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Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels*

Joel Z. Zhang,V. Yarov‐Yarovoy,T. Scheuer,Izhar Karbat,L. Cohen,D. Gordon,M. Gurevitz,W. Catterall

Published 2012 in Journal of Biological Chemistry

ABSTRACT

Background: β-Scorpion toxins enhance activation of voltage-gated sodium (NaV) channels. Results: Four amino acid residues in the IIISS2-S6 extracellular loop contribute to toxin binding and efficacy. Conclusion: The pore module of domain III and the voltage-sensing module of domain II form the receptor site. Significance: Scorpion toxins make a three-point interaction with NaV channels and alter voltage sensor function. Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of NaV channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIVE15A. Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for β-scorpion toxins on NaV channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on NaV channels acts synergistically to modify channel gating and paralyze prey.

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