title: Novel site on sodium channel alpha-subunit responsible for the differential sensitivity of grayanotoxin in skeletal and cardiac muscle.
abstract: We searched for sites on the alpha-subunit of the fast Na(+) channel responsible for the difference in GTX (grayanotoxin) sensitivity of the skeletal- and cardiac-muscle Na(+) current. cDNA clones, encoding the skeletal or cardiac isoforms of the alpha-subunit, were inserted into a mammalian expression vector and transiently transfected into human embryonic kidney cells. The expressed channels were measured using whole-cell patch-clamp techniques and examined for GTX sensitivity. As a measure of GTX sensitivity, we used relative chord conductance (ratio of maximum chord conductance of noninactivating GTX-modified Na(+) currents to that of unmodified peak currents). Wild-type channels from skeletal muscle (mu 1) were more sensitive to GTX modification than wild-type cardiac channels (rH1) by a factor of 1.6. To facilitate exploration of alpha-subunit sites determining GTX sensitivity, we used SHHH, a chimera of skeletal muscle (S) domain D1 and heart muscle (H) domains D2D3D4 with supernormal sensitivity to GTX I (1.5-fold of wild-type mu 1). Successive replacement of Ser-251 (D1S4-S5 intracellular loop) and Ile-433 (D1S6 transmembrane segment), with corresponding rH1 residues Ala and Val, reduced, in a stepwise manner, the GTX sensitivity of the chimera and related mutants to that of wild-type rHl. We concluded that, in addition to Ile-433, known as the GTX-binding site, Ser-251 represents a novel site for GTX modification.
authors.affiliation: Department of Physiology, School of Medicine, Hiroshima University, Kasumi, Hiroshima, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kimura
authors.forename: K
authors.initials: K
authors.lastname: Yamaoka
authors.forename: E
authors.initials: E
authors.lastname: Kinoshita
authors.forename: H
authors.initials: H
authors.lastname: Maejima
authors.forename: T
authors.initials: T
authors.lastname: Yuki
authors.forename: M
authors.initials: M
authors.lastname: Yakehiro
authors.forename: I
authors.initials: I
authors.lastname: Seyama
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D004224:Diterpenes / Q000737:chemistry / Q000494:pharmacology*; D004594:Electrophysiology; D006321:Heart / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000502:physiology; D009206:Myocardium / Q000378:metabolism; D009467:Neuromuscular Depolarizing Agents / Q000737:chemistry / Q000494:pharmacology*; D020033:Protein Isoforms / Q000187:drug effects / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
chemical_list: D004224:Diterpenes; D008565:Membrane Proteins; D009467:Neuromuscular Depolarizing Agents; D020033:Protein Isoforms; D015222:Sodium Channels; D007532:Isoleucine; D012694:Serine; C001311:grayanotoxin I