title: Serine-401 as a batrachotoxin- and local anesthetic-sensing residue in the human cardiac Na+ channel.
abstract: Sequence alignment of four S6 segments in the human cardiac Na+ channel suggests that serine-401 (hNav1.5-S401) at D1S6 along with asparagine-927 (N927) at D2S6, serine-1458 (S1458) at D3S6, and phenylalanine-1760 (F1760) at D4S6 may jointly form a pore-facing S(401)N(927)S(1458)F(1760) ring. Importantly, this pore-facing structure is adjacent to the putative gating-hinge (G(400)G(926)G(1457)S(1759)) and close to the selectivity filter. Within this SNSF ring, only S401 has not yet been identified as a batrachotoxin (BTX) sensing residue. We therefore created S401 mutants with 12 substitutions (S401C,W,P,A,K,F,R,E,L,N,D,G) and assayed their BTX sensitivity. All S401 mutants expressed Na+ currents but often with altered gating characteristics. Ten mutants were found sensitive to 5 muM BTX, which eliminated Na+ channel fast inactivation after repetitive pulses. However, S401K and S401R became BTX resistant. In addition, the block of open and inactivated hNav1.5-S401K Na+ channels by local anesthetic bupivacaine was reduced by approximately 8-10-fold, but not the block of resting Na+ channels. Qualitatively, these ligand-sensing phenotypes of hNav1.5-S401K channels resemble those of S1458K and F1760K channels reported earlier. Together, our results support that residue hNav1.5-S401 at D1S6 is facing the inner cavity and is in close proximity to the receptor sites for BTX and for local anesthetics.
authors.affiliation: Department of Biology, State University of New York at Albany, Albany, NY 12222, USA. sywang@albany.edu
authors.forename: Sho-Ya
authors.initials: SY
authors.lastname: Wang
authors.forename: Denis B
authors.initials: DB
authors.lastname: Tikhonov
authors.forename: Boris S
authors.initials: BS
authors.lastname: Zhorov
authors.forename: Jane
authors.initials: J
authors.lastname: Mitchell
authors.forename: Ging Kuo
authors.initials: GK
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000779:Anesthetics, Local / Q000378:metabolism* / Q000494:pharmacology; D001496:Batrachotoxins / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002045:Bupivacaine / Q000494:pharmacology; D002460:Cell Line; D003198:Computer Simulation; D004558:Electric Stimulation; D004594:Electrophysiology; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
chemical_list: D000779:Anesthetics, Local; D001496:Batrachotoxins; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011994:Recombinant Proteins; C568320:SCN5A protein, human; D015222:Sodium Channels; D012694:Serine; D012964:Sodium; D002045:Bupivacaine