Presence Of Permeant Anions Research Articles

Comparison of Coupled and Uncoupled Currents during Glutamate Uptake by GLT-1 Transporters

The transport of glutamate across the plasma membrane is coupled to the movement of cations (Na+, K+, and H+) that are necessary for glutamate uptake and transporter cycling as well as anions that are uncoupled from the flux of glutamate. Although the relationship between these coupled (stoichiometric) and uncoupled (anion) transporter currents is poorly understood, transporter-associated anion currents often are used to monitor transporter activity. To define the kinetic relationship between these two components, we have recorded transporter currents associated with stoichiometric and anion charge movements occurring in response to the rapid application of l-glutamate to outside-out patches from human embryonic kidney cells expressing GLT-1 transporters. Transporter-associated anion currents were approximately twice as slow to rise and decay as stoichiometric transport currents, but the presence of permeant anions did not slow transporter cycling. A kinetic model for GLT-1 was developed to simulate the behavior of both components of the transporter current and to estimate the capture efficiency of GLT-1. In this model the K+ counter-transport step was defined as rate-limiting, consistent with the slowing of transporter cycling after the substitution of internal K+ with Cs+ or Na+. The model predicts that in physiological conditions approximately 35% of GLT-1 transporters function as buffers, releasing glutamate back into the extracellular space after binding.

Characterization of an Anion Transporter in the Plasma Membrane of Barley Roots

To examine the relationship between H+-ATPase and the transport of anions, we investigated the effects of various inhibitors on the activity of the H+-ATPase, the transport of protons, and the transport of Cl~ ions using plasma membrane vesicles prepared from barley roots. Some inhibitors, namely, 4,4-diisothiocyano-2,2-stilbene disulfonate (DIDS) and Zn2+ ions markedly inhibited H+ATPase activity. Other compounds, such as phenylglyoxal (PGO) and niflumic acid (NIF), inhibited H+-ATPase activity by 20-30%, while anthracene-9-carboxylate (A-9-C) and tetraethylammonium chloride (TEA-C1) had little effect on this activity. The ATP-dependent acidification of the interior of vesicles was strongly dependent on the presence of permeant anions, such as chloride (Cl~) and nitrate (NO3~), and it was completely inhibited by 0.2 mM NIF. Other compounds, namely, A-9-C of 0.1 mM and TEA-C1 of 10 mM, did not affect H+-transport activity. The inhibition of H +-transport activity by NIF was observed even when the activity was assayed in the presence of KC1, KNO3, or bis-tris-prop ane (BTP)-CI. Using 36C1, we quantified Cl~ -transport activity by measuring the uptake of Cl~ ions into the plasma membrane vesicles. The uptake depended on the potential difference across the membrane that was generated by H+-ATPase; it was enhanced by an inside-positive potential gradient. At 0.1 mM, NIF completely blocked the voltage-dependent Cl~-lransport activity. From these properties of the Cl~ transporter and the inhibition of H +transport activity by NIF, we suggest that H+-transport activity across the plasma membrane might be modulated by the transport of anions via a NIF-sensitive anion-permeable transporter that acts to collapse the inside-positive potential generated by H +-ATPase.

Isolation Of H +-Transport-Competent Plasma Membrane Vesicles From Corn Roots By Discontinuous Sucrose Gradient Centrifugation: Effect of Membrane Protectant Agents

Summary Crude KI-washed plasma membrane vesicles isolated from corn roots were further purified on a discontinuous sucrose gradient. Due to the addition of the substrate analogue of phospholipase D, choline, and an inhibitor of the phosphatidic acid phosphatase, sodium glycerol- l-phosphate, to the homogenizing and sucrose gradient media, purified plasma membrane vesicles collected at the 30%/38% interphase conserved a high specific activity of H + -transport. In particular, the presence of choline seemed to be essential to stabilize the ATP-dependent H + -accumulation of membrane vesicles. The H + -transport activity was sensitive to vanadate only. The pH optimum of H + -transport was between 6.0 and 6.5. The H + -transport was highly stimulated by K + without any lag phase, which was consistent with a direct action of K + on the plasma membrane ATPase at the cytoplasmic vesicle surface facing the medium. The K + -stimulation of H + -transport was concentration dependent and increased up to 100 mM K + without achieving saturation. The H + -transport activity depended on the presence of permeant anions. The sequence of anion stimulation followed the order of Br - > Cl - > N0 3 - >> S0 4 - > IDA - and was therefore different from the sequences determined with plasma membrane vesicles isolated from other plant tissues. The lower effectiveness of N0 3 - than Br - and Cl - in stimulating H + -translocation did not originate in tonoplast contamination but appeared rather to be related to genetic differences in corn varieties.

