Lipid rafts determine association of Orai1, STIM1 and the TRPC1 and TRPC6 proteins

Abstract

Different laboratories have shown that proteins involved in Ca2+ entry are localized in discrete plasma membrane lipid rafts, cholesterol and sphingolipid‐enriched membrane domains that scaffold certain signaling molecules while excluding others. Lipid rafts have been shown to determine targeting of STIM1 clusters to endoplasmic reticulum‐plasma membrane junctions upon store depletion, facilitating the interaction of STIM1 with TRPC1 and the activation of store‐operated Ca2+ entry (SOCE). The aim of the study was to investigate the role of lipid rafts in SOCE and the association between Orai1, STIM1 and TRPCs. Treatment of HEK293 cells with thapsigargin (TG; 1 μM) results in association of Orai1 with STIM1, TRPC1 and TRPC6. TG‐induced association of these proteins was significantly attenuated by preincubation for 30 min with 10 mM methyl‐β‐cyclodextrin (CD; p<0.05; Student's t‐test), which depletes cells of cholesterol and disrupts lipid rafts domains, without altering the association of Orai1 with STIM1 or the TRPCs in non‐stimulated cells. Preincubation with CD significantly reduced TG‐induced Ca2+ entry by 20% (p<0.05) without having any effect on Ca2+ release. In conclusion, here we show that lipid raft domains are necessary for the formation of Ca2+ signalling complexes, involving Orai1, TRPCs and STIM1, which are important for SOCE.Supported by MEC grant BFI2007‐60104 and Junta de Extremadura.