L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Integrator 1 (BIN1), in the clustering of Ca v 1.2 channels in ventricular myocytes. Both of these proteins are known to directly interact with Ca v 1.2 channels. AKAP150 is important for local membrane targeting of PKA, PKCa and calcineurin, while Bin1 has established roles in cardiac t-tubule folding and in the trafficking and localization of Ca v 1.2 channels to t-tubules. Using GSD superresolution imaging, we found that clustering of Ca v 1.2 channels in these cells is unaltered by genetic ablation of AKAP79/150 such that the area of Ca V 1.2 clusters was similar in WT (2379 5 43 nm) and AKAP150 -/myocytes (2379 5 43 nm). However, heterozygous deletion of Bin1 significantly reduced Ca v 1.2 channel cluster size. The area of Ca V 1.2 channel clusters was approximately 42% smaller in BIN1 þ/-(1379 5 43 nm) than in WT (2349 5 76 nm 2 ) ventricular myocytes (p< 0.0001). This data suggests that Bin1 is a key regulator of Ca v 1.2 channel clustering in heart.