STIM1 functions as a proton sensor to coordinate cytosolic pH with store-operated calcium entry

Abstract

The meticulous regulation of intracellular pH (pH_i) is crucial for maintaining cellular function and homeostasis, impacting physiological processes such as heart rhythm, cell migration, proliferation, and differentiation. Dysregulation of pH_i is implicated in various pathologies such as arrhythmias, cancer, and neurodegenerative diseases. Here, we explore the role of STIM1, an ER calcium (Ca²⁺) sensor mediating Store Operated Ca²⁺ Entry (SOCE), in sensing pH_i changes. Our study reveals that STIM1 functions as a sensor for pH_i changes, independent of its Ca²⁺-binding state. Through comprehensive experimental approaches including confocal microscopy, FRET-based sensors, and mutagenesis, we demonstrate that changes in pH_i induce conformational alterations in STIM1, thereby modifying its subcellular localization and activity. We identify two conserved histidines within STIM1 essential for sensing pH_i shifts. Moreover, intracellular alkalization induced by agents such as Angiotensin II or NH₄Cl enhances STIM1-mediated SOCE, promoting cardiac hypertrophy. These findings reveal a novel facet of STIM1 as a multi-modal stress sensor that coordinates cellular responses to both Ca²⁺ and pH fluctuations. This dual functionality underscores its potential as a therapeutic target for diseases associated with pH and Ca²⁺ dysregulation.

Keywords: FRET imaging; SOCE; STIM1; calcium signaling; cardiac hypertrophy; intracellular pH (pH(i)).

Figure 1

STIM1 dose-dependently responds to changes in intracellular pH (pH_i), with minimal dependence on adjacent Ca²⁺levels in HeLa cells. Nigericin (10 μM), a H⁺/K⁺ ionophore, was used with high K⁺ buffer solutions at different extracellular pH (pH_e) to adjust pH_i. This allows pH_i to be controlled by matching it with pH_e. A, typical confocal imaging results showing effects of pH_i changes on subcellular distribution of mCherry-STIM1 transiently expressed in HeLa cells at basal or ER Ca²⁺ depleted conditions. Top: cells under basal, or ER Ca²⁺ store replete conditions. Bottom: cells with ER Ca²⁺ store depleted by 5-min bath using 2.5 μM ionomycin (IONO), a Ca²⁺ ionophore (Scale bar: 10 μm, n = 3). B and C, in cellulo responses of PM-localized STIM1 to concurrent alterations in pH_e and pH_i. Schematic representation of the subcellular distribution of the two STIM1 constructs tested for FRET imaging: YFP-STIM1_343-491(SOAR1L) and engineered PM-localized STIM1_1-CC1 (PM-SC1111)-CFP. Cartoon illustration of the subcellular distribution of these two constructs within cells (Refer to Fig. S1 for detailed description of these constructs). B, left: representative curves of FRET responses between YFP-SOAR1L and PM-SC1111–CFP monitored in nominally 0Ca²⁺ and 2 mM Ca²⁺ solution. Middle and left, statistics (C) (ns, p > 0.05, unpaired Student's t test, error bars denote SEM, n = 3). D–F, in situ responses of STIM1 to pH_i changes. Cartoon representation of acid-resistant FRET tools employed for monitoring the activation status of STIM1: mScarlet-SOAR1L and STIM1_1-CC1 (SC)-Tolerance of Lysosomal Environments (TOLLES). TOLLES is an acid-resistant CFP variant (D). FRET responses between SC-TOLLES and mScarlet-SOAR1L in HeLa STIM1-STIM2 double knock-out (SK) cells. The measurements were conducted before altering pH_i, with two conditions represented: with IONO treatment (E) left, representative curve; right, statistics (two-way ANOVA, F (3, 132) = 126.7, p < 0.0001;) (Rest versus pH = 6.0, p < 0.0001; IONO versus pH = 9.0, p = 0.049).and without IONO treatment (one-way ANOVA, F (2, 165) = 53.35, p < 0.0001) (pH = 6.0, p < 0.0001; pH = 9.0, p = 0.001). F, left, representative curve; right, statistics (error bars denote SEM, n = 3). G, in situ H⁺ titrations of FRET responses between transiently co-expressed SC-TOLLES and mScarlet-SOAR1L in HeLa SK cells. Left: typical traces; right: dose-response curves (n = 3).

