Important Role of Asparagines in Coupling the Pore and Votage-Sensor Domain in Voltage-Gated Sodium Channels

Voltage-gated sodium (NaV) channels contain an α-subunit incorporating the channel's pore and gating machinery composed of four homologous domains (DI-DIV), with a pore domain formed by the S5 and S6 segments and a voltage-sensor domain formed by the S1-S4 segments. During a membrane depolarization movement, the S4s in the voltage-sensor domains exert downstream effects on the S6 segments to control ionic conductance through the pore domain. We used lidocaine, a local anesthetic and antiarrhythmic drug, to probe the role of conserved Asn residues in the S6s of DIII and DIV in NaV1.5 and NaV1.4. Previous studies have shown that lidocaine binding to the pore domain causes a decrease in the maximum gating (Qmax) charge of ∼38%, and three-fourths of this decrease results from the complete stabilization of DIII-S4 (contributing a 30% reduction in Qmax) and one-fourth is due to partial stabilization of DIV-S4 (a reduction of 8-10%). Even though substitutions for the Asn in DIV-S6 in NaV1.5, N1764A and N1764C, produce little ionic current in transfected mammalian cells, they both express robust gating currents. Anthopleurin-A toxin, which inhibits movement of DIV-S4, still reduced Qmax by nearly 30%, a value similar to that observed in wild-type channels, in both N1764A and N1764C. By applying lidocaine and measuring the gating currents, we demonstrated that Asn residues in the S6s of DIII and DIV are important for coupling their pore domains to their voltage-sensor domains, and that Ala and Cys substitutions for Asn in both S6s result in uncoupling of the pore domains from their voltage-sensor domains. Similar observations were made for NaV1.4, although substitutions for Asn in DIII-S6 showed somewhat less uncoupling.