PPiase‐Activated ATP‐Dependent H+ Transport at the Tonoplast of Mesophyll Cells of the CAM Plant Kalanchoë daigremontiana*

AbstractThe co‐ordinated action of the two proton‐transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PPiase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PPi‐dependent H+ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H+ pumps were non‐additive in establishing the steady‐state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP‐dependent H+ transport were stimulated enormously if ATP was added a few minutes after pre‐energization of the vesicles with PPi. This stimulation was observed only when the PPiase was active. A similar effect was not found for PPi‐dependent H+ transport after pre‐energization with ATP. Hence, a PPiase‐activated ATP‐dependent H+ transport can be distinguished from the basic ATP‐ and the basic PPi‐dependent H+ transport. In parallel a PPi‐dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PPiase. PPiase‐activated ATP‐dependent H+ transport depends on the presence of permeant anions. It shows properties of both H+ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP‐dependent H+ transport activity, the nitrate stimulation and the KF inhibition of the PPi‐dependent H+ transport activity. Only MgPPi and MgATP were effective as the respective substrates. The PPiase‐activated ATP‐dependent H+ transport had a half life of about 5–9 minutes. It is concluded that the PPiase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed.

Role of Anions in Low pH-induced Translocation of Diphtheria Toxin

Previous work has shown that when Vero cells with surface-bound diphtheria toxin are exposed to low pH, toxin entry across the plasma membrane is induced and that this entry involves two steps, insertion of the B-fragment of the toxin into the membrane and translocation of the enzymatically active A-fragment to the cytosol. Here we have studied the role of permeant anions in this process. It was found that when the B-fragment was inserted into the membrane, part of it, a 25-kDa polypeptide, was shielded from externally added Pronase. This insertion did not require permeant anions. The translocation of the A-fragment was monitored by measuring either its ability to inhibit protein synthesis in the cells or the appearance of radioactively labeled 21-kDa fragment after treatment of the cells with externally applied Pronase. The translocation of the A-fragment was dependent on the presence of permeant anions in the medium. However, when the cells were depleted of Cl- by incubation in Cl- free buffer at high pH, translocation of the A-fragment did not require permeant anions in the medium. The possibility that translocation of the A-fragment is inhibited by an outward directed chloride gradient rather than by the absence of chloride is discussed.

Characterization of a H+-ATPase in rat brain synaptic vesicles: Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.

Hepatic taurine transport: a Na+-dependent carrier on the basolateral plasma membrane.

Highly purified rat basolateral liver plasma membrane vesicles were used to examine the mechanism and the driving forces for hepatic uptake of the beta-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium ("overshoot"). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K+ gradient with valinomycin increased Na+-stimulated taurine uptake. External Cl- stimulated Na+-dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na+-dependent taurine uptake showed a sigmoidal dependence on extravesicular Na+ concentration, suggesting multiple Na+ ions are involved in the translocation of each taurine molecule. Na+-dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol.mg protein-1.min-1 and an apparent Km of 174 microM. [3H]taurine uptake was inhibited by the presence of excess unlabeled taurine, beta-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, we have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for beta-amino acids. Uptake is electrogenic, stimulated by external Cl-, and requires multiple Na+ ions for the translocation of each taurine molecule.

Interactions between diphtheria toxin entry and anion transport in Vero cells. I. Anion antiport in Vero cells.

In sodium-free buffer of low ionic strength, the uptake of chloride and sulfate in Vero cells was found to occur mainly by antiport which was very sensitive to inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Efflux of anions from the cells appeared to energize the uptake. While the uptake of Cl- occurred over a wide pH range, that of SO4(2-) showed a clear maximum at pH 6-7. The rate of efflux of 36Cl- and 35SO4(2-) was strongly increased by the presence of permeant anions in the efflux buffer. Preincubation of the cells at slightly alkaline pH strongly increased the rate of C1- efflux into buffers nominally free of permeant anions, as well as the efflux by exchange. This increase did not occur if the cells were depleted for ATP during the preincubation. Depolarization of the cells reduced the rate of efflux into buffers without permeant anions, indicating that the efflux is at least partly due to net, electrogenic, anion transport. The efflux by antiport was not affected by manipulations of the membrane potential, indicating electroneutral exchange. The uptake and efflux were increased to the same extent with increasing temperature, the activation energies were Ea = 25 kcal/mol of Cl- and Ea = 12 kcal/mol of SO4(2-). Similar anion antiport appears to occur in L, baby hamster kidney, and HeLa S3 cells.

