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      Pre-assembled Ca 2+ entry units and constitutively active Ca 2+ entry in skeletal muscle of calsequestrin-1 knockout mice


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          Mice lacking calsequestrin-1 have reduced levels of releasable Ca 2+ in the sarcoplasmic reticulum of their skeletal muscles. Michelucci et al. reveal that this is compensated by constitutive assembly of STIM1 and Orai1 into Ca 2+ entry units, promoting both constitutive and store-operated Ca 2+ entry.


          Store-operated Ca 2+ entry (SOCE) is a ubiquitous Ca 2+ influx mechanism triggered by depletion of Ca 2+ stores from the endoplasmic/sarcoplasmic reticulum (ER/SR). We recently reported that acute exercise in WT mice drives the formation of Ca 2+ entry units (CEUs), intracellular junctions that contain STIM1 and Orai1, the two key proteins mediating SOCE. The presence of CEUs correlates with increased constitutive- and store-operated Ca 2+ entry, as well as sustained Ca 2+ release and force generation during repetitive stimulation. Skeletal muscle from mice lacking calsequestrin-1 (CASQ1-null), the primary Ca 2+-binding protein in the lumen of SR terminal cisternae, exhibits significantly reduced total Ca 2+ store content and marked SR Ca 2+ depletion during high-frequency stimulation. Here, we report that CEUs are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of sedentary CASQ1-null mice. The higher density of CEUs in EDL (39.6 ± 2.1/100 µm 2 versus 2.0 ± 0.3/100 µm 2) and FDB (16.7 ± 1.0/100 µm 2 versus 2.7 ± 0.5/100 µm 2) muscles of CASQ1-null compared with WT mice correlated with enhanced constitutive- and store-operated Ca 2+ entry and increased expression of STIM1, Orai1, and SERCA. The higher ability to recover Ca 2+ ions via SOCE in CASQ1-null muscle served to promote enhanced maintenance of peak Ca 2+ transient amplitude, increased dependence of luminal SR Ca 2+ replenishment on BTP-2-sensitive SOCE, and increased maintenance of contractile force during repetitive, high-frequency stimulation. Together, these data suggest that muscles from CASQ1-null mice compensate for the lack of CASQ1 and reduction in total releasable SR Ca 2+ content by assembling CEUs to promote constitutive and store-operated Ca 2+ entry.

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          Most cited references53

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          A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.

          Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.
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            STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx.

            Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.
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              STIM1, an essential and conserved component of store-operated Ca2+ channel function

              Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

                Author and article information

                J Gen Physiol
                J Gen Physiol
                The Journal of General Physiology
                Rockefeller University Press
                05 October 2020
                05 August 2020
                : 152
                : 10
                [1 ]Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY
                [2 ]Center for Advanced Studies and Technologies, University G. d’Annunzio of Chieti, Chieti, Italy
                [3 ]Department of Neuroscience, Imaging and Clinical Sciences, University G. d’Annunzio of Chieti, Chieti, Italy
                [4 ]Department of Medicine and Ageing Sciences, University G. d’Annunzio of Chieti, Chieti, Italy
                Author notes
                Correspondence to Robert T. Dirksen: Robert_Dirksen@ 123456URMC.Rochester.edu

                F. Protasi and R.T. Dirksen contributed equally to this article.

                This work is part of the special collection entitled “Electrical Signaling in the Heart and Nervous System: A Joint Meeting of the Society of General Physiologists and Latin American Society of Biophysicists.”

                © 2020 Michelucci et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

                Page count
                Pages: 14
                Funded by: National Institutes of Health, DOI http://dx.doi.org/10.13039/100000002;
                Award ID: AR059646
                Funded by: Ministry of Education, University, and Research, DOI http://dx.doi.org/10.13039/501100003407;
                Award ID: 2015ZZR4W3
                Funded by: Italian Telethon Non-Profit Organization Foundation;
                Award ID: GGP13213
                Funded by: Alfred and Eleanor Wedd Fund;
                Cellular physiology
                SGP/SOBLA Valparaiso Special Issue

                Anatomy & Physiology
                Anatomy & Physiology


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