selectivity over all other channels tested. ShK(L5) suppresses the proliferation of human and rat T(EM) cells and inhibits interleukin-2 production at picomolar concentrations. Naive and central memory human T cells are initially 60-fold less sensitive than T(EM) cells to ShK(L5) and then become resistant to the peptide during activation by up-regulating the calcium-activated K(Ca)3.1 channel. ShK(L5) does not exhibit in vitro cytotoxicity on mammalian cell lines and is negative in the Ames test. It is stable in plasma and when administered once daily by subcutaneous injection (10 mug/kg) attains "steady state" blood levels of approximately 300 pM. This regimen does not cause cardiac toxicity assessed by continuous EKG monitoring and does not alter clinical chemistry and hematological parameters after 2-week therapy. ShK(L5) prevents and treats experimental autoimmune encephalomyelitis and suppresses delayed type hypersensitivity in rats. ShK(L5) might prove useful for therapy of autoimmune disorders.

The Voltage-gated Potassium Channel Kv1.3 is Highly Expressed on Inflammatory Infiltrates in Multiple Sclerosis Brain

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2005  |  Pubmed ID: 16043714

Multiple Sclerosis (MS) is characterized by central nervous system perivenular and parenchymal mononuclear cell infiltrates consisting of activated T cells and macrophages. We recently demonstrated that elevated expression of the voltage-gated potassium channel, Kv1.3, is a functional marker of activated effector memory T (T(EM)) cells in experimental allergic encephalomyelitis and in myelin-specific T cells derived from the peripheral blood of patients with MS. Herein, we show that Kv1.3 is highly expressed in postmortem MS brain inflammatory infiltrates. The expression pattern revealed not only Kv1.3(+) T cells in the perivenular infiltrate but also high expression in the parenchyma of demyelinated MS lesions and both normal appearing gray and white matter. These cells were uniformly chemokine receptor 7 negative (CCR7(-)), consistent with an effector memory phenotype. Using double-labeling immunohistochemistry and confocal microscopy, we demonstrated colocalization of Kv1.3 with CD3, CD4, CD8, and some CD68 cells. The expression patterns mirrored in vitro experiments showing polarization of Kv1.3 to the immunological synapse. Kv1.3 was expressed in low to moderate levels on CCR7(+) central memory T cells from cerebrospinal fluid, but, when these cells were stimulated in vitro, they rapidly became Kv1.3(high)/CCR7(-) T(EM), suggesting that a subset of cerebrospinal fluid cells existed in a primed state ready to become T(EM). These studies provide further rationale for the use of specific Kv1.3 antagonists in MS.

Kv1.3 Channels Are a Therapeutic Target for T Cell-mediated Autoimmune Diseases

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2006  |  Pubmed ID: 17088564

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2, SAP97, ZIP, p56(lck), and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

The D-diastereomer of ShK Toxin Selectively Blocks Voltage-gated K+ Channels and Inhibits T Lymphocyte Proliferation

The Journal of Biological Chemistry. Jan, 2008  |  Pubmed ID: 17984097

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T(EM)); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K(d) 36 nm (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K(+) channels tested. D-allo-ShK inhibited human T(EM) cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-allo-ShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K(+) channel studies; all-d analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.

The Intermediate-conductance Calcium-activated Potassium Channel KCa3.1 Contributes to Atherogenesis in Mice and Humans

The Journal of Clinical Investigation. Sep, 2008  |  Pubmed ID: 18688283

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Imaging of Effector Memory T Cells During a Delayed-type Hypersensitivity Reaction and Suppression by Kv1.3 Channel Block

Immunity. Oct, 2008  |  Pubmed ID: 18835197

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Kv1.3 Potassium Channels As a Therapeutic Target in Multiple Sclerosis

Expert Opinion on Therapeutic Targets. Aug, 2009  |  Pubmed ID: 19538097

We discuss the potential use of inhibitors of Kv1.3 potassium channels in T lymphocytes as therapeutics for multiple sclerosis. Current treatment strategies target the immune system in a non-selective manner. The resulting general immunosuppression, toxic side-effects and increased risk of opportunistic infections create the need for more selective therapeutics. Autoreactive effector-memory T (T(EM)) cells, considered to be major mediators of autoimmunity, express large numbers of Kv1.3 channels. Selective blockers of Kv1.3 inhibit calcium signaling, cytokine production and proliferation of T(EM) cells in vitro, and T(EM) cell-motility in vivo. Kv1.3 blockers ameliorate disease in animal models of multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus and contact dermatitis without compromising the protective immune response to acute infections. Kv1.3 blockers have a good safety profile in rodents and primates.

