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

Toshio Yamagishi, Wei Xiong, Andre Kondratiev, Patricio Vélez, Ailsa Méndez-Fitzwilliam, Jeffrey R. Balser, Eduardo Marbán, Gordon F. Tomaselli

Research output: Contribution to journalArticle

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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 (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 note-worthy 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.

Original languageEnglish (US)
Pages (from-to)861-871
Number of pages11
JournalMolecular Pharmacology
Volume76
Issue number4
DOIs
StatePublished - Oct 1 2009
Externally publishedYes

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Sodium Channels
Local Anesthetics
Cysteine
S 6
Mutation
Site-Directed Mutagenesis
Skeletal Muscle
QX-314

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine

Cite this

Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding. / Yamagishi, Toshio; Xiong, Wei; Kondratiev, Andre; Vélez, Patricio; Méndez-Fitzwilliam, Ailsa; Balser, Jeffrey R.; Marbán, Eduardo; Tomaselli, Gordon F.

In: Molecular Pharmacology, Vol. 76, No. 4, 01.10.2009, p. 861-871.

Research output: Contribution to journalArticle

Yamagishi, T, Xiong, W, Kondratiev, A, Vélez, P, Méndez-Fitzwilliam, A, Balser, JR, Marbán, E & Tomaselli, GF 2009, 'Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding', Molecular Pharmacology, vol. 76, no. 4, pp. 861-871. https://doi.org/10.1124/mol.109.055863
Yamagishi T, Xiong W, Kondratiev A, Vélez P, Méndez-Fitzwilliam A, Balser JR et al. Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding. Molecular Pharmacology. 2009 Oct 1;76(4):861-871. https://doi.org/10.1124/mol.109.055863
Yamagishi, Toshio ; Xiong, Wei ; Kondratiev, Andre ; Vélez, Patricio ; Méndez-Fitzwilliam, Ailsa ; Balser, Jeffrey R. ; Marbán, Eduardo ; Tomaselli, Gordon F. / Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding. In: Molecular Pharmacology. 2009 ; Vol. 76, No. 4. pp. 861-871.
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