Use of Lqh2 mutants (produced at TAU) and rNav1.2a mutants (produced at the US side) for identifying receptor site-3: Based on the fact that binding of scorpion alpha-toxins is voltage-dependent, which suggests toxin binding at the mobile voltage-sensing region, we analyzed which of the toxin bioactive domains (Core-domain or NC-domain) interacts with the DIV Gating-module of rNav1.2a. This analysis was based on the assumption that the dissociation of toxin mutants upon depolarization would vary from that of the unmodified toxin should the substitutions affect a site of interaction with the channel Gating-module. Using a series of toxin mutants (mutations at both domains) and two channel mutants that were shown to reduce the sensitivity to scorpion alpha-toxins, and by comparison of depolarization-driven dissociation of Lqh2 derivatives off their binding site at rNav1.2a mutant channels we found that the toxin Core-domain interacts with the Gating-module of DIV. Details of the experiments and results appear in Guret al (2011). Mapping receptor site 3 at Nav1.2a by extensive channel mutagenesis (Seattle): Since previous studies with photoaffinity labeling and antibody mapping implicated domains I and IV in scorpion alpha-toxin binding, Nav1.2 channel mutants containing substitutions at these extracellular regions were expressed and tested for receptor function by whole-cell voltage clamp. Of a large number of channel mutants, T1560A, F1610A, and E1613A in domain IV had ~5.9-, ~10.7-, and ~3.9-fold lower affinities for the scorpion toxin Lqh2, respectively, and mutant E1613R had 73-fold lower affinity. Toxin dissociation was accelerated by depolarization for both wild-type and mutants, and the rates of dissociation were also increased by mutations T1560A, F1610A and E1613A. In contrast, association rates for these three mutant channels at negative membrane potentials were not significantly changed and were not voltage-dependent. These results indicated that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also showed a ~3.4-fold lower affinity for Lqh2, indicating that this extracellular loop may form a secondary component of the toxin binding site. Analysis with the Rosetta-Membrane algorithm revealed a three-dimensional model of Lqh2 binding to the voltage sensor in a resting state. In this model, amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV that are important for toxin binding interact with amino acid residues on two faces of the wedge-shaped Lqh2 molecule that are important for toxin action. The conserved gating charges in the S4 transmembrane segment are in an inward position and likely form ion pairs with negatively charged amino acid residues in the S2 and S3 segments (Wang et al 2011; Gurevitz 2012; Gurevitzet al 2013).
Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels.
