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Flow cytometric determination of absolute membrane potential of cells.

Membrane potential measurements using fluorescent membrane potential indicator dyes report on relative changes but usually do not result in an absolute value of the measured parameter. The method developed in this paper is based on the assumption that the negatively charged bis-oxonol distributes across the cytoplasmic membrane according to the Nernst equation. It is further supposed that the fluorescence intensity measured from a given stained cell is a single-value function of the intracellular dye concentration. The protocol suggested incorporates the construction of a calibration curve (fluorescence intensity measured from stained cells vs. extracellular dye concentration). This allows the evaluation of the membrane potential in millivolts using fluorescence readings of the cells both in the depolarized state and in the state of interest. Good agreement was found between absolute membrane potential data of human peripheral blood lymphocytes by our method and results of parallel patch clamp measurements.

Animals↗

Oscillations of membrane potential across a polypeptide membrane, induced by an electrical current.

Oscillation of membrane potential across a tri-block copolypeptide membrane composed of (Glu)x-(Leu)y-(Glu)x (x = 0.18 and y = 0.64) was observed under an electrical current, when the membrane was placed between equimolar aqueous salt solutions. The amplitude of the oscillation was influenced by the type of cation and anion in the external salt solution, and the amplitude was in the sequence: K+ > Na+ > Cs+ > Ca2+ and Cl- > Br-. The frequencies of the oscillations were in the range 0.1 to 5 Hz, and were also slightly influenced by the type of cation and anion.

Anions↗

Genome-independent effects of 1,25-dihydroxy vitamin D-3 on membrane potential.

Cell membrane potential, Vm, was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10(-10) M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of Vm (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of Vm in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on Vm, in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.

Animals↗

ARTMEM--an interactive graphical program simulating membrane potential measurements across artificial membranes.

This paper describes ARTMEM, an interactive graphical simulation program, and its use for teaching students the concept of membrane potentials and the measurement of electrical potentials across an ion-selective membrane. The program, written in Borland C++, and specifically designed for IBM-PC-compatible equipment, can also run on fast Macintosh computers using SoftPC emulation software. It demonstrates how an ion-selective membrane separating two different salt solutions automatically generates a potential across the membrane, as well as how such electrical potentials can be measured simply and the underlying permeability ratio determined. The program was developed to replace a previous experiment with actual artificial resin membranes; virtually every feature of the experiment has been simulated (e.g., unstirred-layer effects, solution-mixing mixing contamination, liquid junction potential effects). In addition, a number of these features and the procedure for data fitting are more clearly demonstrated using the computer simulation. The efficacy of such a simulation is discussed in comparison with the real experiment and other types of simulations.

Computer Graphics↗

Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations.

OBJECTIVE: To examine whether a relationship exists between loss of mitochondrial transmembrane potential and plasma membrane translocation of phosphatidylserine (PS) in subpopulations of human spermatozoa of men consulting for infertility. SETTING: A tertiary institutional research center. DESIGN: Prospective observational study. PATIENT(S): Twelve infertile men and five fertile controls were compared. INTERVENTION(S): Sperm subpopulations were compared after density gradient separation. MAIN OUTCOME MEASURE(S): Mitochondrial membrane potential was measured with a cationic dye, translocation of PS was evaluated with Annexin-V binding, and motion parameters were assessed manually. RESULT(S): In both the study and control groups and compared with the high-motility fraction, the low-motility fraction had significantly lower sperm motility and normal morphology, and significantly higher percentage of cells with disrupted mitochondrial membrane potential and plasma membrane PS translocation. There was a positive and significant correlation in both subfertile and control groups between the percentages of Annexin-V+ live cells and cells with mitochondrial membrane potential disruption (r = 0.82 and r = 0.85, respectively). CONCLUSION(S): The correlation of plasma membrane PS translocation and loss of mitochondrial membrane potential is suggestive of an early apoptosis phenotype, as is typically observed in somatic cells identified in sperm subpopulations with percentage of low-motile cells. We speculate that such changes might be used as diagnostic markers of sperm dysfunction(s) and that increased levels found in subfertile men might be indicators of reduced fertility potential.

Annexin A5↗

Membrane potential across anion-exchange membranes in acidic solution system.

