Cysteine Mutagenesis to Probe Site-Specific Interactions in the Voltage Sensor Module of hNaV1.4

Abstract

Cysteine scanning mutagenesis was used to probe interactions of positively charged S4 residues with S1-S3 countercharges in domains I, II and IV of the human skeletal muscle sodium channel hNaV1.4. Single cysteine substitution was used to assess the effect of charge neutralization of residues in the voltage sensor module of these domains. Activation and fast inactivation were most affected by cysteine substitution in domains II and IV respectively, while both of these state transitions were affected by like mutations in domain I. Double cysteine mutations in the VSM of each of these domains did not result in disulfide locking. To probe for specific interactions using these mutations, we employed a metal ion bridge approach using cadmium, and assessed its effect on equilibrium parameters of activation and fast inactivation, and peak current amplitude. In domain I, double cysteine mutations of R219C or R222C with the gating charge transfer glutamate (E171C) significantly affected fast inactivation, with activation midpoint significantly depolarized by several DIS4 cysteine / countercharge mutations incorporating the polar residue substitutions N144C and S134C. Domain II double cysteine mutations R672C / E598C and R675C / E598C also significantly depolarized the activation midpoint with cadmium addition. In domain IV, double cysteine mutations R1448C / E1373C and R1448C / N1389C responded to cadmium with slowed entry into fast inactivation, a depolarized inactivation midpoint and a decreased slope. Our results show that certain domain specific functions of the voltage sensor modules in hNaV1.4 occur with pairwise interactions of S4 positive and S1-S3 countercharge residues. This work was supported by NIH R15NS093579-01A1 to JRG and NIH P20GM103408 (INBRE).