Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP

Summary The ratio of valinomycin-mediated unidirectional K⁺ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K⁺ net efflux and⁴²K influx. The driving force for the net efflux (V _m –E _K ⁺) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K⁺ net efflux has been calculated from the K⁺ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K⁺ transport from unrestricted electrodiffusion, when protonophore is present.In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 m and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.     

Quantitation of corneal endothelial potentials using a carbocyanine dye

The carbocyanine dye, diS-C₃-(5) was used to quantitate the plasma membrane potential of the bullfrog corneal endothelium. It was shown that valinomycin hyperpolarized the endothelial cell and that in the presence of the ionophore the membrane potential largely reflected the K⁺ equilibrium potential. Using calibration curves constructed by changing medium K⁺ concentration in the presence of valinomycin, and nigericin and ouabain to abolish ion gradients and electrogenic pump activity, the cell membrane potential was calculated to be 28.6 ± 4.2 mV. The major source of this potential was a K⁺ diffusion potential, and the membrane Na⁺ conductance reduced the cell potential to less than the apparent K⁺ equilibrium potential of 51.5 ± 5.1 mV. About 20% of the cell potential could be ascribed to the rheogenic (Na⁺+K⁺)-ATPase.     

Mechanism of mitochondrial transport of thallous ions

Rat liver mitochondria were found to swell under nonenergized conditions when suspended in media containing 30–40 mM TINO₃. Respiration on succinate caused a rapid contraction of mitochondria swollen under nonenergized conditions. In the presence of thallous acetate, there was a rapid initial swelling under nonenergized conditions until a plateau was reached; respiration on succinate then caused a further swelling. Trace amounts of²⁰⁴Tl (less than 100 µM) equilibrated fairly rapidly across the mitochondrial membrane. The influx of Tl⁺ was able to promote the decay not only of a valinomycin-induced K⁺-diffusion potential but also of respiration-generated fields in the inner membrane in accordance with the electrophoretic nature of Tl⁺ movement. Efflux of Tl⁺ showed a half-time of about 10 sec at 20°C and was not affected appreciably by the energy state. Efflux was retarded by Mg²⁺ and by lowering the temperature. The data indicate that Tl⁺ when present at high concentrations, 30 mM or more, distributes across the mitochondrial inner membrane both in response to electrical fields and to pH. In energized mitochondria the uptake of Tl⁺ would occur electrophoretically, while Tl⁺/H⁺ exchange would constitute a leak. In the presence of NO ₃ ^– , the movements of Tl⁺ are determined by that of NO ₃ ^– , indicating short-range coupling of electrical forces. At low concentrations of Tl⁺, 5 mM or less, there was no indication of a Tl⁺/H⁺ exchange, which appears to be induced by high concentrations of Tl⁺.     

Mycoplasma membrane potentials determined by potential-sensitive fluorescent dyes

The membrane potentials of mycoplasmas were investigated by using potential-sensitive cyanine dyes. The fluorescence response results from a potential-dependent partition of the dyes between the cells and the extracellular medium. Cell hyperpolarization (inside more negative), e.g., by the addition of valinomycin, results in uptake of the dyes into the cells and, by formation of dye aggregates, in quenching of the fluorescence intensity. The magnitude of the fluorescence change upon addition of valinomycin depended on the external K⁺ concentration. At a defined external K⁺ concentration, no change in fluorescence occurred. The intracellular K⁺ concentration was determined by atomic absorption spectroscopy. Mycoplasma membrane potentials were calculated according to the Nernst equation. The membrane potential of bothMycoplasma mycoides subsp.capri andMycoplasma gallisepticum was −48 mV±10%; the membrane potential ofAcholeplasma laidlawii was −28 mV±20%.     

Tl+ induces both cationic and transition pore permeability in the inner membrane of rat heart mitochondria

Effects of Tl⁺ were studied in experiments with isolated rat heart mitochondria (RHM) injected into 400 mOsm medium containing TlNO₃ and a nitrate salt (KNO₃ or NH₄NO₃) or TlNO₃ and sucrose. Tl⁺ increased permeability of the inner membrane of the RHM to K⁺ and H⁺. This manifested as an increase of the non-energized RHM swelling, in the order of sucrose < K⁺ < NH₄ ⁺, respectively. After succinate administration, the swollen RHM contracted. The Tl⁺-induced opening of the mitochondrial permeability pore (MPTP) in Ca²⁺-loaded rat heart mitochondria increased both the swelling and the inner membrane potential dissipation, as well as decreased basal state and 2,4-dinitrophenol-stimulated respiration. These effects of Tl⁺ were suppressed by the MPTP inhibitors (cyclosporine A, ADP, bongkrekic acid, and n-ethylmaleimide), activated in the presence of the MPTP inducer (carboxyatractyloside) or mitoK_ATP inhibitor (5-hydroxydecanoate), but were not altered in the presence of mitoK_ATP agonists (diazoxide or pinacidil). We suggest that the greater sensitivity of heart and striated muscles, versus liver, to thallium salts in vivo can result in more vigorous Tl⁺ effects on muscle cell mitochondria.     

