title: Batrachotoxin-resistant Na+ channels derived from point mutations in transmembrane segment D4-S6.
abstract: Local anesthetics (LAs) block voltage-gated Na+ channels in excitable cells, whereas batrachotoxin (BTX) keeps these channels open persistently. Previous work delimited the LA receptor within the D4-S6 segment of the Na+ channel alpha-subunit, whereas the putative BTX receptor was found within the D1-S6. We mutated residues at D4-S6 critical for LA binding to determine whether such mutations modulate the BTX phenotype in rat skeletal muscle Na+ channels (mu1/rSkm1). We show that mu1-F1579K and mu1-N1584K channels become completely resistant to 5 microM BTX. In contrast, mu1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization. Furthermore, we demonstrate that cocaine elicits a profound time-dependent block after channel activation, consistent with preferential LA binding to BTX-modified open channels. We propose that channel opening promotes better exposure of receptor sites for binding with BTX and LAs, possibly by widening the bordering area around D1-S6, D4-S6, and the pore region. The BTX receptor is probably located at the interface of D1-S6 and D4-S6 segments adjacent to the LA receptor. These two S6 segments may appose too closely to bind BTX and LAs simultaneously when the channel is in its resting closed state.
authors.affiliation: Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222, USA.
authors.forename: S Y
authors.initials: SY
authors.lastname: Wang
authors.forename: G K
authors.initials: GK
authors.lastname: Wang
mesh_terms: D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D003042:Cocaine / Q000494:pharmacology; D004351:Drug Resistance; D006801:Humans; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D000779:Anesthetics, Local; D001496:Batrachotoxins; D011994:Recombinant Proteins; D015222:Sodium Channels; C041417:batrachotoxin receptor; D003042:Cocaine; D008239:Lysine