Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.     

On the mechanism of generation of electrical potential differences in cell biomembranes]

A statistical model of active ion transport in biomembranes was developed. The model makes it possible to calculate both the value of membrane potential phi zero and the rate of ion concentrations inside and outside the cell. These values depend on the difference of chemical potentials of the ATP-ADP system and the permeability of the biomembrane for ions being transported. The calculated phi zero value approximately 200-250 mV is consistent with the data on proton pumps.     

Intracellular gradients of electrical potential in the epithelial cells of theNecturus gallbladder

Summary When single-barrelled electrodes (5–60 M) were advanced under manual control from the mucosal side of the epithelium the mucosal membrane was on average indented by about 40 m before the microelectrode penetrated the cell. Since this dimpling was comparable with the total depth of the cell, which recovered its original shape within 0.5 sec, the steady intracellular potential was recorded only about 14 m from the basal (serosal) membrane. Fast recording of the associated change in potential revealed an abrupt drop to –26 mV at a mean rate of 84 V/sec, followed by a further slow drop to a steady value of about –50 mV at a mean rate of 0.28 V/sec. The initial level of –26 mV may be regarded as the potential difference across the mucosal membrane. This conclusion was confirmed by mounting the microelectrode on a piezoelectric probe, which delivered 3 m jabs in less than 0.5 msec. With this device in operation to prevent dimpling, the mean potential difference across the mucosal membrane was recorded as –29 mV. In all cases the potential across the basal membrane was recorded as –52 mV. Manual advance of the microelectrode tip within the cytoplasm yielded an intracellular potential gradient of 0.6 mV/m. The same potential profile and membrane potentials were demonstrated on penetrating the epithelium from the serosal side, and measurements with multibarrelled electrodes whose tips were staggered in depth gave roughly the same internal potential gradient. The resistivity of the cytoplasm was determined by a triple-barrelled microelectrode, and varied from 10 times that ofNecturus saline at the mucosal end of the cell to 4 times in the middle and 6 times at the serosal end.     

The membrane potentials of fibroblasts in different environments

The membrane potentials of human embryonic lung fibroblasts have been measured in different cellular environments. Sparse cells on plastic have a mean membrane potential of ?8.5 mV. As the cells progress to dense culture, the mean membrane potential rises to ?14.7 mV. The mean membrane potential of fibroblasts in human embryonic lung fragments by comparison was found to be ?16.5 mV. Sparse cells on collagen, at the same density as the sparse cells on plastic, have mean membrane potentials of ?10.8 mV. Sparse cells on plastic migrating from dense cellular areas, following a cut being made in a thick sheet of cells, have mean membrane potentials of ?5.9 mV. The significance of these results in relation to cellular environments has been discussed.     

Electrical potentials in the ectomycorrhizal fungus Laccaria bicolor after a rainfall event

We measured extracellular bioelectrical activities of the ectomycorrhizal basidiomycete Laccaria bicolor under field conditions to examine its response to environmental factors. Six fruit bodies of L. bicolor in a cluster, to which electrodes were attached, exhibited less electrical potentials at the beginning, probably due to the lack of precipitation for over a week. However, its electrical potential fluctuated after raining, sometimes over 100 mV. The electrical potential of the fruit bodies and its fluctuation were correlated with precipitation. Causality analysis of electrical potential after the rain showed electrical signal transport among fruit bodies, particularly between spatially close ones, with potential directionality. Our preliminary results bring a call for studies on fungal electrical potentials in a more ecological context under field conditions.     

Convenient fluorescence-based methods to measure membrane potential and intracellular pH in the Archaeon Methanobacterium thermoautotrophicum

New and improved methods to determine the membrane potential (Delta Psi) and the Delta pH in methanogenic archaea were developed and tested in Methanobacterium thermoautotrophicum strain Delta H. The Delta pH measurements took advantage of the pH-dependent fluorescence properties of coenzyme F(420), the major intracellular electron carrier in the organism. The protonophore p-nitrophenol did not show any interference with the F(420) fluorescence spectra and was therefore suitable to equalize internal and external pH. The method developed allowed the determination of the intracellular pH with an error of less than 0.05 pH units.Membrane potentials could easily be assessed using the fluorescent probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)) with an accuracy of approximately 10 mV.Both methods were tested with cell suspensions of M. thermoautrophicum incubated at medium pH values between 5.5 and 8. It was found that Delta Psi and Delta pH values remained constant under these conditions. Membrane potentials were about -160 mV and Delta pH was kept at 0.35 pH units (inside minus outside) resulting in a total proton motive force of about -180 mV (inside negative).     

