The 5,5-dimethyloxazolidine-2[14C],4-dione distribution technique and the measurement of intracellular pH in Acer pseudoplatanus cells.

The intracellular pH of suspension-cultured Acer pseudoplatanus cells, was estimated from the distribution of 5,5-dimethyloxazolidine-2[14C],4-dione (DMO) between the culture medium and the cells. The metabolization of DMO in this biological system introduces an error in the calculated intracellular pH value. Three methods are given to overcome this difficulty and to estimate the equilibrium between intracellular and extracellular DMO molecules. A preliminary study has shown that the intracellular pH remains constant about 6.5 when the extracellular pH increases from 5.6 to 7.3.     

Energetics of sucrose transport into protoplasts from developing soybean cotyledons

The accumulation of tetraphenylphosphonium (TPP⁺), 5,5′-dimethyl-oxazolidine-2,4-dione (DMO), and a micro pH electrode were used to measure membrane potential, intracellular and extracellular pH, respectively, upon the addition of exogenous sucrose to soybean cotyledon protoplasts. Addition of sucrose caused a specific and transient (a) depolarization of the membrane potential (measured by TPP⁺ accumulation), (b) acidification of the intracellular pH (measured by DMO accumulation), and (c) alkalization of the external medium (measured by a micro pH electrode). The time course for all these changes was similar (i.e. 5 to 10 minutes). Based on the rate of sucrose uptake and alkalization of the external medium, a stoichiometry of 1.02 to 1.10 for proton to sucrose was estimated. These data strongly support a proton/sucrose cotransporting mechanism in soybean cotyledon cells.     

Osmoregulation in Dunaliella tertiolecta: effects of salt stress,and the external pH on the internal pH

The intracellular pH of the halotolerant green algae Dunaliella tertiolecta, was determined by the distribution of 5,5-dimethyl-2(¹⁴C)-oxalolidine-2,5-dione (DMO) between the cell and the surrounding medium. 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione was not metabolized by the algal cells. The intracellular pH of Dunaliella tertiolecta was 6.8 in the dark and 7.4 in the light. During a salt stress, after two hours, the intracellular pH was increased by 0.2 pH units in both light and dark. The salt stressed cells maintained a constant pH of about 7.5 over the pH range of 6.5 to 8.5. Because of the relatively low permeability coefficient of the plasma membrane for DMO, this technique does not permit rapid pH determinations during the induction period after a salt stress. The magnitude of the salt induced pH changes measured 2 h after the salt stress implies a minor importance of this alkalization in this time range, but does not exclude a larger importance of pH changes for osmoregulation during the induction period.Abbreviations Chl chlorophyll - DMO 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione - PCV packed cell volume - SDS sodium dodecyl sulfate     

Effect of External pH on the Internal pH of Chlorella saccharophila

The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[¹⁴C]oxazolidine-2,4-dione ([¹⁴C]DMO) or [¹⁴C]benzoic acid ([¹⁴C]BA) between the cells and the surrounding medium. [¹⁴C]DMO was used at external pH of 5.0 to 7.5 while [¹⁴C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.     

The intracellular pH of isolated,photosynthetically active Asparagus mesophyll cells

The intracellular pH of isolated, photosynthetically active mesophyll cells of Asparagus sprengeri Regel has been determined, in the light and dark, by the distribution of the weak acid 5,5-dimethyl-[2-¹⁴C]oxazolidine-2,4-dione ([¹⁴C]DMO) between the cells and the liquid medium. [¹⁴C]DMO was taken up rapidly, reaching equilibrium in 7–10 min of incubation, but was not metabolized by the cells, and intracellular binding of the compound was minimal. The intracellular pH, measured at saturating light fluence and 1.5 mM sodium bicarbonate, was found to remain relatively constant at 6.95–7.21 over the external pH range of 5.5–7.2. Illumination of the cells increased the intracellular pH compared to dark controls. The pH of the cytoplasm, excluding and including the chloroplasts (cytoplasmic and bulk cytoplasmic, respectively) was calculated from the experimentally derived intracellular [¹⁴C]DMO concentration and estimates of the vacuolar, chloroplastic and cytoplasmic volumes. The calculated cytoplasmic pH was similar in the light and dark, being 7.75 and 7.74, respectively, while the calculated pH of bulk cytoplasm was 7.85 in the light and 7.49 in the dark. Theoretical analysis indicated that intracellular pH is a good indicator of changes in the bulk cytoplasmic pH but insensitive to changes in vacuolar pH. The external pH optimum for photosynthesis (O₂ evolution) of isolated Asparagus cells was pH 7.2. At pH 8.0 photosynthesis was inhibited by 30% and at pH 5.25 by 45%. Inhibition at alkaline pH may be the result of a decrease in the pH gradient between the cells and the medium, causing CO₂ limitation in the cell. At acid pH, decrease in internal pH caused by substantial accumulation of inorganic carbon may account for the loss in photosynthetic activity.Abbreviations [¹⁴C]DMO 5,5-dimethyl[2-¹⁴C]oxazolidine-2,4-dione - pH_i overall intracellular pH - pH_e pH of external medium     

