Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration.

1. Aerobic respiration by cells of Paracoccus dentrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium. Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation. Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described. 2. If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P. dentrificans respiring aerobically in 100 mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid/NaOH or 100mM-NaH2PO4/NaOH. This potential, however, is decreased by more than 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM-K+. 3. Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150mV in described suspension media. The presence of these species under aerobic conditions, moreover, has negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration. These data indicate that none of these molecules exert a significant uncoupling effect on the protonmotive force. 4. No 204Tl+ uptake into respiring cells was detected. This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore not an indicator of membrane potential as has been proposed. The absence of respiration-driven 204Tl+ uptake indicates that P. denitrificans cells grown under the conditions specified in the present work do not possess K+-transport systems of either the Kdp or TrkA types that have been described in Escherichia coli.     

The transport of univalent thallium across the human erythrocyte membrane

Transport of 204Tl was studied in human erythrocytes incubated in isotonic salt solutions at pH 7.4 and 37 degrees C. 204Tl was rapidly accumulated in cells up to the constant level within a 10 minutes incubation (t0.5 = 3.5 min). The rate of uptake and the distribution ratio decreased in the presence of 0.1 mM ouabain and 0.5-1.0 mM furosemide (t0.5 = 5 min). A broad variability of the coefficient 204Tl distribution was observed in the intact erythrocytes due to a ouabain-sensitive component which was seen to decrease with the increase in Tl+ concentration in the medium (0.005-0.2 mM), and also to depend on the medium ion composition. On the contrary, a passive distribution of 204Tl in the presence of ouabain and furosemide was relatively constant within 1.1-1.5. The steady state distribution of 204Tl was declined after a substitution of Cl- by sucrose in the medium due to depolarization of erythrocyte membrane. On the other side, 204Tl uptake by the cells was raised during hyperpolarization of the membrane in the presence of valinomycin.     

Conversion of Escherichia coli cell-produced metabolic energy into electric form.

The formation of membrane potential in energized E. coli cells has been investigated by means of ionic penetrants. The fluxes of anions and cations in opposite directions have been observed: anions moved out and cations moved into the cells. The energy-linked uptake of cations was stoichiometrically coupled with the outflow of H+ ions from the cells. The value of a membrane potential in the energized cells calculated from a distribution of permanent cations was in the range of -140 mV (inside minus). The uptake of penetrating cations by deenergized cells has been observed following the non-enzymatic generation of a membrane potential. The influx of synthetic and natural (lactose) penetrants collapsed the non-enzymatic membrane potential. The effect of lactose was sensitive to N-ethyl maleimide. These results are in favour of the conception that in the energized E. coli cells an energy-linked H+-pump generates a membrane potential which is a driving force for the transport of synthetic and some natural penetrants.     

Active transport of thallous ions by Streptococcus lactis.

Tl+ ions have been shown to mimic or compete with K+ in a number of membrane systems. We confirmed that in starved, valinomycin-treated cells of Streptococcus lactis 7962, Tl+ ions distributed themselves across the bacterial membrane in response to the potassium diffusion potential. In glucose-energized cells, however, Tl+ was taken up by a system specifically stimulated by sodium salts. The intracellular levels of Tl+ exceeded those attained by [3H]triphenylmethylphosphonium ion, a lipophilic cation which accumulates in response to the membrane potential. The uptake of Tl+ by (Na+ and glucose)-stimulated cells was strongly inhibited by potassium salts. These experiments suggest that metabolic energy is coupled to Tl+ transport by means of a high energy phosphate compound and that Tl+ ions are actively transported by a membrane carrier whose normal substrate is K+. The uptake of Tl+ is not a valid method for determining the streptococcal membrane potential.     

Interconversion of components of the bacterial proton motive force by electrogenic potassium transport.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.     