Correlation between membrane potential, cell shape, and motile activity in Amoeba proteus

The various motile activities and cell shapes of Amoeba proteus grown in Chalkley's solution are correlated with definitive electrical membrane potentials. The same correlations were found when definitive motile activities and cell shapes were experimentally induced by changing the pH of the culture medium. The highest values of membrane potential (−70 mV) were measured in monopodial amebae during active locomotion. In resting cells, which prevail in acid or basic media, the membrane potential decreases to −5 mV. In those resting cells, which also stop internal cytoplasmic movement at basic pH, the membrane potential turns positive (+9 mV − +30 mV).     

Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

The usefulness of a new voltage-sensitive fluorescent dye, the membrane permeant negatively charged oxonol dye diBA-C4-(3)-, was evaluated by measuring the membrane potentials of BICR/M1R-k and L cells with glass microelectrodes and simultaneously recording the fluorescence of the stained cells. The membrane potential of BICR/M1R-k cells was varied between -25 mV and -90 mV by changing the bicarbonate concentration in the medium or by voltage clamping. To avoid any interference by the inserted electrodes with the fluorescence measurement of the cytoplasm, the cells were fused by polyethyleneglycol to form giant cells (homokaryons). These homokaryons also allowed penetration by two glass microelectrodes without causing a serious leakage of the plasma membrane. The slow responding dye diBA-C4-(3)- had a fluorescence response of about 1% per mV. Mathematical analysis of the fluorescence changes after voltage clamping revealed a first-order reaction with a rate constant between 0.1 min-1 and 0.8 min-1, depending on the cell size which was determined by the number of nuclei per homokaryon. A model for the mechanism of the fluorescence changes is proposed.     

Experimental and functional changes in transmembrane potential and zeta potential of single cultured cells

Experiments were performed on single cells to investigate the relations between the total bioelectrical potential difference (PD) across the cell membrane (so-called transmembrane potential) and the net negative surface charge of the cell (zeta potential). The experiments were carried out on FL-cells, leucocytes and ovarian tumour cells. The PD was measured electrophysiologically by means of intracellular glass microelectrodes; the surface charge or the zeta potential was determined using cell electrophoresis. Both measuring methods are critically discussed.Under different conditions (hypothermia, hyperthermia, mitotic blocking agent, cell cycle), the transmembrane potential and zeta potential showed changes in an identical direction and often the response of transmembrane potential was found to be quicker and more intensive than that of the zeta potential. In other experiments (e.g. changing the extracellular Cl^? ion concentration) the reactions of both potentials showed no coincidence. Depending on the type of functionally or experimentally borne changes on the cytoplasmatic membrane, either both potentials or only one of them may be altered.     

The effect of surface and membrane potential on 1-anilino-8-naphthalenesulfonate binding with E. coli membrane

Dependence of ANS fluorescence on the surface potential of E. coli under lowered resistance of the bacterial membrane and after application of the positive diffusion potential inside the cell was investigated. It was shown that in the absence of the latter ANS binding in de-energised bacteria occurs mainly at the outside surface. It may be due to the high negative charge of the inner side of the cytoplasmic membrane. According to produced evaluation the potential of this surface is 120 +/- 25 mV. The data obtained suggest that low ANS fluorescence in intact cells is due to the membrane modification on energisation.     

The membrane potential of Ehrlich ascites tumor cells microelectrode measurements and their critical evaluation

Summary Intracellular potentials were measured, using a piezoelectric electromechanical transducer to impale Ehrlich ascites tumor cells with capillary microelectrodes. In sodium Ringer's, the potential immediately after the penetration was –24±7 mV, and decayed to a stable value of about –8 mV within a few msec. The peak potentials disappeared in potassium Ringer's and reappeared immediately after resuspension in sodium. Ringer's, whereas the stable potentials were only slightly influenced by the change of medium. The peak potential is in good agreement with the Nernst potential for chloride. This is also the case when cell sodium and potassium have been changed by addition of ouabain. It is concluded that the peak potentials represent the membrane potential of the unperturbed cell, and that chloride is in electrochemical equilibrium across the cell membrane.The membrane potential of about –11 mV previously reported corresponds to the stable potential in this study, and is considered as a junction potential between damaged cells and their environment. Similar potential differences were recorded between a homogenate of cells and Ringer's.The apparent membrane resistance of Ehrlich cells was about 70 cm². This is two orders of magnitude less than the value calculated from³⁶Cl fluxes, and may, in part, represent a leak in the cell membrane.For comparison, the influence of an eventual leak on measurements in red cells and mitochondria is discussed.     

