G-protein mediated regulation of voltage gated calcium channel PDZ-scaffolding complexes

Multiple G protein-mediated signaling pathways are known to regulate Cav2.2 (o-conotoxin GVIA sensitive N-type) channel activity, but little is known about how these signaling pathways are integrated, and how channel activity regulation affects downstream pathways within neurotransmitter release sites---known as CAZs (Cytoplasmic Active Zones). To improve our understanding, we applied a multi-disciplinary approach that includes biochemical, microscopy, and computational network analysis. A query of protein-protein databases for interactions between proteins involved in G protein-mediated modulation of voltage-dependent calcium channels activity resulted in a network made of 188 components and 531 interactions. Analysis of this interactome revealed a subnetwork, linking several highly connected nodes (proteins) such as Src (a tyrosine kinase) and calmodulin, as well as several PDZ-domain containing scaffolding proteins to the channel. From this subset of interactions, two PDZ-containing scaffolding proteins CASK (Ca2+ calmodulin-dependent serine protein kinase) and Mint1 (Neuronal Munc18-1- interacting protein 1), and a trans-synaptic adhesion molecule Neurexin, were experimentally analyzed (based on their capability) to interconnect into multi-modular complexes for selective but promiscuous interactions with the channel and other cytoplasmic elements. Our results show that CASK and Mint1 are endocytosed with Cav2.2 channels upon activation of GABAB receptors; whereas Neurexin remains at the membrane. We then proceeded to test whether GABAB activation would promote the trans-colocalization of CASK and Tbr-1 complexes as it has been speculated that CASK can enter the nucleus and co-activate Tbr-1 ( T-brain-1)---a T-box transcription factor also identified by the network analysis. What we found is that upon GABAB receptor activation, the association of CASK and Tbr-1 increases in both cytoplasmic and nuclear regions. This suggests that GABAB activation may not only induce channel sequestration but the trans-co-localization of CASK/Tbr-1 complexes. Together these results suggest that CASK and Mint1 complexes not only participate in the retention and sequestration of Cav2.2 channels, but potentially link channel regulated signaling to transcriptional regulatory changes. Future experiments will determine the molecular mechanisms by which tyrosine kinases regulate Neurexin/scaffolding protein interactions and how calcium channel activity may be affected by these changes.