Ca2+ Channel Antibodies: Subunit-Specific Antibodies as Probes for Structure and Function
Voltage–dependent Ca2+ channels are known to exist in cardiac, skeletal, and smooth muscle cells as well as in neuronal and secretory cells [1, 2]. 1, 4–Dihydropyridines are potent blockers of voltage–dependent Ca2+ channels , and the receptor for 1, 4– dihydropyridines has been found to be highly enriched in the transverse tubular system of skeletal muscle . Curtis and Catterall  were the first to purify the dihydropyridine receptor from rabbit skeletal muscle T–system membranes. Analysis of their preparation of receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) suggested that the dihydropyridine receptor consisted of three subunits: an a subunit of 160000 Da, a β subunit of 50000 Da, and γ y subunit of 32000 Da. The apparent molecular weight of the a subunit in their preparation shifted from 160000 to 130000 upon reduction, whereas the molecular weight of the α and y subunits did not change upon reduction. The dihydropyridine receptor has also been purified from skeletal muscle membranes by Borsotto et al.  and Flockerzi et al. . These groups also identified three subunits in their preparations of dihydropyridine receptor but the exact composition of subunits and molecular weight of the subunits differ from the original report of Curtis and Catterall . Our laboratory has shown that the purified l, 4–dihydropyridine receptor from rabbit skeletal muscle triads contains four protein components of 175 000 Da (α2), 170000 Da (α1), 52000Da (β) and 32000 Da (γ) and that the 170000 Da and 175000 Da components are distinct polypeptides . The 170000 Da polypeptide (a subunit) has been shown by photoaffinity labeling with [3H]azidopine and [3H]PN200–110 to contain the dihydropyridine binding site of the receptor [9,10], and the 170000 Da (α1 subunit) polypeptide and 52000 Da polypeptide (β subunit) have been shown to be substrates for various protein kinases [11–15]. Finally, the primary structure of the α1 subunit shows considerable sequence and structural similarities to the α subunit of the sodium channel .
KeywordsRabbit Skeletal Muscle Dihydropyridine Receptor Transverse Tubular System Skeletal Muscle Membrane High Molecular Weight Subunit
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