A novel method for the determination of electrical potentials across cellular membranes. II. Membrane potentials of acholeplasmas, mycoplasmas, streptococci and erythrocytes

The membrane potentials of Acholeplasma laidlawii, Mycoplasma mycoides subsp. capri, Mycoplasma gallisepticum, Streptococcus faecalis and human erythrocytes have been determined by applying a novel technique. The membrane potentials were calculated simply from potassium concentrations determined by atomic absorption spectroscopy, and gravimetry. The versatility of the new technique is demonstrated by comparing our results with data obtained by different techniques.     

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%.     

Ion diffusion potentials across mycoplasma membranes determined by a novel method using a carbocyanine dye

The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Electric membrane potential was calibrated vs fluorescence intensity of a potential-sensitive carbocyanine dye according to delta psi = (RT/F) X log([aIN(1 - IN) - b]/Kint), where IN = I/I0, I0 = maximal fluorescence intensity (obtained for delta psi----infinity), and a and b are constants. Fluorescence intensity was calibrated vs membrane potential by inducing a K+ diffusion potential. The calibration procedure was based on the assumption that in the presence of valinomycin the membrane potential was determined entirely by K+ diffusion. Then the dependence of fluorescence intensity on the external K+ concentration, Kext, could be described by Ival = I0[1 + a/(Kext + b)]-1. For Mycoplasma mycoides subsp. capri and enterococci, the constants were determined from experimental data using nonlinear least-squares computer-assisted methods. The validity of our assumption was proved using the "null-point" method. Here the Ca2+ ionophore A23187 and varying external Ca2+ concentrations were used to change the membrane potential experimentally. K+ and Na+ diffusion potentials significantly contributed to mycoplasma membrane potential whereas Cl- had no influence. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = -68 mV.     

Simultaneous imaging of cell and mitochondrial membrane potentials.

The distribution of charged membrane-permeable molecular probes between intracellular organelles, the cytoplasm, and the outside medium is governed by the relative membrane electrical potentials of these regions through coupled equilibria described by the Nernst equation. A series of highly fluorescent cationic dyes of low membrane binding and toxicity (Ehrenberg, B., V. Montana, M.-D. Wei, J. P. Wuskell, and L. M. Loew, 1988. Biophys. J. 53:785-794) allows the monitoring of these equilibria through digital imaging video microscopy. We employ this combination of technologies to assess, simultaneously, the membrane potentials of cells and of their organelles in situ. We describe the methodology and optimal conditions for such measurements, and apply the technique to concomitantly follow, with good time resolution, the mitochondrial and plasma membrane potentials in several cultured cell lines. The time course of variations induced by chemical agents (ionophores, uncouplers, electron transport, and energy transfer inhibitors) in either or both these potentials is easily quantitated, and in accordance with mechanistic expectations. The methodology should therefore be applicable to the study of more subtle and specific, biologically induced potential changes in cells.     

Junction potentials,electrode standard potentials,and other problems in interpreting electrical properties of membranes

Summary As background to a detailed analysis of the cation permeation mechanism in rabbit gallbladder epithelium, this paper considers several general problems in interpretation. With regard to liquid junction potentials, the common practice of using saturated KCl bridges was insufficiently accurate for the present purposes because the resulting junctions are time-dependent and poorly understood theoretically. Time-independent and well-defined junction potentials were obtained by arranging all junctions to be of the biionic or single-salt dilution types. The magnitudes of these junction potentials were estimated in three different ways, with good agreement. Recording arrangements using either agar bridges or else Ag/AgCl electrodes also yielded good agreement after appropriate corrections for junction potentials and electrode potentials. The effects of nonelectrolytes on electrode standard potentials were measured. Two experiments were devised to determine whether transepithelially measured electrical properties of the gallbladder refer to a single membrane or to two membranes in series: the potential difference change resulting from a mucosal concentration change was measured as a function of the serosal concentration, and intracellular concentrations were altered by increasing bathing solution osmolalities with an impermeant nonelectrolyte. Both types of experiment indicated that transepithelial measurements are dominated by a single membrane. Small corrections were applied to measured potential differences to take account of unstirred-layer effects with permeant salts.     

The one-electron transfer redox potentials of free radicals. I. The oxygen/superoxide system.

