Clustering of Ca_V 1.3 L-type calcium channels by Shank3

Clustering of L-type voltage-gated Ca²⁺ channels (LTCCs) in the plasma membrane is increasingly implicated in creating highly localized Ca²⁺ signaling nanodomains. For example, neuronal LTCC activation can increase phosphorylation of the nuclear CREB transcription factor by increasing Ca²⁺ concentrations within a nanodomain close to the channel, without requiring bulk Ca²⁺ increases in the cytosol or nucleus. However, the molecular basis for LTCC clustering is poorly understood. The postsynaptic scaffolding protein Shank3 specifically associates with one of the major neuronal LTCCs, the Ca_V 1.3 calcium channel, and is required for optimal LTCC-dependent excitation-transcription coupling. Here, we co-expressed Ca_V 1.3 α1 subunits with two distinct epitope-tags with or without Shank3 in HEK cells. Co-immunoprecipitation studies using the cell lysates revealed that Shank3 can assemble complexes containing multiple Ca_V 1.3 α1 subunits under basal conditions. Moreover, Ca_V 1.3 LTCC complex formation was facilitated by Ca_V β subunits (β3 and β2a), which also interact with Shank3. Shank3 interactions with Ca_V 1.3 LTCCs and multimeric Ca_V 1.3 LTCC complex assembly were disrupted following the addition of Ca²⁺ to cell lysates, perhaps simulating conditions within an activated Ca_V 1.3 LTCC nanodomain. In intact HEK293T cells, co-expression of Shank3 enhanced the intensity of membrane-localized Ca_V 1.3 LTCC clusters under basal conditions, but not after Ca²⁺ channel activation. Live cell imaging studies also revealed that Ca²⁺ influx through LTCCs disassociated Shank3 from Ca_V 1.3 LTCCs clusters and reduced the Ca_V 1.3 cluster intensity. Deletion of the Shank3 PDZ domain prevented both binding to Ca_V 1.3 and the changes in multimeric Ca_V 1.3 LTCC complex assembly in vitro and in HEK293 cells. Finally, we found that shRNA knock-down of Shank3 expression in cultured rat primary hippocampal neurons reduced the intensity of surface-localized Ca_V 1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions.