title: Irreversible block of cardiac mutant Na+ channels by batrachotoxin.
abstract: Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.
authors.affiliation: Department of Biology, State University of New York at Albany, Albany, New York, USA.
authors.forename: Sho-Ya
authors.initials: SY
authors.lastname: Wang
authors.forename: Denis B
authors.initials: DB
authors.lastname: Tikhonov
authors.forename: Jane
authors.initials: J
authors.lastname: Mitchell
authors.forename: Boris S
authors.initials: BS
authors.lastname: Zhorov
authors.forename: Ging Kuo
authors.initials: GK
authors.lastname: Wang
mesh_terms: D000818:Animals; D001216:Asparagine; D001496:Batrachotoxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D002463:Cell Membrane Permeability; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials; D008958:Models, Molecular; D015394:Molecular Structure; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D001496:Batrachotoxins; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; C466180:Scn4a protein, rat; C568322:Scn5a protein, rat; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D001216:Asparagine; D012964:Sodium