Platelet-Derived Growth Factor Maintains Stored Calcium Through a Nonclustering Orai1 Mechanism But Evokes Clustering If the Endoplasmic Reticulum Is Stressed by Store Depletion

Abstract

Rationale: Calcium entry through Orai1 channels drives vascular smooth muscle cell migration and neointimal hyperplasia. The channels are activated by the important growth factor platelet-derived growth factor (PDGF). Channel activation is suggested to depend on store depletion, which redistributes and clusters stromal interaction molecule 1 (STIM1), which then coclusters and activates Orai1. Objective: To determine the relevance of STIM1 and Orai1 redistribution in PDGF responses. Methods and Results: Vascular smooth muscle cells were cultured from human saphenous vein. STIM1 and Orai1 were tagged with green and red fluorescent proteins to track them in live cells. Under basal conditions, the proteins were mobile but mostly independent of each other. Inhibition of sarco-endoplasmic reticulum calcium ATPase led to store depletion and dramatic redistribution of STIM1 and Orai1 into coclusters. PDGF did not evoke redistribution, even though it caused calcium release and Orai1-mediated calcium entry in the same time period. After chemical blockade of Orai1-mediated calcium entry, however, PDGF caused redistribution. Similarly, mutagenic disruption of calcium flux through Orai1 caused PDGF to evoke redistribution, showing that calcium flux through the wild-type channels had been filling the stores. Acidification of the extracellular medium to pH 6.4 caused inhibition of Orai1-mediated calcium entry and conferred capability for PDGF to evoke complete redistribution and coclustering. Conclusions: The data suggest that PDGF has a nonclustering mechanism by which to activate Orai1 channels and maintain calcium stores replete. Redistribution and clustering become important, however, when the endoplasmic reticulum stress signal of store depletion arises, for example when acidosis inhibits Orai1 channels.