Submitted on February 11, 2008
Revised on April 8, 2008
Accepted on April 8, 2008
Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion
Geoffrey E Woodard1, GINES M SALIDO, and Juan Antonio Rosado2*
1 National Institutes of Health
2 University of Extremadura
* To whom correspondence should be addressed. E-mail: jarosado{at}unex.es.
Ca2+ 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 sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+ concentration ([Ca2+]c), as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator that prevented TG-evoked [Ca2+]c elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) SNAP-25 with Botulinum neurotoxin A (BoNT A) impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by BoNT A reduces the maintenance but not the initial stages of SOCE. 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.
