Point mutations in segment I-S6 render voltage-gated Na+ channels resistant to batrachotoxin.
Batrachotoxin (BTX) is a steroidal alkaloid that causes Na+ channels to open persistently. This toxin has been used widely as a tool for studying Na+ channel gating processes and for estimating Na+ channel density. In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine --> lysine substitution of the rat muscle Na+ channel alpha-subunit, mu1-N434K, renders the channel completely insensitive to 5 microM BTX when expressed in mammalian cells. This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, mu1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified mu1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional. This finding implies that the mu1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the mu1-434 position apparently diminishes the efficacy of BTX on eliminating Na+ channel inactivation. Mutants of two adjacent residues, mu1-I433K and mu1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the mu1-I433, mu1-N434, and mu1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor.
Wang
S Y
SY
Department of Biological Sciences, State University of New York, Albany, NY 12222, USA.
Wang
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GK
D000818:Animals; D001216:Asparagine; D001496:Batrachotoxins / Q000633:toxicity*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D006801:Humans; D008239:Lysine; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D015222:Sodium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014701:Veratridine / Q000494:pharmacology
D001496:Batrachotoxins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015222:Sodium Channels; C041417:batrachotoxin receptor; D001216:Asparagine; D014701:Veratridine; D008239:Lysine