The molecular nature of store-operated Ca 2؉ -selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca 2؉ -selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca 2؉ entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca 2؉ entry and Ca 2؉ -selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca 2؉ -selective store-operated Ca 2؉ entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca 2؉ entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca 2؉ sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle.

Store-operated Ca2؉ entry (SOCE) is likely the most common mode of regulated influx of Ca 2؉ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca 2؉ current (I CRAC ). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca 2؉ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and I CRAC -like currents in cells coexpressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than ؉50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca 2؉ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.In many cell types, the activation of phospholipase C through G protein-coupled receptors liberates Ca 2ϩ from the lumen of the endoplasmic reticulum (ER current (I CRAC ), first described in mast cells (3) and since recorded in several cell types (4). Until recently, the mechanism by which I CRAC is activated by store depletion, as well as the channels themselves, was unknown. However, the discoveries of both STIM1 (5, 6) and Orai1 (CRACM1) (7-9) have revealed two key molecular components of the I CRAC -signaling pathway.It is now clear that STIM1 functions as the Ca 2ϩ sensor within the ER, whereas members of the family of Orai proteins (including Orai1, -2, and -3) function as pore-forming subunits of CRAC channels in the plasma membrane. When intracellular Ca 2ϩ stores are depleted, STIM1 rearranges from a fibrillar localization that depends on microtubules to discrete punctate structures near the plasma membrane (6, 10 -12). Remarkably, Orai1 channels also rearrange into punctate structures, in response to store depletion, that coincide with those formed by STIM1 (13-15). Thus, highly orchestrated molecular rearrangements underlie I CRAC activation.Overexpression of Orai1 together with STIM1 in HEK293 cells produces unusually large currents with biophysical properties similar to I CRAC (9, 16 -18), suggesting that either these two proteins are sufficien...

Peri-operative SARS-CoV-2 infection increases postoperative mortality. The aim of this study was to determine the optimal duration of planned delay before surgery in patients who have had SARS-CoV-2 infection. This international, multicentre, prospective cohort study included patients undergoing elective or emergency surgery during October 2020. Surgical patients with pre-operative SARS-CoV-2 infection were compared with those without previous SARS-CoV-2 infection. The primary outcome measure was 30-day postoperative mortality. Logistic regression models were used to calculate adjusted 30-day mortality rates stratified by time from diagnosis of SARS-CoV-2 infection to surgery. Among 140,231 patients (116 countries), 3127 patients (2.2%) had a pre-operative SARS-CoV-2 diagnosis. Adjusted 30-day mortality in patients without SARS-CoV-2 infection was 1.5% (95%CI 1.4-1.5). In patients with a pre-operative SARS-CoV-2 diagnosis, mortality was increased in patients having surgery within 0-2 weeks, 3-4 weeks and 5-6 weeks of the diagnosis (odds ratio (95%CI) 4.1 (3.3-4.8), 3.9 (2.6-5.1) and 3.6 (2.0-5.2), respectively). Surgery performed ≥ 7 weeks after SARS-CoV-2 diagnosis was associated with a similar mortality risk to baseline (odds ratio (95%CI) 1.5 (0.9-2.1)). After a ≥ 7 week delay in undertaking surgery following SARS-CoV-2 infection, patients with ongoing symptoms had a higher mortality than patients whose symptoms had resolved or who had been asymptomatic (6.0% (95%CI 3.2-8.7) vs. 2.4% (95%CI 1.4-3.4) vs. 1.3% (95%CI 0.6-2.0), respectively). Where possible, surgery should be delayed for at least 7 weeks following SARS-CoV-2 infection. Patients with ongoing symptoms ≥ 7 weeks from diagnosis may benefit from further delay.