title: Residues in Na(+) channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities.
abstract: 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 alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM 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 approximately 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 mu1-S1276 and mu1-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.
authors.affiliation: Department of Biology, State University of New York, Albany, New York 12222, USA. wang@zeus.bwh.harvard.edu
authors.forename: S Y
authors.initials: SY
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Nau
authors.forename: G K
authors.initials: GK
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D051381:Rats; D016415:Sequence Alignment; D015222:Sodium Channels / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
chemical_list: D000779:Anesthetics, Local; D001496:Batrachotoxins; D015222:Sodium Channels