Subunit Composition of Brain Voltage-gated Potassium Channels Determined by Hongotoxin-1, a Novel Peptide Derived fromCentruroides limbatus Venom*
Abstract
Five novel peptidyl inhibitors ofShaker-type (Kv1) K+ channels have been purified to homogeneity from venom of the scorpionCentruroides limbatus. The complete primary amino acid sequence of the major component, hongotoxin-1 (HgTX1), has been determined and confirmed after expression of the peptide inEscherichia coli. HgTX1 inhibits125I-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 inhibiting125I-margatoxin binding to rat brain membranes as HgTX1 (IC50 values ∼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 (K d values 0.1–0.25 pm, respectively) but with lower affinity to Kv1.6 channels (K d 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.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A preliminary report of this work has been presented in abstract form (6).
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↵¶ Supported by Austrian Research Foundation Grants S6611-MED and P-11187-MED, Austrian National Bank Foundation Grant 6239, and the European Union BIOMED 2 program Grant BMH4-CT96–2118. To whom correspondence should be addressed. Tel.: 43-512-507-3156; Fax: 43-512-507-2858; E-mail: hans.g.knaus{at}uibk.ac.at.
- Received October 16, 1997.
- Revision received November 15, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
