Lipid rafts modulate the activation but not the maintenance of store-operated Ca2+ entry

Different studies have reported that proteins involved in Ca2+ entry are localized in discrete plasma membrane domains known as lipid rafts, which have been suggested to support store-operated Ca2+ 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 Ca2+ entry with two components, an early, La3+-sensitive, component and a late component that shows both La3+-sensitive and -insensitive constituents. Preincubation with methyl-β-cyclodextrin (MβCD) prevented TG-induced activation of Ca2+ entry but, in contrast, enhanced this process after its activation. Addition of MβCD after store depletion did not modify the La3+-sensitive store-operated divalent cation entry but increased La3+-insensitive non-capacitative Ca2+ 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 MβCD, without altering surface expression of Orai1 or TRPCs. In contrast, the association of Orai1 with STIM1 or TRPC1 was unaffected when MβCD was added after store depletion with TG. Addition of MβCD to TG-treated cells promoted dissociation between Orai1 and TRPC6, as well as non-capacitative Ca2+ entry. TRPC6 expression silencing indicates that MβCD-enhanced non-capacitative Ca2+ 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 Ca2+ signalling complexes involving Orai1, TRPCs and STIM1.