Residues in Na⁺ channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities

Batrachotoxin (BTX) alters the gating of voltage-gated Na⁺ channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na⁺ conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na⁺ channel α-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle μ1 Na⁺ channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, μ1-S1276K and μ1-L1280K, when expressed in mammalian cells, become completely resistant to 5 μM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display ~10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues μ1-S1276 and μ1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na⁺ channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.