Rationale: Calcium entry through Orai1 channels drives vascular smooth muscle cell migration and neointimal hyperplasia. The channels are activated by the important growth factor PDGF. Channel activation is suggested to depend on store-depletion which redistributes and clusters STIM1 which then co-clusters 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 co-clusters. 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 non-clustering mechanism to activate Orai1 channels and maintain calcium stores replete. Redistribution and clustering become important, however, when the ER stress signal of store-depletion arises, for example when acidosis inhibits Orai1 channels.
37th Congress of IUPS (Birmingham, UK) (2013) Proc 37th IUPS, PCA386
Poster Communications: PDGF maintains stored calcium through a non-clustering Orai1 mechanism but evokes clustering if the ER is stressed by store depletion
L. McKeown1,2, N. K. Moss1,2, P. Turner1,3, J. Li1,2, N. Heath1, D. Burke3, O. David3, M. S. Gilthorpe4, P. E. Karen1,2, D. J. Beech1,2
1. School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom. 2. Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, United Kingdom. 3. Leeds Teaching Hospitals, Leeds General Infirmary, Leeds, United Kingdom. 4. Faculty of Medicine & Health, University of Leeds, Leeds, United Kingdom.
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Where applicable, experiments conform with Society ethical requirements.