The Functional Network of Ion Channels in T Lymphocytes

Immunological Reviews. Sep, 2009  |  Pubmed ID: 19754890

For more than 25 years, it has been widely appreciated that Ca2+ influx is essential to trigger T-lymphocyte activation. Patch clamp analysis, molecular identification, and functional studies using blockers and genetic manipulation have shown that a unique contingent of ion channels orchestrates the initiation, intensity, and duration of the Ca2+ signal. Five distinct types of ion channels--Kv1.3, KCa3.1, Orai1+ stromal interacting molecule 1 (STIM1) [Ca2+-release activating Ca2+ (CRAC) channel], TRPM7, and Cl(swell)--comprise a network that performs functions vital for ongoing cellular homeostasis and for T-cell activation, offering potential targets for immunomodulation. Most recently, the roles of STIM1 and Orai1 have been revealed in triggering and forming the CRAC channel following T-cell receptor engagement. Kv1.3, KCa3.1, STIM1, and Orai1 have been found to cluster at the immunological synapse following contact with an antigen-presenting cell; we discuss how channels at the synapse might function to modulate local signaling. Immuno-imaging approaches are beginning to shed light on ion channel function in vivo. Importantly, the expression pattern of Ca2+ and K+ channels and hence the functional network can adapt depending upon the state of differentiation and activation, and this allows for different stages of an immune response to be targeted specifically.

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

The Journal of Biological Chemistry. Mar, 2010  |  Pubmed ID: 19965868

Peptide toxins found in a wide array of venoms block K(+) channels, causing profound physiological and pathological effects. Here we describe the first functional K(+) channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23(TxD)) that is evolutionarily related to peptide toxins from sea anemones. MMP23(TxD) shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K(+) channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K(+) channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K(+) channels by co-localizing with and trapping MMP23(TxD)-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.

Genetics and the Environment Converge to Dysregulate N-glycosylation in Multiple Sclerosis

Nature Communications. , 2011  |  Pubmed ID: 21629267

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice, N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis, spontaneous inflammatory demyelination and neurodegeneration, the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3), including genetic variants in interleukin-7 receptor-α (IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3), optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation.

18F-Fallypride PET of Pancreatic Islets: in Vitro and in Vivo Rodent Studies

Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine. Jul, 2011  |  Pubmed ID: 21680697

Islet cell loss in the pancreas results in diabetes. A noninvasive method that measures islet cell loss and also tracks the fate of transplanted islets would facilitate the development of novel therapeutics and improve the management of diabetes. We describe a novel dopamine D(2)/D(3) receptor (D(2)/D(3)R)-based PET method to study islet cells in the rat pancreas and in islet cell transplantation.

Therapeutic Potential of Peptide Toxins That Target Ion Channels

Inflammation & Allergy Drug Targets. Oct, 2011  |  Pubmed ID: 21745182

Traditional healthcare systems in China, India, Greece and the Middle East have for centuries exploited venomous creatures as a resource for medicines. This review focuses on one class of pharmacologically active compounds from venom, namely peptide toxins that target ion channels. We highlight their therapeutic potential and the specific channels they target. The field of therapeutic application is vast, including pain, inflammation, cancer, neurological disorders, cardioprotection, and autoimmune diseases. One of these peptides is in clinical use, and many others are in various stages of pre-clinical and clinical development.

Development of a Sea Anemone Toxin As an Immunomodulator for Therapy of Autoimmune Diseases

Toxicon : Official Journal of the International Society on Toxinology. Aug, 2011  |  Pubmed ID: 21867724

Electrophysiological and pharmacological studies coupled with molecular identification have revealed a unique network of ion channels-Kv1.3, KCa3.1, CRAC (Orai1 + Stim1), TRPM7, Cl(swell)-in lymphocytes that initiates and maintains the calcium signaling cascade required for activation. The expression pattern of these channels changes during lymphocyte activation and differentiation, allowing the functional network to adapt during an immune response. The Kv1.3 channel is of interest because it plays a critical role in subsets of T and B lymphocytes implicated in autoimmune disorders. The ShK toxin from the sea anemone Stichodactyla helianthus is a potent blocker of Kv1.3. ShK-186, a synthetic analog of ShK, is being developed as a therapeutic for autoimmune diseases, and is scheduled to begin first-in-man phase-1 trials in 2011. This review describes the journey that has led to the development of ShK-186.