The method of determination of Redox potentials of radicals, using the pulse radiolysis technique, is outlined. The method is based on the determination of equilibrium constants of electron transfer reactions between the radicals and appropriate acceptors. The limitations of this technique are discussed. The redox potentials of several quinones-semiquinones are calculated, as well as the standard redox potential of the peroxy radical. EO2/O2=-0.33 V and the redox oxidation properties of the peroxy radical in various systems and pH are discussed. The value determined for the redox potentials of O2/O2 is higher by more than 0.2 V than earlier estimates, which has important implications on the possible role of O2 in biological processes of O2 fixation.     

Voltage-sensitive dye recording of action potentials and synaptic potentials from sympathetic microcultures.

Given the appropriate multicell electrophysiological techniques, small networks of cultured neurons (microcultures) are well suited to long-term studies of synaptic plasticity. To this end, we have developed an apparatus for optical recording from cultured vertebrate neurons using voltage-sensitive fluorescent dyes (Chien, C.-B., and J. Pine. 1991. J. Neurosci. Methods. 38:93-105). We evaluate here the usefulness of this technique for recording action potentials and synaptic potentials in microcultures of neurons from the rat superior cervical ganglion (SCG). After extensive dye screening and optimization of conditions, we chose the styryl dye RH423, which gave fast linear fluorescence changes of approximately 1%/100 mV for typical recordings. The root mean square noise of the apparatus (limited by shot noise) was typically 0.03%, equivalent to 3 mV of membrane potential. Illumination for at least 100 flashes of 100 ms each caused no noticeable photodynamic damage. Our results show that voltage-sensitive dyes can be used to record from microcultures of vertebrate neurons with high sensitivity. Dye signals were detected from both cell bodies and neurites. Signals from presumptive dendrites showed hyperpolarizations and action potentials simultaneous with those in the cell body, while those from presumptive axons showed delayed propagating action potentials. Subthreshold synaptic potentials in the cell body were occasionally detectable optically; however, they were usually masked by signals from axons passing through the same pixel. This is due to the complex anatomy of SCG microcultures, which have many crisscrossing neurites that often pass over cell bodies. Given a simpler microculture system with fewer neurites, it should be possible to use dye recording to routinely measure subthreshold synaptic strengths.     

Extracellular potentials from single spinal motoneurons

Extracellular action potentials found close to the surface of motoneurons are related to the intracellular spikes. Evidence is cited to support the assumption that the extracellular spikes have the same time course as the membrane current at the site of recording. Simultaneously recorded intracellular and extracellular spikes are compared. Intracellular spikes are transformed, by means of a circuit which is equivalent to the extracellular recording situation, into transients that are like those appearing extracellularly. Evidence is given that the recordings are from the cell bodies of motoneurons. The results show that the membrane at the extracellular recording site does not produce a spike since the time course of the extracellular potentials is determined by the passive properties of the membrane.     

Na(+) action potentials in human photoreceptors

Mammalian photoreceptors are hyperpolarized by a light stimulus and are commonly thought to be nonspiking neurons. We used the whole-cell patch-clamp technique on surgically excised human retina to examine whether human photoreceptors can elicit action potentials. We discovered that human rod photoreceptors express voltage-gated Na(+) channels, and generate Na(+) action potentials, in response to membrane depolarization from membrane potentials of -60 or -70 mV. Na(+) spikes in human rods were elicited at the termination of a light response that hyperpolarized the potential well below -50 mV. This served to amplify the release of a neurotransmitter when a bright light is turned off, and thus selectively amplify the off response to the light signal.     

The effect of insulin on plasma-membrane and mitochondrial-membrane potentials in isolated fat-cells.

1. A recently developed technique for the measurement of plasma-membrane and mitochondrial-membrane potentials in intact cells by using the distribution of 86Rb+ and [3H]methyltriphenylphosphonium+ has enabled us to characterize a novel insulin effect on fat-cell mitochondria. For control cells the plasma-membrane and mitochondrial-membrane potentials were 75 mV and 152 mV respectively. Insulin (10 mu units/ml) caused a 9 mV hyperpolarization of the plasma membrane and a 19 mV depolarization of the mitochondrial membrane. 2. The insulin-dependent mitochondrial depolarization was observed at physiological insulin concentrations (10 mu units/ml) and was apparent when the cells metabolized a wide variety of substrates. 3. Evidence from the uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione by fat-cells was interpreted as indicating that the mitochondrial pH gradient was increased by insulin. 4. Insulin alters the balance between the electrical and pH-gradient components that form the mitochondrial protonmotive force. A model is proposed.     