Membrane Potential Measurements in Cultured Intestinal Villi

Fragmented epithelia of newborn rat small intestine were successfully cultured for periods of up to 4 weeks. Stable intracellular recordings of membrane potential were obtained from these cultured cells. Membrane resting potential varied according to cell location along a villus. The potentials ranged from -70 to -15 mV, being highest at the tip of the villus. The mean resting potential and membrane resistance were -72.4 mV and 8.6 M Ω, respectively. The membrane potential was markedly dependent on the extracellular K⁺ concentration ([K]₀], but not significantly on [Na]₀ and [Cl]₀-Deprivation of Ca²⁺ from the surrounding medium depolarized the membrane by 20 mV. When the cells were cooled down to 6°C, membrane potential was reduced by 40 mV. Based on these data, basic mechanisms underlying the resting potential are discussed in connection with cell differentiation or maturation.     

Energy flux modulation on the outer membrane of mitochondria by metabolically-derived potential

Voltage-dependent anion channels (VDACs) are the porins in the outer mitochondrial membrane allowing metabolite flux between mitochondria and the cytoplasm. The permeabilities of the VDACs to ATP(-4), ADP(3-), creatine phosphate2-, Pi2-, Pi-, and other charged metabolites depend on the membrane potential. But neither the existence of the electrical potential across the outer membrane of mitochondria, nor its generation mechanisms have been experimentally shown. In this work, the concept of metabolically-derived potential that could be generated on the outer membrane was developed further. The computational study of the quantitative models shows that a steady-state membrane potential above 40 mV may be generated across a membrane with VDACs, if the VDACs are considered to be non-permeable to K+ and Cl-. Free permeability of VDACs to these inorganic ions, mimicking VDACs biological behavior, decreases the potential to nearly 12 mV. This decrease does not result from the electrical shortening of the potential by K+ and Cl- fluxes, but is caused by the electrodynamic compartmentation of the charged metabolites influencing the Goldman fluxes and the enzyme activity determining the fluxes. The interaction of two cyclic steady-state fluxes of charged metabolites due to the synergetic superposition of the potentials generated by each of these fluxes was obtained, and the effect of amplification of one flux by the other was theoretically demonstrated. These calculations based on VDACs' known permeability-voltage characteristics indicate that there is a certain possibility that the cell energy metabolism is regulated on the outer membrane of mitochondria by the electrical potential generated by various metabolically-dependent mechanisms.     

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.     

Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.     

Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.     

Redox properties of human transferrin bound to its receptor

Virtually all organisms require iron, and iron-dependent cells of vertebrates (and some more ancient species) depend on the Fe(3+)-binding protein of the circulation, transferrin, to meet their needs. In its iron-donating cycle, transferrin is first captured by the transferrin receptor on the cell membrane, and then internalized to a proton-pumping endosome where iron is released. Iron exits the endosome to enter the cytoplasm via the ferrous iron transporter DMT1, a molecule that accepts only Fe(2+), but the reduction potential of ferric iron in free transferrin at endosomal pH (approximately 5.6) is below -500 mV, too low for reduction by physiological agents such as the reduced pyridine nucleotides with reduction potentials of -284 mV. We now show that in its complex with the transferrin receptor, which persists throughout the transferrin-to-cell cycle of iron uptake, the potential is raised by more than 200 mV. Reductive release of iron from transferrin, which binds Fe(2+) very weakly, is therefore physiologically feasible, a further indication that the transferrin receptor is more than a passive conveyor of transferrin and its iron.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

Membrane properties of identified embryonic nerve and glial cells of the leech central nervous system

Summary The membrane potential of identified nerve (Retzius) cells and neuropil glial cells from 11 (±1) day-old embryos of the leechHirudo medicinalis was recorded using conventional intracellular microelectrodes. At this stage all ganglia of the segmental nervous system are formed. The membrane potential of Retzius cells was –68±4 mV (±SD,n=8), and showed a slope of 42 mV between 10 mM and 100 mM external K concentration. Retzius cells were able to fire action potentials which had a fast Na-dependent component, and, under appropriate conditions, also generated slow Ca (Ba) action potentials. The mean membrane potential of the neuropil glial cell at physiological K concentration (4 mM) was –83±5 mV (±SD,n=10), and showed a dependence of 56 mV for a tenfold change in the external K concentration (> 4mM). Neuropil glial cells showed no signs of voltage-activated excitability, but they repeatedly depolarized in the presence of 0.1 mM 5-HT.