title: Native STIM2 and ORAI1 proteins form a calcium-sensitive and thapsigargin-insensitive complex in cortical neurons.
abstract: In non-excitatory cells, stromal interaction molecule 1 (STIM1) and STIM2 mediate store-operated calcium entry via an interaction with ORAI1 calcium channels. However, in neurons, STIM2 over-expression appears to play a role in calcium homeostasis that is different from STIM1 over-expression. The aim of this study was to establish the role and localization of native STIM2 in the neuronal cell. Co-immunoprecipitation experiments revealed that the interaction between endogenous STIM2 and ORAI1 was greater in a low-calcium medium than in a high-calcium medium. Using a Proximity Ligation Assay (PLA), the number of apparent complexes of endogenous STIM2 with ORAI1 was quantified. No change in the number of PLA signals was observed in the presence of thapsigargin, which depletes calcium from the endoplasmic reticulum (ER). However, the number of apparent STIM2-ORAI1 complexes increased when intracellular and subsequently ER calcium concentrations were decreased by BAPTA-AM or a low-calcium medium. Both Fura-2 acetoxymethyl ester calcium imaging and PLA in the same neuronal cell indicated that the calcium responses correlated strongly with the number of endogenous STIM2-ORAI1 complexes. The small drop in calcium levels in the ER caused by decreased intracellular calcium levels appeared to initiate the calcium-sensitive and thapsigargin-insensitive interaction between STIM2 and ORAI1. We show in neuronal somata the formation of endogenous complexes of stromal interaction molecule 2 (STIM2) with ORAI1 calcium channels. Their number increased when intracellular Ca²⁺ concentrations were decreased by the Ca²⁺ chelator BAPTA-AM or a low-calcium medium (EGTA), but did not in the presence of thapsigargin (TG). We conclude that the small drop of Ca²⁺ level in endoplasmic reticulum, due to the decreased level of intracellular Ca²⁺, is sufficient to trigger STIM2-ORAI1 complex formation in a thapsigargin-insensitive manner.
authors.affiliation: Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Gruszczynska-Biegala
authors.forename: Jacek
authors.initials: J
authors.lastname: Kuznicki
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D007091:Image Processing, Computer-Assisted; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D000071740:ORAI1 Protein; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D000071738:Stromal Interaction Molecule 2; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; C560435:STIM2 protein, rat; D000071738:Stromal Interaction Molecule 2; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D002118:Calcium
keywords: ORAI1; STIM2; calcium signaling; neurons; proximity ligation assay; store-operated calcium entry