Voltage-sensitive calcium channels mediate calcium influx and calcium-dependent depolarization in response to changes of membrane potential in many excitable tissues. In the heart, calcium influx mediated by these channels is responsible for the plateau phase of the cardiac action potential and initiates excitation-contraction coupling. The positive inotropic and chronotropic of norepinephrine and other Beta-adrenergic agents mediated by cAMP-dependent protein phosphorylation is due in part to an increase in the number of calcium channels activated during the cardiac action potential. In this research, radiolabeled organic calcium antagonists such as [H3]-nitrendipine will be used as probes to monitor purification of the protein components of the calcium antagonist receptor of the voltage-sensitive calcium channel. The calcium antagonist receptor will be solubilized with digitonin and purified by conventional and affinity chromatographic procedures. Preliminary results show that the purified calcium antagonist receptor consists of a complex of three subunits. The purified receptor will be characterized with respect to subunit size, composition, stoichiometry, and general biochemical properties. The purified calcium antagonist receptor will be incorporated into phospholipid vesicles and its functional activity in ion transport will be assessed by isotopic flux measurements to determine whether the purified receptor contains all the components required to mediate voltage-sensitive calcium conductance. The subunits will be separated under native conditions and the functional role of the individual subunits assessed by reconstitution. Sites of phosphorylation of the subunits of the calcium channel will be identified both in vitro and in vivo and the functional effects of pholphorylation will be analyzed. The structure of the calcium channel will be investigated further by preparation and molecular cloning of cDNA encoding the channel subunits. These cDNA clones will then be sequenced and the amino acid sequence and secondary structure of the calcium channel subunits will be derived from the nucleotide sequence to give a detailed structural model of the calcium channel. These results will provide a basis for analysis of the molecular basis of calcium channel function and physiological regulation.
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