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.     

Measurements of the Cytoplasmic pH of Chara corallina using Double-barrelled pH Micro-electrodes

A range of polymeric compounds was examined for their suitabilityas pressure-stabilizing agents in liquid membrane pH micro-electrodesfor intracellular use in plant cells. Of the compounds tested,mixtures of liquid proton sensor and nitrocellulose were foundto be superior to epoxy resins, polyvinylchloride and ethylcellulose. The electrical resistance of silicone rubber mixtureswas too high for micro-electrodes with tip diameters of 1.0µm. Double-barrelled micro-electrodes containing nitrocellulosemaintained excellent pH sensitivity for up to 1.0 impalementsof charophyte cells. Measurements of cytoplasmic pH were madein both internodal and whorl cells of Chora corallina over arange of experimental conditions. The response of cytoplasmicpH to rapid changes in external pH or illumination occurredover several minutes. The advantages of the use of double-barrelledpH micro-electrodes over other methods of intracellular pH measurementsuch as the distribution of weak acids (DMO), ³¹P-NMR and single-barrelledmicro-electrodes is discussed. Key words: pH micro-electrodes, cytoplasmic pH, charophytes     

Measurement of the pH of frozen buffer solutions by using pH indicators.

A method was established to estimate the pH change of several buffers solutions on freezing by using a combination of pH indicators. Among more than 30 buffers solutions examined, almost half exhibited a pH change in the temperature range between freezing point and 220 degrees K; the results were tabulated. Glycerol was found to suppress the pH changes because of its "salt buffer" effect.     

Effect of Temperature and External pH on the Cytoplasmic pH of Chara corallina

Cytoplasmic pH (pH_c) in Chara corallina was measured (from [¹⁴C]stribution)as a function of external pH (pH₀)and temperature. With pH₀near 7, pH_c at 25?C is 7.80; pH_cincreases by 0.005 pH units?C^–1 temperature decrease, i.e. pH_c at 5 ?C is 7.90. WithpH? near 5.5, the increase in pH_c with decreasing temperatureis 0.015 units ?C^–1 between 25 and 15?C, but 0.005 units?C^–1 between 15 and 5?C. This implies a more precise regulationof pH_c with variations in pH_o at 5 or 15 ?C compared with 25?C. The observed dp H_c/dT is generally smaller than the –0.017units ?C^–1 needed to maintain a constant H⁺/OH^–1,or a constant fractional ionization of histidine in protein,with variation in temperature. It is closer to that needed tomaintain the fractional ionization of phosphorylated compoundsor of CO₂–HCO₃^– The value of dpH_c/dT has importantimplications for several regulatory aspects of cell metabolism.These include (all as a function of temperature) the rates ofenzyme reactions, the _H+ at the plasmalemma(and hence the energy available for cotransport processes),and the mechanism for pH_c regulation by the control of bidirectionalH⁺ fluxes at the plasmalemma.     

Modifying the pH sensitivity of OmpG nanopore for improved detection at acidic pH

The outer membrane protein G (OmpG) nanopore is a monomeric β-barrel channel consisting of seven flexible extracellular loops. Its most flexible loop, loop 6, can be used to host high-affinity binding ligands for the capture of protein analytes, which induces characteristic current patterns for protein identification. At acidic pH, the ability of OmpG to detect protein analytes is hampered by its tendency toward the closed state, which renders the nanopore unable to reveal current signal changes induced by bound analytes. In this work, critical residues that control the pH-dependent gating of loop 6 were identified, and an OmpG nanopore that can stay predominantly open at a broad range of pHs was created by mutating these pH-sensitive residues. A short single-stranded DNA was chemically tethered to the pH-insensitive OmpG to demonstrate the utility of the OmpG nanopore for sensing complementary DNA and a DNA binding protein at an acidic pH.     

