Calcium (Ca2+) is a physiological key factor, and the precise modulation of free cytosolic Ca2+ levels regulates multiple cellular functions. Store‐operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ homeostasis, and is mediated by the concerted activity of the Ca2+ sensor STIM1 and the Ca2+ channel ORAI1. Dominant gain‐of‐function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss‐of‐function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation‐dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non‐muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca2+ homeostasis and leading to muscle dysfunction.

Key points Gain‐of‐function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM‐associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK‐7975A prevents the constitutive activity of TAM‐associated channels and might be used in therapy for patients suffering from TAM. Abstract Skeletal muscle differentiation relies on store‐operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+‐permeable ORAI channels. Gain‐of‐function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK‐293T cells or human primary myoblasts, the mutated channels increased basal and store‐operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK‐7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1‐V107M constitutive activity and revealed larger STIM1‐gated V107M‐ and T184M‐mediated currents with conserved fast and slow Ca2+‐dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1−/−/Stim2−/− mouse embryonic fibroblasts expressing the binding‐deficient STIM1‐F394H mutant, indicating a higher sensitivity for STIM1‐mediated gating, with ORAI1‐T184M gain‐of‐function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain‐of‐function mutations in ORAI1.

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca2+ entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca2+ influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca2+ imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1−/−/Stim2−/− fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca2+ elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.

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Twenty-seven Lactobacillus pentosus strains, and the undefined starter for table olives from which they were isolated, were characterised for their technological properties: tolerance to low temperature, high salt concentration, alkaline pH, and olive leaf extract; acidifying ability; oleuropein degradation; hydrogen peroxide and lactic acid production. Two strains with appropriate technological properties were selected. Then, table olive fermentation in vats, with the original starter, the selected strains, and without starter (spontaneous fermentation) were compared. Starters affected some texture profile parameters. The undefined culture resulted in the most effective Enterobacteriaceae reduction, acidification and olive debittering, while the selected strains batch showed the lowest antioxidant activity. Our results show that the best candidate strains cannot guarantee better fermentation performance than the undefined biodiverse mix from which they originate.