STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.

Center for Nanobiology and Structural Biology Institute of Microbiology Academy of Sciences of the Czech Republic CZ 373 33 Nové Hrady Czech Republic

Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London Ontario N6A 5C1 Canada

Institute of Biophysics Johannes Kepler University of Linz Gruberstrasse 40 4020 Linz Austria

Institute of Biophysics Medical University of Graz Harrachgasse 21 4 8010 Graz Austria

Princess Margaret Cancer Centre and Department of Medical Biophysics University of Toronto Toronto Ontario M5G 1L7 Canada

See more in PubMed

Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: Dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4:517–529. PubMed

Zweifach A, Lewis RS. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci U S A. 1993;90:6295–6299. PubMed PMC

Lewis RS. Store-operated calcium channels. Adv Second Messenger Phosphoprotein Res. 1999;33:279–307. PubMed

Liou J, Kim ML, Heo WD, Jones JT, Myers JW, Ferrell JE, Jr, Meyer T. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr Biol. 2005;15:1235–1241. PubMed PMC

Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, Stauderman KA, Cahalan MD. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature. 2005;437:902–905. PubMed PMC

Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel S-H, Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441:179–185. PubMed

Vig M, Peinelt C, Beck A, Koomoa DL, Rabah D, Koblan-Huberson M, Kraft S, Turner H, Fleig A, Penner R, Kinet J-P. CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science. 2006;312:1220–1223. PubMed PMC

Zhang SL, Yeromin AV, Zhang XH-F, Yu Y, Safrina O, Penna A, Roos J, Stauderman KA, Cahalan MD. Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity. Proc Natl Acad Sci U S A. 2006;103:9357–9362. PubMed PMC

Calloway N, Vig M, Kinet J-P, Holowka D, Baird B. Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions. Mol Biol Cell. 2009;20:389–399. PubMed PMC

Barr VA, Bernot KM, Srikanth S, Gwack Y, Balagopalan L, Regan CK, Helman DJ, Sommers CL, Oh-Hora M, Rao A, Samelson LE. Dynamic movement of the calcium sensor STIM1 and the calcium channel Orai1 in activated T-cells: Puncta and distal caps. Mol Biol Cell. 2008;19:2802–2817. PubMed PMC

Muik M, Frischauf I, Derler I, Fahrner M, Bergsmann J, Eder P, Schindl R, Hesch C, Polzinger B, Fritsch R, Kahr H, et al. Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J Biol Chem. 2008;283:8014–8022. PubMed

Derler I, Madl J, Schütz G, Romanin C. Structure, regulation and biophysics of ICRAC, STIM/Orai1. Adv Exp Med Biol. 2012;740:383–410. PubMed

Hou X, Pedi L, Diver MM, Long SB. Crystal structure of the calcium release– activated calcium channel Orai. Science. 2012;338:1308–1313. PubMed PMC

Park CY, Hoover PJ, Mullins FM, Bachhawat P, Covington ED, Raunser S, Walz T, Garcia KC, Dolmetsch RE, Lewis RS. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell. 2009;136:876–890. PubMed PMC

Derler I, Plenk P, Fahrner M, Muik M, Jardin I, Schindl R, Gruber HJ, Groschner K, Romanin C. The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1. J Biol Chem. 2013;288:29025–29034. PubMed PMC

Rothberg BS, Wang Y, Gill DL. Orai channel pore properties and gating by STIM: Implications from the Orai crystal structure. Sci Signal. 2013;6:pe9. PubMed PMC

Feske S, Prakriya M, Rao A, Lewis RS. A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients. J Exp Med. 2005;202:651–662. PubMed PMC

Derler I, Fahrner M, Carugo O, Muik M, Bergsmann J, Schindl R, Frischauf I, Eshaghi S, Romanin C. Increased hydrophobicity at the N terminus/membrane interface impairs gating of the severe combined immunodeficiency-related ORAI1 mutant. J Biol Chem. 2009;284:15903–15915. PubMed PMC

Derler I, Schindl R, Fritsch R, Romanin C. Gating and permeation of Orai channels. Front Biosci (Landmark Ed) 2012;17:1304–1322. PubMed

Hogan PG, Rao A. Store-operated calcium entry: Mechanisms and modulation. Biochem Biophys Res Commun. 2015;460:40–49. PubMed PMC

Srikanth S, Jew M, Kim K-D, Yee M-K, Abramson J, Gwack Y. Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1) Proc Natl Acad Sci U S A. 2012;109:8682–8687. PubMed PMC

Palty R, Raveh A, Kaminsky I, Meller R, Reuveny E. SARAF inactivates the store operated calcium entry machinery to prevent excess calcium refilling. Cell. 2012;149:425–438. PubMed