Oxidation-reduction potentials and absorption spectra of two b-type cytochromes from the halophilic archaebacterium, Halobacterium halobium

The oxidation-reduction midpoint potentials were determined for two b-type cytochromes, which had been solubilized from the membrane of Halobacterium halobium and partially purified. The two b-type cytochromes have oxidation-reduction midpoint potentials of 175 and 7 mV, respectively. These b-type cytochromes could also be resolved by difference absorption spectroscopy, which revealed one b-type cytochrome with absorption maximum (alpha-peak) at 558 nm, reducible by ascorbate-tetramethyl-p-phenylenediamine, and the other with absorption maximum (alpha-peak) at 560 nm, reducible by dithionite. Different substrates such as succinate, NADH, and alpha-glycerophosphate were used to study the b-type cytochromes in situ when bound to the membrane in a functional state. Reducing equivalents from succinate and alpha-glycerophosphate appear to enter the respiratory chain at the 175 mV b-type cytochrome. Cytochrome a3 is spectrophotometrically shown to be present in the membrane of H. halobium.     

Discontinuous feedback amplifier: principles and application to the measurement of transmembrane potentials and currents of frog atrial fibers

The study and achievement of a discontinuous feedback amplifier to measure membrane potentials and currents in frog atrial fibres using the double sucrose gap technique was achieved. It was shown that, with the present device, the effects of the resistance in series with the membrane resistance and the membrane capacity on the measure of cardiac membrane potentials and fast currents are markedly reduced.     

"Action potentials" in Neurospora crassa, a mycelial fungus.

Occasional spontaneous "action potentials" are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5-20 mV depolarized from the normal resting potential (near-180 mV). The "action potentials" are long-lasting, 1-2 min and at the peak reach a membrane potential near-40 mV. A 2-to 8-fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H+ or Cl- and (b) periodic decreases in activity of the major electrogenic pump (H+) or the Neurospora membrane, coupled with a nonlinear (inverse signoid) current-boltage relationship. Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.     

Generation of action potentials in a mathematical model of corticotrophs.

Corticotropin-releasing hormone (CRH) is an important regulator of adrenocorticotropin (ACTH) secretion from pituitary corticotroph cells. The intracellular signaling system that underlies this process involves modulation of voltage-sensitive Ca2+ channel activity, which leads to the generation of Ca2+ action potentials and influx of Ca2+. However, the mechanisms by which Ca2+ channel activity is modulated in corticotrophs are not currently known. We investigated this process in a Hodgkin-Huxley-type mathematical model of corticotroph plasma membrane electrical responses. We found that an increase in the L-type Ca2+ current was sufficient to generate action potentials from a previously resting state of the model. The increase in the L-type current could be elicited by either a shift in the voltage dependence of the current toward more negative potentials, or by an increase in the conductance of the current. Although either of these mechanisms is potentially responsible for the generation of action potentials, previous experimental evidence favors the former mechanism, with the magnitude of the shift required being consistent with the experimental findings. The model also shows that the T-type Ca2+ current plays a role in setting the excitability of the plasma membrane, but does not appear to contribute in a dynamic manner to action potential generation. Inhibition of a K+ conductance that is active at rest also affects the excitability of the plasma membrane.     

Comparison of double layer potentials in lipid monolayers and lipid bilayer membranes

Summary It is shown that the Gouy-Chapman double layer analysis adequately describes the variation of the surface potential of monolayers of acidic natural lipids over a wide range of surface charge density and salt concentration. It is also shown that the potential which initially appears when an electrolyte gradient is rapidly imposed across a bilayer membrane is due to a difference in the double layer potentials on the two sides of the membrane. This conclusion follows from the fact that the observed bilayer potentials arise much more rapidly than can be accounted for by charge migration across the membrane and from the observation that the bilayer membrane concentration potentials, when measured immediately after establishment of a gradient, are equal to the surface potential change observed when the subphase concentration of a monolayer of the same lipid is changed by an amount equal to the gradient across the bilayer. The bilayer potential and monolayer potential changes, so measured, agree in a number of different electrolyte solutions over a wide range of electrolyte concentrations and surface charge densities. Because of this agreement and the applicability of the Gouy theory to monolayers, initial bilayer potentials may be calculated if the composition of the mixture used to form the membrane is known, provided that the pK's and areas of such components are available. In the absence of this information, membrane potentials may be calculated from electrophoretic data on the membrane lipid mixture; the conditions under which the latter approach is possible have been determined. The experimental results indicate that the composition of monolyers and bilayers spread from the same lipid mixture in decane are very similar, that the composition of the two types of film closely resembles the composition of the solution used to generate them, and that bilayer membranes are close-packed. The evidence further indicates that if any hydrocarbon solvent remains in these bilayers, it must be so situated that it contributes little, if anything, to the surface area. The steady state potential in the bilayer membrane system is frequently not identical with the initial potential which supports the hypothesis that in many cases only a fraction of the electrical conductance of unmodified membranes is caused by the ions which constitute the bulk electrolyte. An expression for the relationship between diffusion and double layer potentials has been derived which shows that, in the absence of any intrinsic selectivity of the hydrocarbon region of the membrane for hydrogen, hydroxyl, or impurity, the two potentials should be identical.     