Increased Potassium Conductance of Brain Mitochondria Induces Resistance to Permeability Transition by Enhancing Matrix Volume

Modulation of K⁺ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K⁺ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca²⁺ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K⁺ or H⁺ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK_ATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K⁺ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H₂O₂-induced protein kinase Cϵ activation. Rather, increased K⁺ conductance reduced H₂O₂ generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.     

RAPID EFFECTS OF VERATRIDINE, TETRODOTOXIN, GRAMICIDIN D, VALINOMYCIN AND NaCN ON THE NA+, K+ AND ATP CONTENTS OF SYNAPTOSOMES

Abstract— The effects of brief exposures of a number of depolarizing agents on ²⁴Na⁺ influx and on the Na⁺, K⁺ and ATP contents of synaptosomes were studied using a Millipore filtration technique to terminate the reaction. When synaptosomes were incubated in normal medium, there was a rapid influx of ²⁴Na⁺ and a gain in Na’contents; neither the ²⁴Na⁺ influx nor the Na⁺ gain were blocked by tetrodotoxin suggesting that this Na⁺ entry did not involve Na⁺-channels. Veratridine markedly increased the rate of ²⁴Na⁺ influx into synaptosomes and also increased the Na⁺ content and decreased the K⁺ content of synaptosomes within the first 10s of exposure. The normal ion contents were reversed by 1 min. The effects of veratridine on Na⁺ influx and on synaptosomal ion contents were prevented by tetrodotoxin and required Na⁺ in the medium. The ionophores gramicidin D and valinomycin also rapidly reversed the Na⁺ and K⁺ contents of synaptosomes, but these effects could not be blocked by tetrodotoxin. The reducing effect of gramicidin D on synaptosomal K⁺ content required Na’in the medium, whereas valinomycin caused a fall in the K⁺ content of synaptosomes in a Na⁺-free medium. Veratridine and gramicidin D, at concentrations known to reverse the synaptosomal ion contents, did not affect synaptosomal ATP levels. In contrast, valinomycin and NaCN caused an abrupt fall in synaptosomal ATP levels. The above findings suggest that veratridine quickly alters synaptosomal Na⁺ and K⁺ contents by opening Na ⁺-channels in the presynaptic membrane, and provide direct evidence for the existence of Na⁺-channels in synaptosomes. In contrast, gramicidin D and valinomycin appear to act independently of Na ⁺-channels, possibly by their ionophoric effects and, in the case of valinomycin, by diminishing synaptosomal ATP contents and hence diminishing Na⁺-pump activity. The rapid reversals of Na⁺ and K⁺ contents by these drugs could affect the resting membrane potentials, Na⁺-Ca²⁺ exchange across the synaptosomal membrane, and the release, synthesis and uptake of neurotransmitters by synaptosomes.     

Anilinonaphthalenesulfonate fluorescence changes induced by non-enzymatic generation of membrane potential in mitochondria and submitochondrial particles

Changes in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS⁻) accompanying non-enzymatic generation of the membrane potential in mitochondria and sonicated submitochondrial particles have been demonstrated. Generation of the membrane potential was induced by addition of an ionophore (valinomycin for K⁺, or tetrachlorotri-fluoromethylbenzimidazole for H⁺) under conditions where there existed K⁺ (or H⁺) gradients across the mitochondrial membrane. The ANS⁻ fluorescence decreased when the mitochondrial (or particle) interior became more negative, and increased when it became more positive. Collapse of the membrane potential reversed the ANS⁻ responses. A hypothesis is put forward to explain the energy-dependent ANS⁻ responses in mitochondria and particles by the membrane potential-induced redistribution of ANS⁻ between the membrane and water phases.     

