STIM2 drives Ca2+ oscillations through store-operated Ca2+ entry caused by mild store depletion

Abstract

Abstract Agonist-induced Ca(2+) oscillations in many cell types are triggered by Ca(2+) release from intracellular stores and driven by store-operated Ca(2+) entry. Stromal cell-interaction molecule (STIM) 1 and STIM2 serve as endoplasmic reticulum Ca(2+) sensors that, upon store depletion, activate Ca(2+) release-activated Ca(2+) channels (Orai1-3, CRACM1-3) in the plasma membrane. However, their relative roles in agonist-mediated Ca(2+) oscillations remain ambiguous. Here we report that while both STIM1 and STIM2 contribute to store-refilling during Ca(2+) oscillations in mast cells (RBL), T cells (Jurkat) and human embryonic kidney (HEK293) cells, they do so dependent on the level of store depletion. Molecular silencing of STIM2 by siRNA or inhibition by G418 suppresses store-operated Ca(2+) entry and agonist-mediated Ca(2+) oscillations at low levels of store depletion, without interfering with STIM1-mediated signals induced by full store depletion. Thus, STIM2 is preferentially activated by low-level physiological agonist concentrations that cause mild reductions in endoplasmic reticulum Ca(2+) levels. We conclude that with increasing agonist concentrations, store-operated Ca(2+) entry is mediated initially by endogenous STIM2 and incrementally by STIM1, enabling differential modulation of Ca(2+) entry over a range of agonist concentrations and levels of store depletion.

Figure 1. Vanadate effects in RBL-2H3 cells

[Ca²⁺]_i was clamped to 150 nm with 10 mm BAPTA and 4 mm CaCl₂. Data represent average leak-subtracted current densities (pA/pF ± s.e.m.) evoked by 50 ms voltage ramps from –100 to +100 mV extracted at –80 mV. A, average CRAC current densities with 5 μm (n = 9), 50 μm (n = 8), 5 mm (n = 10) vanadate or 20 μm IP₃ (n = 5). B, average I–V relationships of CRAC currents extracted from representative cells shown in (A) at 200 s. C, average CRAC current densities elicited by 5 μm vanadate in the absence (n = 7) and presence (n = 9) of 10 μm G418. D, same as C but using 50 μm vanadate in the absence (n = 6) or presence (n = 9) of 100 μm G418. E, average CRAC current densities elicited by 5 μm vanadate in the absence (n = 10) and presence (n = 13) of 10 μg ml⁻¹ C-terminal STIM2. F, same as E but in the absence (n = 10) or presence (n = 12) of 10 μg ml⁻¹ N-terminal STIM2 antibody. Ab, antibodies; CRAC, Ca²⁺ release-activated Ca²⁺; IP₃, inositol trisphosphate; STIM, stromal cell-interaction molecule.

Figure 2. Effects of STIM2 knockdown on vanadate-induced I_CRAC

[Ca²⁺]_i was clamped to 150 nm. Data represent average leak-subtracted current densities (pA/pF ± s.e.m.) evoked by 50 ms voltage ramps from −100 to +100 mV extracted at −80 mV. Average CRAC current densities induced in RBL-2H3 by (A) 5 μm vanadate (control, n = 13; STIM2 kd cells, n = 14); (B) 20 μm IP₃ (control, n = 6; STIM2 kd, n = 5); and (C) 500 μm vanadate (control, n = 6; STIM2 kd, n = 5) on day 1 post-transfection. D, same as A, RBL-2H3 control (n = 6) and STIM2 kd cells (n = 6) induced by 5 μm vanadate on day 2 post-transfection. E, STIM2 kd cells stimulated with 5 μm vanadate in the absence (n = 6, same as in D) and presence (n = 8) of 10 μm G418. F, STIM2 siRNA specificity was assayed by immunoprecipitation and Western blot. RBL-2H3 cells were transfected with siRNA targeting STIM2 or control for 0, 1 and 2 days. Equal amounts of protein from total lysates were immunoprecipitated with anti-STIM2 C-terminal antibody. Immune complexes were resolved on SDS-PAGE gel and immunoblotted with anti-STIM2 C-terminal antibody. As control, equal amounts of total lysates from transfected RBL-2H3 cells were immunoblotted with anti-GAPDH antibody. G, endoplasmic reticulum Ca²⁺ levels were measured by the magnitude of Ca²⁺ release following addition of the SERCA inhibitor thapsigargin (2 μm) in Ca²⁺-free solution on day 1 post-transfection. H and I, the relative ER Ca²⁺ levels based on Ca²⁺ transient amplitudes (Δpeak) on day 1 (H) and day 2 (I) in RBL-2H3 cells transfected with siRNA specific for STIM2. Ab, antibodies; CRAC, Ca²⁺ release-activated Ca²⁺; IP₃, inositol trisphosphate; kd, knockdown; STIM, stromal cell-interaction molecule.

Figure 3. Inhibition of IgE-mediated Ca²⁺ signals by G418 in RBL-2H3 cells and LTC₄-mediated Ca²⁺ signals by G418 in RBL-1 cells

RBL-2H3 (A–D) and RBL-1 (F–H) were preincubated for 1 h in the absence or presence of G418 (500 μg ml⁻¹). The arrow indicates the application of DNP-BSA or LTC₄. Error bars indicate s.e.m. A, average intracellular Ca²⁺ changes (F₃₄₀/F₃₈₀) in RBL-2H3 cells elicited by 100 ng ml⁻¹ DNP-BSA in control (n = 110) or preincubated cells (n = 131). B, same as A (10 ng ml⁻¹) DNP-BSA stimulation in control (n = 784) or preincubated cells (n = 1139). C and D, average number of spikes during 1200 s (P < 0.001) after 100 ng ml⁻¹ (C) or 10 ng ml⁻¹ (D) DNP-BSA stimulation in the same cells as shown in A and B. E and F intracellular Ca²⁺ changes (F₃₄₀/F₃₈₀) in a representative RBL-1 cell after stimulation with 160 nm LTC₄ in control (E) or preincubated with G418 (F). G, average number of spikes during the first 340 s (P = 0.64) and 400–1200 s (P < 0.005) into the experiment after LTC₄ stimulation (160 nm). kd, knockdown; LTC₄, leukotriene C₄; STIM, stromal cell-interaction molecule.

Figure 4. Effects of G418 and STIM knockdown on MeCh-induced Ca²⁺ oscillations

A, intracellular Ca²⁺ changes (F₃₄₀/F₃₈₀) after 5 μm MeCh stimulation in two single representative HEK293 cells in the absence or preincubated for 1 h with 500 μg ml⁻¹ G418. B, average rate of spikes during 1200 s (P < 0.001) after MeCh stimulation (5 μm) for cells in the absence or preincubated for 1 h with 500 μg ml⁻¹ G418 and performed in the absence or presence of external Ca²⁺. Error bars indicate s.e.m. C, similar experiments were performed when cells were transfected with control siRNA or siRNA directed against STIM1 or STIM2 and analysed on days 1, 2 and 3 post-transfection. kd, knockdown; MeCh, metacholine; STIM, stromal cell-interaction molecule.