The membrane potential across anion-exchange membranes in H2SO4 and Na2SO4 solutions was measured, and the experimental results were fitted to the theory in the 2-1 electrolyte system based on the Donnan equilibrium and the Nernst-Planck flux equations. For the Na2SO4 solution, the Donnan potential makes a significant contribution to the membrane potential, but for the H2SO4 solution, the diffusion potential significantly contributes to the membrane potential. The diffusion potential has a greater contribution to the membrane potential across AEM-2 with a high water content than that across AEM-1. These results suggest that a proton with a high mobility can move without substantial influence of electrostatic interaction in a positively charged membrane.

Journal Article↗

Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential.

Traditionally, neuronal operations in the cerebral cortex have been viewed as occurring through the interaction of synaptic potentials in the dendrite and soma, followed by the initiation of an action potential, typically in the axon. Propagation of this action potential to the synaptic terminals is widely believed to be the only form of rapid communication of information between the soma and axonal synapses, and hence to postsynaptic neurons. Here we show that the voltage fluctuations associated with dendrosomatic synaptic activity propagate significant distances along the axon, and that modest changes in the somatic membrane potential of the presynaptic neuron modulate the amplitude and duration of axonal action potentials and, through a Ca2+-dependent mechanism, the average amplitude of the postsynaptic potential evoked by these spikes. These results indicate that synaptic activity in the dendrite and soma controls not only the pattern of action potentials generated, but also the amplitude of the synaptic potentials that these action potentials initiate in local cortical circuits, resulting in synaptic transmission that is a mixture of triggered and graded (analogue) signals.

Action Potentials↗

Cevadine-induced changes of membrane potential and sodium transport of muscle membrane in a chloride-free solution.

Cevadine-induced changes in membrane potential, sodium transport, intracellular Na, K, and water content were investigated in sartorius muscles incubated in chloride-free (glutamate) Ringer. Cevadine sensitivity of muscles incubated in glutamate Ringer was about five times greater than that of muscles incubated in normal Ringer. Therefore, even 0.005 mmol/l cevadine could induce depolarization and membrane potential oscillations. The membrane potential oscillations were recorded much longer from muscles incubated in chloride-free Ringer (even in the 15th hour of treatment) than in normal Ringer. Depolarization and membrane potential oscillations reversed more slowly in cevadine-free glutamate Ringer than in alkaloid-free normal Ringer. The rhythmic activity could be recorded even in the 10th-15th hour of incubation in cevadine-free glutamate Ringer. Cevadine increased the 24Na uptake of muscles incubated in glutamate Ringer by an average of 230%. In comparison, the cevadine-induced increase of 24Na uptake of muscles incubated in normal Ringer was approximately 350%. In the presence of cevadine the 24Na loss of muscles incubated either in glutamate or in normal Ringer increased to the same degree, i.e. three times. The increase of 24Na loss developed faster in glutamate Ringer than in the presence of chloride. The water content of muscles incubated in cevadine containing, chloride-free (glutamate) Ringer did not increase significantly. Muscles incubated in normal Ringer with cevadine showed a 42.7% increase of water content in 2 hours. Intracellular Na content and Na concentration increased by about 60% during a 2-hour-treatment with cevadine in a chloride-free environment. At the same time, cevadine treatment increased the intracellular Na content and Na concentration of muscles incubated in normal Ringer by about 160% and 80%, respectively. The cevadine-induced decrease of intracellular K content and concentration of muscles incubated in glutamate Ringer was 5% and 10%, respectively, in 2 hours. On the other hand, the decrease of intracellular K concentration in muscles incubated in cevadine-containing normal Ringer occasionally reached 30% due to the increase of water content of the muscles. The cevadine-induced increase of the wet weight of muscles incubated in normal Ringer was practically irreversible. It was not possible to eliminate the increase of wet weight even by washout lasting for 10-15 hours.

Animals↗

DCCD-sensitive, Na+-dependent H+-influx process coupled to membrane potential formation in membrane vesicles of Halobacterium halobium.

