Dynamic L-type Ca_V1.2 channel trafficking facilitates Ca_V1.2 clustering and cooperative gating

L-type Ca_V1.2 channels are key regulators of gene expression, cell excitability and muscle contraction. Ca_V1.2 channels organize in clusters throughout the plasma membrane. This channel organization has been suggested to contribute to the concerted activation of adjacent Ca_V1.2 channels (e.g. cooperative gating). Here, we tested the hypothesis that dynamic intracellular and perimembrane trafficking of Ca_V1.2 channels is critical for formation and dissolution of functional channel clusters mediating cooperative gating. We found that Ca_V1.2 moves in vesicular structures of circular and tubular shape with diverse intracellular and submembrane trafficking patterns. Both microtubules and actin filaments are required for dynamic movement of Ca_V1.2 vesicles. These vesicles undergo constitutive homotypic fusion and fission events that sustain Ca_V1.2 clustering, channel activity and cooperative gating. Our study suggests that Ca_V1.2 clusters and activity can be modulated by diverse and unique intracellular and perimembrane vesicular dynamics to fine-tune Ca²⁺ signals.