Point mutations at N434 in D1-S6 of mu 1 Na+ channels modulate binding affinity and stereoselectivity of local anesthetic enantiomers

Voltage-gated Na+ channels are the primary targets of local anesthetics (LA s). Amino acid residues in domain 4, transmembrane segment 6 (D4-S6) form p art of the LA binding site. LAs inhibit binding of the neurotoxin batrachot oxin (BTX). Parts of the BTX binding site are located in D1-S6 and D4-S6. T he affinity of BTX-resistant Na+ channels mutated in D1-S6 (mu 1-N434K, mu 1-N437K) toward several LAs is significantly decreased. We have studied how residue mu 1-N434 influences LA binding. By using site-directed mutagenesi s, we created mutations at mu 1-N434 that vary the hydrophobicity, aromatic ity, polarity, and charge and investigated their influence on state-depende nt binding and stereoselectivity of bupivacaine. Wild-type and mutant chann els were transiently expressed in human embryonic kidney 293t cells and inv estigated under whole-cell voltage-clamp. For resting channels, bupivacaine enantiomers showed a higher potency in all mutant channels compared with w ild-type channels. These changes were not well correlated with the physical properties of the substituted residues. Stereoselectivity was small and al most unchanged. In inactivated channels, the potency of bupivacaine was inc reased in mutations containing a quadrupole of an aromatic group (mu 1-N434 F, mu 1-N434W, mu 1-N434Y), a polar group (mu 1-N434C), or a negative charg e (mu 1-N434D) and was decreased in a mutation containing a positive charge (mu 1-N434K). In mutation mu 1-N434R, containing the positively charged ar ginine, the potency of S(-)-bupivacaine was selectively decreased, resultin g in a stereoselectivity (stereopotency ratio) of 3. Similar results were o bserved with cocaine but not with RAC 109 enantiomers. We propose that in i nactivated channels, residue mu 1-N434 interacts directly with the positive ly charged moiety of LAs and that D1-S6 and D4-S6 form a domain-interface s ite for binding of BTX and LAs in close proximity.