Association of Ca_v1.3 L-type calcium channels with shank

Neurons express multiple types of voltage-gated calcium (Ca²⁺) channels. Two subtypes of neuronal L-type Ca²⁺ channels are encoded by Ca_v1.2 and Ca_v1.3 pore-forming subunits. Both Ca _v1.2 and Ca_v1.3 subunits contain class I PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-binding consensus at their C termini. In yeast two-hybrid screen of rat brain cDNA library with the C-terminal bait of Ca_v1.3a (long C-terminal splice variant) L-type Ca²⁺ channel subunit, we isolated multiple clones of postsynaptic adaptor protein Shank. We demonstrated a specific association of Ca _v1.3a C termini, but not of Ca_v1.2 C termini, with Shank PDZ domain in vitro. We further demonstrated that the proline-rich region present in C termini of Ca_v1.3a subunit binds to Shank Src homology 3 domain. We established that Ca_v1.3a and Shank localized to postsynaptic locations in cultured rat hippocampal neurons. By expressing epitope-tagged recombinant Ca_v1.3 subunits in rat hippocampal neuronal cultures, we demonstrated that the presence of Shank-binding motifs in Ca_v1.3a sequence is both necessary and sufficient for synaptic clustering of Ca_v1.3 L-type Ca²⁺ channels. In experiments with dominant-negative peptides and dihydropyridine-resistant Ca_v1.3a mutants, we demonstrated an importance of Shank-binding motif in Ca _v1.3a sequence for phosphorylated cAMP response element-binding protein (pCREB) signaling in cultured hippocampal neurons. Our results directly link Ca_v1.3 neuronal L-type Ca²⁺ channels to macromolecular signaling complex formed by Shank and other modular adaptor proteins at postsynaptic density and provide novel information about the role played by Ca_v1.3 L-type Ca²⁺ channels in pCREB signaling.