Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3.

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.

Subunit composition of brain voltage-gated potassium channels determined by hongotoxin-1, a novel peptide derived from Centruroides limbatus venom.

Five novel peptidyl inhibitors of Shaker-type (Kv1) K+ channels have been purified to homogeneity from venom of the scorpion Centruroides limbatus. The complete primary amino acid sequence of the major component, hongotoxin-1 (HgTX1), has been determined and confirmed after expression of the peptide in Escherichia coli. HgTX1 inhibits 125I-margatoxin binding to rat brain membranes as well as depolarization-induced 86Rb+ flux through homotetrameric Kv1.1, Kv1. 2, and Kv1.3 channels stably transfected in HEK-293 cells, but it displays much lower affinity for Kv1.6 channels. A HgTX1 double mutant (HgTX1-A19Y/Y37F) was constructed to allow high specific activity iodination of the peptide. HgTX1-A19Y/Y37F and monoiodinated HgTX1-A19Y/Y37F are equally potent in inhibiting 125I-margatoxin binding to rat brain membranes as HgTX1 (IC50 values approximately 0.3 pM). 125I-HgTX1-A19Y/Y37F binds with subpicomolar affinities to membranes derived from HEK-293 cells expressing homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels and to rat brain membranes (Kd values 0.1-0.25 pM, respectively) but with lower affinity to Kv1.6 channels (Kd 9.6 pM), and it does not interact with either Kv1.4 or Kv1.5 channels. Several subpopulations of native Kv1 subunit oligomers that contribute to the rat brain HgTX1 receptor have been deduced by immunoprecipitation experiments using antibodies specific for Kv1 subunits. HgTX1 represents a novel and useful tool with which to investigate subclasses of voltage-gated K+ channels and Kv1 subunit assembly in different tissues.

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

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.

Chemical synthesis and structure-function studies of margatoxin, a potent inhibitor of voltage-dependent potassium channel in human T lymphocytes.

The 39 amino acid peptide, margatoxin (MgTX), a potent inhibitor of the voltage-activated potassium channel (Kv 1.3) in human T lymphocytes, was synthesized by a solid phase technique. Formation of the disulfide bridges was rapid at pH 8.2. The final product was purified to homogeneity and was physically and biologically indistinguishable from the toxin prepared biosynthetically. The disulfide bridge pairing was similar to that found previously for the related toxin-charybdotoxin (3): from Cys7 to Cys29, from tested for inhibition of 125I margatoxin binding to voltage-activated potassium channels. The results indicate that the three C-terminal residues of MgTX are important for the efficient toxin binding to Kv1.3.