Changes In Proton Concentration Research Articles

NONLINEAR DYNAMICS NEAR THE CELL MEMBRANE OFCHARA CORALLINA

The phenomenon of patterned distribution of pH near the cell membrane of the algae Chara corallina upon illumination is well-known. In this paper, we develop a mathematical model, based on the detailed kinetic analysis of proton fluxes across the cell membrane, to explain this phenomenon. The model yields two coupled nonlinear partial differential equations which describe the spatial dynamics of proton concentration changes and transmembrane potential generation. The experimental observation of pH pattern formation, its period and amplitude of oscillation, and also its hysteresis in response to changing illumination, are all reproduced by our model. A comparison of experimental results and predictions of our theory is made. Finally, a mechanism for pattern formation in Chara corallina is proposed.

A step-by-step procedure for pH model construction in aquatic systems

Abstract. We present, by means of a simple example, a comprehensive step-by-step procedure to consistently derive a pH model of aquatic systems. As pH modelling is inherently complex, we make every step of the model generation process explicit, thus ensuring conceptual, mathematical, and chemical correctness. Summed quantities, such as total inorganic carbon and total alkalinity, and the influences of modeled processes on them are consistently derived. The different time scales of processes involved in the pH problem (biological and physical reactions: days; aquatic chemical reactions: fractions of seconds) give rise to a stiff equation system. Subsequent reformulations of the system reduce its stiffness, accepting higher non-linear algebraic complexity. The model is reformulated until numerically and computationally simple dynamical solutions, like a variation of the operator splitting approach (OSA) and the direct substitution approach (DSA), are obtained. As several solution methods are pointed out, connections between previous pH modelling approaches are established. The final reformulation of the system according to the DSA allows for quantification of the influences of kinetic processes on the rate of change of proton concentration in models containing multiple biogeochemical processes. These influences are calculated including the effect of re-equilibration of the system due to a set of acid-base reactions in local equilibrium. This possibility of quantifying influences of modeled processes on the pH makes the end-product of the described model generation procedure a powerful tool for understanding the internal pH dynamics of aquatic systems.

Subunit–subunit interactions are critical for proton sensitivity of ROMK: Evidence in support of an intermolecular gating mechanism

The tetrameric K channel ROMK provides an important pathway for K secretion by the mammalian kidney, and the gating of this channel is highly sensitive to changes in cytosolic pH. Although charge-charge interactions have been implicated in pH sensing by this K channel tetramer, the molecular mechanism linking pH sensing and the gating of ion channels is poorly understood. The x-ray crystal structure KirBac1.1, a prokaryotic ortholog of ROMK, has suggested that channel gating involves intermolecular interactions of the N- and C-terminal domains of adjacent subunits. Here we studied channel gating behavior to changes in pH using giant patch clamping of Xenopus laevis oocytes expressing WT or mutant ROMK, and we present evidence that no single charged residue provides the pH sensor. Instead, we show that N-C- and C-C-terminal subunit-subunit interactions form salt bridges, which function to stabilize ROMK in the open state and which are modified by protons. We identify a highly conserved C-C-terminal arginine-glutamate (R-E) ion pair that forms an intermolecular salt bridge and responds to changes in proton concentration. Our results support the intermolecular model for pH gating of inward rectifier K channels.

Potentiometric pH sensors based on electrodeposited polymers

Five potentiometric pH sensors based on thin polymer films, obtained from electropolymerization of their respective monomers: 1,3-diaminopropane, diethylenetriamine, pyrrole, p-phenylenediamine and aniline, were compared. All these molecules have been chosen since they contain amino groups, which are sensitive to proton concentration changes. The resulting sensors consist in a polymer-coated smooth platinum wire protruded from glass sheath. Their potentiometric responses appeared linear in the range from pH 2 to 11. These electrodes present also a good reversibility and a good stability in time over a period of 1 month. The response mechanisms are also examined.

