Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin

beta -Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depo larizing prepulse to fully activate the channels. Their receptor site inclu des the S3-S4 loop at the extracellular end of the S4 voltage sensor in dom ain II of the alpha subunit. Here, we probe the role of gating charges in t he IIS4 segment in beta -scorpion toxin action by mutagenesis and functiona l analysis of the resulting mutant sodium channels. Neutralization of the p ositively charged amino acid residues in the IIS4 segment by mutation to gl utamine shifts the voltage dependence of channel activation to more positiv e membrane potentials and reduces the steepness of voltage-dependent gating , which is consistent with the presumed role of these residues as gating ch arges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850Q, R853Q, and R853C markedl y enhances beta -scorpion toxin action, whereas mutations R856Q K859Q and K 862Q have no effect. In contrast to wild-type, the beta -scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of muta nts R853Q and R853C without a depolarizing prepulse at holding potentials f rom -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethios ulfonate causes a positive shift of the voltage dependence of activation an d restores the requirement for a depolarizing prepulse for Css IV action. E nhancement of sodium channel activation by Css IV causes large tail current s upon repolarization, indicating slowed deactivation of the IIS4 voltage s ensor by the bound toxin. Our results are consistent with a voltage-sensor- trapping model in which the P-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhanc ing subsequent channel activation. Evidently, neutralization of R850 and R8 53 removes kinetic barriers to binding of the IIS4 segment by Css IV, and t hereby enhances toxin-induced channel activation.