Interactions between diphtheria toxin entry and anion transport in Vero cells. IV. Evidence that entry of diphtheria toxin is dependent on efficient anion transport.

Entry of prebound diphtheria toxin at low pH occurred rapidly in the presence of isotonic NaCl, NaBr, NaSCN, NaI, and NaNO3, but not in the presence of Na2SO4, 2-(N-morpholino)ethanesulfonic acid neutralized with Tris, or in buffer osmotically balanced with mannitol. SCN- was the most efficient anion to facilitate entry. Uptake studies with radioactively labeled anions showed that SCN- was transported into cells 3 times faster than Cl-, while the entry of SO2-4 occurred much more slowly. The anion transport inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and piretanide inhibited entry at low pH even in the presence of permeant anions. When cells with bound toxin were exposed to low pH in the absence of permeant anions, then briefly exposed to neutral pH and subsequently exposed to pH 4.5 in the presence of isotonic NaCl, toxin entry was induced. The data indicate that efficient anion transport at the time of exposure to low pH is required for entry of surface-bound diphtheria toxin into the cytosol. Since insertion of diphtheria toxin into the membrane occurs even in the absence of permeant anions, the results indicate that low pH is required not only for insertion of fragment B into the membrane, but also for the subsequent entry of fragment A into the cytosol.

Uptake and transport of mannosylated ligands by alveolar macrophages. Studies on ATP-dependent receptor-ligand dissociation.

During endocytosis, mannosylated ligands enter vesicles which have a density intermediate between that of the plasma membrane and secondary lysosomes. Mannosylated ligands are transferred from these vesicles to lysosomes. A solubilization-precipitation assay was used to study the dissociation of mannosylated ligands from their receptor. In whole cells dissociation was rapid (t 1/2 (37 degrees C) = 8 min) and took place before delivery of the ligand to lysosomes. Receptor-ligand dissociation within membrane vesicles, washed free of cytosol, could be induced by addition of ATP and GTP but not ADP. Receptor-ligand dissociation caused by manipulating the pH of the vesicles suggested that the pH within endosomes was lowered to 5.5 by addition of ATP. Dissociation was blocked by proton ionophores and Zn2+, but was unaffected by inhibitors of the F1, Fo-ATPase or the Na+,K+-ATPase. Dissociation did not require Na+ or K+ and was blocked by anion transport inhibitors. Dissociation was slowed in the absence of permeant anions (Cl-). Receptor-ligand complexes within vesicles isolated as early as 2 min following ligand internalization responded to addition of ATP. The results suggest that receptor-ligand dissociation in endosomes requires ATP, possibly to power endosomal acidification via an ATP-dependent proton pump. Dissociation is enhanced in the presence of permeant anions, suggesting the involvement of an anion channel or carrier.

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses.

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.

Interaction of Ca2+ with Blowfly Flight Muscle Mitochondria

Abstract Blowfly flight muscle mitochondria differ markedly from mammalian mitochondria in their interaction with Ca2+. State 4 respiration of blowfly mitochondria is not stimulated by Ca2+, nor does Ca2+ induce a reversible shift in the oxidation-reduction state of cytochrome b. Nevertheless, Ca2+ is accumulated by respiring blowfly mitochondria, but with a low efficiency; the Ca2+: ∼ accumulation ratio is about 0.6 with pyruvate plus proline, and about 0.2 with glycerol 1-phosphate, compared with values of about 2.0 observed in mammalian mitochondria. Ca2+ uptake is blocked by respiratory chain inhibitors and by uncouplers, but not by oligomycin. The presence of permeant anions in the medium is essential for Ca2+ uptake by blowfly mitochondria. At high levels of Ca2+, phosphate supports Ca2+ uptake better than acetate, but at low Ca2+ levels, phosphate and acetate are equally effective. Since acetate penetrates blowfly mitochondria passively it is probable that respiration-coupled uptake of Ca2+ in blowfly mitochondria must be accompanied by passive entry of matching anions. The accumulation of Ca2+ by blowfly mitochondria is very slow and does not exhibit saturation kinetics; moreover, no high affinity Ca2+ sites characteristic of a specific Ca2+ carrier could be detected. For these reasons it is concluded that the respiration-dependent accumulation of Ca2+ in blowfly mitochondria is not mediated by a specific carrier, but occurs through slow, concentration-dependent, physical diffusion through the membrane, in response to an electrochemical gradient generated by electron transport. The number of low affinity Ca2+-binding sites is surprisingly low, about one-tenth of that found in mammalian mitochondrial, and has been related to the peculiar phospholipid composition of these mitochondria. The findings described strongly suggest that the mitochondria of blowfly flight muscle do not actively segregate Ca2+ at the low concentrations at which it is presumed to occur in the sarcoplasm in vivo.