Effects of pH on the kinetics of light-dependent proton flux in thylakoids isolated from lettuce leaves

The pH ranged from 5.5 to 7.5 influences the kinetics of light-dependent proton concentration decrease in the suspension of isolated thylakoids. Irradiation of dark-adapted thylakoid suspension increased of the initial medium pH equal to 5.5, 6.5 or 7.5 with a velocity of 10.9, 3.5 or 1.4×10−3Us−1, respectively. On contrary, turning the light off led to acidification of the medium with initial rates found to be 2.7, 2.2 and 1.4×10−3Us−1 at pH 5.5, 6.5 and 7.5, respectively. Biphasic kinetics were found both in the light-induced deacidification and dark-induced acidification of the medium. These pH changes disappeared in the presence of uncouplers (valinomycin plus nigericin), or were reduced considerably in the presence of DCMU. DCCD, an inhibitor of proton channels, enchanced the velocity of the medium deacidification. Analysis of the chlorophyll fluorescence parameters led to conclusion that at pH 5.5 the PQ pool was more oxidised than at pH 6.5 or 7.5. Thus, the data obtained indicate that the pH of the medium affect significantly the proton flux across the thylakoid membranes and suggest that under physiological condition the proton concentration changes in stroma, induced by light–dark transition, might regulate also the protonation of reduced plastoquinone at QB site.

Acid potentiation of the capsaicin receptor determined by a key extracellular site

The capsaicin receptor, VR1, is a sensory neuron‐specific ion channel that serves as a detector of pain‐producing chemical and physical stimuli. The response of VR1 to capsaicin or heat is dynamically potentiated by extracellular protons within a pH range encountered during tissue acidosis, such as that associated with arthritis, ischemia or tumor growth. A molecular determinant for this activity was localized to an extracellular Glu residue (E600) in the region linking the fifth transmembrane domain with the pore region of the channel. This residue serves as a key regulatory site of the receptor by setting sensitivity to other noxious stimuli in response to changes in proton concentration. We also show that protons, vanilloids, and heat promote channel opening through distinct pathways, because mutations at a second site (E648) selectively abrogate proton‐evoked channel activation without diminishing responses to other noxious stimuli. Our findings provide molecular evidence for stimulus‐specific steps in VR1 activation and offer strategies for the development of analgesic agents.

Metabolic Modulation

featured in this issue of Circulation presents data showingthat pyruvate, which dose-dependently improved perfor-mance in myocardium from failing human hearts, may be onesuch agent.A number of abnormalities in both energy metabolism andcontractile function have been identified in failing cardiacmuscle. Metabolic changes include decreases in high energyphosphorylation potential, decreases in mitochondrial oxida-tive phosphorylation capacity, and a switch to a more fetalglycolytic metabolism.

Shifts of intracellular pH distribution as a part of the signal mechanism leading to the elicitation of benzophenanthridine alkaloids . Phytoalexin biosynthesis in cultured cells of eschscholtzia californica

Cultured cells of Eschscholtzia californica (Californian poppy) respond to a yeast elicitor preparation or Penicillium cyclopium spores with the production of benzophenanthridine alkaloids, which are potent phytoalexins. Confocal pH mapping with the probe carboxy-seminaphthorhodafluor-1-acetoxymethylester revealed characteristic shifts of the pH distribution in challenged cells: within a few minutes after elicitor contact a transient acidification of cytoplasmic and nuclear areas occurred in parallel with an increase of the vacuolar pH. The change of proton concentration in the vacuole and in the extravacuolar area showed a nearly constant relation, indicating an efflux of vacuolar protons into the cytosol. A 10-min treatment with 2 mM butyric or pivalic acid caused a transient acidification of the cytoplasm comparable to that observed after elicitor contact and also induced alkaloid biosynthesis. Experimental depletion of the vacuolar proton pool reversibly prevented both the elicitor-triggered pH shifts and the induction of alkaloid biosynthesis. pH shifts and induction of alkaloid biosynthesis showed a similar dependence on the elicitor concentration. Net efflux of K+, alkalinization of the outer medium, and browning of the cells were evoked only at higher elicitor concentrations. We suggest that transient acidification of the cytoplasm via efflux of vacuolar protons is both a necessary and sufficient step in the signal path toward biosynthesis of benzophenanthridine alkaloids in Californian poppy cells.

