Venomous animals have evolved a vast array of peptide toxins for prey capture and defence. These peptides are directed against a wide variety of pharmacological targets, making them an invaluable source of ligands for studying the properties of these targets in different experimental paradigms. A number of these peptides have been used in vivo for proof-of-concept studies, with several having undergone preclinical or clinical development for the treatment of pain, diabetes, multiple sclerosis and cardiovascular diseases. Here we survey the pharmacology of venom peptides and assess their therapeutic prospects.

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Therapeutic potential of venom peptides

The human genome contains 40 voltage-gated potassium channels (KV) which are involved in diverse physiological processes ranging from repolarization of neuronal or cardiac action potentials, over regulating calcium signaling and cell volume, to driving cellular proliferation and migration. KV channels offer tremendous opportunities for the development of new drugs for cancer, autoimmune diseases and metabolic, neurological and cardiovascular disorders. This review first discusses pharmacological strategies for targeting KV channels with venom peptides, antibodies and small molecules and then highlights recent progress in the preclinical and clinical development of drugs targeting KV1.x, KV7.x (KCNQ), KV10.1 (EAG1) and KV11.1 (hERG) channels.

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Voltage-gated potassium channels as therapeutic targets

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.

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The functional network of ion channels in T lymphocytes

An assembly for loading a collapsible embolic protection filter 1 into a catheter 2 , comprises a catheter 2 defining a reception space at a distal end of the catheter 2 for receiving a collapsed embolic protection filter 1 ; and a separate removable pushing device 8 for delivering the embolic protection filter 1 into the reception space. The pushing device 8 comprises an elongate stem 71 with a proximal stop 72 for engagement with the filter 1 . A separate loading device 7 to collapse the embolic protection filter 1 is also provided. The loading device 7 defines an inlet end and an outlet end, the outlet end being configured for co-operative alignment with the reception space.

Embolic protection system

Over the past decade, a variety of ion channels have been identified and characterized in lymphocytes by use of the patch-clamp technique. This review discusses biophysical and regulatory aspects of lymphocyte potassium and calcium channels with the aim of understanding the role of these channels in lymphocyte functions. Lymphocytes express both voltage-dependent potassium [K(V)] channels and calcium-activated potassium [K(Ca)] channels, and each is upregulated as cells progress toward division following mitogenic stimulation. The genes encoding two K(V) channels, Kv1.3 (type n) and Kv3.1 (type l), have been cloned. Mutational analysis is revealing functionally important regions of these channel proteins. Exogenous expression studies and the use of highly specific channel blockers have helped to establish the roles of type n K(V) channels in sustaining the resting membrane potential, in regulating cell volume, and in enabling lymphocyte activation. Blockade of K(V) and K(Ca) channels effectively inhibits the antigen-driven activation of lymphocytes, probably by inducing membrane depolarization and thereby diminishing calcium influx. A prolonged rise in intracellular calcium ([Ca2+]i) is a required signal for lymphocyte activation by antigen or mitogens. Single-cell fluorescence measurements have revealed underlying [Ca2+]i oscillations that are linked closely to the opening and closing of Ca2+ and K+ channels. Sustained Ca2+ signaling and oscillations depend absolutely on plasma-membrane Ca2+ channels that are activated by the depletion of intracellular calcium stores. Under physiological conditions these channels open as a consequence of store depletion induced by inositol 1,4,5-trisphosphate (IP3), but they can also be activated experimentally by several agents that empty the stores without generating IP3, such as the microsomal Ca(2+)-ATPase inhibitor thapsigargin. The intricate causal relationships among ion channels, membrane potential, [Ca2+]i, and lymphokine gene expression can now be pursued at the single-cell level with patch-clamp recording, calcium-dependent dyes, reporter genes, and fluorescence video techniques. These approaches will help to clarify the essential roles of ion channels in the molecular pathways subserving activation and other lymphocyte behaviors.

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Potassium and calcium channels in lymphocytes.

Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels.

The solution structure of the 39-residue peptide margatoxin, a scorpion toxin that selectively blocks the voltage-gated potassium-channel Kv1.3, has been determined by NMR spectroscopy. The toxin was isotopically labeled with 13C and 15N and studied using two-dimensional homonuclear and three- and four-dimensional heteronuclear NMR spectroscopy. The final structure was determined using 501 constraints, comprising 422 NOE constraints, 60 dihedral angle constraints, 9 disulfide constraints, and 10 hydrogen bond constraints. Structures were initially determined with the program PEGASUS and subsequently refined with X-PLOR. The average rms deviation from a calculated average structure for the backbone atoms of residues 3-38 is 0.40 A. A helix is present from residues 11 to 20 and includes two proline residues at positions 15 and 16. A loop at residues 21-24 leads into a two-strand antiparallel sheet from residues 25 to 38 with a turn at residues 30-33. Residues 3-6 run adjacent to the 33-38 strand but do not form a canonical beta-strand. The two additional residues of margatoxin, relative to the related toxins charybdotoxin and iberiotoxin, insert in a manner that extends the beta-sheet by one residue. Otherwise, the global structure is very similar to that of these two other toxins. The longer sheet may have implications for channel selectivity.

