title: [3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes.
abstract: Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [3H]PN200-110, and the alkaloid, [3H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling.
authors.affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: S L
authors.initials: SL
authors.lastname: Hamilton
authors.forename: R M
authors.initials: RM
authors.lastname: Alvarez
authors.forename: M
authors.initials: M
authors.lastname: Fill
authors.forename: M J
authors.initials: MJ
authors.lastname: Hawkes
authors.forename: K L
authors.initials: KL
authors.lastname: Brush
authors.forename: W P
authors.initials: WP
authors.lastname: Schilling
authors.forename: E
authors.initials: E
authors.lastname: Stefani
mesh_terms: D000470:Alkaloids / Q000378:metabolism*; D000818:Animals; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D004095:Dihydropyridines / Q000494:pharmacology; D004586:Electrophoresis / Q000379:methods; D017275:Isradipine; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism*; D015221:Potassium Channels / Q000502:physiology; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000648:ultrastructure; D014316:Tritium
chemical_list: D000470:Alkaloids; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018118:Chloride Channels; D002712:Chlorides; D004095:Dihydropyridines; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010069:Oxadiazoles; D015221:Potassium Channels; D014316:Tritium; D012433:Ryanodine; C038806:1,4-dihydropyridine; D017275:Isradipine