Figure 2

Mapping critical regions essential for STIM1 in sensing changes in pH_iwithin HeLa SK cells.A and B, perturbations in pH_e adjacent to EF-SAM region of PM-SC1111–CFP had no substantial impact on its FRET responses with YFP-SOAR1L. Recordings were carried out using cells immersed in nominally Ca²⁺ free solution (A) left: cartoon depiction of constructs used; Middle: representative curves; right: statistics (one-way ANOVA, F (2, 390) = 0.39, p = 0.67) (pH = 6.0, p = 0.65; pH = 9.0, p = 0.91), (error bars denote SEM, n = 3) or buffers containing 2 mM Ca²⁺ (B). Left: cartoon depiction; middle: representative curves; right: statistics (one-way ANOVA, F (2, 333) = 0.19, p = 0.83) (pH = 6.0, p = 0.92; pH = 9.0, p = 0.83), (error bars denote SEM, n = 3). C and D, under extracellular conditions with a high concentration of Ca²⁺ (30 mM), replacing the CC1-domain and SOAR1L with those of STIM2 (PM-SC1112-CFP and YFP-SOAR2L) had minimal impact on STIM1's response to changes in adjacent pH (Plasmid construction strategy: see Fig. S1). Schematic representation of STIM1/2 constructs tested (PM-SC1111-CFP + YFP-SOAR2L or PM-SC1112-CFP + YFP-SOAR1L). C, typical traces (left) and statistics (middle and right) (D) (ns, p > 0.05, unpaired Student's t test, error bars denote SEM, n = 3). E and F, STIM1-H240N mutation had no effect on in situ FRET responses between STIM1_1-261(SCS)-TOLLES and mScarlet-SOAR1L. Schematic representation of STIM1 constructs tested (E). Representative traces (left) and statistics (middle and right) (F) (ns, p > 0.05, unpaired Student's t test, error bars denote SEM, n = 3).

Figure 3

Mutations at SOAR1L-H355-H407 minimally impact STIM1's coupling with Orai1 but significantly diminish its pH-sensing ability.A, FRET responses between SCS-TOLLES and mScarlet-SOAR1L WT (black), or its H407N (pink), H355N (orange), H355N-H407N mutant (2HN) (blue) in HeLa SK cells. Left most: typical curves; bar charts on the right: statistics of FRET signals. Middle left, basal FRET; middle right and rightmost, changes in FRET signals induced by acidification or alkalization, normalized against those resulting from store depletion. (Middle left: one-way ANOVA, F (3, 238) = 9.88, p < 0.0001) (H355N, p = 0.006; H407N, p = 0.922; 2HN, p = 0.91), (middle right: one-way ANOVA, F (3, 242) = 80.36, ∗∗∗∗p < 0.0001; ∗∗∗p = 0.0001, ∗∗p = 0.0096), (right: one-way ANOVA, F (3, 238) = 110.2, ∗∗∗∗p < 0.0001; ∗∗∗p = 0.0003) (error bars denote SEM, n = 3). B, a diagram illustrating the location of H355 and H407 residue on SOAR1 structure. C, FRET responses mediated by PM-SC1111-CFP and YFP-SOAR1L-2HN in HeLa SK cells. Left, representative traces; middle & right, statistics of pH-induced changes in FRET signal (middle: one-way ANOVA, F (2, 102) = 1.615, p = 0.2) (rest versus pH = 9.0, p = 0.99; pH = 9.0 versus pH = 6.0, p = 0.31) (left: one-way ANOVA, F = 3.14 p = 0.0453, rest versus pH = 9.0, p = 0.9643; pH = 9.0 versus pH = 6.0, p = 0.1074) (error bars denote SEM, n = 3). D–F, effects of 2HN mutation on the coupling of STIM1 with Orai1 and corresponding Ca²⁺ responses examined in HEK SK cells stably expressing Orai1-CFP (SKO cells). D, co-localization between Orai1-CFP (red) and transiently expressed STIM1-YFP or STIM1-H355N-H407N (2HN)-YFP (green) with Orai1-CFP (red) in HEK SKO cells pre- and post-store depletion. Store depletion induced by a 5-min bath application of 2.5 μM IONO. Left, typical confocal images. (Uppermost and lower middle row on the left: Store repleted; upper middle lowest row: Store depleted). (Scale bar: 10 μm, n = 3). Bar chart on the right, statistics. (n = 3, ∗∗∗∗p < 0.0001, unpaired Student's t test, error bars represent SEM). E, FRET responses mediated by Orai1-CFP and YFP-STIM1 or YFP-STIM1-2HN. Store depletion was induced by 2.5 μM IONO (C). Left, representative traces; right, statistics of IONO-induced increases in FRET signal (ns, p > 0.05, unpaired Student's t test, error bars denote SEM, n = 3). F, Ca²⁺ responses induced by thapsigargin (TG, 1 μM) in SKO cells transiently co-expressing R-GECO1.2, a low affinity red GECI, and YFP-STIM1 or YFP-STIM1-2HN. TG is a blocker of the ER Ca²⁺ pump to induce depletion of ER Ca²⁺ store and subsequent SOCE. Left, typical traces; right, statistics of TG-induced SOCE (n = 3, ∗∗∗∗p < 0.0001, unpaired Student's t test, error bars represent SEM).

