Stimulated association of STIM1 and Orai1 is regulated by the balance of PtdIns(4,5)P₂ between distinct membrane pools

Abstract

We have previously shown that PIP5KIβ and PIP5KIγ generate functionally distinct pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] important for antigen-stimulated Ca(2+) entry in mast cells. In the present study, we find that association of the endoplasmic reticulum (ER) Ca(2+) sensor, STIM1, and the store-operated Ca(2+) channel, Orai1, stimulated by thapsigargin-mediated ER store depletion, is enhanced by overexpression of PIP5KIβ and inhibited by overexpression of PIP5KIγ. These different PIP5KI isoforms cause differential enhancement of PtdIns(4,5)P(2) in detergent-resistant membrane (DRM) fractions, which comprise ordered lipid regions, and detergent-solubilized membrane (DSM) fractions, which comprise disordered lipid regions. Consistent with these results, the inositol 5-phosphatase L10-Inp54p, which is targeted to ordered lipids, decreases PtdIns(4,5)P(2) in the DRM fraction and inhibits thapsigargin-stimulated STIM1-Orai1 association and store-operated Ca(2+) entry, whereas the inositol 5-phosphatase S15-Inp54p, which is targeted to disordered lipids, decreases PtdIns(4,5)P(2) in the DSM fraction and enhances STIM1-Orai1 association. Removal of either the STIM1 C-terminal polylysine sequence (amino acids 677-685) or an N-terminal polyarginine sequence in Orai1 (amino acids 28-33) eliminates this differential sensitivity of STIM1-Orai1 association to PtdIns(4,5)P(2) in the distinctive membrane domains. Our results are consistent with a model of PtdIns(4,5)P(2) balance, in which store-depletion-stimulated STIM1-Orai1 association is positively regulated by the ordered lipid pool of PtdIns(4,5)P(2) and negatively regulated by PtdIns(4,5)P(2) in disordered lipid domains.

Fig. 1.

Thapsigargin-stimulated SOCE and STIM1–Orai1 FRET following cholesterol depletion and PI4K inhibition. (A) Representative Ca²⁺ responses to thapsigargin (TG) in control RBL cells (black circles) and cells treated with 4 mM MβCD for 20 minutes (red circles). Sensitivity to 1 μM Gd³⁺ is shown by addition at the indicated time points. (B) Effect of cholesterol reduction on thapsigargin-stimulated FRET between AcGFP–Orai1 and STIM1–mRFP. Cells were untreated (black) or incubated with 5 mM MβCD for 10 minutes (red) before stimulation by thapsigargin. Error bars show s.e.m. (C) Representative Ca²⁺ responses in control RBL cells (black), and cells treated with 10 μM wortmannin for 10 minutes (green) before stimulation by thapsigargin. (D) Thapsigargin-stimulated FRET between AcGFP–Orai1 and STIM1–mRFP in unperturbed cells (black) and cells treated with 10 μM wortmannin (green). Error bars show s.e.m.

Fig. 2.

Differential effects of PIP5KI isoforms and targeted inositol 5-phosphatases on thapsigargin-stimulated association between AcGFP–Orai1 and STIM1–mRFP and PtdIns(4,5)P₂ concentrations. (A) Thapsigargin (TG)-stimulated FRET between STIM1 and Orai1 in control cells (black), cells coexpressing PIP5KIβ (cyan) and cells coexpressing PIP5KIγ (red). Error bars show s.e.m. (B) Stimulated FRET between STIM1 and Orai1 in control cells (black), cells coexpressing the DRM-targeted inositol 5-phosphatase L10-Inp54p (green) and cells coexpressing the DSM-targeted inositol 5-phosphatase S15-Inp54p (pink). Error bars show s.e.m. (C) PIP5KI isoforms differentially enhance PtdIns(4,5)P₂ levels in DRMs and DSMs. Values are normalized to the total PtdIns(4,5)P₂ level in control cells from each experiment. Error bars show s.d. Unpaired, one-tailed Student's t-test between indicated populations are: *P=0.03, **P=0.05, ***P=0.05 (n=3 for each type of sample). (D) Targeted inositol 5-phosphatases selectively hydrolyze PtdIns(4,5)P₂. Values are represented as in C; *P=0.001, **P=0.007 (n=6).

Fig. 3.

Effects of targeted inositol 5-phosphatases on thapsigargin-stimulated Ca²⁺ responses. Cells were transfected with L10-Inp54p–mRFP or S15-Inp54p–mRFP and loaded with Fluo-4 before analysis of individual cells with confocal microscopy. Thapsigargin (TG) was added at t=0. Each trace corresponds to Ca²⁺ responses as averaged over at least 12 cells of each type; the control is the untransfected cells in the same fields. Error bars show s.e.m.

Fig. 4.

Removal of either the polybasic sequences on STIM1 (STIM1ΔK) or Orai1 (Orai1ΔR) alters PtdIns(4,5)P₂ pool selectivity. (A,B) Thapsigargin (TG)-stimulated FRET between AcGFP–Orai1 and STIM1ΔK–mRFP in control cells (black, A,B), cells expressing L10-Inp54p (light green, A) and cells expressing S15-Inp54p (pink, A), cells expressing PI5KIβ (cyan, B), and cells expressing PI5KIγ (bright red, B). FRET between wild-type (wt) STIM1 and Orai1 in the presence of L10-Inp54p (dark green, A; reproduced from Fig. 2B) or in the presence of PIP5KIγ (dark red, B; reproduced from Fig. 2A). (C,D) Thapsigargin-stimulated FRET between AcGFP–Orai1ΔR and STIM1–mRFP in control cells (black, C,D), cells expressing L10-Inp54p (light green, C) and cells expressing S15-Inp54p (pink, C), and cells expressing PIP5KIβ (cyan, D) and cells expressing PIP5KIγ (red, D). FRET between STIM1 and wild-type Orai1 in the presence of S15-Inp54p (purple, B; reproduced from Fig. 2B) or in the presence of PIP5KIβ (blue, D; reproduced from Fig. 2A). Error bars show s.e.m.

Fig. 5.

Proposed scheme for PtdIns(4,5)P₂ regulation of thapsigargin-stimulated association of STIM1 and Orai1. (A) In cells coexpressing the wild-type proteins STIM1 and Orai1, SOCE is initiated by the translocation of STIM1 to PtdIns(4,5)P₂ associated with ordered lipid subregions of the plasma membrane (L_O, blue; ER membrane omitted for clarity). This is followed by PtdIns(4,5)P₂-dependent redistribution of Orai1 from disordered to ordered lipid subregions (L_D, red) to facilitate the binding interaction with STIM1. (B) In cells expressing STIM1ΔK and wild-type Orai1, STIM1 lacks the polylysine sequence directing it to interact with PtdIns(4,5)P₂, allowing STIM1 to directly engage Orai1 that is primarily localized to disordered lipid subregions of the PM. This results in somewhat slower timecourses of association, which are larger in magnitude at longer times. (C) In cells expressing wild-type STIM1 and Orai1ΔR, Orai1 lacks the capacity for PtdIns(4,5)P₂-mediated redistribution between membrane pools. In this case, stimulated association of these proteins is more limited in magnitude.