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STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca(2+) 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 Ca(2+) influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca(2+) 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 Ca(2+) 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., (© 2015. Published by The Company of Biologists Ltd.)

Cytosolic Ca(2+) signals encoded by repetitive Ca(2+) releases rely on two processes to refill Ca(2+) stores: Ca(2+) reuptake from the cytosol and activation of a Ca(2+) influx via store-operated Ca(2+) entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca(2+) sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca(2+) channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca(2+) channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca(2+) signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca(2+) sensor for their Ca(2+) homeostasis and intracellular signaling.

Our recent work identified store-operated Ca(2+) entry (SOCE) as the critical Ca(2+) source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370-5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca(2+) mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca(2+) content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca(2+)-content during repeated KCl-induced Ca(2+) releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca(2+)](in) involving store-operated Ca(2+) entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca(2+) sensor activating Orai Ca(2+) channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca(2+)](in) and intracellular Ca(2+) store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K(+) channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.