Rabbit skeletal muscle microsomes contain two distinct phenylalkylamine-binding sites.
Lu49888, a photoaffinity analog of verapamil, was used to identify specific binding sites for phenylalkylamines of calcium channels present in rabbit skeletal muscle microsomes. Direct binding equilibrium measurements and displacement curves of Lu49888 by its non-radioactive analog yielded an apparent single class of binding sites with Kd and Bmax values of 16.5 nM and 7.5 pmol/mg respectively. Lu49888 was specifically incorporated into three proteins of apparently 165 kDa, and 33 kDa. Incorporation into the 55-kDa protein was blocked by 10--50-fold higher concentrations of unlabeled phenylalkylamines compared to incorporation into the 165-kDa protein, suggesting that the 165-kDa and 55-kDa proteins contain a high and a low-affinity verapamil-binding site respectively. The photoaffinity-labeled proteins were solubilized by 1% digitonin or 1% Chaps in roughly equal amounts. The 165-kDa protein bound to wheat-germ-agglutinin(WGA)--Sepharose and sedimented in sucrose density gradients with the same constant as the purified dihydropyridine receptor, which has been reconstituted to a functional calcium channel. The 55-kDa membrane protein did not bind to the WGA-Sepharose column and sedimented in sucrose density gradients with a lower s value than the 165-kDa protein. The 165-kDa but not the 55-kDa membrane protein was specifically labeled by azidopine, the photoaffinity analogue of dihydropyridines. The 55-kDa protein of the purified dihydropyridine receptor was not significantly labeled by Lu49888 showing that the 55-kDa protein of the membrane is unrelated to the purified high-affinity dihydropyridine receptor.
D000345:Affinity Labels; D000588:Amines / Q000032:analysis*; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D007473:Ion Channels / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010777:Photochemistry; D011817:Rabbits; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D000588:Amines; D001386:Azides; D007473:Ion Channels; C051537:LU 49888; D014700:Verapamil