title: Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1.
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.affiliation: Department of Physiology and Pharmacology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Alla D
authors.initials: AD
authors.lastname: Lyfenko
authors.forename: Robert T
authors.initials: RT
authors.lastname: Dirksen
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D004558:Electric Stimulation / Q000379:methods; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D032446:Myoblasts / Q000502:physiology*; D009200:Myocardial Contraction / Q000502:physiology*; D000071740:ORAI1 Protein; D000071737:Stromal Interaction Molecule 1
chemical_list: D015220:Calcium Channels; D008562:Membrane Glycoproteins; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1; D002118:Calcium
references.citation: J Physiol. 2006 Aug 15;575(Pt 1):69-81
references.pmid: 16777939
references.citation: Nat Immunol. 2008 Jan;9(1):89-96
references.pmid: 18059270
references.citation: Cell Calcium. 2007 Aug;42(2):205-11
references.pmid: 17517433
references.citation: Am J Respir Cell Mol Biol. 2008 Jun;38(6):744-9
references.pmid: 18239188
references.citation: Curr Biol. 2006 Oct 24;16(20):2073-9
references.pmid: 16978865
references.citation: Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2941-4
references.pmid: 12601149
references.citation: Nat Cell Biol. 2002 May;4(5):379-83
references.pmid: 11988740
references.citation: J Biol Chem. 2005 Jul 1;280(26):24804-15
references.pmid: 15878845
references.citation: J Biol Chem. 2008 May 9;283(19):12935-40
references.pmid: 18326500
references.citation: J Biol Chem. 2006 Nov 3;281(44):33477-86
references.pmid: 16945924
references.citation: Proc Natl Acad Sci U S A. 2004 Nov 2;101(44):15793-8
references.pmid: 15505226
references.citation: Mol Pharmacol. 2008 Apr;73(4):1203-12
references.pmid: 18171728
references.citation: J Biol Chem. 2007 Dec 28;282(52):37471-8
references.pmid: 17942409
references.citation: Nature. 2006 Sep 14;443(7108):230-3
references.pmid: 16921383
references.citation: J Gen Physiol. 2001 Sep;118(3):277-90
references.pmid: 11524458
references.citation: Proc Natl Acad Sci U S A. 2004 Jun 22;101(25):9387-92
references.pmid: 15199180
references.citation: Physiol Rev. 2005 Apr;85(2):757-810
references.pmid: 15788710
references.citation: Mol Cell. 2000 Aug;6(2):421-31
references.pmid: 10983988
references.citation: Physiol Genomics. 2005 Sep 21;23(1):72-8
references.pmid: 15998745
references.citation: J Physiol. 2007 Aug 15;583(Pt 1):25-36
references.pmid: 17615095
references.citation: Nature. 2006 May 11;441(7090):179-85
references.pmid: 16582901
references.citation: Nat Cell Biol. 2008 Jun;10(6):688-97
references.pmid: 18488020
references.citation: Science. 2006 May 26;312(5777):1220-3
references.pmid: 16645049
references.citation: Nat Rev Immunol. 2007 Sep;7(9):690-702
references.pmid: 17703229
references.citation: J Physiol. 2001 May 15;533(Pt 1):185-99
references.pmid: 11351027
references.citation: Exp Gerontol. 2007 Apr;42(4):309-19
references.pmid: 17174053
references.citation: J Physiol. 2007 Aug 15;583(Pt 1):81-97
references.pmid: 17569733
references.citation: J Cell Biol. 2005 May 9;169(3):435-45
references.pmid: 15866891
references.citation: Curr Biol. 2005 Jul 12;15(13):1235-41
references.pmid: 16005298
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):2895-900
references.pmid: 18287061