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) on light-induced H+-transport and transmembrane electric potential (delta phi) formation were studied in the membrane vesicles of Halobacterium halobium R1M1. In accordance with our previous finding of the existence of two DCCD-binding components in vesicle membrane using 14C-DCCD (Konishi & Murakami FEBS Lett. 169, 283-286 (1984)), DCCD inhibited the H+-influx process biphasically; that is, the H+-influx process which is electrically silent was initially inhibited at concentrations below 30 nmol of DCCD/mg vesicle protein, while another H+-influx process which is coupled to delta phi formation was secondarily inhibited above this concentration of DCCD. The latter H+-influx process was highly dependent on the Na+ concentration. The extents of Na+-dependent recovery of delta phi formation and H+-influx were quantitatively correlated. From these results, it was concluded that the second DCCD-sensitive H+-influx process which is coupled to delta phi formation is due to the hypothetical Na+/H+-antiporter postulated by Lanyi and MacDonald (Biochemistry 15, 4608-4614 (1976)). It was also found that Li+ can be substituted for Na+ in this system, as is the case with Na+/H+-antiporters found in other organisms.

Carrier Proteins↗

Membrane potential can influence the rate of membrane photomodification.

Though cellular photomodification has been shown to change cellular resting membrane potential, an effect of membrane potential on the rate of photomodification has never been reported. Here we demonstrate that the rate of photomodification of potassium channels in frog atrial cells is voltage dependent. The rate of potassium channel photomodification using negatively charged Rose Bengal as the photosensitizer is about 2.5 times greater at the resting membrane potential of -70 mV compared to +40 mV. Similar results are obtained using the positively charged photosensitizer methylene blue. On the other hand, the rate of photomodified increase of leak current in the same cells does not significantly change in this voltage range with Rose Bengal as photosensitizer, but demonstrates a voltage dependence like that of potassium current when methylene blue is the photosensitizer. These observations cannot be explained based on voltage-dependent partitioning of the sensitizer, as similar effects on potassium current were obtained using either a positively charged or negatively charged sensitizer.

Animals↗

[Trans-membrane potential in Entamoeba].

The membrane potential in Entamoeba is an important driving force for the uptake of substrates. In Entamoeba invadens PZ a membrane potential of -36 mV was obtained when Nernst equation was applied to the distribution at equilibrium of 86Rb+ in the presence of valinomycin. This could explain the levels of accumulation of up to 4 times found for positively charged substrates. Membrane potential was diminished by depolarizing conditions (high external K+ concentration in the presence of valinomycin). Moreover, we recorded continuously the membrane potential of Entamoeba invadens PZ and Entamoeba histolytica HM1 using the fluorescent lipophilic cation diisopropylthiodicarbocyanine. It was found that the uptake of this cation by the amoebae was fast in both species, conditions that modify the membrane potential (hyperpolarization and depolarization) produced changes in the fluorescence of the dye in agreement with its reported capability to detect variations in membrane potential. It can be concluded that these microorganisms have a membrane potential negative inside them.

Animals↗

Effect of proton concentration on membrane potential across a weak amphoteric polymer membrane.

An amphoteric membrane consists of both positively and negatively fixed charge groups chemically bound to the polymer chains. If the external solution is changed from alkali to acid, it is possible to obtain an experimental result in which the membrane potential changes from positive to negative through the isoelectric point. It was characterized by examining the relationship between membrane potential and proton concentration (pH) obtained from both experimental and theoretical considerations. The Nernst-Planck flux equation and the Donnan equilibrium theory were also solved for a four-component system combined with the dissociation constant, in order to discuss the pH dependence of membrane potential in a weak amphoteric membrane by comparing the experimental results with the calculated results. It was proven that the calculated results substantially deviated from the theoretical results despite a similar tendency. Such a deviation was caused by the fact that the original theory disregarded the activity coefficient and the ionic mobility, which were dependent on the fixed charge concentration in a membrane. The original theoretical model was modified by adding the effect of a fixed charge group to the activity coefficient and ionic mobility. The calculated results using the modified model explained well the experimental results if the parameter called charge effectiveness, phi, was introduced into the equations. Introduction of phi into the prediction of membrane potential was already done by Kobatake et al. in a system of a strong polyelectrolyte monopolar membrane/salt aqueous solution. In this study, it was proved that phi can also be introduced into a weak amphoteric polymer membrane/salt aqueous solution system. Finally it was also concluded that the Donnan equilibrium and the Nernst-Planck flux equation were still applicable for examining the transport phenomena for the system of a weak amphoteric charged membrane and electrolyte solutions at various pH.

Journal Article↗