Purification of the dihydropyridine receptor of the voltage-dependent Ca2+ channel from skeletal muscle transverse tubules using (+) [3H]PN 200-110.
The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels; D007473:Ion Channels / Q000032:analysis*; D017275:Isradipine; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism*; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000302:isolation & purification* / Q000378:metabolism
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D007473:Ion Channels; D010069:Oxadiazoles; D011978:Receptors, Nicotinic; D002118:Calcium; D017275:Isradipine