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Jeffrey Balser
Vice Chancellor for Health Affairs

Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding.

Yamagishi T, Xiong W, Kondratiev A, Vélez P, Méndez-Fitzwilliam A, Balser JR, Marbán E, Tomaselli GF
Mol Pharmacol. 2009 76 (4): 861-71

PMID: 19620257 · PMCID: PMC2769053 · DOI:10.1124/mol.109.055863

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.

MeSH Terms (9)

Anesthetics, Local Animals Ion Channel Gating Kinetics Muscle, Skeletal Muscle Proteins Mutagenesis, Site-Directed Rats Sodium Channels

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