Activity dependent Clustering of Neuronal L-Type Calcium Channels by CaMKII

Neuronal excitation-transcription (E-T) coupling pathways can be initiated by local increases of Ca ²⁺ concentrations within a nanodomain close to the L-type voltage-gated Ca ²⁺ channel (LTCC). However, molecular mechanisms controlling LTCC organization within the plasma membrane that help creation these localized signaling domains remain poorly characterized. Here, we report that neuronal depolarization increases Ca _V 1.3 LTCC clustering in cultured hippocampal neurons. Our previous work showed that binding of the activated catalytic domain of Ca ²⁺ /calmodulin-dependent protein kinase II (CaMKII) to an RKR motif in the N-terminal cytoplasmic domain of Ca _V 1.3 is required for LTCC-mediated E-T coupling. We tested whether multimeric CaMKIIα holoenzymes can bind simultaneously to co-expressed Ca _V 1.3 α1 subunits with two different epitope tags. Co-immunoprecipitation assays from HEK293T cell lysates revealed that CaMKIIα assembles multimeric Ca _V 1.3 LTCC complexes in a Ca ²⁺ /calmodulin-dependent manner. CaMKII-dependent assembly of multi-Ca _V 1.3 complexes is further facilitated by co-expression of the CaMKII-binding LTCC β2a subunit, relative to the β3 subunit, which cannot bind directly to CaMKII. Moreover, clustering of surface localized Ca _V 1.3 α1 subunits in intact HEK293 cells was increased by pharmacological LTCC activation, but only in the presence of co-expressed wild-type CaMKIIα. Moreover, depolarization-induced clustering of surface-expressed Ca _V 1.3 LTCCs in cultured hippocampal neurons was disrupted by suppressing the expression of CaMKIIα and CaMKIIβ using shRNAs. The CaMKII-binding RKR motif is conserved in the N-terminal domain of Ca _V 1.2 α1 subunits and we found that activated CaMKIIα promoted the assembly of Ca _V 1.2 homomeric complexes, as well as Ca _V 1.3-Ca _V 1.2 heteromeric complexes in vitro . Furthermore, neuronal depolarization enhanced the clustering of surface-expressed Ca _V 1.2 LTCCs, and enhanced the colocalization of endogenous Ca _V 1.2 LTCCs with surface-expressed Ca _V 1.3, by CaMKII-dependent mechanisms. This work indicates that CaMKII activation-dependent LTCC clustering in the plasma membrane following neuronal depolarization may be essential for the initiation of a specific long-range signal to activate gene expression.