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 .
KeywordsAmide Fluoride Myeloma Tate Coomassie
Unable to display preview. Download preview PDF.
- 4.Fosset M, Jaimovich E, Delpont E, Lazdunski M (1983) pH]Nitrendipine receptors in skeletal muscle: properties and preferential localization in transverse tubules. J Bioi Chern 258:6086–6092Google Scholar
- 6.Borsotto M, Barhanin J, Fosset M, Lazdunski M (1985) The lA–dihydropyridine receptor associated with the skeletal muscle voltage–dependent Ca2+ channel: purification and subunit composition. J Bioi Chern 290:14255–14263Google Scholar
- 8.Leung AT, Imagawa T, Campbell KP (1987) Structural characterization of the 1,4 dihydropyridine receptor of the voltage–dependent Ca2+ channel from rabbit skeletal muscle: evidence for two distinct high molecular weight subunits. J Biol Chern 262:7943–7946Google Scholar
- 9.Sharp AH, Imagawa T, Leung AT, Campbell KP (1987) Identification and characterization of the dihydropyridine–binding subunit of the skeletal muscle dihydropyridine receptor. J Biol Chern 262:12309–12315Google Scholar
- 11.Imagawa T, Leung AT, Campbell KP (1987) Phosphorylation of the 1,4–dihydropyridine receptor of the voltage–dependent Ca2+ channel by an intrinsic protein kinase in isolated triads from rabbit skeletal muscle. J Biol Chern 262:8333–8339Google Scholar
- 15.Leung AT, Imagawa T, Block B, Franzini-Armstrong C, Campbell KP (1988) Biochemical and ultrastructural characterization of the lA-dihydropyridine receptor from rabbit skeletal muscle: evidence for a 52000–Da subunit. J Biol Chern 263:994–1001Google Scholar
- 17.Rosemblatt M, Hidago C, Vergara C, Ikemoto N (1981) Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle. J Biol Chern 256:8140–8148Google Scholar
- 22.Kennett RH (1980) Fusion by centrifugation of cells suspended in polyethylene glycol. In: Kennett RH, McKearn TJ, Bechtol KG (eds) Monoclonal antibodies, Plenum, New York, pp 365–367Google Scholar
- 27.Vilven J, Leung AT, Imagawa T, Sharp AH, Campbell KP and Coronado R (1988) Interaction of calcium channels of skeletal muscle with monoclonal antibodies specific for its dihydropyridine receptor. Biophysical J 53:556aGoogle Scholar