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 Ca2+ channel 1.2 and the plasma membrane Ca2+ ATPase 4b (PMCA4b), we used PDZ domain arrays probing for 124 PDZ domains. We confirmed this by GST pull-downs and immunoprecipitations. In PDZ arrays, strongest interactions with 1.2 and PMCA4b were found for the PDZ domains of SAP-102, MAST-205, MAGI-1, MAGI-2, MAGI-3, and ZO-1. We observed binding of the 1.2 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 1.2 and PMCA4b with NHERF1/2, CASK, MAST-205 and MAGI-3 via immunoprecipitation. We also verified the interaction of 1.2 and nNOS and hypothesized that nNOS overexpression might reduce Ca2+ influx through 1.2. To address this, we measured Ca2+ currents in HEK 293 cells co-expressing 1.2 and nNOS and observed reduced voltage-dependent 1.2 activation. Taken together, we conclude that 1.2 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. 1. Introduction PDZ domains are protein interaction motifs that play a crucial role in cellular signaling and bind specifically to cytoplasmatic carboxyl (C-) terminal sequences of their interacting proteins, which often belong to transmembrane receptor and ion channel families. This motif typically spans 90–100 amino acids and was first found in three polypeptides: the mammalian protein postsynaptic density-95 (PSD-95), the Drosophila melanogaster epithelial tumor suppressor protein Discs Large (Dlg), and the mammalian epithelial tight junction protein Zonula occludens-1 (ZO-1) [1–7]. Typical ligands for PDZ domains are the high voltage-activated L-type calcium channel (LTCC) Cav1.2 and the plasma membrane calcium ATPase (PMCA) 4b, both essential for calcium homeostasis in excitable and nonexcitable cells. Voltage-gated calcium channels (Cav) allow the cellular entry of calcium ( ) ions and initiate muscle excitation-contraction coupling, neurotransmitter release, gene expression, or hormone secretion. Cav1.2 channels play a major role for the voltage-dependent influx. Its overexpression augmented influx into cardiomyocytes, thereby increasing cardiac contractile force [8, 9]. Further studies demonstrated the importance of Cav1.2 in
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