Voltage-gated calcium (Ca) channels initiate excitation-contraction coupling in cardiac myocytes. Stimulation of the sympathetic nervous system activates -adrenergic receptors, adenylyl cyclase, and cAMP-dependent protein kinase (PKA). PKA phosphorylates Cav1.2 channels and increases their activity, which contributes to increased beating rate and contractile force in response to exercise, stress, and fear. Cav1.2 channel activity is also regulated by voltage-dependent potentiation and Ca-dependent facilitation, and phosphorylation by PKA and Ca/calmodulin-dependent protein kinase II (CaMKII) is involved in this regulation. Our recent research has revealed unexpected complexity in regulation of Cav1.2 channels by PKA. First, in acutely dissociated ventricular myocytes, an A Kinase Anchoring Protein (AKAP) is required for anchoring of PKA to the distal C- terminal domain (DCT). Second, in vivo proteolytic processing severs the C-terminus near its center, potentially separating the DCT from the Cav1.2 channel. Third, the proteolytically processed DCT binds noncovalently to the proximal C-terminal domain and inhibits Cav1.2 channel activity. This autoinhibitory signaling complex with noncovalently bound DCT, AKAP, and PKA is the primary substrate for regulation PKA, which phosphorylates the channel near the site of interaction of these two halves of the C-terminus and disinhibits channel activity. We have used proteomic methods to identify novel Ser/Thr residues that are phosphorylated in vivo in response to -adrenergic receptor/PKA signaling. Phosphorylation of Thr1704 by casein kinase II is important for setting basal Cav1.2 channel activity, whereas Ser1700 is required for regulation by PKA. Mutation of these phosphorylation sites in mice prevents -adrenergic regulation of Cav1.2 channels in ventricular myocytes. Moreover, mice in which the DCT is deleted have marked hypertrophy and heart failure, indicating that this autoinhibitory signaling complex is required for normal cardiovascular function in vivo. Our proposed experiments will address three aims. 1. We will use unbiased co-immunoprecipitation and proteomic methods to identify AKAPs that bind to Cav1.2 in the heart, and the functional role of these AKAPs in channel regulation will be determined. 2. We will analyze voltage-dependent potentiation and CaMKII-dependent facilitation of full-length, truncated, and truncated+DCT channels in transfected cells using mutants at the Ser1700 site to determine its functional role, and we will define the functional role of this site in vivo using S1700A mice. 3. We will examine changes in the levels of full-length, truncated, and truncated+DCT Cav1.2 channels and their interactions with AKAPs in the -adrenergic hyperstimulation model of heart failure, in which our preliminary studies reveal substantial molecular remodeling of Cav2.1. We will use our S1700A mice to define the role of phosphorylation of Ser1700 in hypertrophy and heart failure in vivo. These studies will increase understanding of regulation of the heart by the sympathetic nervous system and give essential new insight into the molecular and functional changes in the Cav1.2 signaling complex in heart failure.
The contractility of the heart is controlled by the sympathetic nervous system, which acts through adrenalin and noradrenalin to cause phosphorylation of the cardiac calcium channel. Our studies will further elucidate the mechanism through which cardiac calcium channels are regulated by protein phosphorylation and will determine how this key regulatory process is altered in heart failure.
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