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

1 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and 2 Department 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 Ca 2+ (CRAC) channels are activated when free Ca 2+ concentration in the intracellular stores is substantially reduced and mediate sustained Ca 2+ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca 2+ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca 2+ -ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca 2+ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca 2+ chelator that prevented TG-evoked cytosolic free Ca 2+ 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 Ca 2+ 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 Ca 2+ entry. Orai1; synaptosomal-associated protein-25; Ca 2+ entry Address for reprint requests and other correspondence: J. A. Rosado, Dept. of Physiology, Univ. of Extremadura, Cáceres 10071, Spain (e-mail: jarosado{at}unex.es )