Determination of the Three-Dimensional Structure of Margatoxin by 1H, 13C, 15N Triple-Resonance Nuclear Magnetic Resonance Spectroscopy

Voltage-gated potassium (K(V)) channels play key roles in setting the resting potential and in the activation cascade of human peripheral T lymphocytes. Margatoxin (MgTX), a 39-amino acid peptide from Centruroides margaritatus, is a potent inhibitor of lymphocyte K(V) channels. The binding of monoiodotyrosinyl margatoxin ([125I]MgTX) to plasma membranes prepared from either Jurkat cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, K(V)1.3, has been used to investigate the properties of lymphocyte K(V) channels. These data were compared with [125I]MgTX binding to heterotetrameric K(V) channels in rat brain synaptic plasma membranes [Knaus, H. G., et al. (1995) Biochemistry 34, 13627-13634]. The affinity for [125I]MgTX is 100-200 fM in either Jurkat or CHO/K(V)1.3 membranes, and the receptor density is 20-120 fmol/mg in Jurkat membranes or 1000 fmol/mg in CHO/K(V)1.3 membranes. In contrast to rat brain, [125I]MgTX binding to Jurkat and CHO/K(V)1.3 membranes exhibits an absolute requirement for K+, with no potentiation of binding by Na+. K(V)1.3 was the only K(V)1 series channel present in either CHO/K(V)1.3 or Jurkat plasma membranes as determined by immunoprecipitation of [125I]MgTX binding or by Western blot analyses using sequence-specific antibodies prepared against members of the K(V)1 family. The relative potencies of a series of peptidyl K(V) channel inhibitors was essentially the same for inhibition of [125I]MgTX binding to Jurkat, CHO, or rat brain membranes and for blocking 86Rb+ efflux from the CHO/K(V)1.3 cells, except that alpha-dendrotoxin was more potent at blocking binding to rat brain membranes than in the other assays. The characteristics of [125I]MgTX binding, the antibody profiles, and the effects of the peptidyl K(V) inhibitors all indicate that the [125I]MgTX receptor in Jurkat lymphocytes is comprised of a homomultimer of K(V)1.3, unlike the heteromultimeric arrangement of the receptor in rat brain.

Margatoxin binds to a homomultimer of K(V)1.3 channels in Jurkat cells. Comparison with K(V)1.3 expressed in CHO cells.

A thirty-nine amino acid peptide, Margatoxin (MgTX), is purified to homogeneity from venom of the scorpion Centruroides margaritatus. The gene encoding MgTX is constructed and this gene is expressed in E. coli, to produce recombinant MgTX. MgTX is a potent and selective inhibitor of a voltage-dependent K+ channel present in human lymphocytes. MgTX exhibits immunosuppressant activity with human T-lymphocytes, and is useful as an immunosuppressant, in modeling nonpeptidyl K+ channel blockers, and in establishing biochemical assays based on ligand binding or other protocols with which to screen for other novel modulators of voltage dependent K+ channels in lymphocytes and other tissues including the brain. As an immunosuppressant, MgTX is useful in the treatment of autoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.

Scorpion peptide margatoxin with immunosuppressant activity

A thirty-nine amino acid peptide, Margatoxin (MgTX), is purified to homogeneity from venom of the scorpion Centruroides margaritatus. The gene encoding MgTX is constructed and this gene is expressed in E. coli, to produce recombinant MgTX. MgTX is a potent and selective inhibitor of a voltage-dependent K+ channel present in human lynphocytes. MgTX exhibits immunosuppressant activity with human T-lymphocytes, and is useful as an immunosuppressant, in modeling nonpeptidyl K+ channel blockers, and in establishing biochemical assays based on ligand binding or other protocols with which to screen for other novel modulators of voltage dependent K+ channels in lymphocytes and other tissues including the brain. As an immunosuppressant, MgTX is useful in the treatment ofautoimmune diseases, the prevention of rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.

Scott P. Stevens

Papers

Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels.

Determination of the Three-Dimensional Structure of Margatoxin by 1H, 13C, 15N Triple-Resonance Nuclear Magnetic Resonance Spectroscopy

Margatoxin binds to a homomultimer of K(V)1.3 channels in Jurkat cells. Comparison with K(V)1.3 expressed in CHO cells.

Scorpion peptide margatoxin with immunosuppressant activity

Scorpion peptide with immunosuppressant activity