The Mechanism of Onset of the Stationary Phase in Euglena gracilis Grown with 10 mM Succinate: Intracellular pH Values*

SYNOPSIS. When Euglena gracilis were grown with 10mM succinate at pH 3.5 the extracellular pH averaged 3.62 and the cultures had produced 6 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 30 μliters/10⁶ cells/hr. Total protein and dry weights per cell remained constant during the logarithmic phase and began to decline when the late logarithmic phase was reached. Added succinate caused the cultures in stationary phase to commence logarithmic growth once more. Onset of the stationary phase in cultures grown at pH 3.5 was due to depletion of succinate. When cultures were grown at pH 6.9 the extracellular pH averaged 7.62 and the cultures produced 3 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 20 μliters/10⁶ cells/hr during the logarithmic phase. The decline in total protein and dry weights per cell began at the beginning of the logarithmic phase and continued into the stationary phase of growth. Cultures grown at pH 3.5 should produce a larger number of cells/ml than cultures grown at pH 6.9 if the cells are responding to the unionized moiety of succinate and not the ionized moiety. At pH 3.5 83% of the succinate is unionized, whereas at pH 6.9 0.20% of the succinate is unionized. The onset of the stationary phase in cultures grown at pH 3.5 and pH 6.9 is due to lack of an adequate amount of extracellular unionized succinate. Intracellular pH values were determined in cultures grown at pH 6.9 using the weak acid DMO (5.5-dimethyl-2,4-oxazolidinedione). As the extracellular pH increased from 6.90 to 7.62, the intracellular pH increased from 5.89 to 6.89. As the extracellular pH increased from 7.62 to 8.44, the intracellular pH increased from 6.89 to 7.50.     

Use of salicylic acid to measure the apparent intracellular pH in the ehrlich ascites-tumor cell and Escherichia coli

The distribution of salicylic acid between the intracellular and extracellular phases has been used to estimate the intracellular pH in the Ehrlich cell and Escherichia coli. The validity of the method was established by: (i) comparison of the results obtained with salicylic acid with those obtained with 5,5-dimethyloxazolidine-2,4-dione; (ii) by following changes of the apparent intracellular pH under circumstances in which such changes are predictable, e.g., the addition of weak acids or proton conductors to the incubation medium during incubation at acidic pH; (iii) by comparison of the apparent intracellular pH changes with the uptake of H⁺ by the cells estimated from the changes of the medium pH. Optimal results are obtained with this indicator when the extracellular pH is below 5.5, because in this case the indicator is to a sufficient extent in its penetrating form, so that its movement can reflect intracellular pH changes occurring in less than 30 s. When the intracellular pH falls below 5.2 measurable binding of salicylic acid to the intracellular material of the Ehrlich cell takes place, but above this pH no binding has been found.The Ehrlich cell and cells of Escherichia coli behaved similarly under various experimental circumstances tested, but striking differences were found in the inherent permeability of the membrane to H⁺ and in the changes in this parameter by lowering the temperature to 2°C.     

Short-term Measurements of the Cytoplasmic pH of Chara corallina Derived from the Intracellular Equilibration of 5,5-Dimethyloxazolidine-2,4-Dione (DMO)

