LYSINE POINT MUTATIONS IN NA-S6 REDUCE INACTIVATED CHANNEL BLOCK BY LOCAL-ANESTHETICS( CHANNEL D4)

Voltage-gated Na+ channels are a primary target for local anesthetics (LAs). Open or inactivated Na+ channels usually have a severalfold hig her affinity for LAs than do resting channels. Hille's modulated recep tor hypothesis attributed the changes in LA affinity to state-dependen t alterations in the conformation of the LA receptor. We expressed wil d-type and mutant rat skeletal muscle (mu 1) Na+ channels in human emb ryonic kidney cells to investigate the state-dependent modulation of L A receptor affinity. As an alternative approach to using alanine for p oint mutation, we substituted lysine (a hydrophilic residue) for nativ e residues in the putative LA receptor located in D4-S6 of the mu 1 Na + channel. Lysine mutation at Y1586 did not alter resting channel affi nity for cocaine but did reduce resting affinity at F1579K and N1584K by 2- and 3-fold, respectively. Compared with mu 1, 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-pi electron interacti ons between particular moieties on the LA and lysine. Surprisingly, ly sine substitution at these residues allowed the channels to undergo st eady state fast inactivation yet reduced inactivated channel block by cocaine by up to 27-fold and reduced the benzocaine-induced leftward s hift in the h(infinity) curve by up to 22 mV. Our data suggest that tr ansitions 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.