Novel Molecular Determinants in the Pore Region of Sodium Channels Regulate Local Anesthetic Binding

Abstract

The pore of the Na ϩ 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 Nterminal 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.Sodium channels are transmembrane proteins that rapidly conduct electrical impulses throughout nerve and muscle. The channels have a modular structure, with distinct regions mediating gating and permeation. The pore-lining (P) segments, formed by the S5-S6 linkers, are the major determinants for selective ion flux, whereas the S4 transmembrane domain and the cytoplasmic III-IV interdomain linker figure prominently in activation and fast inactivation gating, respectively. The structural separation of function is not clearcut; mutations in the P segments are known to alter gating (Tomaselli et al

Mechanism of sodium channel block by local anesthetics, antiarrhythmics, and anticonvulsants

“…cationic and sodium-bound electroneutral ligands in the channel. (A) The triethylammonium group of QX-314 is close to the Na III site, the aromatic ring binds between F 4i15 and Y 4i22 , and the ligand also interacts with Q 1p49 and F 3p49 in agreement with mutational data (Yamagishi et al, 2009). (B) The bulky moiety of quinidine fits the hotspot at the Na III site, whereas the hydroxyl group donates an H bond to 3p48 O=C.…”

“…Mutations in the selectivity filter (the DEKA locus) also affect the action, strongly suggesting electrostatic interactions with the selectivity filter (Sunami et al, 1997). More ligand-sensing residues are found at the P-loops (Tsang et al, 2005;Yamagishi et al, 2009), but the data on roles of these residues are fragmental. Thus, mutational studies unambiguously show the ligand-binding region and suggest candidate residues that contribute to receptors of various ligands, but they do not provide a complete picture of the channel interactions with the ligands.…”

Opposing Effects on Na V 1.2 Function Underlie Differences Between SCN2A Variants Observed in Individuals With Autism Spectrum Disorder or Infantile Seizures

“…S2C–D). Mutation of a homologous residue in rat Na V 1.4 to cysteine had modest effects on channel function (38), suggesting that the observed change in pore hydrophobicity is critical at this residue.…”

A Molecular Switch between the Outer and the Inner Vestibules of the Voltage-gated Na+ Channel

“…Later, the same group demonstrated that the drug-specific kinetics of binding to slow inactivated states by lidocaine and bupivacaine closely matched the kinetics of sulfhydryl modification of a cysteine engineered to the outer vestibule ( 58 ). Yamagishi et al ( 59 ) observed that application of external QX314 (a membrane-impermeant quaternary derivative of lidocaine) did not block rNa V 1.4 channels but nevertheless slowed recovery from internal block by QX314, suggesting an allosteric effect of external QX314 binding on recovery from internal block. In the rNa V 1.2 channel a potential interaction between Trp-1716 in the DIV-P-loop (corresponding to Trp-1531 in rNa V 1.4) and Phe-1764 in the DIV-S6 segment (corresponding to Phe-1579 in rNa V 1.4) was recently reported, being closely related to anticonvulsant and/or local anesthetic binding ( 60 ).…”