Cholesterol modulates the cellular localization of Orai1 channels and its disposition among membrane domains

Abstract

Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE. In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern (unobstructed diffusion) opposite to basal cholesterol conditions were most of Orai1 channels moves in a confined space, as assessed by Fluorescence Correlation Spectroscopy, Cav1 overexpression inhibited these alterations maintaining Orai1 into a confined and partially confined movement. These results not only highlight the complex effect of cholesterol regulation on SOCE, but also indicate a direct regulatory effect on Orai1 localization and compartmentalization by this lipid.

Keywords: Caveolae; Cholesterol; Fluorescence Correlation Spectroscopy (FCS); Orai1; SOCE.

Figure 1. SOCE reduction is induced by Orai1 internalization

(A) Calcium response measurements of cells with basal, low concentration of cholesterol and cholesterol replenished at the plasma membrane. (B) Area under the curve (AUC) of calcium entry after depletion of the ER with TG. (C) SPIM measurements of increment of fluorescent signal in the cytoplasm due to the redistribution of mCherry-Orai1. The black ROI define cytoplasm (D) Kaede-Orai1 is photoconverted (from green to red emission) in PM and change localization to the cytoplasm of cells exposed to MβCD. (E) Amount of Orai1 in PM of cells with basal concentration and low concentration of cholesterol at the plasma membrane. Black: Basal cholesterol, Red: Low cholesterol, Blue: cholesterol replenished. n ≥ 20 cells, n ≥ 10 independent biotinylation experiments, error bars: S.E.M. ***p < 0.001 or **p < 0.01.

Figure 2. Cav1 overexpression prevents the effects of cholesterol depletion on SOCE

(A) Calcium response measurements of cells overexpressing Cav1-GFP, with basal concentration (black line) low concentration of cholesterol at the plasma membrane (red line) and cells with only endogenous Cav1-GFP (green line). (B) Area under the curve (AUC) of calcium entry in response to ER depletion induced by TG. (C) Localization of mCherry-Orai1 in control cells (Basal) and low concentration of cholesterol at the plasma membrane (MβCD). (D) Change of fluorescent signal (mCherry-Orai1) localization measurements in cytoplasm measured with confocal microscopy. (E) Amount of biotinylated Orai1 in control cells and low concentration of cholesterol at the PM. All the cells overexpressed Cav1-GFP. Black: Basal cholesterol, Red: Low cholesterol. n ≥ 20 cells, n ≥ 10 independent biotinylation experiments, error bars: S.E.M.

Figure 3. Orai1 and Cav1 interaction is influenced by SOCE activation

(A) Representative images of FRET efficiency measurements using acceptor photobleaching methodology between mCherry-Orai1 and Cav1-GFP obtained by TIRF. Top panels show the pre photobleaching, bottom post photobleaching. Yellow circle: control with no photobleaching protocol, yellow square: area with acceptor photobleaching protocol (B) FRET efficiency plot at different conditions, from left to right; Basal, TG, MβCD, and MβCD + TG. (C) Top panel shows representative western blot membranes, upper and bottom membrane, show respectively co-immunoprecipitated Cav1-GFP (47 kDa) and Orai1 (50 kDa) at different conditions, from left to right; Basal, MβCD, TG and MβCD + TG. Lower panel shows the signal of co-immunoprecipitated Cav1, normalized with Orai1 concentration at each condition. Basal (black), MβCD (red), TG (dark blue), and MβCD + TG (light blue). FRET n ≥ 70 cells, Pseudocolor scale maps showing the FRET efficiency (%), obtained in the control (yellow circle, eff = 0) area and the acceptor photobleached (yellow square) area. CoIP n ≥ 10 independent assays. Error bars: S.E.M. ***p < 0.001, **p < 0.01 or *p < 0.05.

Figure 4. Cholesterol depletion reduces TG-induced whole-cell currents

(A) Example of whole cell patch clamp recordings, left panel, endogenous Orai1 and STIM1, right panel overexpressing Orai1 and STIM1, at basal (blue line, n = 54), Cav1 overexpressed (pink line, n = 56 cells) and cholesterol depleted conditions (red line, n = 61 cells), cholesterol depleted conditions overexpressing Cav1 (black line, n = 58 cells). (B) Bar graph summarizing current densities measured at −100 mV, same color code as A. (C) Current-voltage relationships (I/V) for TG induced currents. Number of cells explored is indicated at each plot. Error bars: S.E.M. ***p < 0.001.

Figure 5. Cholesterol depletion and Cav1 overexpression influence Orai1 diffusion and cluster size

(A) Change of mCherry-Orai1 diffusion models at basal cholesterol conditions (left panel) compared with low cholesterol conditions (right panel). (B) mCherry-Orai1 diffusion models in basal cholesterol conditions when Cav1 is overexpressed. The diffusion models are presented as percentage of total analyzed cells, partially confined (orange), confined (red), linear (green). For the models used please refer to Material and Methods. (C) Change in mCherry-Orai1 aggregate size (D) Changes in mCherry-Orai1 confinement size at different conditions. Basal (black), MβCD (red), Cav1 overexpressed (green). n ≥ 50 cells, in 6 independent experiments. Error bars: S.E.M. ***p < 0.001, **p < 0.01 or *p < 0.05.