A glucose-sensing polymer

We have prepared a robust polymer that can be used to measure glucose concentrations in complex biological media. At alkaline pH, the metal-complexing polymer binds glucose and instantly releases protons in proportion to the glucose concentration over a clinically relevant range (0 to 25 mM). The inexpensive polymer is sufficiently selective to provide an easily measurable response to glucose in porcine plasma. The polymer's ability to function at nonphysiological pH (at which the buffer capacity of biological samples is small) makes it possible to design simple and inexpensive sensing devices based on measurement of changes in proton concentration.

Electrochemical adsorption method for the determination of the p K of ionizable groups of lipid membranes

The intrinsic p K value (p K i) of the phosphate group of cardiolipin liposomes was found using an original method which resembles the electrochemical adsorption technique conventionally used in studies of the electrical double layer. Our method is based on the measurement of changes in proton concentration Δ c H + in the diffuse layer adjacent to the charged lipid membrane, as a result of stepwise changes of monovalent salt concentration in the bulk solution. p K i(PO 4 −) = 2.5 (± 0.1) was found by fitting the theoretical Δ c H + vs. log c curves to the corresponding experimental values in the range 10 −3−10 −1 M sodium chloride. Changes in the membrane charge density at various p K i are presented. The advantages and limitations of the suggested method are also discussed.

Use of reversed-phase h.p.l.c. for determination of the hydrolysis rate constants of dichloro(1,2-diarylethylenediamine)platinum(II) complexes

The rates of the Pt-Cl hydrolysis reactions for a series of dichloro(1,2-diarylethylenediamine)platinum(II) complexes were measured by means of a reversed-phase h.p.l.c. assay. The compounds were hydrolysed at rates comparable to those published for [PtCl2(en)]. The configuration of the 1,2-diarylethylenediamine ligand (meso versus racem), as well as the type of substituent (i.e. OMe, CN or F) in the para-position of the aromatic rings had only a marginal influence on the rates of hydrolysis; however, the introduction of ortho-chloro substituents onto the aromatic rings markedly slowed the hydrolysis reactions. In contrast to the reported pH-dependency of the hydrolysis rates of cisplatin, changes in proton concentration had no effect on the hydrolysis rates of the title compounds over a pH range of 2.2–7.2. The activation parameters ΔH ≠ and ΔS ≠ of the Pt-Cl hydrolysis rates were comparable to those reported for similar PtII compounds.

Direct Measurement of ATP-Dependent Proton Concentration Changes and Characterization of a K+-Stimulated ATPase in Pea Chloroplast Inner Envelope Vesicles.

Inner envelope membrane vesicles prepared from pea (Pisum sativum L. var Laxton's Progress No. 9) chloroplasts have K+-stimulated ATPase activity with a pH optimum of 8.4. ATP addition to inner envelope vesicles loaded with pyranine caused a decrease in pyranine fluorescence that was consistent with internal acidification. The transmembrane pH change induced by the addition of 5 mM ATP was about 0.4 unit. Measurement of phosphate released by ATP hydrolysis paralleled the pH change, indicating that intravesicular acidification was linked to ATPase activity. Vanadate, molybdate, N-ethylmaleimide, and dithiothreitol inhibited ATP-dependent vesicle acidification completely, whereas ATPase activity was only partially inhibited. These data indicate that pea chloroplast inner envelope vesicles contain a proton translocating ATPase and that the pyranine-loading method can be utilized to study directly ATP-dependent H+ transport across these membranes.