Functional identification of ATP-sensitive K<Superscript>+</Superscript> uniporter in mitochondria from sugar beet taproot

Mitochondria isolated from sugar beet (Beta vulgaris L.) taproot were shown to swell spontaneously after the transfer from a sucrose-containing isolation medium to isoosmotic potassium chloride solutions. The kinetics of this process was strongly retarded after the replacement of potassium with sodium in the incubation medium and was substantially stimulated by the electron-transport chain activity and valinomycin. At neutral pH of the incubation medium, the rate of K⁺-dependent swelling of mitochondria decreased by 30–50% after adding 1 mM ATP but was insensitive to other nucleotides (GTP, UTP, and CTP). In the medium acidified to pH 6.0, the addition of ATP caused shrinkage of mitochondria that had been swollen in the KCl medium. In the absence of this nucleotide, the kinetics of K⁺-dependent swelling of mitochondria was considerably decelerated upon the acidification of the incubation medium. The effects of ATP were independent of the presence or absence of oligomycin and atractyloside. However, the ATP-dependent shrinkage of mitochondria was inhibited in the presence of quinine, and this agent also inhibited K⁺-dependent swelling of organelles in potassium acetate solutions. The presence of K⁺ ions in the incubation medium caused a rapid dissipation of the mitochondrial membrane potential () that was generated during succinate oxidation. The addition of ATP to the reaction medium resulted in the oligomycin-insensitive restoration of . The results are regarded as evidence that the membrane of taproot mitochondria is endowed with functionally active ATP-sensitive K⁺ uniporter. This system is likely to represent a K⁺ channel that catalyzes the electrogenic transfer of potassium ions to the mitochondrial matrix. It is supposed that the membrane of taproot mitochondria also contains a quinine-sensitive K⁺/H⁺ antiporter that catalyzes the efflux of potassium from the matrix or, on the contrary, the accumulation of K⁺ in the presence of potassium acetate.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 209–215.Original Russian Text Copyright © 2005 by Shugaev, Andreev, Vyskrebentseva.This revised version was published online in April 2005 with a corrected cover date.     

Thallium interaction with the gastric (K,H)-ATPase

Summary The gastric (K,H)-ATPase has been shown to catalyze an electroneutral H⁺ for K⁺ exchange. Tl⁺ is able to substitute for K⁺ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 m as compared to about 870 m for K⁺. The ability of Tl⁺ to participate in transport is shown by the development of pH gradients in the presence of Tl⁺ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl⁺ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl⁺ concentrations above 3.0mm. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl⁺ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl⁺ concentrations, following the initial Tl⁺ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl⁺ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t _1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl⁺ concentrations in excess of 1 to 4mm are able to dissipate intravesicular proton gradients by an electrically coupled H⁺ for Tl⁺ exchange. Thus, although Tl⁺ is able to activate the gastric ATPase by mimicking K⁺, the permeability of this cation in lipid bilayers tends to uncouple H⁺ transport at concentrations high enough to generate detectable proton gradients.     

Activation of potassium-dependent H+ efflux from mitochondria by cadmium and phenylarsine oxide

Addition of Cd²⁺ or phenylarsine oxide (PhAsO) to respiring rat liver mitochondria results first in acidification of the medium (H⁺ efflux) followed by disappearance of H⁺ (discharge of the pH gradient or uncoupling). The first phase of H⁺ efflux is dependent upon the presence of K⁺ in the medium, and is not seen in the presence of valinomycin, which is consistent with the conclusion that H⁺ efflux is linked to membrane potential-dependent uptake of K⁺. These effects are abolished by low levels of 2,3-dimercaptopropanol but potentiated by excess of 2-mercaptoethanol, showing involvement of a dithiol type of group in the response. Mersalyl produces only the H⁺ efflux, and subsequent addition of Cd²⁺ or PhAsO produces collapse of the pH.Abbreviations BAL British Anti-Lewisite or 2,3-dimercaptopropanol - 2-ME 2-mercaptoethanol - PhAsO phenylarsine oxide - FCCP carbonylcyanide trifluoromethoxyphenylhydrazone - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

The membrane potential modulates the ATP-dependent Ca pump of cardiac sarcolemma

The effect of membrane potential on the activity of the ATP-dependent Ca²⁺ pump of isolated canine ventricular sarcolemmal vesicles were investigated. The membrane potential was controlled by the intravesicular and extravesicular concentration of K⁺, and the initial rates of Ca²⁺ uptake both in the presence and the absence of valinomycin were determined. The rate of Ca²⁺ uptake was stimulated by a inside-negative potential induced in the presence of valinomycin. The valinomycin-dependent stimulation was enhanced by the addition of K⁺ channel blocker, tetraethylammonium ion or Ba²⁺. The electrogenicity of cardiac sarcolemmal ATP-dependent Ca²⁺ pump is suggested from the increase of Ca²⁺ uptake by negative potential induced by valinomycin.     