Srikanth S, Gwack Y. Orai1, STIM1, and their associating partners. J Physiol. 2012;590:4169–4177. PubMed PMC

Walsh CM, Chvanov M, Haynes LP, Petersen OH, Tepikin AV, Burgoyne RD. Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry. Biochem J. 2010;425:159–168. PubMed PMC

Calloway N, Owens T, Corwith K, Rodgers W, Holowka D, Baird B. Stimulated association of STIM1 and Orai1 is regulated by the balance of PtdIns(4,5)P2 between distinct membrane pools. J Cell Sci. 2011;124:2602–2610. PubMed PMC

Cao X, Choi S, Maléth JJ, Park S, Ahuja M, Muallem S. The ER/PM microdomain, PI(4,5)P2 and the regulation of STIM1-Orai1 channel function. Cell Calcium. 2015;58:342–348. PubMed PMC

Abu-Arish A, Pandzic E, Goepp J, Matthes E, Hanrahan JW, Wiseman PW. Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells. Biophys J. 2015;109:85–94. PubMed PMC

Gimpl G, Gehrig-Burger K. Cholesterol reporter molecules. Biosci Rep. 2007;27:335–358. PubMed

Dar O, Pecht I. Fcε receptor mediated Ca2+ influx into mast cells is modulated by the concentration of cytosolic free Ca2+ ions. FEBS Lett. 1992;310:123–128. PubMed

Kovarova M, Wassif CA, Odom S, Liao K, Porter FD, Rivera J. Cholesterol deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness. J Exp Med. 2006;203:1161–1171. PubMed PMC

Epand RM. Proteins and cholesterol-rich domains. Biochim Biophys Acta. 2008;1778:1576–1582. PubMed

Zhou Y, Meraner P, Kwon HT, Machnes D, Oh-hora M, Zimmer J, Huang Y, Stura A, Rao A, Hogan PG. STIM1 gates the store-operated calcium channel ORAI1 in vitro. Nat Struct Mol Biol. 2010;17:112–116. PubMed PMC

Liu Y, Zheng X, Mueller GA, Sobhany M, DeRose EF, Zhang Y, London RE, Birnbaumer L. Crystal structure of calmodulin binding domain of Orai1 in complex with Ca2+•calmodulin displays a unique binding mode. J Biol Chem. 2012;287:43030–43041. PubMed PMC

Mullins FM, Park CY, Dolmetsch RE, Lewis RS. STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels. Proc Natl Acad Sci U S A. 2009;106:15495–15500. PubMed PMC

Srikanth S, Jung H-J, Kim K-D, Souda P, Whitelegge J, Gwack Y. A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells. Nat Cell Biol. 2010;12:436–446. PubMed PMC

Li H, Papadopoulos V. Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology. 1998;139:4991–4997. PubMed

Muik M, Fahrner M, Derler I, Schindl R, Bergsmann J, Frischauf I, Groschner K, Romanin C. A cytosolic homomerization and a modulatory domain within STIM1 C terminus determine coupling to ORAI1 channels. J Biol Chem. 2009;284:8421–8426. PubMed PMC

Klein U, Gimpl G, Fahrenholz F. Alteration of the myometrial plasma membrane cholesterol content with β-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry. 1995;34:13784–13793. PubMed

Raghuraman H, Chattopadhyay A. Interaction of melittin with membrane cholesterol: A fluorescence approach. Biophys J. 2004;87:2419–2432. PubMed PMC

Hölttä-Vuori M, Uronen R-L, Repakova J, Salonen E, Vattulainen I, Panula P, Li Z, Bittman R, Ikonen E. BODIPY-cholesterol: A new tool to visualize sterol trafficking in living cells and organisms. Traffic. 2008;9:1839–1849. PubMed

Liao Y, Plummer NW, George MD, Abramowitz J, Zhu MX, Birnbaumer L. A role for Orai in TRPC-mediated Ca2+ entry suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+ entry. Proc Natl Acad Sci U S A. 2009;106:3202–3206. PubMed PMC

Dionisio N, Galán C, Jardín I, Salido GM, Rosado JA. Lipid rafts are essential for the regulation of SOCE by plasma membrane resident STIM1 in human platelets. Biochim Biophys Acta. 2011;1813:431–437. PubMed

Galan C, Woodard GE, Dionisio N, Salido GM, Rosado JA. Lipid rafts modulate the activation but not the maintenance of store-operated Ca2+ entry. Biochim Biophys Acta. 2010;1803:1083–1093. PubMed

Jardin I, Salido GM, Rosado JA. Role of lipid rafts in the interaction between hTRPC1, Orai1 and STIM1. Channels. 2008;2:401–403. PubMed

