title: Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding.
abstract: The pore of the Na+ 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 N-terminal 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.
authors.affiliation: Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Toshio
authors.initials: T
authors.lastname: Yamagishi
authors.forename: Wei
authors.initials: W
authors.lastname: Xiong
authors.forename: Andre
authors.initials: A
authors.lastname: Kondratiev
authors.forename: Patricio
authors.initials: P
authors.lastname: Vélez
authors.forename: Ailsa
authors.initials: A
authors.lastname: Méndez-Fitzwilliam
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Balser
authors.forename: Eduardo
authors.initials: E
authors.lastname: Marbán
authors.forename: Gordon F
authors.initials: GF
authors.lastname: Tomaselli
mesh_terms: D000779:Anesthetics, Local / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D015640:Ion Channel Gating; D007700:Kinetics; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000779:Anesthetics, Local; D009124:Muscle Proteins; C466180:Scn4a protein, rat; D015222:Sodium Channels