Augmentation and suppression of action potentials by estradiol in the myometrium of pregnant rat.

The purpose of this study was to investigate the actions of estradiol on spontaneous and evoked action potentials in the isolated longitudinal smooth muscle cells of the pregnant rat. Single cells were obtained by enzymatic digestion from pregnant rat longitudinal myometrium. Action potentials and currents were recorded by whole-cell current-clamp and voltage-clamp methods, respectively. The acute effects of 17beta-estradiol on action potentials and inward and outward currents were investigated. The following results were obtained. The average resting membrane potential of single myometrial cells was -54 mV (n = 40). In many cells, an electrical stimulation evoked a membrane depolarization, and action potentials were superimposed on the depolarization. In some cells, spontaneous action potentials were observed. Estradiol (30 microM) slightly depolarized the membrane (ca. 5 mV) and attenuated the generation of action potentials by reducing the frequency and amplitude of the spikes. Afterhyperpolarization was also attenuated by estradiol (30 microM). On the other hand, in 5 of 35 cells, estradiol increased the first spike amplitude and action potential duration, while frequency of the spike generation and afterhyperpolarization were inhibited. In voltage-clamped muscle cells, estradiol inhibited both inward and outward currents. Acute inhibition or augmentation of spike generation by estradiol is due to the balance of inhibition of inward and outward currents. Inhibition of both currents also prevented afterhyperpolarization, causing potential-dependent block of Ca spikes.     

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.     

Dog red blood cells: Na and K diffusion potentials with extracellular ATP

External ATP causes a prompt increase in the Na and K permeability of dog red blood cells. By manipulating intra- and extracellular ion composition it is possible to observe ATP-induced net fluxes which can be explained in terms of the contribution of Na or K diffusion potentials to the membrane potential. Measurements of membrane voltage by a fluorescent dye technique confirm the existence of such potentials. A rough calculation of chloride permeability gives a value of the order of 10(-8) cm/s, which agrees with results in other species. The cells appear to be somewhat more permeable to bromide than to chloride.     

Relation of unit spike discharges and evoked potentials in the auditory cortex of cats.

Extracellular microelectrode recordings were made from the auditory cortex of anaesthetized cats during acoustic click stimulation. The microelectrode of low resistance allowed to record evoked field potentials and unit discharges simultaneously. In distant extracellular leads the relation of unit discharges and field potentials was equivocal. Near extracellular leads revealed that the antidromic invasion of the somadendritic membrane by excitation is a frequency dependent process (just as evoked field potentials) while spike potentials can reliably be elicited from the initial segment at high frequencies. It is assumed that the excitation spreading from the initial segment to the soma-dendritic membrane represents an important component of the evoked potentials, and their frequency dependence may be traced back to inhibitions activated by afferent impulses.     

Donnan potentials from striated muscle liquid crystals. Sarcomere length dependence.

Donnan potentials from A-bands and I-bands were measured as a function of sarcomere length in skinned long-tonic muscle fibers of the crayfish. These measurements were made using standard electrophysiological technique. Simultaneously, the relative cross-sectional area of the fibers was determined. Lattice plane spacings and hence unit-cell volumes were determined by low-angle x-ray diffraction. At a sarcomere length at which the myosin filaments and actin filaments nominally do not overlap, measurements of potential, relative cross-sectional area, and unit-cell volume were used in conjunction with Donnan equilibrium theory to calculate the effective linear charge densities along the myosin filament (6.6 X 10(4) e-/mu) and actin filament (6.8 X 10(3) e-/mu). Using these linear charge densities, unit-cell volumes and Donnan equilibrium theory, an algorithm was developed to predict A-band and I-band potentials at any sarcomere length. Over the range of sarcomere lengths investigated, the predicted values coincide with the experimental data. The ability of the model to predict the data demonstrates the applicability of Donnan equilibrium theory to measurements of electrochemical potential from liquid-crystalline systems.