title: Lipid rafts modulate the activation but not the maintenance of store-operated Ca(2+) entry.
abstract: Different studies have reported that proteins involved in Ca(2+) entry are localized in discrete plasma membrane domains known as lipid rafts, which have been suggested to support store-operated Ca(2+) entry by facilitating STIM1 clustering in endoplasmic reticulum-plasma membrane junctions as well as the interaction of STIM1 with TRPC1. Here we report that treatment of HEK293 cells with thapsigargin (TG) results in the activation of Ca(2+) entry with two components, an early, La(3+)-sensitive, component and a late component that shows both La(3+)-sensitive and -insensitive constituents. Preincubation with methyl-beta-cyclodextrin (MbetaCD) prevented TG-induced activation of Ca(2+) entry but, in contrast, enhanced this process after its activation. Addition of MbetaCD after store depletion did not modify the La(3+)-sensitive store-operated divalent cation entry but increased La(3+)-insensitive non-capacitative Ca(2+) entry. Cell stimulation with TG results in a transient increase in Orai1 co-immunoprecipitation with STIM1, TRPC1 and TRPC6. TG-induced association of these proteins was significantly attenuated by preincubation for 30 min with MbetaCD, without altering surface expression of Orai1 or TRPCs. In contrast, the association of Orai1 with STIM1 or TRPC1 was unaffected when MbetaCD was added after store depletion with TG. Addition of MbetaCD to TG-treated cells promoted dissociation between Orai1 and TRPC6, as well as non-capacitative Ca(2+) entry. TRPC6 expression silencing indicates that MbetaCD-enhanced non-capacitative Ca(2+) entry was mediated by TRPC6. In conclusion, lipid raft domains are necessary for the activation but not the maintenance of SOCE probably due to the support of the formation of Ca(2+) signalling complexes involving Orai1, TRPCs and STIM1.
authors.affiliation: Department of Physiology (Cell Physiology Research Group), University of Extremadura, 10071 Caceres, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Galan
authors.forename: Geoffrey E
authors.initials: GE
authors.lastname: Woodard
authors.forename: Natalia
authors.initials: N
authors.lastname: Dionisio
authors.forename: Gines M
authors.initials: GM
authors.lastname: Salido
authors.forename: Juan A
authors.initials: JA
authors.lastname: Rosado
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002413:Cations, Divalent / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D000071740:ORAI1 Protein; D011485:Protein Binding; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000378:metabolism / Q000502:physiology; D000074287:TRPC6 Cation Channel; D019284:Thapsigargin / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; 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; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C096351:transient receptor potential cation channel, subfamily C, member 1; D019284:Thapsigargin; D002118:Calcium