The pore of the Na ϩ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the Nterminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na V 1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5-and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.Sodium channels are transmembrane proteins that rapidly conduct electrical impulses throughout nerve and muscle. The channels have a modular structure, with distinct regions mediating gating and permeation. The pore-lining (P) segments, formed by the S5-S6 linkers, are the major determinants for selective ion flux, whereas the S4 transmembrane domain and the cytoplasmic III-IV interdomain linker figure prominently in activation and fast inactivation gating, respectively. The structural separation of function is not clearcut; mutations in the P segments are known to alter gating (Tomaselli et al
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