Ca2+ influx through single L-type Ca2+ channels provides the trigger for the larger release of Ca2+ from sarcoplasmic reticulum (SR) stores that leads to muscle contraction. As such, factors that influence L-type Ca2+ channel gating are key determinants of cardiac excitation- contraction (EC) coupling. Chief among such factors are the alpha1 and beta subunit composition of the channel, and up-regulation of channel activity by protein kinase A (PKA)-mediated phosphorylation. Observed changes in the density or complement of alpha1 and beta subunits in heart failure (HF) may underlie altered L-type Ca2+ channel gating, and reduced sensitivity to PKA modulation. Therefore, such changes in channel subunit expression loom as candidate molecular mechanisms for observed EC coupling abnormalities, and loss of beneficial effects of beta-adrenergic agonists in HF. However, fundamental ambiguities regarding the exact identity and configuration of alpha1C (Cav1.2) and beta subunits of cardiac L-type channels hinder efforts to rigorously assess how their differential expression and dysregulation may contribute to cardiac pathophysiology. These gaps in knowledge seriously limit opportunities for development of novel therapeutic strategies against HF. Hence, the long-term objective of this proposal is to deepen understanding of the molecular identity and configuration of cardiac L-type channel subunits, and to bridge this basic knowledge to a new appreciation of the functional operation and modulation of these new channels in heart. Electrophysiology, recombinant Ca2+ channels, and molecular manipulation of native heart Ca2+ channels, and molecular manipulation of cardiac L-type channel structure and function. 1. Clarify the role of a post-translationally cleaved C-terminus fragment of alpha1C in turning the gating of cardiac L-type channels. 2. Elucidate molecular determinants and mechanisms of PKA modulation of cardiac L-type channels. 3. Determine functional consequences of molecular diversity of Ca2+ channel beta2 subunits generated by alternative splicing. 4. Determine the functionally dominant beta2 splice variant expressed in heart utilizing an antisense strategy.
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