Chantôme A, Potier-Cartereau M, Clarysse L, Fromont G, Marionneau-Lambot S, Guéguinou M, Pagès J-C, Collin C, Oullier T, Girault A, Arbion F, et al. Pivotal role of the lipid Raft SK3–Orai1 complex in human cancer cell migration and bone metastases. Cancer Res. 2013;73:4852–4861. PubMed

Miller CM, Brown AC, Mittal J. Disorder in cholesterol-binding functionality of CRAC peptides: A molecular dynamics study. J Phys Chem B. 2014;2014:13169–13174. PubMed PMC

Li H, Yao Z-x, Degenhardt B, Teper G, Papadopoulos V. Cholesterol binding at the cholesterol recognition/ interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide. Proc Natl Acad Sci U S A. 2001;98:1267–1272. PubMed PMC

Scolari S, Muller K, Bittman R, Herrmann A, Muller P. Interaction of mammalian seminal plasma protein PDC-109 with cholesterol: Implications for a putative CRAC domain. Biochemistry. 2010;49:9027–9031. PubMed

Rosenhouse-Dantsker A, Noskov S, Durdagi S, Logothetis DE, Levitan I. Identification of novel cholesterol-binding regions in Kir2 channels. J Biol Chem. 2013;288:31154–31164. PubMed PMC

Picazo-Juárez G, Romero-Suárez S, Nieto-Posadas A, Llorente I, Jara-Oseguera A, Briggs M, McIntosh TJ, Simon SA, Ladrón-de-Guevara E, Islas LD, Rosenbaum T. Identification of a binding motif in the S5 helix that confers cholesterol sensitivity to the TRPV1 ion channel. J Biol Chem. 2011;286:24966–24976. PubMed PMC

Kucerka N, Nieh MP, Pencer J, Sachs JN, Katsaras J. What determines the thickness of a biological membrane. Gen Physiol Biophys. 2009;28:117–125. PubMed

Nyholm TKM, van Duyl B, Rijkers DTS, Liskamp RMJ, Killian JA. Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain. Biophys J. 2011;101:1959–1967. PubMed PMC

Yuan C, O’Connell RJ, Jacob RF, Mason RP, Treistman SN. Regulation of the gating of BKCa channel by lipid bilayer thickness. J Biol Chem. 2007;282:7276–7286. PubMed

Tong J, Briggs MM, McIntosh TJ. Water permeability of aquaporin-4 channel depends on bilayer composition, thickness, and elasticity. Biophys J. 2012;103:1899–1908. PubMed PMC

Kato N, Nakanishi M, Hirashima N. Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells. Biochemistry. 2003;42:11808–11814. PubMed

Mahammad S, Parmryd I. Cholesterol depletion using methyl-β-cyclodextrin. Methods Mol Biol. 2015;1232:91–102. PubMed

Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–3450. PubMed

Singh A, Hamedinger D, Hoda J-C, Gebhart M, Koschak A, Romanin C, Striessnig J. C-terminal modulator controls Ca2+-dependent gating of Cav 1.4 L-type Ca2+ channels. Nat Neurosci. 2006;9:1108–1116. PubMed

Derler I, Hofbauer M, Kahr H, Fritsch R, Muik M, Kepplinger K, Hack ME, Moritz S, Schindl R, Groschner K, Romanin C. Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+-dependent inactivation. J Physiol. 2006;577:31–44. PubMed PMC

Xia Z, Liu Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J. 2001;81:2395–2402. PubMed PMC

Berney C, Danuser G. FRET or no FRET: A quantitative comparison. Biophys J. 2003;84:3992–4010. PubMed PMC

Muik M, Fahrner M, Schindl R, Stathopulos P, Frischauf I, Derler I, Plenk P, Lackner B, Groschner K, Ikura M, Romanin C. STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation. EMBO J. 2011;30:1678–1689. PubMed PMC

Schindl R, Fritsch R, Jardin I, Frischauf I, Kahr H, Muik M, Riedl MC, Groschner K, Romanin C. Canonical transient receptor potential (TRPC) 1 acts as a negative regulator for vanilloid TRPV6-mediated Ca2+ influx. J Biol Chem. 2012;287:35612–35620. PubMed PMC

Bisaillon JM, Motiani RK, Gonzalez-Cobos JC, Potier M, Halligan KE, Alzawahra WF, Barroso M, Singer HA, Jourd’heuil D, Trebak M. Essential role for STIM1/Orai1-mediated calcium influx in PDGF-induced smooth muscle migration. Am J Physiol Cell Physiol. 2010;298:C993–C1005. PubMed PMC

Woodard GE, Salido GM, Rosado JA. Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion. Am J Physiol Cell Physiol. 2008;294:C1323–C1331. PubMed

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