PT  - JOURNAL ARTICLE
AU  - Nau, Carla
AU  - Wang, Sho-Ya
AU  - Wang, Ging Kuo
TI  - Point Mutations at L1280 in Na<sub>v</sub>1.4 Channel D3-S6 Modulate  Binding Affinity and Stereoselectivity of Bupivacaine Enantiomers
AID  - 10.1124/mol.63.6.1398
DP  - 2003 Jun 01
TA  - Molecular Pharmacology
PG  - 1398--1406
VI  - 63
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/63/6/1398.short
4100  - http://molpharm.aspetjournals.org/content/63/6/1398.full
SO  - Mol Pharmacol2003 Jun 01; 63
AB  - Local anesthetics (LAs) block voltage-gated sodium channels. Parts of the LA binding site are located in the pore-lining transmembrane segments 6 of domains 1, 3, and 4 (D1-S6, D3-S6, D4-S6). We suggested previously that residue N434 in D1-S6 interacts directly with bupivacaine enantiomers in inactivated channels because side-chain properties of different residues substituted at N434 correlated with changes in blocking potencies of bupivacaine enantiomers. Furthermore, mutation N434R exhibited significant stereoselectivity for block of inactivated channels that resulted from a selective decrease in block by S(–)-bupivacaine. In the present study, we analyzed the role of residue L1280 in D3-S6 of the rat skeletal muscle Nav1.4 channel in interactions with the enantiomers of bupivacaine. We substituted native leucine at L1280 with amino acids of different physicochemical properties. Wild-type and mutant channels were expressed transiently in human embryonic kidney 293t cells and were investigated under whole-cell voltage clamp. Block of resting mutant channels by bupivacaine enantiomers revealed little difference compared with wild-type channels. Block of inactivated channels was increased in a mutation containing an aromatic group (L1280W) and decreased in mutations containing a positive charge (L1280K, L1280R). Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels. More surprisingly, stereoselectivity resulted from a selective decrease in block by R(+)-bupivacaine, in contrast to mutation N434R in D1-S6. We propose that in inactivated channels, residues L1280 in D3-S6 and N434 in D1-S6 interact directly with LAs and thereby face each other in the ion-conducting pore.