Smith, F. A. 1986. Short-term measurements of the cytoplasmicpH of Chara corallina derived from the intracellular equilibrationof 5,5-dimethyloxazolidine-2,4-dione (DMO).—J. exp. Bot.37: 1733–1745. Measurements of the time-course of influx of ¹⁴C-labelled 5,5-dimethyloxazolidine-2,4-dione(DMO) into the cytoplasm and vacuole of internodal cells ofChara corallina, and of efflux of DMO into non-radioactive solutions,have shown that exchange of DMO across the tonoplast is veryrapid compared with exchange across the plasma membrane. Thishas made possible calculations of cytoplasmic pH from distributionof DMO between cytoplasm and vacuole over short periods (5 or10 min) even when intracellular DMO is not at flux equilibriumwith external DMO. Using this new method, estimates have beenmade of the rates and magnitude of: (i) acidification of thecytoplasm caused by acidic growth regulators (IAA and NAA) andby metabolic inhibitors (azide, DNP, CCCP and DCMU), and (ii)alkalinization caused by uptake of ammonium and methylammoniumions. The potential application of the method to future studiesof membrane transport in charophyte cells is assessed. Key words: Charophyles, cytoplasmic pH.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Distribution and effect of the weak acids 5,5-dimethyl-2,4-oxazolidinedione (DMO) and 2,4-dinitrophenol (DNP) in membrane vesicles of Micrococcus denitrificans.

The effects of 5,5-dimethyl-2,4-oxazolidinedione (DMO) and 2,4-dinitrophenol (DNP) on membrane vesicles of Micrococcus denitrificans were compared. DMO did not affect the ability of these vesicles to accumulate glycine in the presence of the substrate l-lactate. Both glycine transport and l-lactate oxidation were inhibited by DNP; the concentration of DNP required for inhibition of respiration was fortyfold higher than that required for inhibition of transport. Using the technique of equilibrium dialysis with membrane residues from which the lipid had been extracted, no binding of [¹⁴C]DMO to membrane protein was detected. However, [¹⁴C]DNP did bind to membrane protein. At 100 μm DNP, 12% of the [¹⁴C]DNP was bound, equivalent to 1.56 nmol/mg protein. The pH inside vesicles respiring on l-lactate was calculated from the distribution of [¹⁴C]DMO and was found not to differ from the pH of the suspending buffer. The mechanism of action of DNP on active transport in M. denitrificans vesicles appears not to involve proton conduction.     

A Highly Temperature-sensitive Proton Current in Mouse Bone Marrow–derived Mast Cells

Proton (H⁺) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow–derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly activated a voltage-dependent outward conductance with Q₁₀ of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24°C and Q₁₀ was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K⁺, Cl⁻, and Na⁺), but depended on the pH gradient, suggesting that H⁺ (equivalents) is a major ion species carrying the current. The H⁺ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H⁺ current was reversibly inhibited by Zn²⁺ but not by bafilomycin A₁, an inhibitor for a vacuolar type H⁺-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn²⁺, and depletion of extracellular K⁺, but not by bafilomycin A₁. These results suggest that the H⁺ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H⁺ conductance.     

Nitric oxide and pH modulation in gynaecological cancer

Nitric oxide plays several roles in cellular physiology, including control of the vascular tone and defence against pathogen infection. Neuronal, inducible and endothelial nitric oxide synthase (NOS) isoforms synthesize nitric oxide. Cells generate acid and base equivalents, whose physiological intracellular concentrations are kept due to membrane transport systems, including Na⁺/H⁺ exchangers and Na⁺/HCO₃^? transporters, thus maintaining a physiological pH at the intracellular (~7.0) and extracellular (~7.4) medium. In several pathologies, including cancer, cells are exposed to an extracellular acidic microenvironment, and the role for these membrane transport mechanisms in this phenomenon is likely. As altered NOS expression and activity is seen in cancer cells and because this gas promotes a glycolytic phenotype leading to extracellular acidosis in gynaecological cancer cells, a pro‐inflammatory microenvironment increasing inducible NOS expression in this cell type is feasible. However, whether abnormal control of intracellular and extracellular pH by cancer cells regards with their ability to synthesize or respond to nitric oxide is unknown. We, here, discuss a potential link between pH alterations, pH controlling membrane transport systems and NOS function. We propose a potential association between inducible NOS induction and Na⁺/H⁺ exchanger expression and activity in human ovary cancer. A potentiation between nitric oxide generation and the maintenance of a low extracellular pH (i.e. acidic) is proposed to establish a sequence of events in ovarian cancer cells, thus preserving a pro‐proliferative acidic tumour extracellular microenvironment. We suggest that pharmacological therapeutic targeting of Na⁺/H⁺ exchangers and inducible NOS may have benefits in human epithelial ovarian cancer.     

