CaMKII clusters neuronal LTCCs in biomolecular condensates to gate excitation-transcription coupling

Neuronal depolarization activates L-type voltage-gated Ca²⁺ channels (LTCCs), increasing local Ca²⁺ concentrations to initiate excitation-transcription (E-T) coupling. We show that depolarization enhances clustering of Ca_V1.2 and Ca_V1.3 LTCCs in cultured hippocampal neurons, coinciding with increased nuclear CREB phosphorylation. LTCC clustering and LTCC-dependent CREB phosphorylation are selectively disrupted by 1,6-hexanediol, implicating biomolecular condensation. Activated CaMKIIα holoenzymes assemble complexes containing multiple Ca_V1.2 and/or Ca_V1.3 α1 subunits. Complex assembly is facilitated by co-expression of CaMKII-binding β2a subunits and Shank3 and selectively disrupted by 1,6-hexanediol. In HEK293 cells, pharmacological LTCC activation enhances clustering only when wild-type CaMKIIα is co-expressed. A CaMKII mutant that cannot bind LTCC N-terminal domains fails to support LTCC subunit complex formation in vitro and LTCC clustering in HEK293 cells. In neurons, the knockdown of CaMKII expression disrupts depolarization-induced (co-)clustering of Ca_V1.2 and Ca_V1.3. Together, these findings indicate that CaMKII-dependent clustering of plasma membrane LTCCs via biomolecular condensation is essential for initiating long-range signaling to activate gene expression following neuronal depolarization.