Gramicidin induced ion transport in brain mitochondria preparations.

Abstract— —(1) Gramicidin at low concentrations induces an uptake of K+ and Na+ in brain mitochondria in a manner similar to that observed with liver mitochondria.(2) The cation uptake is energy dependent, and is accompanied by an ejection of H+ ions and a slight increase in respiration in the absence of added permeant anion.(3) The cation uptake and hydrogen ion release are both inhibited by agents which inhibit electron transport. Barbiturates and chlorpromazine inhibit the transport phenomenon by inhibiting electron transport.(4) In the presence of permeant anions (phosphate and acetate) respiration is stimulated quite significantly.(5) At high gramicidin concentrations there is a release of Na+ and K+ from the mitochondria and uptake of H+. There is also a cyclic reduction‐oxidation of the nicotinamide adenine dinucleotides, which is believed to be due to the release from the mitochondria of the reduced dinucleotides followed by their subsequent oxidation.(6) The effect of high gramicidin on the mitochondrial nicotinamide‐adenine dinucleotides and cation distribution is irreversible and is not blocked by individual inhibitors of respiration and of phosphorylation, but is prevented by prior addition of a mixture of these inhibitors.(7) Gramicidin is therefore believed to have a bimodal function; one on the mitochondrial membrane per se, and the other on the energy dependent ion accumulation apparatus.(8) A model of induced mitochondrial ion accumulation is presented.

Acid-Base Exchanges in Mitochondria and Suspending Medium during Respiration-linked Accumulation of Bivalent Cations

Abstract When rat liver mitochondria accumulate Ca++ during succinate-linked respiration in the absence of permeant anions, there is ejection of H+ into the suspending medium and an apparent alkalinization of the mitochondria. The alkalinization is estimated by direct pH measurements and microtitration of suspensions of Ca++-loaded mitochondria in distilled H2O or in dilute Lubrol solutions, in which their dissociating groups become accessible to measurement. The increase in pH of Ca++-loaded mitochondria suspended in this way may exceed 1.5 pH units following uptake of 80 nmoles of Ca++ per mg of protein. From such measurements it is concluded that for each pair of electrons passing each energy-conserving site, about 2 Ca++ ions are accumulated, 2 H+ ions are ejected into the medium, and an increase of titratable alkalinity that is equivalent to 2.0 OH- ions occurs in the mitochondria, in the absence of permeant anions. Alkalinization of the mitochondria is inhibited by antimycin A, 2,4-dinitrophenol, gramicidin in the presence of NaCl, and valinomycin in the presence of KCl but not by oligomycin. The increase in titratable alkalinity of the mitochondria is linear with the amount of Ca++ accumulated over a wide range; alkalinization also occurs during accumulation of Sr++ and Mn++. Permeant anions such as phosphate and acetate diminish mitochondrial alkalinization during Ca++ uptake in proportion to the decrease in H+ ejected. The alkalinization is also diminished when β-hydroxybutyrate and α-ketoglutarate are present, due to internal acidification of the mitochondria by side reactions associated with these substrates. Exposure of Ca++-loaded mitochondria to Lubrol or to sonic irradiation in the absence of respiratory substrates caused loss of Ca++ to the medium, accompanied by alkalinization of the latter. However, when a respiratory substrate was present during sonic treatment, the separated membrane fraction retained all the accumulated Ca++, and no titratable alkalinity appeared in the soluble fraction. It is concluded that there may be no intrinsic alkalinization of mitochondria following Ca++ uptake; titratable alkalinization becomes manifest only in experimental conditions, such as interruption of electron transport or the presence of permeant anions, that cause loss of bound Ca++ from the membrane with corresponding stoichiometric uptake of H+ from the aqueous phase, leaving it alkaline. These properties are best explained by occurrence of a reversible Ca++-H+ exchange diffusion carrier in the membrane.

