L-TYPE Ca channels containing Cav1.2 alpha1 subunits are responsible for Ca entry that initiates contraction in cardiac and smooth muscle, and they are therefore the final common pathway for regulation of contractile force by many different effectors such as neurotransmitters, hor5moines and drugs. Because of they key role in regulation of contraction, many different intracellular second messengers converge on these Ca channels and regulate their function, including cAMP, Ca, calmodulin, diacylglycerol, and ATP. Recent studies show that the sites of action of many of in these intracellular second messengers are in the large intracellular C-terminal domain, which is comprised of more than 660 amino acid residues representing 30% of the mass of the alpha1subunit, and in the smaller interacting intracellular N-terminal domain. In addition, the C-terminal domain is subject to regulated proteolysis, which modulates its function. Thus, the N-terminal and C-terminal domains interact with each other and integrate many kinds of cellular regulatory signals, which together comprise an intracellular signaling network controlling Ca channel activity. We propose to analyze the functional interactions among the regulatory sites for cAMP-dependent protein kinase, protein kinase C, A kinase anchoring protein 15 (AKAP-15) and ATP in the N-terminal and distal C-terminal domains of Cav1.2 Ca channels, to examine the cellular mechanism and functional significance of regulated proteolysis of the C-terminal domain of Cav1.2 channels, to define the molecular basis for interaction of Ca and calmodulin with the C-terminal domain of Cav1.2 channels, to define the molecular basis for interaction of Ca and calmodulin with the C-terminal domain and their regulation of Ca channel function, and to determine the structural basis for functional interactions among multiple regulatory pathways impinging on the C-terminus of Cav1.2 channels. Our experiments will use a combination of structural, biochemical, cell biological, electrophysiological, and mouse genetic approaches to reveal the molecular basis for Ca channel regulation and its role in cardiovascular physiology. The results will shed new light on the mechanisms of regulation of cardiac beating rate and contractility through an intracellular signaling network impinging on the L-type Ca channels.
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