Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1

Abstract

Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membrane-associated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by ∼50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1.

Keywords: calcium release; calcium release-activated calcium current; endoplasmic reticulum; permeability; transient receptor potential protein of the canonical subfamily.

Fig. 1.

Thapsigargin activates concentration-dependent calcium entry in pulmonary artery endothelial cells (PAECs). A: PAECs were loaded with the ratiometric intracellular calcium indicator fura 2-AM, and cytosolic calcium was continuously monitored in cell populations (average fluorescence from ∼20 cells). Thapsigargin was added to cells in a low-calcium bath solution, then calcium chloride was added to achieve the final concentration of 10 μM (n = 3), 100 μM (n = 8), 500 μM (n = 7), 1 mM (n = 8), 5 mM, (n = 5), and 10 mM (n = 7), respectively. Values are means ± SE. [Ca²⁺]_e, extracellular calcium concentration; [Ca²⁺]_i, intracellular calcium concentration. B: peak store-operated calcium entry level from each calcium dose. Values are means ± SE. Data were analyzed using 1-way ANOVA with Bonferroni's post hoc test. *Significantly different from 10 μM. #Significantly different from 100 μM. C: data in B replotted to illustrate dose-response relationship. Half-maximal effect (EC₅₀) was ∼400 μM.

Fig. 2.

Orai1 silencing does not significantly reduce the magnitude of the store-operated calcium entry response in PAECs. A: PAECs were engineered for doxycycline-inducible expression of Orai1 shRNA, as described previously (15). Cells were treated with doxycycline (3 μg/ml) every 24 h for a total of 72 h. After doxycycline exposure, cell lysates were collected and subjected to Western blot analysis. Conditional expression of Orai1 shRNA silenced Orai1 protein by 72 h (left), an effect that resulted in an ∼80% decrease in protein as determined by densitometry (middle, n = 20 experiments). Doxycycline treatment induced uniform mCherry expression, consistent with uniform Orai1 shRNA expression (right). B: Orai1 expression was not a determinant of the cytosolic calcium response to thapsigargin in the recalcification protocol following readdition of 100 μM [+Orai1 (n = 4) and −Orai1 (n = 8)], 500 μM [+Orai1 (n = 6) and −Orai1 (n = 10)], and 5 mM [+Orai1 (n = 7) and −Orai1 (n = 9)] calcium, respectively. ns, Not significant. C and D: cytosolic calcium response to 30 nM, 100 nM, and 1 μM thapsigargin in the presence of 2 mM extracellular calcium in the presence and absence of Orai1. C: Orai1 silencing delayed the rise in cytosolic calcium. Pearson P < 0.05 at 30 and 100 nM thapsigargin. *P < 0.05. D: averaged responses following 30 nM [+Orai1 (n = 6) and −Orai1 (n = 7)], 100 nM [+Orai1 (n = 7) and −Orai1 (n = 8)], and 1 μM [+Orai1 (n = 9) and −Orai1 (n = 7)] thapsigargin, respectively.

Fig. 3.

Sodium induces a dose-dependent increase in Asante NaTRIUM Green-2 (ANG 2) fluorescence in intact PAECs. PAECs were loaded with ANG 2-AM. A: baseline fluorescence was measured, and gramicidin A (3 μM) was added in the relative absence of extracellular sodium. After a baseline equilibration period, extracellular sodium was replenished at ascending concentrations. Data represent average fluorescence response from 3 separate experiments. B: raw data in A plotted as a dose-response curve to demonstrate the half-maximal fluorescence signal at ∼11 mM cytosolic sodium. C: representative fluorescence images from a confluent PAEC monolayer exposed to ascending cytosolic sodium concentrations as described in A. [Na⁺]_e, extracellular sodium concentration; [Na⁺]_i, intracellular sodium concentration.

Fig. 4.

Orai1 suppresses basal sodium leak in the presence of 2 mM extracellular calcium. PAECs were treated with doxycycline to silence Orai1, as described in Fig. 2 legend. Cells were loaded with the intracellular sodium dye ANG 2-AM, and the signal ratio (GFP/F₀) was repeatedly measured over time in the presence of 2 mM or low extracellular calcium. Reducing extracellular calcium (A) and silencing Orai1 (B) resulted in an increase in cytosolic sodium, characteristic of increased basal sodium leak. For studies conducted in the presence of both Orai1 and calcium, n = 6 experiments in which 67 cells were analyzed; for studies conducted in the presence of Orai1 and the absence of Ca²⁺, n = 8 experiments in which 88 cells were analyzed. For studies conducted in the absence of both Orai1 and Ca²⁺, n = 6 experiments in which 65 cells were analyzed; for studies conducted in the presence of Orai1 and the absence of Ca²⁺, n = 6 experiments in which 67 cells were analyzed. *P < 0.01.

Fig. 5.

Orai1 suppresses thapsigargin-induced sodium entry through activated store-operated calcium entry channels. PAECs were prepared and treated with doxycycline as described in Fig. 2 legend. Cells were loaded with ANG 2-AM and then imaged for cytosolic sodium. Thapsigargin was applied in the presence (A) and absence (B) of extracellular calcium. A: thapsigargin increased cytosolic sodium in the presence and absence of Orai1. For studies conducted in the presence of Orai1, n = 6 experiments in which 69 cells were analyzed; for studies conducted in the absence of Orai1, n = 10 experiments in which 101 cells were analyzed. B: thapsigargin effect was more pronounced in the absence of extracellular calcium. For studies conducted in the presence of Orai1, n = 5 experiments in which 55 cells were analyzed; for studies conducted in the absence of Orai1, n = 5 experiments in which 55 cells were analyzed. Addition of extracellular calcium in the recalcification protocol resulted in an abrupt reduction in cytosolic sodium. Values are means ± SE. *P < 0.05.