Figure 4

Angiotensin II (Ang II) or NH₄Cl may induce cardiac hypertrophy by enhancing STIM1-mediated SOCE through pH_ialkalization in HL-1 Cells.A–D, Ang II-induced cardiac hypertrophy was accompanied with pH_i alkalization and enhancement of SOCE. Hypertrophy was induced by 24 h-incubation with 0.1 μM Ang II in serum-free medium. Control cells were compared with those treated with 0.1 μM Ang II alone, or with the combination of 1 μM cariporide, an inhibitor of Na⁺/H⁺ exchange inhibitor 1 (NHE1). Statistics of mRNA level of atrial natriuretic factor (ANF) (one-way ANOVA, F (2, 6) = 29.89, p = 0.0039) (Ctrl versus Ang II, p = 0.01; Ctrl versus AngII + cariporide, p = 0.986) (A) and cell surface area (B) mRNA levels were normalized to GAPDH (one-way ANOVA, F (2, 6) = 4.952, p = 0.0085) (Ctrl versus Ang II, p = 0.0231; Ang II versus AngII + cariporide, p = 0.260). Statistics of pH_i, measured with a genetically encoded ratiometric pH sensor, pHmScarlet-mTurquoise2 (C). (One-way ANOVA, F (2, 166) = 9.283, p = 0.0002) (Ang II, p = 0.0016; AngII + cariporide, p = 0.6532). TG-induced Ca²⁺ responses showed by a cytosolic Ca²⁺ indicator, Fura-2. Left, typical traces; right, statistics of SOCE responses (D) (one-way ANOVA, F (2, 321) = 105.6, p < 0.0001) (Ctrl versus Ang II, p < 0.0001; Ang II versus AngII + cariporide, p < 0.0001). (n = 3, error bars denote SEM). E, statistics showing effects of 24 h-treatment with 40 mM NH₄Cl on pH_i. pH_i was measured using pHmScarlet-mTurquoise2. (n = 3, ∗∗∗∗p < 0.0001, unpaired Student's t test, error bars represent SEM). F–H, TG-induced Ca²⁺ responses or cardiac hypertrophic responses in controls, or cells following 24 h-treatment with 40 mM NH₄Cl alone, or in combination with 1.5 μM celastrol (CEL), a specific inhibitor of SOCE. TG-induced Ca²⁺ signals left: representative curves; right: statistics of SOCE responses (one-way ANOVA, F (2, 389) = 43.78, p < 0.0001) (Ctrl versus NH₄Cl, p < 0.0001; Ctrl versus NH₄Cl + CEL, p < 0.0001) (F). Statistics of ANF mRNA level (one-way ANOVA, F (2, 8) = 17.58, p = 0.0012) (Ctrl versus NH₄Cl, p < 0.0013; Ctrl versus NH₄Cl + CEL, p = 0.5735) (G), cell surface area (one-way ANOVA, F (2, 282) = 33.04, p < 0.0001) (Ctrl versus NH₄Cl, p = 0.002; Ctrl versus NH₄Cl + CEL, p < 0.0001) (H). (n = 3, error bars denote SEM).