SUMMARY The cardiac L-type Ca2+ channel plays a key role in cardiac excitation-contraction coupling, action potential duration, and gene expression. Abnormalities in CaV1.2 function, including increased long-opening-mode gating and blunted adrenergic responsiveness, are associated with heart failure and hypertrophy. The increased activation of CaV1.2, in turn, triggers Ca2+-responsive signaling pathways, which contribute to the pathogenesis of heart failure and hypertrophy. Proper targeting of CaV1.2 to distinct surface sites, and hormonal regulation of their activity, is vital for normal cardiac physiology. Cav1.2 in heart is associated with large supramolecular complexes that impact on channel trafficking, localization, turnover, and function. Much of the prevailing dogma relating to mechanisms underlying CaV1.2 trafficking and modulation is derived from studies using recombinant channels reconstituted in heterologous expression systems. Unfortunately, however, at least some of the most critical questions regarding trafficking and regulation by -adrenergic agonists in cardiomyocytes cannot be assessed using heterologous expression, likely because the unique intracellular environment of cardiomyocytes is not reproduced elsewhere. In the previous funding period, we developed and implemented innovative methods to probe determinants underlying CaV1.2 trafficking and β- adrenergic regulation directly in cardiomyocytes. Using this approach, our findings that β-less α1C in adult cardiomyocytes generate CaV1.2 channels with normal basal activity that are not regulated by PKA provide the first opportunity to probe the relative contribution of this modulation to sympathetic regulation in the fight or flight response. We propose three Aims that build on our previous findings, and designed to deepen mechanistic understanding of CaV1.2 regulation in heart: (1) Determine the role of CaV binding to 1C in regulating cardiac contractility in vivo; (2) To determine the mechanism(s) by which subunits enable - adrenergic regulation of CaV1.2. (3) To elucidate the mechanisms of β-dependent and β-independent CaV1.2 channel trafficking. The three Aims, which should provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding cardiac pathologies and the molecular mechanisms responsible for cardiac excitation-contraction coupling and adrenergic modulation of the cardiac Ca2+ channel.