Highly cooperative and hysteretic response of the skeletal muscle ryanodine receptor to changes in proton concentrations

Ryanodine receptors are key molecules in excitation-contraction coupling of skeletal muscle. They form the pore of the calcium release channel, which is regulated by Ca and ATP. Multiple proton titration sites are involved in controlling the different open states of the channel, as indicated by the following: i) the channel had a biphasic response to changes in proton concentrations around neutral pH; ii) the activities of the channel were inhibited by acidic pHs in a highly cooperative manner; and iii) the channel exhibited pronounced hysteresis to changes in pH. Four distinct conductance states can be identified in the single ryanodine-activated calcium release channel. The distribution of the multiple conductance states depends on the level of [Ca], ATP, and pH in the recording solution. The data are consistent with the multimeric structure of the skeletal muscle ryanodine receptor.

A chemically and electrochemically switchable molecular shuttle

THE developing field of nanotechnology has generated wide interest across a broad range of scientific disciplines1. In particular, the realization of nanoscale switching devices might have far-reaching implications for computing and biomimetic engineering2–4. But miniaturization of existing semiconductor technology may not be the best approach to the fabrication of structures whose dimensions are smaller than the wavelength of the radiation used in optical lithography and etching techniques5. The approach observed in the natural world, whereby nanostructures are built up through the self-assembly6–9 of smaller molecular entities, holds substantial promise. Nature abounds with molecular switching devices which perform a variety of functions, such as the transport of metabolites across cell membranes or the signalling of nerve impulses. These processes are commonly controlled by stimuli such as changes in ion concentrations and electrical potentials. Here we report the synthesis of a supramolecular structure (compound 1-[PF6]4, Fig. 1A) that can be reversibly switched between two states by proton concentration changes or by electrochemical means. The super-molecule is a rotaxane comprising a molecular ring threaded on an axle containing two ‘docking points’. We can effect controlled switching of the ring from one of these positions to the other. We use 1H NMR and ultra violet/visible spectroscopy to characterize the dynamics of the bead's movement along the thread before and after switching.

Relationship between intracellular proton buffering capacity and intracellular pH.

In a recent publication the widely held view that the intracellular proton buffering power [defined as the amount of acid or base that has to be added to the cytosol to change the intracellular pH (pHi) by one pH unit] increases as the intracellular pH decreases, has been challenged, with the opposite relationship being proposed. In that publication, buffering was defined not in terms of pH change, but in terms of the change in proton concentration. The reason for this re-definition was the fear that the conventional analysis, using as it does a logarithmic function (pHi), could bias the outcome in favor of an increasing buffering power with decreasing pHi. The new system uses a "buffering co-efficient," defined as the number of protons necessary to be added to the cytosol to change the intracellular proton concentration by 1 mM. We report the use of both of these methods to analyze the relationship of pHi and buffering power, using human peripheral leucocytes loaded with the pH-sensitive fluorophore BCECF examined over a very wide range of pHi values (pHi 6.0 to 7.5). The most common method for pHi perturbation for the measurement of buffering is used, the rapid diffusion of ammonia across the cell membrane. In this study, analysis for both a bicarbonate-containing "open" system and for a Hepes-buffered "closed" system was performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the T-type cardiac Ca channel by changes in proton concentration.

