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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.

We have shown previously that prenylamine, a calcium channel blocker, has potent local anesthetic activity in vivo and in vitro. We now characterize the tonic and use-dependent block of prenylamine on wild-type human cardiac voltage-gated sodium channels (hNav1.5) transiently expressed in human embryonic kidney 293t cells under whole-cell voltage-clamp condition. We also determine whether prenylamine and local anesthetics interact with a common binding site on the Nav1.5 channel by analyzing prenylamine block on mutant hNav1.5 channels that have substitution mutations in amino acids at the putative local anesthetic binding sites. Prenylamine exhibits tonic block at both hyperpolarizing and depolarizing potentials on hNav1.5 channels with 50% inhibitory concentrations of 9.67 +/- 0.25 microM and 0.72 +/- 0.02 microM, respectively. Substitutions of the amino acids at the putative local anesthetic binding site (i.e., F1760, N1765, Y1767, and N406) with lysine had much lesser effects on prenylamine block of the mutant hNav1.5 channels compared with local anesthetic block. The affinity of prenylamine was reduced at most by 5.8-fold, whereas that of bupivacaine, a known local anesthetic, was reduced by as much as 68-fold compared with wild-type by the mutations at the local anesthetic receptor site. Furthermore, equilibrium results between prenylamine-bupivacaine mixtures suggest two independent receptors. Thus, the data demonstrate that prenylamine has both tonic and use-dependent block of hNav1.5 channels similar to that of local anesthetics, but the location of the prenylamine binding site on hNav1.5 differs from that of the local anesthetic binding site.

Batrachotoxin (BTX) stabilizes the voltage-gated Na(+) channels in their open conformation, whereas local anesthetics (LAs) block Na(+) conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha-subunit Na(+) channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na(+) channel ((mu)1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na(+) currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na(+) currents during repetitive pulses. Two of these four mutants, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na(+) channels (K(I)) of approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.

Voltage-gated Na(+) channels are the primary targets of local anesthetics (LAs). Amino acid residues in domain 4, transmembrane segment 6 (D4-S6) form part of the LA binding site. LAs inhibit binding of the neurotoxin batrachotoxin (BTX). Parts of the BTX binding site are located in D1-S6 and D4-S6. The affinity of BTX-resistant Na(+) channels mutated in D1-S6 (mu1-N434K, mu1-N437K) toward several LAs is significantly decreased. We have studied how residue mu1-N434 influences LA binding. By using site-directed mutagenesis, we created mutations at mu1-N434 that vary the hydrophobicity, aromaticity, polarity, and charge and investigated their influence on state-dependent binding and stereoselectivity of bupivacaine. Wild-type and mutant channels were transiently expressed in human embryonic kidney 293t cells and investigated under whole-cell voltage-clamp. For resting channels, bupivacaine enantiomers showed a higher potency in all mutant channels compared with wild-type channels. These changes were not well correlated with the physical properties of the substituted residues. Stereoselectivity was small and almost unchanged. In inactivated channels, the potency of bupivacaine was increased in mutations containing a quadrupole of an aromatic group (mu1-N434F, mu1-N434W, mu1-N434Y), a polar group (mu1-N434C), or a negative charge (mu1-N434D) and was decreased in a mutation containing a positive charge (mu1-N434K). In mutation mu1-N434R, containing the positively charged arginine, the potency of S(-)-bupivacaine was selectively decreased, resulting in a stereoselectivity (stereopotency ratio) of 3. Similar results were observed with cocaine but not with RAC 109 enantiomers. We propose that in inactivated channels, residue mu1-N434 interacts directly with the positively charged moiety of LAs and that D1-S6 and D4-S6 form a domain-interface site for binding of BTX and LAs in close proximity.

Local anesthetics (LAs) are noncompetitive antagonists of batrachotoxin (BTX) in voltage-gated Na+ channels. The putative LA receptor has been delineated within the transmembrane segment S6 in domain IV of voltage-gated Na+ channels, whereas the putative BTX receptor is within segment S6 in domain I. In this study, we created BTX-resistant muscle Na+ channels at segment I-S6 (micro1-N434K, micro1-L437K) to test whether these residues modulate LA binding. These mutant channels were expressed in transiently transfected human embryonic kidney 293T cells, and their sensitivity to lidocaine, QX-314, etidocaine, and benzocaine was assayed under whole-cell, voltage-clamp conditions. Our results show that LA binding in BTX-resistant micro1 Na+ channels was reduced significantly. At -100 mV holding potential, the reduction in LA affinity was maximal for QX-314 (by 17-fold) and much less for neutral benzocaine (by 2-fold). Furthermore, this reduction was residue specific; substitution of positively charged lysine with negatively charged aspartic acid (micro1-N434D) restored or even enhanced the LA affinity. We conclude that micro1-N434K and micro1-L437K residues located near the middle of the I-S6 segment of Na+ channels can reduce the LA binding affinity without BTX. Thus, this reduction of the LA affinity by point mutations at the BTX binding site is not caused by gating changes induced by BTX alone. We surmise that the BTX receptor and the LA receptor within segments I-S6 and IV-S6, respectively, may align near or within the Na+ permeation pathway.