Abstract 105: Tuning EC-Coupling: β -Adrenergic Receptor Activation Stimulates Dynamic Augmentation of Ca V 1.2 Channel Sarcolemmal Abundance and Cooperativity

Voltage-gated L-type Ca V 1.2 channels play an essential role in cardiac excitation-contraction coupling. These channels decorate the z-lines of ventricular myocytes where they form discrete clusters on the t-tubule membrane. We have previously reported that groups of channels within these clusters are capable of influencing each other’s gating behavior and undergo dynamic, cooperative interactions. This occurs via Ca 2+ -calmodulin-dependent, physical interactions between the C-terminal tails of adjacent channels. The interactions increase the open probability ( P o ) of adjoined channels, and thus amplify Ca 2+ influx into the myocytes. It has long been known that activation of β -adrenergic receptors during the ‘fight or flight’ response, leads to increased mean open time and P o of Ca V 1.2 channels, and thus enhanced Ca 2+ influx. Given our previous finding that Ca V 1.2 channel cooperativity is triggered by Ca 2+ -influx, we predicted that β -AR stimulation could affect Ca V 1.2 channel cooperativity in ventricular myocytes. To test this hypothesis, we investigated the effect of the β -agonist isoproterenol (ISO) on Ca V 1.2 channel clustering and cooperativity using a combination of optical and electrophysiological approaches. Ground State Depletion super-resolution nanoscopy revealed ‘super-clustering’ of Ca V 1.2 channels in ISO-stimulated adult mouse ventricular myocytes. Channels within these super-clusters display enhanced physical interactions compared to control unstimulated cells as revealed by Bimolecular fluorescence complementation. To visualize this process, we performed live cardiomyocyte imaging and observed a robust, dynamic increase in sarcolemmal Ca V 1.2 channel abundance in response to ISO. Functionally, optical and electrophysiologicaI recordings of Ca V 1.2 channel activity revealed that ISO-stimulated cells display enhanced Ca V 1.2 channel cooperativity, and amplified Ca 2+ influx. On the basis of these data, we propose that stimulation of β ARs initiates dynamic augmentation of Ca V 1.2 channel abundance, and enhanced cooperative gating of Ca V 1.2 channels, in the sarcolemma of ventricular myocytes, to amplify Ca 2+ influx into these cells and tune EC-coupling in times of high metabolic or hemodynamic demand.