title: How batrachotoxin modifies the sodium channel permeation pathway: computer modeling and site-directed mutagenesis.
abstract: A structural model of the rNav1.4 Na+ channel with batrachotoxin (BTX) bound within the inner cavity suggested that the BTX pyrrole moiety is located between a lysine residue at the DEKA selectivity filter (Lys1237) and an adjacent phenylalanine residue (Phe1236). We tested this pyrrole-binding model by site-directed mutagenesis of Phe1236 at D3/P-loop with 11 amino acids. Mutants F1236D and F1236E expressed poorly, whereas nine other mutants either expressed robust Na+ currents, like the wild-type (F1236Y/Q/K), or somewhat reduced current (F1236G/A/C/N/W/R). Gating properties were altered modestly in most mutant channels, with F1236G displaying the greatest shift in activation and steady-state fast inactivation (-10.1 and -7.5 mV, respectively). Mutants F1236K and F1236R were severely resistant to BTX after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz), whereas seven other mutants were sensitive but with reduced magnitudes compared with the wild type. It is noteworthy that rNav1.4-F1236K mutant Na+ channels remained highly sensitive to block by the local anesthetic bupivacaine, unlike several other BTX-resistant mutant channels. Our data thus support a model in which BTX, when bound within the inner cavity, interacts with the D3/P-loop directly. Such a direct interaction provides clues on how BTX alters the Na+ channel selectivity and conductance.
authors.affiliation: Department of Biology, State University of New York at Albany, USA.
authors.forename: Sho-Ya
authors.initials: SY
authors.lastname: Wang
authors.forename: Jane
authors.initials: J
authors.lastname: Mitchell
authors.forename: Denis B
authors.initials: DB
authors.lastname: Tikhonov
authors.forename: Boris S
authors.initials: BS
authors.lastname: Zhorov
authors.forename: Ging Kuo
authors.initials: GK
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D002045:Bupivacaine / Q000494:pharmacology; D003198:Computer Simulation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics
chemical_list: D001496:Batrachotoxins; D009124:Muscle Proteins; C466180:Scn4a protein, rat; D015222:Sodium Channels; D010649:Phenylalanine; D002045:Bupivacaine