Thallium and rubidium permeability of human and rat erythrocyte membrane

Transport of Tl+ and Rb+ in human and rat erythrocytes was investigated in the presence of ouabain. The chloride-dependent cotransport of Tl+, Rb+ and Na+ was precluded by replacement of Cl- by NO3-. The inward and outward rate constants for the residual fluxes of the cations were determined by measuring the transport of 204Tl and 86Rb in double label experiments. The rate of passive transport of Tl+ exceeded that of Rb+ by one-two orders of magnitude in human as well as rat erythrocytes. The membrane barrier which contributes to the maintenance of ion gradients was shown not to be a barrier for Tl+ which easily penetrates the membrane by an unknown mechanism. In rat erythrocytes the barrier for Rb+ was 10-15 times weaker than that in human red blood cells, while the corresponding ratio of rat/human Tl+ permeabilities was about 1.8-2.0. It follows that Tl+ permeability is only slightly affected by factors modifying the permeability to alkali cations. The increase of temperature from 20 degrees to 37 degrees C resulted in a three-fourfold stimulation of the passive transport of Tl+ both in human and rat erythrocytes. The movement of Tl+ and Rb+ through the erythrocyte membrane differed substantially from their diffusion along the excitable membrane channels characterized both by poor Tl+/K+ selectivity and weak temperature dependence.     

A novel crystallization method for visualizing the membrane localization of potassium channels.

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.     

Factors affecting the relative magnitudes of the ouabain-sensitive and the ouabain-insensitive fluxes of thallium ion in erythrocytes

A maximal rate of the ouabain-sensitive 204-Tl influx in human erythrocytes can be attained at trace concentrations of Tl+ in Mg2+ isotonic media free of K+ and Na+. The maximal influx of Tl+ from isotonic Mg(NO3)2 at 20 degrees C and pH 7.4 was 0.45 mM.l(-1).h-1 with a Km of 0.025 mM. In contrast to the active influx of Tl+, the passive Tl+ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl+ and K+ studied. The rate constants of Tl+ passive fluxes in human and cat erythrocytes can be related to pH by the equation log kin(OUT)= -A + B.pH, where A and B are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl+ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl+ is about two orders of magnitude faster than those of K+ or Rb+. An extra non-Coulombic interaction between Tl+ and membrane ligands appears to be involved providing an accumulation of Tl+ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl+ in both directions.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

Oxidation--reduction potentials of molybdenum and iron--sulphur centres in nitrate reductase from Escherichia coli.

The potentials of the couples Mo(IV)--(Mo(V) and Mo(V)--Mo(VI) in nitrate reductase from Escherichia coli K12 were measured as + 180 mV and + 220 mV respectively at pH 7.14. The potentials associated with two other e.p.r. signals, believed to be due to iron--sulphur centres, were measured as + 50 mV and + 80 mV.     

Thallous ion is accumulated by potassium transport systems in Escherichia coli.

The accumulation of 204T1+ by Escherichia coli occurs primarily via either of two K+ transport systems called Kdp and TrkA. T1+ influx is inhibited and T1+ efflux is stimulated by the addition of K+ to the assay medium. Mutants defective in both the Kdp and TrkA systems accumulate little T1+. Uptake of triphenylmethylphosphonium, a lipid-soluble cation whose distribution is widely used to estimate the membrane electrical potential in bacteria, occurs to about the same extent in mutants that accumulate little T1+ as in strains that accumulate T1+ to high levels. These findings indicate that T1+ may be useful as a probe of bacterial K+ transport systems but is not a reliable indicator of the membrane electrical potential in E. coli.     

Measurement of plasma membrane potential in isolated rat hepatocytes using the lipophilic cation, tetraphenylphosphonium: correction of probe intracellular binding and mitochondrial accumulation.

The lipophilic cation tetraphenylphosphonium (TPP+) has been extensively utilized as the probe for the membrane potential (Vm) in various cells. For application to mammalian cells, however, two serious problems require resolution: (1), correction of TPP+ binding to intracellular constituents and (2), estimation of the considerable TPP+ accumulation in mitochondria. We propose here a simple corrective method for the TPP+ binding and its accumulation. TPP+ distribution is assumed as: (1), two compartments (a cytosolic and a mitochondrial space); (2), a proportional relationship between TPP+ bound amount and its unbound concentration in each compartment. We theoretically derived the simple equation: Vm = - RT/F ln(C/Mphys ratio/C/Mabol ratio) where R, T and F have their usual thermodynamic significance. Here, the C/M ratio is defined as the ratio of TPP+ concentration of apparent intracellular to extracellular space. The suffixes phys and abol, respectively, mean the physiological and solely Vm-abolished conditions. This equation was checked with hepatocytes, because estimating hepatocytes Vm with TPP+ distribution is not considered possible because of the relatively high mitochondrial content. The selective Vm abolition was achieved by permeabilization with 20 microM of amphotericin B. The Vm value was, thus, estimated to be -38.6 +/- 0.3 mV, compatible with those obtained with microelectrodes in other laboratories. Vm in hepatocytes is composed of transmembrane K+ diffusion potential (-20.6 +/- 0.3 mV) and electrogenic Na+/K(+)-ATPase (-19.6 +/- 0.4 mV). Addition of rheogenic L-alanine caused a transient but significant depolarization (from control to -34 +/- 0.3 mV). These results taken together indicate that hepatocyte Vm can be accurately determined with the present simple method, so that it may possibly be applicable to the evaluation of Vm in other mammalian cells.     

Dependence of cytoplasmic calcium transients on the membrane potential in isolated nerve endings of the guinea pig

The relation of changes in internal, free Ca2+, measured with arsenazo III, to the membrane potential, measured with the cyanine dye di-S-C2(5) or 86Rb+ distribution ratio, was studied in isolated guinea pig cortical nerve endings. Depolarization of the plasma membrane with veratridine or gramicidin as well as addition of ionophore A23187 led to an increase in cytosolic Ca2+. Only the response to veratridine was inhibited by tetrodotoxin. The dependence of the depolarization-induced increase in intraterminal, free Ca2+ on the membrane potential between about -50 to 0 mV was sigmoidal. A maximal increase in cytosolic Ca2+ was reached when the membrane potential was depolarized from the resting level, about -64 mV, to about -40 mV. These results show that in isolated nerve endings the activation of voltage-sensitive Ca2+ channels concomitantly leads to an increase in cytosolic, free Ca2+. Comparison of the results of the present study with the previous electrophysiological observations indicate that Ca2+ channels in synaptosomes, presynaptic nerve terminals of the squid giant synapse and cardiac cells have essentially similar voltage dependency.     

Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase

Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E. coli aerobic respiratory chain. In this paper, the role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles. The enzymes used in this reconstitution are the flavoprotein and the recently purified E. coli cytochrome d terminal oxidase. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E. coli membranes. It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(tri-chloromethoxy)phenylhydrazone and valinomycin. A trans-membrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein. It is concluded that (1) the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer, (2) menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain, and (3) the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a "coupling site" in the E. coli aerobic respiratory chain. These investigations suggest a relatively simple organization for the E. coli respiratory chain.     

The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.     

The membrane potential of mouse ascites-tumour cells studied with the fluorescent probe 3,3''-dipropyloxadicarbocyanine. Amplitude of the depolarization caused by amino acids.

1. The magnitude of the K+ gradient across the plasma membrane, which was in equilibrium with the membrane potential (E) of the tumour cells, was determined by the "null point" procedure of Hoffman & Laris (1974) [J. Physiol. (London) 239, 519--552] in which the fluorescence of the dye serves as an indicator of changes in the magnitude of E. 2. A mixture of oligomycin, 2,4-dinitrophenol and antimycin was used to stop the mitochondria from interfering with the fluorescence signal. Transport functions at the plasmalemma were maintained under these conditions in the presence of glucose. 3. Physiological circumstances were found in which incubation with glycine or with glucose changed the "null point" value of E within the range--20mV to--100mV. The fluorescence intensity at the "null point" was an approximately linear function of E over that range. The procedure enabled E to be inferred form the fluorescence intensity in circumstances where titration to the "null point" was not feasible. 4. The rapid depolarization caused by l-methionine or glycine was shown in this way to have a maximum amplitude of about 60mV. A mathematical model of this process was devised. 5. The electrogenic Na+ pump hyperpolarized the cells up to about --80mV when the cellular and extracellular concentrations of K+ were roughly equal. 6. The observations show that the factors generating the membrane potential represent a major source of energy available for the transport of amino acids in this system.     