Single-channel measurements and whole-cell experiments with the two suction electrode, voltage clamp technique were used to investigate the effects of external and internal proton concentrations on T-type Ca channels in heart muscle cells of the guinea pig. As in the L-type Ca channel, an increase in the external proton concentration decreases T-type currents, while external alkalinization enlarges the currents. In contrast to the L-type Ca channel, however, a change in the internal proton concentration does not modulate T-type Ca currents. The T-type Ca channel is much more sensitive to variations in pHo than the L-type Ca channel. By the combination of single-channel and whole-cell experiments we can conclude that the observed changes in macroscopic currents are due to (a) changes in the single-channel conductance and in the probability of the T-type Ca channel being open, and (b) the titration of the negative surface charges in the neighborhood of the T-type Ca channel with shifts of both the activation and inactivation processes of the channel. The pHo-induced changes in the maximal conductance (gmax) of the T-type Ca channel show an apparent pKa in the range of 7.1-7.5, while the titration of the negative surface charges near the channel shows an apparent pKa of 7.1 with a concomitant surface potential of -24.6 mV at 5.4 mM [Ca]o. These pKa values, less acid than the pKa values found for the pHo-induced, L-type Ca channel modulation, might imply a physiological importance of this novel type of channel modulation.

Proton-induced chloride current and voltageactivated Na + and Ca 2+ currents in embryonic neurons from the medicinal leech ( Hirudo medicinalis)

Voltage-activated ionic currents and currents induced by step changes in proton concentration (pH 7.9 6.7) were studied in early embryonic neurons from Hirudo medicinalis. Ganglia were dissociated at embryonic day (E) 8 15, and the largest neurons were investigated using whole-cell patch clamp recording. All cells studied displayed voltage-activated Na + and Ca 2+ currents. Step changes in pH induced sustained currents which reversed at the equilibrium potential for Cl − and were blocked by substituting Cl − with acetate or sulfate. These currents thus differ in their ion selectivity from proton-induced currents in vertebrate neurons which are carried by Na +.

Steady-state characteristics of the proton receptor in the somatic membrane of rat sensory neurons

Steady-state characteristics of proton-activated permeability of the somatic membrane of rat sensory neurons were studied. Transmembrane ionic currents arising in response to application of solutions with low pH were measured during intracellular perfusion and simultaneous voltage clamping. A rapid change in proton concentration in the external medium was produced by a modified rapid application method. Steady-state desensitization of proton-induced permeability was found to develop in the region of weakly alkaline values (pKa 7.15). The mechanism of desensitization is highly cooperative: Hill's parameter for dose-effect dependences was close to 7. Activation also is cooperative, but its Hill's parameters is about 2. Calcium and magnesium cations compete with protons, changing pKa of dose-effect relationships for desensitization and activation, but they have only a weak effect on the value of Hill's parameter.

Conformational changes of apigenin 7-O-(6-O-malonylglucoside), a vacuolar pigment from parsley, with solvent composition and proton concentration.

Malonylated apigenin 7-O-glucoside was prepared from malonyl-CoA and apigenin 7-O-glucoside using a malonyltransferase from parsley cell cultures. The enzyme product, as well as the flavonoid substrate, was analyzed by high-resolution nuclear magnetic resonance spectroscopy, confirming that malonic acid had been attached to the primary hydroxyl of the glucose moiety of apigenin 7-O-glucoside. During these studies in several solvents, it became apparent that, in particular, the chemical shift of H-6" A and the coupling constants J5",6"A and J5",6"B were dependent on solvent composition and proton concentration. Similar changes of sugar proton resonance frequencies were also observed with methyl 6-O-malonyl-beta-D-glucopyranoside, but not with apigenin 7-O-[6-O-(4-coumaroyl)glucoside]. The change of coupling constants is ascribed to a conformational modification of the sugar portion in the malonylglucosides. Malonylated flavonoid glycosides are exclusively located within the vacuoles of parsley cells. We propose that acylation of flavonoid glycosides which are synthesized in the cytoplasm facilitates transport of the substrates into the vacuole. The conformational modification which is induced by changes in proton concentration may provide a mechanism to trap flavonoids in situ.

A simple spectrophotometric method for determining proton release from yeast cells

A simple spectrophotometric acid-base indicator method was developed to determine the rate of proton extrusion after addition of a fermentable hexose to a yeast cell suspension. Unlike the conventional pH-electrode measurements the method gives linear response to changes in proton concentration and makes rapid kinetic determinations possible.