PICK1/Calcineurin Suppress ASIC1-mediated Ca2+ Entry in Rat Pulmonary Arterial Smooth Muscle Cells.
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Acid sensing ion channel 1 (ASIC1) contributes to Ca(2+) influx and contraction in pulmonary arterial smooth muscle cells (PASMC). ASIC1 binds the PDZ (PSD-95/Dlg/ZO-1) domain of the protein interacting with C kinase 1 (PICK1) and this interaction is important for the subcellular localization and/or activity of ASIC1. Therefore, we first hypothesized that PICK1 facilitates ASIC1 dependent Ca(2+) influx in PASMC by promoting plasma membrane localization. Using Duolink to determine protein-protein interactions and a biotinylation assay to assess membrane localization, we demonstrated that the PICK1 PDZ domain inhibitor, FSC231, diminished the co-localization of PICK1 and ASIC1, but did not limit ASIC1 plasma membrane localization. Although stimulation of store-operated Ca(2+) entry (SOCE) greatly enhanced co-localization between ASIC1 and PICK1, both FSC231 and shRNA knockdown of PICK1 largely augmented SOCE. These data suggest PICK1 imparts a basal inhibitory effect on ASIC1 Ca(2+) entry in PASMC and led to an alternative hypothesis that PICK1 facilitates the interaction between ASIC1 and negative intracellular modulators, namely PKC and/or the calcium-calmodulin-activated phosphatase, calcineurin. FSC231 limited PKC-mediated inhibition of SOCE, supporting a potential role for PICK1 in this response. Additionally, we found PICK1 inhibits ASIC1-mediated SOCE through an effect of calcineurin to dephosphorylate the channel. Furthermore, it appears PICK1/calcineurin-mediated regulation of SOCE opposes PKA phosphorylation and activation of ASIC1. Together our data suggest PKA and PICK1/calcineurin differentially regulate ASIC1-mediated SOCE and these modulatory complexes are important in determining downstream Ca(2+) signaling.Copyright © 2015, American Journal of Physiology - Cell Physiology.