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

Mol Pharmacol. 2009 Oct;76(4):861-71. doi: 10.1124/mol.109.055863. Epub 2009 Jul 20.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Anesthetics, Local / metabolism
  • Anesthetics, Local / pharmacology*
  • Animals
  • Ion Channel Gating
  • Kinetics
  • Muscle Proteins / chemistry
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism*
  • Muscle, Skeletal / drug effects*
  • Muscle, Skeletal / metabolism
  • Mutagenesis, Site-Directed
  • Rats
  • Sodium Channels / chemistry
  • Sodium Channels / genetics
  • Sodium Channels / metabolism*

Substances

  • Anesthetics, Local
  • Muscle Proteins
  • Scn4a protein, rat
  • Sodium Channels