Monoclonal Antibody Characterization of the 1,4‐Dihydropyridine Receptor of Rabbit Skeletal Muscle

Abstract

The 1,4-dihydropyridine receptor (DHPR) of the voltage-dependent Ca channel has been purified from transverse tubular membranes of skeletal muscle. Curtis and Catterall have shown that it consists of three polypeptides of 160,000 daltons, 50,000 Da, and 32,000 Da and that under reducing conditions the apparent molecular weight of the 160,000 Da subunit shifted to 130,000. Borsotto et al. have identified three polypeptides of 142,000 Da, 33,000 Da, and 32,000 Da in their preparation of the dihydropyridine receptor. Furthermore, they have shown by immunoblotting with polyclonal antibodies that the 142,000 Da and 32,000 Da subunits are produced by the reduction of a 170,000 Da polypeptide. We report here the identification and characterization with monoclonal antibodies (MAb) of an additional high molecular weight subunit of the DHPR which is distinct from that described by Curtis and Catterrall and Borsotto et al. Monoclonal antibodies capable of specifically immunoprecipitating the [H]PN200-110-labeled DHPR of rabbit skeletal muscle were produced by immunizing BALB/c mice initially with skeletal muscle triad vesicles followed by booster immunizations with purified DHPR from skeletal muscle triads. Hybridoma supernatants were screened for the production of anti-DHPR antibodies with an immunodot assay. Supernatants that reacted positively against the partially purified DHPR were then tested for their ability to immunoprecipitate the [H]PN200-110-labeled receptor from digitonin-solubilized triads (see TABLE 1). All three MAbs (IIC12, IIF7, IIID5) that were capable of immunoprecipitating the DHPR recognized a protein with an Mr of 170,000 on nitrocellulose transfers of skeletal muscle triads and transverse tubular membranes separated on sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions (FIGURE la). Wheat-germ agglutinin (WGA) peroxidase stained a 175,000 Da protein on similar nitrocellulose transfers. Neither the 170,000 Da polypeptide nor the 175,000 Da polypeptide was detected in light sarcoplasmic reticulum vesicles, a preparation devoid of DHPR. Under reducing conditions, the Mr of the 170,000 Da