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 (K(v)1) K+ channels have been purified to homogeneity from venom of the scorpion Centruroides limbatus, The complete primary amino acid sequence of the major compon ent, hongotoxin-1 (HgTX1), has been determined and confirmed after exp ression of the peptide in Escherichia coil, HgTX1 inhibits I-125-marga toxin binding to rat brain membranes as well as depolarization-induced Rb-86(+) flux through homotetrameric K(v)1.1, K(v)1.2, and K(v)1.3 ch annels stably transfected in HEK-293 cells, but it displays much lower affinity for K(v)1.6 channels, A HgTX1, double mutant (HgTX1-A19Y/Y37 F) was constructed to allow high specific activity iodination of the p eptide, HgTX1-A19Y/Y37F and monoiodinated HgTX1-A19Y/Y37F are equally potent in inhibiting I-125-margatoxin binding to rat brain membranes a s HgTX1 (IC50 values similar to 0.3 pM), I-125-HgTX1-A19Y/Y37F binds w ith subpicomolar affinities to membranes derived from HEK-293 cells ex pressing homotetrameric K(v)1.1, K(v)1.2, and K(v)1.3 channels and to rat brain membranes (K-d values 0.1-0.25 phl, respectively) but with l ower affinity to K(v)1.6 channels (K-d 9.6 PM), and it does not intera ct with either K(v)1.4 or K(v)1.5 channels, Several subpopulations of native K(v)1 subunit oligomers that contribute to the rat brain HgTX1 receptor have been deduced by immunoprecipitation experiments using an tibodies specific for K(v)1 subunits, HgTX1, represents a novel and us eful tool with which to investigate subclasses of voltage-gated K+ cha nnels and K(v)1 subunit assembly in different tissues.