Monoclonal antibody specific for the transverse tubular membrane of skeletal muscle activates the dihydropyridine-sensitive Ca2+ channel.
In skeletal muscle, dihydropyridine receptors and dihydropyridine-sensitive Ca2+ channels are preferentially localized in the transverse tubular membranes. Starting with an antigenic membrane fraction enriched in rabbit muscle transverse tubules (T-tubules), several monoclonal antibodies were produced by a fusion of spleen cells from an immunized BALB/c mouse with P3 X 63Ag.8.6.5.3 mouse myeloma cells. Antibodies were screened according to a scheme designed to select IgG immunoglobins that recognized a determinant specifically associated with the T-tubule membrane. Antibodies that fulfilled the screening criteria were used in in vitro planar bilayer recording of the activity of the dihydropyridine-sensitive Ca2+ channel present in T-tubules. Cells producing one antibody (Ab 21) survived cloning dilution and stably produced a monoclonal antibody (mAb21-4) that increased the rate of single channel opening when interacting with the internal side of the channel protein. mAb21-4 immobilized by covalent crosslinking on beads (Affi-Gel 10) consistently immunoprecipitated polypeptide bands with the following electrophoretic mobility: Mr values of greater than or equal to 175,000; 90,000; 55,000; and 34,000.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002118:Calcium / Q000378:metabolism*; D004095:Dihydropyridines; D007074:Immunoglobulin G / Q000276:immunology; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000276:immunology*; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004095:Dihydropyridines; D007074:Immunoglobulin G; D007473:Ion Channels; D011725:Pyridines; C038806:1,4-dihydropyridine; D002118:Calcium