Abstract
Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) 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 CssIV(E15A). 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 Na(V) channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.
Highlights
Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. -Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of NaV channels
These results led us to hypothesize that the single receptor site for the -scorpion toxin CssIV in Nav channels (8) is formed by IIS1-S2, IIS3-S4, and IIISS2-S6 extracellular loops
The Receptor Site for -Scorpion Toxins Includes Amino Acid Residues in the IIISS2-S6 Loop—In our previous experiments, we mapped the molecular determinants of voltage-sensor trapping in domain II (8, 9, 11)
Summary
CssIV, IV of Centruroides suffusus suffusus; IVST, voltage-sensor trapping current. Receptor Site for -Scorpion Toxins on NaV Channels to open the central ion conduction pore. Three of them markedly decreased voltage-sensor trapping, whereas two increased voltage-sensor trapping These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with bound toxin. Our data show that IIS3-S4 plays a primary role, whereas IIS1-S2 and IIISS2-S6 play important secondary roles, in determining binding affinity and efficacy at a single -scorpion toxin receptor site. They indicate that the SS2-S6 loop in domain III is in close proximity to the voltage-sensing module of domain II in mammalian NaV channels, as suggested by the structure of bacterial NaV channels (5)
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