Identification of PDZ Domain Containing Proteins Interacting with Cav1.2 and PMCA4b

PDZ (PSD-95/Disc large/Zonula occludens-1) protein interaction domains bind to cytoplasmic protein C-termini of transmembrane proteins. In order to identify new interaction partners of the voltage-gated L-type Ca channel Cav1.2 and the plasma membrane Ca ATPase 4b (PMCA4b), we used PDZ domain arrays probing for 124 PDZ domains.We confirmed this byGST pulldowns and immunoprecipitations. In PDZ arrays, strongest interactions with Cav1.2 and PMCA4bwere found for the PDZdomains of SAP-102, MAST-205, MAGI-1, MAGI-2, MAGI-3, and ZO-1. We observed binding of the Cav1.2 C-terminus to PDZ domains of NHERF1/2, Mint-2, and CASK. PMCA4b was observed to interact with Mint-2 and its known interactions with Chapsyn-110 and CASK were confirmed. Furthermore, we validated interaction of Cav1.2 and PMCA4b with NHERF1/2, CASK, MAST-205 and MAGI-3 via immunoprecipitation.We also verified the interaction ofCav1.2 andnNOS andhypothesized that nNOSoverexpression might reduce Ca influx through Cav1.2. To address this, we measured Ca 2+ currents in HEK 293 cells co-expressing Cav1.2 and nNOS and observed reduced voltage-dependent Cav1.2 activation. Taken together, we conclude that Cav1.2 and PMCA4b bind promiscuously to various PDZ domains, and that our data provides the basis for further investigation of the physiological consequences of these interactions.