Valinomycin inhibition of insulin release and alteration of the electrical properties of pancreatic B cells

The effects of valinomycin on insulin release and rubidium efflux from perifused isolated rat islets were investigated and correlated with its effects on the electrical properties of mouse B cells studied with microelectrode techniques. Valinomycin produced a (1 · 10^?9–1 · 10^?6 M) dose- and time-dependent inhibition of (10–15 mM) glucose-stimulated insulin release but did not affect basal secretion. This inhibitory effect rapidly followed addition of the ionophore and equally affected the two phases of glucose-stimulated secretion. It was not reversible by simple washing of the islets, but could be reversed transiently by tetraethylammonium or high extracellular potassium ion levels. At low or high glucose, valinomycin rapidly augmented the rate of ⁸⁶Rb⁺ efflux from preloaded islets. Amplitude and rapidity of this effect were dose-dependent and it was antagonized by tetraethylammonium. Glucose metabolism by islet cells was reduced only slightly (15%) by 1 · 10^?7 M valinomycin. During the first 6 to 8 min of valinomycin addition, the membrane potential of B cells augmented slowly but the typical bursts of spikes disappeared rapidly. Later on, B cells hyperpolarized more quickly to a stable value of approx. ?70 mV. Increasing extracellular K⁺ immediately depolarized B cells and the linear relationship found between the logarithm of K⁺ concentration and the membrane potential was characterized by a slope of 58 mV for a ten-fold increase in extracellular K⁺.These results suggest that valinomycin interferes with the insulin releasing effect of glucose by increasing the potassium permeability of the B cell membrane.     

Unmasking the mitochondrial K/H exchanger: swelling-induced K+-loss.

Matrix swelling induces a rapid, transient, energy-independent potassium efflux in rat liver mitochondria. Swelling-induced K⁺-loss is electroneutral; therefore it does not reflect electrophoretic diffusion secondary to increased membrane permeability. Matrix swelling unmasks an endogenous $\text{K}\text{H}$ transport mechanism in the mitochondrial membrane, providing a valuable experimental approach to the study of K⁺ transport in mitochondria.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Electroneutral H+-K+ exchange in liver mitochondria Regulation by membrane potential

The paper analyzes the factors affecting the H⁺-K⁺ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg²⁺ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K⁺ efflux following addition of A23187 in low-K⁺ media. (1) The H⁺-K⁺ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of ΔpH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, Δψ. Maximal rate of K⁺ efflux is observed at 180–190 mV, whereas K⁺ efflux is inhibited below 140–150 mV. (2) Activation of H⁺-K⁺ exchange leads to depression of ΔpH but not of Δψ. Respiration is only slightly stimulated by the onset of H⁺-K⁺ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the Δψ control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H⁺-K⁺ exchange, while leaving unaffected the level of Mg²⁺ depletion. This type of activation results in partial ‘uncoupling’ from the Δψ control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high Δψ. (4) The available evidence suggests that the activity of the H⁺-K⁺ exchanger is modulated by the electrical field across the inner mitochondrial membrane.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

Active K+ transport in Mycoplasma mycoides var. Capri. Relationships between K+ distribution,electrical potential and ATPase activity

The addition of 5 · 10^?5 M or less of dicyclohexylcarbodiimide to Mycoplasma mycoides var. Capri preferentially influences K⁺ influx rather than efflux and reduces by 30–40% the activity of the membrane-bound Mg²⁺-ATPase. Adding valinomycin to metabolizing cells does not markedly affect K⁺ distribution but induces a rapid and complete loss of intracellular K⁺ in non-metabolizing cells. Uncoupling agents such as dinitrophenol, carbonyl cyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$, dissipate the K⁺ concentration gradient only when combined with valinomycin.Variations in the merocyanine fluorescence intensity indicate that a transmembrane electrical potential (Δψ) is generated on cell energization. This Δψ, not affected by valinomycin or uncouplers when used alone, is collapsed by a mixture of both. No change in fluorescence intensity can be detected when glucose is added to dicyclohexylcarbodiimide treated organisms.These experiments suggest that the membrane-bound Mg-ATPase activity controls K⁺ distribution in these organisms through the generation of a transmembrane electrical potential difference.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient

Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K⁺]_{intracellular}/[K⁺]_{extracellular}) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (¹⁴C) tetraphenylphosphonium (TPP⁺). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of ¹²C-TTP⁺ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K⁺ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP⁺ Tetra phenylphosphonium bromide - DTE Dithioerythritol - TCS 3,5,3,4-Tetrachlorosalycylanilide