The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel.
The dihydropyridine receptor purified from rabbit skeletal muscle yields in the presence of dithiothreitol and sodium dodecyl sulfate on polyacrylamide gels bands of apparent molecular mass 165 +/- 5, 130 +/- 5, 55 +/- 3, 32 +/- 2 and 28 +/- 1 kDa (chi +/- SEM, n = 12). Under nonreducing conditions, the 130 kDa and 28-kDa peptides migrate as a single peptide of 165 kDa. These peptides were separated on a HPLC size-exclusion column. The specific absorption coefficients of the isolated peptides were determined. From these a stoichiometry of 1:1.7 +/- 0.2:1.4 +/- 0.3 (chi +/- SEM of 12 experiments with three different preparations) was calculated for the 165-kDa, 55-kDa and 32-kDa peptides. The relative amount of the 130/28-kDa peptide varied with different preparations. Tryptic, chymotryptic and V-8 protease peptides of the isolated proteins suggested that the 130/28-kDa peptide was not related to the 165-kDa peptide. The dihydropyridine photoaffinity analog (+/-)-azidopine was specifically incorporated only into the 165-kDa peptide with an efficiency of about 2.4%. The azido analog of desmethoxyverapamil, LU 49888, was specifically incorporated into the same peptide with an efficiency of 1.5%. These results suggest that only the 165-kDa peptide contains the regulatory sites detected so far in the voltage-operated L-type calcium channel. They suggest further that the 130/28-kDa peptide, which migrates as a 165-kDa peptide under nonreducing conditions, does not contain high-affinity binding sites for the calcium channel blockers.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D004095:Dihydropyridines; D007473:Ion Channels / Q000378:metabolism*; D017275:Isradipine; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D010069:Oxadiazoles / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000032:analysis*; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D015220:Calcium Channels; D004095:Dihydropyridines; D007473:Ion Channels; D010069:Oxadiazoles; D011725:Pyridines; D011978:Receptors, Nicotinic; C051537:LU 49888; C041130:azidopine; C043184:4-desmethoxyverapamil; D014700:Verapamil; D002118:Calcium; D017275:Isradipine