Abstract

Voltage-gated Na⁺ channels are a primary target for local anesthetics (LAs). Open or inactivated Na⁺ channels usually have a severalfold higher affinity for LAs than do resting channels. Hille’s modulated receptor hypothesis attributed the changes in LA affinity to state-dependent alterations in the conformation of the LA receptor. We expressed wild-type and mutant rat skeletal muscle (μ1) Na⁺ channels in human embryonic kidney cells to investigate the state-dependent modulation of LA receptor affinity. As an alternative approach to using alanine for point mutation, we substituted lysine (a hydrophilic residue) for native residues in the putative LA receptor located in D4-S6 of the μ1 Na⁺channel. Lysine mutation at Y1586 did not alter resting channel affinity for cocaine but did reduce resting affinity at F1579K and N1584K by 2- and 3-fold, respectively. Compared with μ1, resting benzocaine block did not change at F1579K, decreased at N1584K, and increased at Y1586K. These effects on resting block could largely be accounted for by either steric/charge interference or cation-π electron interactions between particular moieties on the LA and lysine. Surprisingly, lysine substitution at these residues allowed the channels to undergo steady state fast inactivation yet reduced inactivated channel block by cocaine by up to 27-fold and reduced the benzocaine-induced leftward shift in the h_∞ curve by up to 22 mV. Our data suggest that transitions in channel state indeed invoke conformational changes in the LA receptor and that lysine mutations in the LA receptor region alter such conformational changes during the transition to the inactivated state.