Fig. 6.

Thapsigargin induced a transient rise in cytosolic sodium prior to endoplasmic reticulum calcium release. PAECs were treated with doxycycline as described in Fig. 2 legend and then loaded with ANG 2-AM or fura 2-AM to measure intracellular sodium or calcium, respectively. Thapsigargin was applied in the absence of extracellular calcium, and cytosolic sodium and calcium responses were evaluated. Raw data were synchronized according to the time at which thapsigargin was added and converted to a maximal response scale (0–100% of maximal response), and sodium and calcium release phases were compared. For studies conducted in the presence of Orai1, [Na⁺]_i data included 17 experiments in which 182 cells were analyzed and [Ca²⁺]_i data included 19 experiments; for studies conducted in the absence of Orai1, [Na⁺]_i data included 15 experiments in which 163 cells were analyzed and [Ca²⁺]_i data included 26 experiments. Data reveal that the sodium peak occurred prior to the calcium peak but was of shorter duration. Similar results were obtained in the presence (A) and absence (B) of Orai1. Mean value from these experiments is shown. Orai1 silencing did not change peak [Na⁺]_i or [Ca²⁺]_i signal (P = not significant).

Fig. 7.

Store-operated calcium entry and calcium release-activated calcium channel inhibitors 2-aminoethoxydiphenyl borate (2-APB) and YM-58483 decrease basal sodium leak and thapsigargin-induced sodium entry only in the presence of Orai1. PAECs were treated with doxycycline as described in Fig. 2 legend and loaded with ANG 2-AM or fura 2-AM. A: baseline sodium. In separate experiments, 2-APB or YM-58483 was added. Orai1 and extracellular calcium (2 mM) together potently limit basal sodium leak. 2-APB (75 μM) and YM-58483 (10 μM) suppress cytosolic sodium, but only in the presence of Orai1. The following studies were conducted in the absence of [Ca²⁺]_e: for studies conducted in the absence of Orai1, data represent 6 experiments in which 67 cells were analyzed; for studies conducted in the presence of Orai1, data represent 8 experiments in which 88 cells were analyzed; for studies conducted in the presence of Orai1 and 2-APB, data represent 7 experiments in which 77 cells were analyzed; for studies conducted in the presence of Orai1 and YM-58483, data represent 5 experiments in which 55 cells were analyzed. The following studies were conducted in the presence of [Ca²⁺]_e: for studies conducted in the absence of Orai1, data represent 6 experiments in which 65 cells were analyzed; for studies conducted in the presence of Orai1, data represent 6 experiments in which 67 cells were analyzed; for studies conducted in the presence of Orai1 and 2-APB, data represent 5 experiments in which 54 cells were analyzed; for studies conducted in the presence of Orai1 and YM-58483, data represent 6 experiments in which 63 cells were analyzed. Data were analyzed using 1-way ANOVA with Bonferroni's post hoc test: *P < 0.0001; #P < 0.01. B–E: after baseline sodium and calcium signals were measured, 2-APB (B and C) or YM-58483 (D and E) was added, followed by thapsigargin in the presence or absence of extracellular calcium. In the presence of 2 mM extracellular calcium, 2-APB (for studies conducted in the presence of Orai1, data represent 5 experiments in which 53 cells were analyzed; for studies conducted in the absence of Orai1, data represent 6 experiments in which 66 cells were analyzed) and YM-58483 (for studies conducted in the presence of Orai1, data represent 6 experiments in which 66 cells were analyzed; for studies conducted in the absence of Orai1, data represent 5 experiments in which 55 cells were analyzed) inhibited thapsigargin-induced sodium entry, but only in the presence of Orai1. In recalcification studies, 2-APB (for studies conducted in the presence of Orai1, data represent 6 experiments in which 66 cells were analyzed; for studies conducted in the absence of Orai1, data represent 5 experiments in which 55 cells were analyzed) and YM-58483 (for studies conducted in the presence of Orai1, data represent 5 experiments in which 55 cells were analyzed; for studies conducted in the absence of Orai1, data represent 7 experiments in which 77 cells were analyzed) again inhibited the thapsigargin-induced increase in cytosolic sodium, only in the presence of Orai1. 2-APB (for studies conducted in the presence of Orai1, data represent 4 experiments; for studies conducted in the absence of Orai1, data represent 4 experiments) and YM-58483 (for studies conducted in the presence of Orai1, data represent 5 experiments; for studies conducted in the absence of Orai1, data represent 7 experiments) decreased the thapsigargin-induced increase in cytosolic calcium, independent of Orai1. Values are means ± SE. F: transient thapsigargin-induced increases in cytosolic sodium and calcium were rescaled as percentage of maximal response. 2-APB and YM-58483 reduced the peak cytosolic sodium response to thapsigargin, whereas only 2-APB decreased the peak cytosolic calcium response to thapsigargin. For [Na⁺]_i peaks: *P < 0.0001; ^P < 0.001; #P < 0.01. For [Ca²⁺]_i peaks, P = not significant. G and H: extracellular sodium was replaced with choline, and the thapsigargin-induced cytosolic sodium response was evaluated. Choline substitution abolished thapsigargin-induced sustained increase in cytosolic sodium (G) but did not alter the transient increase in cytosolic sodium (H).