Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation

Abstract

Ca(2+)-release-activated Ca(2+) (CRAC) channels generate sustained Ca(2+) signals that are essential for a range of cell functions, including antigen-stimulated T lymphocyte activation and proliferation. Recent studies have revealed that the depletion of Ca(2+) from the endoplasmic reticulum (ER) triggers the oligomerization of stromal interaction molecule 1 (STIM1), the ER Ca(2+) sensor, and its redistribution to ER-plasma membrane (ER-PM) junctions where the CRAC channel subunit ORAI1 accumulates in the plasma membrane and CRAC channels open. However, how the loss of ER Ca(2+) sets into motion these coordinated molecular rearrangements remains unclear. Here we define the relationships among [Ca(2+)](ER), STIM1 redistribution and CRAC channel activation and identify STIM1 oligomerization as the critical [Ca(2+)](ER)-dependent event that drives store-operated Ca(2+) entry. In human Jurkat leukaemic T cells expressing an ER-targeted Ca(2+) indicator, CRAC channel activation and STIM1 redistribution follow the same function of [Ca(2+)](ER), reaching half-maximum at approximately 200 microM with a Hill coefficient of approximately 4. Because STIM1 binds only a single Ca(2+) ion, the high apparent cooperativity suggests that STIM1 must first oligomerize to enable its accumulation at ER-PM junctions. To assess directly the causal role of STIM1 oligomerization in store-operated Ca(2+) entry, we replaced the luminal Ca(2+)-sensing domain of STIM1 with the 12-kDa FK506- and rapamycin-binding protein (FKBP12, also known as FKBP1A) or the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR, also known as FRAP1). A rapamycin analogue oligomerizes the fusion proteins and causes them to accumulate at ER-PM junctions and activate CRAC channels without depleting Ca(2+) from the ER. Thus, STIM1 oligomerization is the critical transduction event through which Ca(2+) store depletion controls store-operated Ca(2+) entry, acting as a switch that triggers the self-organization and activation of STIM1-ORAI1 clusters at ER-PM junctions.

Figure 1. The [Ca²⁺]_ER-response relation for the CRAC channel

Simultaneous measurements of [Ca²⁺]_ER and I_CRAC in individual Jurkat T cells. a, Non-stationary measurements of I_CRAC and [Ca²⁺]_ER. Store depletion with 20 μM CPA induces an increase in I_CRAC (top) that follows a decrease in [Ca²⁺]_ER (middle) monitored with YC4.2er. The I-V relationship shows the inward rectification typical of I_CRAC (bottom). In this cell, a small inward current through outwardly-rectifying Cl⁻ channels is also present initially but disappears before I_CRAC is induced. b, Recordings of I_CRAC (top) and [Ca²⁺]_ER (middle) under steady-state conditions. Each cell was treated with the indicated CPA concentration for 8–15 min prior to recording, and CPA was maintained throughout the experiment. I-V relations are typical for I_CRAC (bottom). c, Steady-state I_CRAC and [Ca²⁺]_ER are plotted for 40 cells after treatment with 0.5–20 μM CPA. A fit of the Hill equation with a K_1/2 of 169 μM and Hill coefficient of 4.2 is superimposed on the data. Squares: mean ± s.e.m. of 3–12 cells. Circles: single cells (see Supplementary Information).

Figure 2. The [Ca²⁺]_ER dependence of STIM1 redistribution determines the [Ca²⁺]_ER-response relation of the CRAC channel

a,Widefield epifluorescence images of a cell expressing Cherry-STIM1 at rest (top) and following store depletion with 3 μM CPA (middle) and TG (bottom). The redistribution of Cherry-STIM1 in single cells was monitored as the ratio of the mean Cherry fluorescence in the most peripheral 0.5 μm of the cell (F_P, right) to the mean fluorescence of the entire cell (F_TOT, left). Scale bar = 2μm. b, In the same cell, STIM1 redistribution represented by F_P/F_TOT normalized to the maximum ratio with TG (blue). F_P/F_TOT increases as [Ca]_ER (red) declines. Individual data points (open symbols) and the mean response (bars) are shown. c, STIM1 redistribution (F_P/F_TOT, blue) plotted against [Ca²⁺]_ER after treatment with 0–3 μM CPA (means ± s.e.m. of 3–4 cells; 41 cells total). A fit of the Hill equation (blue line) indicates a K_1/2 of 187 μM and Hill coefficient of 3.8. Steady-state I_CRAC data fitted with the Hill equation are re-plotted from Fig. 1 (red).

Figure 3. STIM1 oligomerization induces the accumulation of STIM1 at ER-plasma membrane junctions

a, The cartoon depicts the oligomerization of F-STIM1 induced by rapalog (R). At rest, FKBP-STIM1 and FRB-STIM1 are expected to form homo- and heterodimers; only intermolecular crosslinks between homodimers are shown here. Abbreviations: EF (EF hand), SAM (sterile-α motif), CC (coiled-coil), S/P (serine-proline-rich), K (lysine-rich), CH (mCherry). b, BN-PAGE and Western blot of transiently expressed F-STIM1 harvested from HEK293 cells. Untreated (left) and rapalog-treated (right) F-STIM1 was detected using a monoclonal anti-STIM1 antibody. c, Rapalog induces a time-dependent peripheral redistribution of F-STIM1 (n=10 cells). d, Peripheral redistribution of Cherry-STIM1 by TG (red bars; n=31 for each) and redistribution of F-STIM1 by rapalog (black bars; n=39, rest; n=42, rapalog). Values expressed as mean ± s.e.m. (c, d). e–h, TIRF images of Jurkat cells, scale bar = 2 μm. e, F-STIM1 before (left) and after (right) incubation with rapalog. f, Cherry-STIM1 before (left) and after (right) store depletion with TG. g, Cherry-STIM1 before (left) and after (right) incubation with rapalog. h, F-STIM1 (top row), GFP-STIM1 (middle row) and merged images (bottom row) from a single cell after rapalog treatment (left column) and subsequent store depletion with TG (right column). Cherry and GFP intensities are scaled to the maximal intensity of each fluorophore after TG treatment.

Figure 4. STIM1 oligomerization activates Ca²⁺ entry through CRAC channels

a, In rapalog-treated cells expressing F-STIM1 (black, n=45), resting [Ca²⁺]_i is elevated and sensitive to the removal of extracellular Ca²⁺, indicating constitutive Ca²⁺ entry. In contrast, resting Ca²⁺ influx was largely absent in untreated F-STIM1-expressing cells (red, n=61) and in wt Jurkat cells with (green, n=617) or without (blue, n=517) rapalog. TG-induced Ca²⁺ release in rapalog-treated cells was similar to that of untreated cells. b, I_CRAC development during whole-cell recording from rapalog-treated (black, n=9) and untreated (red, n=9) cells expressing F-STIM1. I_CRAC was measured beginning within 5 s of break-in. c, I–V relations upon break-in, showing the inward rectification typical of I_CRAC in the rapalog-treated cell (black) and the absence of current in the untreated cell (red). Values expressed as mean ± s.e.m. (a,b).