Modulation of TRPM2 by acidic pH and the underlying mechanisms for pH sensitivity

TRPM2 is a Ca²⁺-permeable nonselective cation channel that plays important roles in oxidative stress–mediated cell death and inflammation processes. However, how TRPM2 is regulated under physiological and pathological conditions is not fully understood. Here, we report that both intracellular and extracellular protons block TRPM2 by inhibiting channel gating. We demonstrate that external protons block TRPM2 with an IC₅₀ of pH_o = 5.3, whereas internal protons inhibit TRPM2 with an IC₅₀ of pH_i = 6.7. Extracellular protons inhibit TRPM2 by decreasing single-channel conductance. We identify three titratable residues, H958, D964, and E994, at the outer vestibule of the channel pore that are responsible for pH_o sensitivity. Mutations of these residues reduce single-channel conductance, decrease external Ca²⁺ ([Ca²⁺]_o) affinity, and inhibit [Ca²⁺]_o-mediated TRPM2 gating. These results support the following model: titration of H958, D964, and E994 by external protons inhibits TRPM2 gating by causing conformation change of the channel, and/or by decreasing local Ca²⁺ concentration at the outer vestibule, therefore reducing [Ca²⁺]_o permeation and inhibiting [Ca²⁺]_o-mediated TRPM2 gating. We find that intracellular protons inhibit TRPM2 by inducing channel closure without changing channel conductance. We identify that D933 located at the C terminus of the S4-S5 linker is responsible for intracellular pH sensitivity. Replacement of Asp⁹³³ by Asn⁹³³ changes the IC₅₀ from pH_i = 6.7 to pH_i = 5.5. Moreover, substitution of Asp⁹³³ with various residues produces marked changes in proton sensitivity, intracellular ADP ribose/Ca²⁺ sensitivity, and gating profiles of TRPM2. These results indicate that D933 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM2 channel gating. Collectively, our findings provide a novel mechanism for TRPM2 modulation as well as molecular determinants for pH regulation of TRPM2. Inhibition of TRPM2 by acidic pH may represent an endogenous mechanism governing TRPM2 gating and its physiological/pathological functions.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

pH measurements in ultranarrow immobilized pH gradients

It is possible to measure pH values in immobilized pH gradients (IPG) when the polyacrylamide matrix is made to contain an additional, carrier ampholyte-generated pH gradient. After an IPG run, 5 mm gel segments, along the separation axis, are cut and eluted in 300 microliter of 10 mM KCl and the pH read with a standard pH meter. When using ultranarrow pH gradients, larger gel segments (ca. 265 microliter) are eluted in 900 microliter of 100 mM KCl and the pH assessed with a differential pH meter. In the latter case, either internal or external standards are used as a reference, or starting point, to convert delta pH values into an actual pH curve. The reproducibility of the system is better than +/- 0.05 pH units, with a ca. 15% error over a 0.3 pH unit span. In ultranarrow pH gradients, it is imperative to use mixtures of all commercially available carrier ampholytes, so as to smoothen conductivity and buffering capacity gaps. By the present method, it is also possible to convert a wide (2-3 pH unit) carrier ampholyte interval into a narrow (0.2-0.3 pH unit) one.     

Regulation of the Cytoplasmic pH of Chara corallina: Response to Changes in External pH

Effects of external pH (pH_o) on the cytoplasmic pH (pH_c) ofChara corallina have been measured with the weak acid 5, 5-dimethyloxazolidine-2,4-dione (DMO) following standardized pretreatment of cells insolutions at pHo 4.5, 6.3 and 8.3. Irrespective of pH_c duringpretreatment, pH_o responded to pHo during the experimental periodsof 150–180 min or (in one experiment) 90–110 min.There were increases or decreases of about 0.5 in pHo when cellswere transferred from pH_o 4.5 to 8.3 or vice versa. In the darkpH_c was 0.2–0.3 units lower than the corresponding valuein the light. The results are discussed in relation to the factorsinvolved in the regulation of pH_c in C. corallina, which maybegin to break down below about pH_o4.5, as indicated by relativelylarge decreases in pH_c at low pH_o. Key words: Chara corallina, Cytoplasmic pH, External pH, DMO     

Determinants of the pH of the Golgi complex

The factors contributing to the establishment of the steady state Golgi pH (pH(G)) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K(+) in the lumen of the Golgi using a null point titration method. The [K(+)] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH(G). Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H(+) pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH(G). The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH(G). In fact, active H(+) pumping was detectable even below the resting pH(G). A steady state pH was attained when the rate of pumping was matched by the passive backflux of H(+) (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H(+)/cation antiporters or symporters were found to contribute to the net loss of H(+) from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn(2+), resembling the H(+) conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H(+) pump determines the pH at which the Golgi lumen attains a steady state.