title: The batrachotoxin receptor on the voltage-gated sodium channel is guarded by the channel activation gate.
abstract: Batrachotoxin (BTX), from South American frogs of the genus Phyllobates, irreversibly activates voltage-gated sodium channels. Previous work demonstrated that a phenylalanine residue approximately halfway through pore-lining transmembrane segment IVS6 is a critical determinant of channel sensitivity to BTX. In this study, we introduced a series of mutations at this site in the Na(v)1.3 sodium channel, expressed wild-type and mutant channels in Xenopus laevis oocytes, and examined their sensitivity to BTX using voltage clamp recording. We found that substitution of either alanine or isoleucine strongly reduced channel sensitivity to toxin, whereas cysteine, tyrosine, or tryptophan decreased toxin action only modestly. These data suggest an electrostatic ligand-receptor interaction at this site, possibly involving a charged tertiary amine on BTX. We then used a mutant channel (mutant F1710C) with intermediate toxin sensitivity to examine the properties of the toxin-receptor reaction in more detail. In contrast to wild-type channels, which bind BTX almost irreversibly, toxin dissociation from mutant channels was rapid, but only when the channels were open, not when they were closed. These data suggest the closed activation gate trapped bound toxin. Although BTX dissociation required channel activation, it was, paradoxically, slowed by strong membrane depolarization, suggesting additional state-dependent and/or electrostatic influences on the toxin binding reaction. We propose that BTX moves to and from its receptor through the cytoplasmic end of the open ion-conducting pore, in a manner similar to that of quaternary local anesthetics like QX314.
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors.forename: Hong-Ling
authors.initials: HL
authors.lastname: Li
authors.forename: David
authors.initials: D
authors.lastname: Hadid
authors.forename: David S
authors.initials: DS
authors.lastname: Ragsdale
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids; D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D003545:Cysteine / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D000596:Amino Acids; D001496:Batrachotoxins; D015222:Sodium Channels; C041417:batrachotoxin receptor; D010649:Phenylalanine; D003545:Cysteine