β-adrenergic-mediated dynamic augmentation of sarcolemmal CaV1.2 clustering and co-operativity in ventricular myocytes

Voltage-dependent L-type Ca(V)1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the -adrenergic receptor (AR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced Ca(V)1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. Ca(V)1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+-dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether AR signalling could alter Ca(V)1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the AR agonist, isoproterenol (ISO), promotes enhanced Ca(V)1.2-Ca(V)1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of Ca(V)1.2 super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal Ca(V)1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of Ca(V)1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of AR-mediated regulation of Ca(V)1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal Ca(V)1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands.