Resting State of S4 Identified for Each Domain of Nav1.2 using Omega Current Technique

Abstract

During gating transitions the voltage sensor S4 slides through the narrow so-called gating pore. The positively charged amino acids of S4, arginine (R) or lysine (K) sense the transmembranal electric field and promote S4 in or out. If one or more long R or K is replaced by the short neutral glutamine (Q), a leak (omega-) pore is created through which a leak current, the omega current will flow when S4 is at the appropriate position. The occupancy of this leaky position can then be electrically monitored. Rat brain sodium channels Nav1.2 were studied at high expression in X. laevis oocytes with two-electrode voltage clamping at strong hyperpolarization to force S4 into the resting state. Mutant channels with single gaps R1Q, R2Q or R3Q as well as with double gaps RRn,n+1QQ were tested for the presence of omega leaks. We found unambiguous omega currents for double gaps in domain DI (RR12QQ), DII (RR12QQ), DIII (RR23QQ) and DIV (RR12QQ), indicating the resting position of S4 in each domain. These findings are in contrast to skeletal muscle sodium channels Nav1.4 where single gap omega leaks are reported for DII and DIII. However, our single gap mutants in Nav1.2 produced only very small leak currents similar to artifacts sometimes also occurring at wild-type channels at very strong hyperpolarizing pulses and at least ten times smaller than those with double gaps. Based on this study, we currently use our double gap channel constructs as a tool to selectively investigate which S4 of the four domains I-IV are immobilized by inactivation.