Ca channel inhibitors, which are also termed Ca antagonists, are novel drugs for the treatment of several cardiovascular disorders such as angina pectoris, supraventricular arrhythmias and hypertension. These drugs inhibit the influx of Ca into cells of excitable tissues heart, vascular smooth muscle and brain. Pharmacological, biophysical and biochemical studies localized the site of action of these drugs to voltage-dependent Ca channels (VDCC). The long-term objective of our study is to determine the molecular mechanism of action of this structurally heterogeneous group of drugs on VDCC. To attain this goal, pharmacologically relevant receptors must be identified, characterized and their relation to VDCC must be understood. Most recent biochemical studies indicate that the 1,4-dihydropyridine-sensitive skeletal muscle Ca channel contains five distinct subunits termed alpha 1, alpha 2, beta, gamma and delta. The one which has the largest apparent molecular weight on SDS-polyacrylamide gel after disulfide reduction is the alpha 1. subunit. This is a 1,4- dihydropyridine and phenylalkylamine binding polypeptide. Diltiazem (benzothiazepine) binding to this polypeptide has not been demonstrated. Our specific goals are to identify and characterize pharmacologically relevant receptors for calcium channel inhibitors in cardiac muscle, coronary smooth muscle, and in brain. The following specific experiments will be done to accomplish these goals: 1. Photoaffinity labeling using tritiated aryl azide derivatives of all three chemical groups of Ca channel inhibitors i.e. 1,4-dihydropyridines, phenylalkylamines and benzothiazepines. 2. Identification of drug binding domains. 3. Protein phosphorylation. 4. Immunochemical experiments with monoclonal antibodies directed against the alpha 1 subunit of the skeletal muscle Ca channel. 5. Glycoprotein staining. 6. Hydrophobic labeling. These studies are complementary to the protein chemical, immunological and molecular biological experiments being carried out in this and other departments for determining the primary structure of cardiac, coronary smooth muscle and brain VDCC subunits.
