Abstract
Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca(2+) entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca(2+) store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1(D76A), which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1(D76A) puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE.
Highlights
Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca2؉ entry (SOCE)
We report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1dependent SOCE are determined by lipid raft domains (LRD)
Recent data suggest that STIM1 puncta in the peripheral region of the cells marks the location where the protein functionally interacts with plasma membrane SOCE channels and regulates Ca2ϩ entry
Summary
Cell Culture, Transfection, and Reagents—HSG and HEK293 cells were cultured in MEM and Dulbecco’s modified Eagle’s medium, respectively, supplemented with 10% fetal bovine serum and antibiotics. A similar distribution of proteins in the BF and HF fractions was observed in HEK293 cells as well (Fig. 1C) These findings were further confirmed using a detergent-free method to obtain the raft and non-raft fractions. A, presence of proteins including STIM1 and TRPC1 density gradient fractions isolated from HSG cells demonstrate partitioning into raft and non-raft domains. The role of LRD in the association of TRPC1 and STIM1 [16, 17, 30] was assessed by immunoprecipitation using the raft (BF) and non-raft (HF) fractions collected from the from high resolution currents in response to voltage ramps gradient. (Input levels of the proteins in the respective fractions are shown, adjacent panel.) Overall, these results indicate that STIM1 and TRPC1 partition into LRD and interact with each other preferentially within this domain. Triton X-100 soluble (non-raft, S) and insoluble (raft, R) fractions were isolated from
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