[3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes.
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.
Hamilton
S L
SL
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Alvarez
R M
RM
Fill
M
M
Hawkes
M J
MJ
Brush
K L
KL
Schilling
W P
WP
Stefani
E
E
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
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