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 Ca2+ concentrations within a nanodomain close to the L-type voltage-gated Ca2+ 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 CaV1.3 LTCC clustering in cultured hippocampal neurons. Our previous work showed that binding of the activated catalytic domain of Ca2+/calmodulin-dependent protein kinase II (CaMKII) to an RKR motif in the N-terminal cytoplasmic domain of CaV1.3 is required for LTCC-mediated E-T coupling. We tested whether multimeric CaMKII holoenzymes can bind simultaneously to co-expressed CaV1.3 1 subunits with two different epitope tags. Co-immunoprecipitation assays from HEK293T cell lysates revealed that CaMKII assembles multimeric CaV1.3 LTCC complexes in a Ca2+/calmodulin-dependent manner. CaMKII-dependent assembly of multi-CaV1.3 complexes is further facilitated by co-expression of the CaMKII-binding LTCC {beta}2a subunit, relative to the {beta}3 subunit, which cannot bind directly to CaMKII. Moreover, clustering of surface localized CaV1.3 1 subunits in intact HEK293 cells was increased by pharmacological LTCC activation, but only in the presence of co-expressed wild-type CaMKII. Moreover, depolarization-induced clustering of surface-expressed CaV1.3 LTCCs in cultured hippocampal neurons was disrupted by suppressing the expression of CaMKII and CaMKII{beta} using shRNAs. The CaMKII-binding RKR motif is conserved in the N-terminal domain of CaV1.2 1 subunits and we found that activated CaMKII promoted the assembly of CaV1.2 homomeric complexes, as well as CaV1.3-CaV1.2 heteromeric complexes in vitro. Furthermore, neuronal depolarization enhanced the clustering of surface-expressed CaV1.2 LTCCs, and enhanced the colocalization of endogenous CaV1.2 LTCCs with surface-expressed CaV1.3, 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.