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 concentrations within a nanodomain close to the L-type voltage-gated Ca 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 Ca1.3 LTCC clustering in cultured hippocampal neurons. Our previous work showed that binding of the activated catalytic domain of Ca/calmodulin-dependent protein kinase II (CaMKII) to an RKR motif in the N-terminal cytoplasmic domain of Ca1.3 is required for LTCC-mediated E-T coupling. We tested whether multimeric CaMKIIα holoenzymes can bind simultaneously to co-expressed Ca1.3 α1 subunits with two different epitope tags. Co-immunoprecipitation assays from HEK293T cell lysates revealed that CaMKIIα assembles multimeric Ca1.3 LTCC complexes in a Ca/calmodulin-dependent manner. CaMKII-dependent assembly of multi-Ca1.3 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 Ca1.3 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 Ca1.3 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 Ca1.2 α1 subunits and we found that activated CaMKIIα promoted the assembly of Ca1.2 homomeric complexes, as well as Ca1.3-Ca1.2 heteromeric complexes . Furthermore, neuronal depolarization enhanced the clustering of surface-expressed Ca1.2 LTCCs, and enhanced the colocalization of endogenous Ca1.2 LTCCs with surface-expressed Ca1.3, 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.