Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC128

Poster Communications

SNAP-25-dependent exocytosis regulates plasma membrane insertion of Orai1 and contributes to store-operated Ca2+ influx

J. A. Rosado1, G. E. Woodard2, G. M. Salido1

1. PHYSIOLOGY, UNIVERSITY OF EXTREMADURA, Caceres, Spain. 2. 2National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, USA.

Ca2+ release-activated Ca2+ (CRAC) channels are activated when free Ca2+ concentration in the intracellular Ca2+ stores is substantially reduced and mediate sustained Ca2+ entry in a number of cell types. Although the molecular identity of CRAC channels has not been precisely determined, recent findings have identified Orai1 as a CRAC channel subunit. SNARE proteins have been shown to be important for the insertion of Ca2+ channels of the TRPC family in the plasma membrane, whose surface expression is determined by a recycling-type of trafficking mechanism in a number of cells (Singh et al., 2004). The aim of the present study was to investigate whether Orai1 surface expression is regulated by depletion of the intracellular Ca2+ stores and the possible involvement of the SNARE protein SNAP-25 in this process. Human HeLa and embryonic kidney 293T (HEK-293T) cells were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium. Cytosolic free Ca2+ concentration ([Ca2+]i) measurement, Western blotting and biotinylation of cell-surface proteins were performed as previously described (Rosado et al., 2000a; Lopez et al., 2006). Passive Ca2+ store depletion using the inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (TG), enhances the surface expression of Orai1 to 246 ± 22% and 193 ± 20% of control (resting cells), in HeLa and HEK cells, respectively (mean ± SEM; P<0.05, Student's t-test; n=4). This effect depends on rises in [Ca2+]i, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator that prevented TG-evoked elevation in [Ca2+]i. 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. BoNT A also decreased TG-induced Ca2+ entry by 60%. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves the protein SNAP-25, a process that contributes to store-dependent Ca2+ entry.

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