Many voltage-activated ion channels are proposed to be regulated by phosphorylation. An extensively studied example involves L-type Ca channels in cardiac cells. Although extensive electrophysiological studies have provided compelling evidence that these channels are regulated by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), a shortcoming of this system is that the protein phosphorylation reactions that regulate the channels remain undefined. However, studies of the homologous L-type Ca channels from skeletal muscle (SkM) have succeeded and led to the proposal that the key phosphorylation reactions occur on the pore-forming, """"""""signature"""""""" alpha1-subunit. With available new tools, it is now possible to directly test if this hypothesis will apply to the well-characterized cardiac Ca channels. Importantly, the alpha1-subunits of the cardiac and SkM channels possess unique and conserved potential phosphorylation sites, suggesting that both similar and unique events may be involved in the regulation of these homologous but distinct channels. The overall goal of this proposal is to provide a detailed model, which defines the phosphorylation reactions and their functional consequences, of the phosphorylation-mediated regulation of cardiac and skeletal muscle L-type Ca channels. The first specific aim is to identify the protein phosphorylation reactions that regulate cardiac L-type channels in intact cells. The isoforms of the cardiac Ca channel subunits that combine to form functional channels will be defined using newly developed antibodies. Phosphorylation will be studied in cultured Cardiac cells, and in heterologous cells expressing the subunits of cardiac Ca channels from their cDNAs; the latter will permit detailed studies previously not possible. Effects of phosphorylation on channel function will be determined with biochemical and electrophysiological analyses using whole cell and reconstitution approaches.
The second aim will be to compare the events involved in the regulation of the homologous but distinct cardiac and SkM L-type Ca channels. Studies will test which phosphorylation reactions occur in intact SkM cells; expression systems will be developed to allow for biochemical and electrophysiological studies of the wild type and mutated SkM channels by PKA and PKC.
The third aim i s to determine the structure/function relationships underlying the regulation of L-type Ca channels by phosphorylation. Site-directed phosphorylation mutants of the cardiac and SkM alpha1-subunits and accessory beta-subunits will be prepared. These will be analyzed to determine which mutations result in the loss of the ability of the channel proteins to undergo phosphorylation and regulation by PKA and PKC. The roles of subunits in the regulation of L-type Ca channels by protein phosphorylation will be defined. The results will provide new insights into the reactions that result in regulation of Ca channels by protein phosphorylation.
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