CaMKII clusters neuronal LTCCs in biomolecular condensatesto gate excitation-transcription coupling

Neuronal depolarization activates L-type voltage-gated Ca{superscript 2} channels (LTCCs), increasing local Ca{superscript 2} concentrations to initiate excitation-transcription (E-T) coupling. We show that depolarization enhances clustering of CaV1.2 and CaV1.3 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 CaMKII holoenzymes assemble complexes containing multiple CaV1.2 and/or CaV1.3 1 subunits. Complex assembly is facilitated by co-expression of CaMKII-binding {beta}2a subunits and Shank3 and selectively disrupted by 1,6-hexanediol. In HEK293 cells, pharmacological LTCC activation enhances clustering only when wild-type CaMKII 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 CaV1.2 and CaV1.3. 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.