Super-stoichiometric Ratios between Ion Movements and Electron Transport in Rat Liver Mitochondria

Abstract The number of Ca++ ions accumulated per pair of electrons passing through each energy-conserving site of the respiratory chain during Ca++-induced respiratory jumps in isolated rat liver mitochondria (i.e. the Ca++: ∼ ratio) is normally approximately 2.0 when the suspending medium contains 80 mm KCl or NaCl at pH 7.4, but no permeant anion. However, when the concentration of impermeant salts is raised to as high as 240 mm, or when the pH is raised to above 8.0, the Ca++ ∼ ratio increases to values exceeding 4.0 to 5.0, primarily because of a decrease in the amount of extra oxygen uptake evoked by addition of Ca++. The ratio H+ ejected: ∼ also increases in proportion to the Ca++: ∼ ratio. Increase in sodium chloride concentration does not influence the adenosine diphosphate to oxygen ratio of oxidative phosphorylation. Such super-stoichiometric ratios of Ca++ uptake are almost completely dependent on electron transport, and occur in the presence of high concentration of impermeant anions such as chloride, bromide, iodide, and thiocyanate, but not when such salts are replaced with sucrose. In the presence of permeant anions, such as phosphate, the Ca++: ∼ ratio remains constant at about 2.0. The possible causes of the apparently flexible stoichiometry of Ca++ uptake and H+ ejection with electron transport in the absence of phosphate are discussed.

Hydrogen Ion Concentration Changes in Mitochondrial Membranes

1. Bromthymol blue added to rat liver mitochondria at concentrations in the range 0.4 to 4.0 mµmoles per mg of protein is tightly bound as indicated by centrifugation and washing of the mitochondria. 2. The absorbance of the dye is increased on adding Ca++ or Mn++ to the mitochondria in the absence of a permeant anion. The extent of increase is 0.03 cm-1 for 167 µ m Ca++ at 618 mµ for 0.5 mµmole of bromthymol blue per mg of protein. 3. The spectrum of the indicator bound to the mitochondria has shifted only slightly to longer wave lengths than in the solution. The indicator shows very nearly the same absorbance changes on alkalinization of the mitochondrial suspension as it does upon addition of small amounts of calcium to the mitochondria. 4. The absorbance increase is independent of the pH of the suspension medium (6.5 to 7.4) and of its buffer capacity from 2.0 m m to 0.3 m sodium succinate. 5. At low protein concentrations (1 to 2 mg per ml), the kinetics of the absorbance increase is compared with the glass electrode recordings and indicates an equal and opposite pH change in the suspension medium of a similar time course. 6. At higher protein concentrations (5 to 10 mg per ml), a similar phenomenon is observed, but only after several prior additions of Ca++; endogenous material (termed Hi+) present, in terms of Ca++, at levels of approximately 40 mµmoles per mg of protein counteracts the initial absorbance increase. A small amount of added phosphate readily serves as Hi+, and endogenous phosphate at 8 mµmoles per mg of protein would afford an appropriate source of Hi+. However, the endogenous material may be phospholipid. 7. The absorbance increase on calcium additions with bromthymol blue as an indicator can be counteracted and even reversed with a 30-fold excess of bovine serum albumin, suggesting that the indicator can be removed from the mitochondrial membranes. 8. The Ca++-induced absorbance increase is nullified rapidly by additions of permeant anions such as phosphate or acetate. Ammonium ions at a 3 m m concentration do not prevent the acidification caused by acetate, and 0.1 m concentrations only slightly facilitate the alkalinization on addition of Ca++. 9. It is concluded that rapid accumulation of a cation such as Ca++ in the absence of a permeant anion can result in an intramitochondrial alkalinization and a pH gradient of about 1 pH unit across the mitochondrial membrane. This gradient develops only as a consequence of cation accumulation in the absence of a permeant anion. In the presence of permeant anions, particularly phosphate under the conditions of oxidative phosphorylation, there is no evidence of a measurable pH gradient across the mitochondrial membrane. 10. The intramitochondrial location of the indicator and of the site of calcium accumulation is tentatively assigned to the intracristal space because of the insensitivity of the bromthymol blue response to changes of osmolarity from 0.1 to 0.8 m . 11. The reaction mechanism for cation accumulation based upon the reaction with the X ∼ I intermediate of energy transfer meets the needs of the available experimental data.