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.