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Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1.
J Physiol 2008; 586(20):4815-24JP

Abstract

In non-excitable cells, agonist-induced depletion of intracellular Ca(2+) stores triggers Ca(2+) influx via a process termed store-operated Ca(2+) entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca(2+) sensor and Orai1 functions as the Ca(2+)-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca(2+) entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca(2+) entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca(2+) entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca(2+) entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca(2+) measurements and Mn(2+) quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.

Authors+Show Affiliations

Department of Physiology and Pharmacology, University of Rochester, Rochester, NY 14642, USA.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

18772199

Citation

Lyfenko, Alla D., and Robert T. Dirksen. "Differential Dependence of Store-operated and Excitation-coupled Ca2+ Entry in Skeletal Muscle On STIM1 and Orai1." The Journal of Physiology, vol. 586, no. 20, 2008, pp. 4815-24.
Lyfenko AD, Dirksen RT. Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1. J Physiol (Lond). 2008;586(20):4815-24.
Lyfenko, A. D., & Dirksen, R. T. (2008). Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1. The Journal of Physiology, 586(20), pp. 4815-24. doi:10.1113/jphysiol.2008.160481.
Lyfenko AD, Dirksen RT. Differential Dependence of Store-operated and Excitation-coupled Ca2+ Entry in Skeletal Muscle On STIM1 and Orai1. J Physiol (Lond). 2008 Oct 15;586(20):4815-24. PubMed PMID: 18772199.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1. AU - Lyfenko,Alla D, AU - Dirksen,Robert T, Y1 - 2008/09/04/ PY - 2008/9/6/pubmed PY - 2009/1/10/medline PY - 2008/9/6/entrez SP - 4815 EP - 24 JF - The Journal of physiology JO - J. Physiol. (Lond.) VL - 586 IS - 20 N2 - In non-excitable cells, agonist-induced depletion of intracellular Ca(2+) stores triggers Ca(2+) influx via a process termed store-operated Ca(2+) entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca(2+) sensor and Orai1 functions as the Ca(2+)-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca(2+) entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca(2+) entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca(2+) entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca(2+) entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca(2+) measurements and Mn(2+) quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities. SN - 1469-7793 UR - https://www.unboundmedicine.com/medline/citation/18772199/Differential_dependence_of_store_operated_and_excitation_coupled_Ca2+_entry_in_skeletal_muscle_on_STIM1_and_Orai1_ L2 - https://doi.org/10.1113/jphysiol.2008.160481 DB - PRIME DP - Unbound Medicine ER -