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 component, hongotoxin-1 (HgTX1), has been determined and confirmed after expression of the peptide in Escherichia coli, HgTX1 inhibits I-125-margatoxin 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 channels stably transfected in HEK-293 cells, but it displays much lower affinity for K(v)1.6 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 I-125-margatoxin binding to rat brain membranes as HgTX1 (IC50 values similar to 0.3 pM), I-125-HgTX1-A19Y/Y37F binds with subpicomolar affinities to membranes derived from HEK-293 cells expressing 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 lower affinity to K(v)1.6 channels (K-d 9.6 PM), and it does not interact 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 antibodies specific for K(v)1 subunits, HgTX1, represents a novel and useful tool with which to investigate subclasses of voltage-gated K+ channels and K(v)1 subunit assembly in different tissues.