Clustering of Ca_V1.3 L‐type calcium channels by Shank3

Abstract

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 CaV1.3 calcium channel, and is required for optimal LTCC‐dependent excitation‐transcription coupling. Here, we co‐expressed CaV1.3 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 CaV1.3 α1 subunits under basal conditions. Moreover, CaV1.3 LTCC complex formation was facilitated by CaVβ subunits (β3 and β2a), which also interact with Shank3. Shank3 interactions with CaV1.3 LTCCs and multimeric CaV1.3 LTCC complex assembly were disrupted following the addition of Ca2+ to cell lysates, perhaps simulating conditions within an activated CaV1.3 LTCC nanodomain. In intact HEK293T cells, co‐expression of Shank3 enhanced the intensity of membrane‐localized CaV1.3 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 CaV1.3 LTCCs clusters and reduced the CaV1.3 cluster intensity. Deletion of the Shank3 PDZ domain prevented both binding to CaV1.3 and the changes in multimeric CaV1.3 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 CaV1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions.image

Nanoscale architecture and dynamics of CaV1.3 channel clusters in cardiac myocytes revealed by single channel nanoscopy

“…This leads us to a comparison of putative protein interactions on the proximal versus extended (full-length) CTT to relate our overall findings to possible clustering mechanisms. The extended CTT of Ca V 1.3 42 contains binding sites for JPH2, AKAP family and PDZ-binding proteins, which are relevant scaffolds for LTCC and may spatially define cluster nanodomains or mediate channel tethering (Choi et al, 2022; Sahu et al, 2019; Stanika et al, 2016; Yang et al, 2023). While these interactions may enhance clustering, none of them appear to be strictly required for clustering, given that we showed cluster formation for the short CTT sequence of Ca V 1.3 42A .…”

“…LTCC form subdiffraction-sized clusters, which facilitate calcium release, cooperative gating and protein interactions (Dixon et al, 2022). Previous studies on Ca V 1.3 clustering in neurons showed that alternative splicing affects Ca V 1.3 function and cluster formation possibly through C-terminal protein interactions with Calmodulin (CaM; Moreno et al, 2016), PDZ-binding proteins (Jenkins et al, 2010; Stanika et al, 2016; Yang et al, 2023) and Junctophilin isoforms (Sahu et al, 2019). These findings point towards analogous modulatory mechanisms governing Ca V 1.3 channel function in cardiomyocytes.…”