RT Journal Article
SR Electronic
T1 Novel Molecular Determinants in the Pore Region of Sodium Channels Regulate Local Anesthetic Binding
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 861
OP 871
DO 10.1124/mol.109.055863
VO 76
IS 4
A1 Toshio Yamagishi
A1 Wei Xiong
A1 Andre Kondratiev
A1 Patricio Vélez
A1 Ailsa Méndez-Fitzwilliam
A1 Jeffrey R. Balser
A1 Eduardo Marbán
A1 Gordon F. Tomaselli
YR 2009
UL http://molpharm.aspetjournals.org/content/76/4/861.abstract
AB 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 (NaV1.4) 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. © 2009 The American Society for Pharmacology and Experimental Therapeutics