title: Capacitative Ca2+ entry via Orai1 and stromal interacting molecule 1 (STIM1) regulates adenylyl cyclase type 8.
abstract: Capacitative Ca(2+) entry (CCE), which occurs through the plasma membrane as a result of Ca(2+) store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca(2+) store content, and the pore-forming component Orai1. However, additional factors, such as C-type transient receptor potential (TRPC) channels, may also participate in the CCE apparatus. To explore whether the store-dependent Ca(2+) entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca(2+)-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca(2+) entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca(2+) entry mediated by previous CCE candidates, diacylglycerol-activated TRPC channels, does not activate AC8. Here, we expressed Orai1 and STIM1 in HEK293 cells and saw a robust increment in CCE, and a proportional increase in CCE-stimulated AC8 activity. Inhibitors of the CCE assembly process ablated the effects on cyclase activity in both AC8-overexpressing HEK293 cells and insulin-secreting MIN6 cells endogenously expressing Ca(2+)-sensitive AC isoforms. AC8 is believed to be closely associated with the source of CCE; indeed, not only were AC8, Orai1, and STIM1 colocalized at the plasma membrane but also all three proteins occurred in lipid rafts. Together, our data indicate that Orai1 and STIM1 can be integral components of the cAMP and CCE microdomain associated with adenylyl cyclase type 8.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Agnes C L
authors.initials: AC
authors.lastname: Martin
authors.forename: Debbie
authors.initials: D
authors.lastname: Willoughby
authors.forename: Antonio
authors.initials: A
authors.lastname: Ciruela
authors.forename: Laura-Jo
authors.initials: LJ
authors.lastname: Ayling
authors.forename: Mario
authors.initials: M
authors.lastname: Pagano
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wachten
authors.forename: Anders
authors.initials: A
authors.lastname: Tengholm
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000071740:ORAI1 Protein; D051381:Rats; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000378:metabolism / Q000502:physiology
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8