Tl+-induced micros gating of current indicates instability of the MaxiK selectivity filter as caused by ion/pore interaction

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app). The analysis of the amplitude histograms by beta distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = I(true)/I(app) = (k(CO) + k(OC))/k(CO) could be described by exponential functions with different characteristic voltages above or below 50 mM Tl(+). The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of I(true) were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.     

Characterization and phenotypic control of the cytochrome content of Escherichia coli

1. Electron-transport particles derived from Escherichia coli grown aerobically contain three b-type cytochromes with mid-point oxidation-reduction potentials at pH7 of +260mV, +80mV and -50mV, with n=1 for each. The variation of these values with pH was determined. 2. E. coli develops a different set of b-type cytochromes when grown anaerobically on glycerol with fumarate or nitrate as terminal electron acceptor. Electron-transport particles of fumarate-grown cells contain b-type cytochromes with mid-point potentials at pH7 of +140mV and +250mV (n=1). These two cytochromes are also present in cells grown with nitrate as terminal acceptor, where an additional cytochrome b with a mid-point potential of +10mV (n=1) is developed. 3. The wavelengths of the alpha-absorption-band maxima of the b-type cytochromes at 77K were: (a) for aerobically grown cells, cytochrome b (E(m7) +260mV), 556nm and 563nm, cytochrome b (E(m7) +80mV), 556nm and cytochrome b (E(m7)-50mV), 558nm; (b) for anaerobically grown cells, cytochrome b (E(m7) +250mV), 558nm, cytochrome b (E(m7) +40mV), 555nm and cytochrome b (E(m7) +10mV), 556nm. 4. Cytochrome d was found to have a mid-point potential at pH7 of +280mV (n=1). 5. Cytochrome a(1) was resolved as two components of equal magnitude with mid-point potentials of +260mV and +160mV (n=1). 6. Redox titrations performed in the presence of CO showed that one of the b-type cytochromes in the aerobically grown cultures was reduced, even at the upper limits of our range of electrode potentials (above +400mV). Cytochrome d was also not oxidizable in the presence of CO. Neither of the cytochromes a(1) was affected by the presence of CO.     

Effect of positive redox potentials (>+400mV) on the expression of anaerobic respiratory enzymes in Escherichia coli

The expression of fumarate reductase and other enzymes of anaerobic respiration in Escherichia coli was studied as a function of the redox potential (Eh) in the medium. Redox potentials up to +300 mV allowed full expression of fumarate reductase (frd) genes. Higher values resulted in decreased expression. The relationship between Eh and expression of frd could be approximated by the Nernst equation, assuming a redox couple with a midpoint potential Eo' = +400 mV to 440 mV. At Eh values greater than +510 mV (generated anaerobically by hexacyanoferrate(III] the degree of repression was the same as that obtained by O2. Hexacyanoferrate(III) also caused decreased activities of dimethylsulphoxide (DMSO), nitrite and nitrate reductases. Since expression of these enzymes depends on FNR, the gene activator of anaerobic respiratory genes, it is suggested that the function of FNR is controlled by a redox couple of Eo' = +400 mV to 440 mV.     

A quantitative assessment of the use of 36Cl- distribution to measure plasma membrane potential in isolated hepatocytes

The plasma membrane potential of isolated rat hepatocytes was clamped at different values between 0 and -68 mV by addition of valinomycin in the presence of different extracellular concentrations of K+, and measured by the distribution of 86Rb+ between cells and medium. 36Cl- distribution came to steady state in 10-15 min. This steady-state distribution was compared to the plasma membrane potential over a range of values. 36Cl- distribution provided an accurate measurement of plasma membrane potential between -4 and -40 mV. At higher potentials intracellular chloride concentration is less than 20% of the extracellular concentration and errors due to uncertainties in the measurement of intracellular volume and of the contamination of cell pellets by extracellular medium precluded accurate determination of membrane potential: thus in our experiments 36Cl- underestimated the plasma membrane potential at -68 mV by 8 mV.