Endogenous STIM2 and endogenous ORAI1 form a calcium-sensitive and thapsigargin-insensitive complex in cortical neurons

• Published in: Acta neurobiologiae experimentalis Vol. 73; no. 1; pp. 189 - 190
• Main Authors: Gruszczynska-Biegala, J, Kuznicki, J
• Format: Journal Article
• Language: English
• Published: 01-01-2013
• ISSN: 0065-1400

ER calcium sensors (STIM1, STIM2) and calcium channel-ORAI1 interaction is crucial for store-operated calcium entry (SOCE) in non-excitable cells, but in neurons their localization and dynamics are not clear. We showed earlier that in neurons STIM1 is involved in thapsigargin induced SOCE, while STIM2 is active after EGTA-driven depletion of extracellular Ca super(2+) (Klejman et al. 2009, Gruszczynska-Biegala et al. 2011). To confirm that this is not due to the overexpression of exogenous proteins we used Proximity Ligation Assay to analyze activities of endogenous proteins. Cortical neurons were cultured in 2 mM CaCL sub(2), 2 mM EGTA or 2 mu M thapsigargin, fixed and incubated with primary antibodies anti-STIM2 and anti-ORAI1. The pairs of appropriate secondary antibodies with conjugated oligonucleotides were then added and Duolink II was performed to create the fluorescent products. We detected in situ the endogenous STIM2/ORAI1 complexes in somata and quantified in single neurons the number of hetero- and homo-complexes. The amount of hetero-complexes increased up to 10-fold in response to calcium depletion by EGTA. The number of STIM2/ORAI1 endogenous complexes correlated well with the number of overexpressed YFP-STIM2/ORAI1 complexes formed under the same conditions (Gruszczynska-Biegala et al. 2011). By co-immunoprecipitation we confirmed the in situ interaction between endogenous STIM2 and ORAI1 and that the interaction is increased after Ca super(2-) depletion in the medium. In conclusion, the present study provides a novel finding that endogenous STIM2 can physically interact and form hetero-complexes with endogenous ORAI1 in TG-insensitive manner, suggesting that the proteins are key molecules that underlie the regulation of basal calcium levels in neurons and constitutive calcium entry.