Photosynthesis and Inorganic Carbon Accumulation in the Acidophilic Alga Cyanidioschyzon merolae

The intracellular pH and membrane potential were determined in the acidophilic algae Cyanidoschyzon merolae as a function of extracellular pH. The alga appear to be capable of maintaining the intracellular pH at the range of 6.35 to 7.1 over the extracellular pH range of 1.5 to 7.5. The membrane potential increase from −12 millivolts (negative inside) to −71 millivolts and thus ΔH⁺ decreased from −300 to −47 millivolts over the same range of extracellular pH. It is suggested that the ΔH⁺ may set the upper and lower limits of pH for growth. Photosynthetic performance was also determined as a function of pH. The cells appeared to utilize CO₂ from the medium as the apparent K_m(co2) was 2 to 3 micromolar CO₂ over the pH range of 1.5 to 7.5 C. merolae appear to possess a `CO₂ concentrating' mechanism.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP⁺) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP⁺ was used as an index of the electrical potential difference, Δψ.Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP⁺ (10^?3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP⁺ uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, , amounts under these conditions to about 140 mV.The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Measurement of Cytoplasmic pH by the DMO Technique in Hydrodictyon africanum

The 5, 5-dimethyl-[2-¹⁴C]oxazolidine-2, 4-dione (DMO) distributiontechnique for the measurement of intracellular pH has been appliedto giant cells of Hydrodictyon africanum. Significant metabolism of DMO was found in this alga; the free[DMO + DMO–] in subcellular samples is thus derived fromthe total label in cells equilibrated in [¹⁴C]DMO solutionsby measuring and subtracting the label in metabolic productsof DMO. A further problem arises from the observation that theDMO concentration in the vacuolar sap is always lower than thatpredicted by the transmembrane equilibration of undissociatedDMO from the bathing medium. This is interpreted in terms ofa finite permeability to the anion DMO–. Since the effectof P_DMO– on the DMO distribution is much smaller at thetonoplast (where the transmembrane electrical potential differenceis small) than at the plasmalemma, the values of cytoplasmicpH are computed assuming equilibration of undissociated DMOacross the tonoplast. At an external pH of 7.0 the cytoplasmic pH is about 7.4; decreaseor increase of external pH by 1 unit causes a decrease and anincrease in cytoplasmic p11 respectively of about 0.2 pH units.Determinations of _vo at pH 6, 7, and 8, together with an assumedconstant value of _cv, permit calculations of µ_H+ at theplasmalemma and tonoplast. The values are relatively independentof external pH in the range pH 6–8 at 21–25 and12–14 kJ mol^–1 respectively. The significance ofthese results for the regulation of intracellular pH, and forthe regulation and energising of the fluxes of ions, is discussed.     

The effect of short-term fluctuations in pH on NO−3 uptake and intracellular constituents in Skeletonemacostatum (Grev.) Cleve

Measurements of uptake rates, intracellular nitrogen pools, and other key intracellular constituents were made during exponential growth in Skeletonema costatum (Grev.) Cleve under varying pH levels. An understanding of the overall effects of extracellular pH on the above mentioned cellular parameters is crucial in order to ascertain the degree to which pH must be regulated and monitored in laboratory experiments with marine phytoplankton.It was found that uptake rates and intracellular pool sizes of NO^?₃ were directly influenced by the extracellular pH level, whereas, other cellular compounds remained relatively unchanged. Therefore, nitrogen uptake and intracellular nitrogen storage are dependent on key H⁺ and OH_? ion transport mechanisms that are associated with phytoplankton metabolism. These findings reiterate the fact that investigators examining nitrogen uptake and assimilatory mechanisms in marine phytoplankton must be conscious of cellular H ⁺ and OH^? fluxes that contribute to intracellular pH regulation and changes in extracellular pH levels, both of which interact to affect phytoplankton metabolic processes.     

Modulation of Ca2+ Influx in Leech Retzius Neurons. I. Effect of Extracellular pH

We investigated the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca²⁺]_i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K⁺ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca²⁺]_i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca²⁺]_i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca²⁺]_i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca²⁺]_i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane. Received: 23 January 2001/Revised: 15 June 2001     

Regulation of endogenous and heterologous Ca release-activated Ca currents by pH

Deviations from physiological pH (∼pH 7.2) as well as altered Ca²⁺ signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca²⁺ influx is the store-operated Ca²⁺ entry (SOCE) or Ca²⁺ release-activated Ca²⁺ current (I_CRAC), which is activated upon depletion of intracellular Ca²⁺ stores. We here show that extracellular and intracellular changes in pH regulate both endogenous I_CRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous I_CRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP₃-induced I_CRAC. In the absence of IP₃, external alkalization did not elicit endogenous I_CRAC but was able to activate heterologous I_CRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP₃-induced activation of endogenous and heterologous I_CRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of I_CRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates I_CRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate I_CRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.