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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca²⁺ store depletion

¹National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and ²Department of Physiology (Cell Physiology Research Group), University of Extremadura, Cáceres, Spain

Submitted 11 February 2008 ; accepted in final form 8 April 2008

Ca⁺ release-activated Ca²⁺ (CRAC) channels are activated when free Ca²⁺ concentration in the intracellular stores is substantially reduced and mediate sustained Ca²⁺ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca²⁺ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca²⁺-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca²⁺ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca²⁺ chelator that prevented TG-evoked cytosolic free Ca²⁺ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca²⁺ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca²⁺ entry.

Orai1; synaptosomal-associated protein-25; Ca²⁺ entry