Mapping of RGS12-Cav2.2 channel interaction.
The alpha1 (pore-forming) subunit of the Cav2.2 (N-type) channel is tyrosine phosphorylated by Src kinase upon activation of GABAB receptors. The tyrosine-phosphorylated form of the alpha1 subunit of the Cav2.2 channel becomes a target for the binding of RGS12, a GTPase-accelerating protein. Binding of the phosphotyrosine-binding domain of RGS12 to the tyrosine-phosphorylated channel alters the kinetics of the termination of GABA-mediated inhibition of the calcium current. Using a combination of biochemical and electrophysiological approaches, we have determined that the SNARE binding or "synprint" region of the Cav2.2 binds to RGS12. This article describes the protocols used to map the interaction using primary neuronal cultures.
Richman
Ryan W
RW
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Diversé-Pierluissi
María A
MA
D000818:Animals; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D004594:Electrophysiology; D005727:Ganglia, Spinal / Q000166:cytology; D006624:Hippocampus / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020710:RGS Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats
D020864:Calcium Channels, N-Type; D010455:Peptides; D020710:RGS Proteins; C491401:Rgs12 protein, rat