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PubMed2004

Membrane potential and cation channels in rat juxtaglomerular cells.

The relationship between membrane potential and cation channels in juxtaglomerular (JG) cells is not well understood. Here we review electrophysiological and molecular studies of JG cells demonstrating the presence of large voltage-sensitive, calcium-activated potassium channels (BK(Ca)) of the ZERO splice variant, which is also activated by cAMP. These channels explain the hyperpolarization, which has been observed after stimulation of renin release with cAMP. In addition, there is now evidence that JG cells express functional L-type voltage-dependent calcium channels (Ca(v) 1.2), which in situations with strong depolarization lead to calcium influx and inhibition of renin release. In most in vivo situations the membrane potential is probably protected against depolarization by the BK(Ca) channels.

Animals↗
PubMed2003

P-glycoprotein, expressed in multidrug resistant cells, is not responsible for alterations in membrane fluidity or membrane potential.

Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors. However, the physiological role of P-gp in normal tissues is not well understood. Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity and membrane potential associated with P-gp expression, but interpretation of these studies is difficult, because most experimental cells have been selected for long periods in the presence of cytotoxic drugs and may have other host alterations. Therefore, we created two cell lines in which a transfected human MDR1 cDNA is repressed by tetracycline and induced in the absence of tetracycline. One cell line was derived from a mouse embryonic fibroblast cultured from a double (mdr1a/1b) knockout mouse, and the other was from a human HeLa cell line. Analysis of the kinetics of expression of P-gp showed that the mRNA had a half-life of approximately 4 h, and the protein had a half-life of approximately 16 h. P-gp cell surface expression (measured with monoclonal antibody MRK-16) and P-gp function (measured with a fluorescent substrate, rhodamine 123) was characterized by using fluorescence-activated cell sorting. No differences in membrane potential using the fluorescent probe oxonol or in membrane "fluidity" using fluorescent anisotropy probe or electron spin resonance probe were observed in the tet-repressible P-gp-expressing cells. In contrast, several drug-selected cells that express P-gp showed an increase in membrane fluidity and membrane potential. These results suggest that expression of P-gp per se has little effect on membrane fluidity or membrane potential, and it does not have H(+) pump activity. The changes in these parameters observed in drug-selected cells must reflect other host adaptations to drug selection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

[Experimental analysis of membrane potential changes during maturation of Bufo bufo gargarizans oocytes].

The full-grown oocytes obtained from toad (bufo bufo gargarizans) submitted in hibernation state or reared at 25-30 degrees C for several months, named hibernation oocyte or high temperature oocyte, had a membrane potential of -41.51 +/- 0.77 mV and -43.83 +/- 1.39 mV in Ringer's solution respectively. The hibernation oocytes underwent GVBD (germinal vesicle breakdown) and membrane depolarization at 19 +/- 1 degree C after progesterone stimulation. The membrane potential was about -20 mV at the period of GVBD, and -10 mV or so at 20 hours after the hormone treatment. However, the high temperature oocytes did not undergo GVBD, their membrane potential decreased before the fourth hour after treatment with progesterone and then recovered. If the hibernation oocytes were preincubated at 37-38 degrees C for 13 hours prior to the culture in the medium containing progesterone (10(-6)M, 37-38 degrees C), no GVBD was observed and the membrane depolarized before the fourth hour after treatment with progesterone then recovered, but MPF was detectable in the cytoplasm (unpublished). Both GVBD and membrane depolarization appeared in the hibernation oocytes and high temperature oocytes after injection of MPF. The time required for the hibernation oocytes injected MPF to attain the membrane potential about -20 mV was 4 hours earlier than that of progesterone treatment. It was just the time required for the appearance of MPF in the cytoplasm of oocytes treated with the hormone. It was noticed in our precedent article that a factor which appeared in the cytoplasm of high temperature oocytes differed from MPF. The factor was called Hibernation Oocyte Mature Promoting Factor (HOMPF).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Effect of membrane potential and cellular ATP on glutathione efflux from isolated rat hepatocytes.

total glutathione (GSH) efflux was studied in isolated rat hepatocyte suspensions at repleted GSH content (45-55 nmol/10(6) cells). The increase in concentrations of medium K+ in place of Na+ caused a parallel fall in membrane potential and total GSH efflux. Ouabain (1 mM) and replacement of Na+ with choline caused a gradual fall in membrane potential and GSH efflux. Hyperpolarization of hepatocytes with lipophilic anions, thiocyanate, and nitrate was associated with significantly increased efflux. Total GSH efflux was inhibited by increasing concentrations of fructose, antimycin A, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and there was a direct relationship between the rate of efflux and cellular ATP. Changes in total GSH efflux were paralleled by changes in GSH determined by high-performance liquid chromatography. Vanadate markedly inhibited efflux but caused only a modest decrease in cellular ATP. Fructose, antimycin A, and vanadate did not affect membrane potential or cell volume under the conditions at which efflux was inhibited. These results suggest independent requirements for both membrane potential and ATP in the transport of GSH.

Adenosine Triphosphate↗
PubMed1997

Theoretical considerations on the role of membrane potential in the regulation of endosomal pH.

Na+,K(+)-ATPase has been observed to partially inhibit acidification of early endosomes by increasing membrane potential, whereas chloride channels have been observed to enhance acidification in endosomes and lysosomes. However, little theoretical analysis of the ways in which different pumps and channels may interact has been carried out. We therefore developed quantitative models of endosomal pH regulation based on thermodynamic considerations. We conclude that 1) both size and shape of endosomes will influence steady-state endosomal pH whenever membrane potential due to the pH gradient limits proton pumping, 2) steady-state pH values similar to those observed in early endosomes of living cells can occur in endosomes containing just H(+)-ATPases and Na+,K(+)-ATPases when low endosomal buffering capacities are present, and 3) inclusion of active chloride channels results in predicted pH values well below those observed in vivo. The results support the separation of endocytic compartments into two classes, those (such as early endosomes) whose acidification is limited by attainment of a certain membrane potential, and those (such as lysosomes) whose acidification is limited by the attainment of a certain pH. The theoretical framework and conclusions described are potentially applicable to other membrane-enclosed compartments that are acidified, such as elements of the Golgi apparatus.

Buffers↗
PubMed2005

Modulation of membrane potential in mesothoracic moto- and interneurons during stick insect front-leg walking.

During walking, maintenance and coordination of activity in leg motoneurons requires intersegmental signal transfer. In a semi-intact preparation of the stick insect, we studied membrane potential modulations in mesothoracic (middle leg) motoneurons and local premotor nonspiking interneurons that were induced by stepping of a front leg on a treadmill. The activity in motoneurons ipsi- and contralateral to the stepping front leg was recorded from neuropilar processes. Motoneurons usually exhibited a tonic depolarization of < or =5 mV throughout stepping sequences. This tonic depolarization depended on membrane potential and was found to reverse in the range of -32 to -47 mV. It was accompanied by a mean membrane resistance decrease of approximately 12%. During front-leg stepping, an increased spike activity to depolarizing current pulses was observed in 73% of contralateral flexor motoneurons that were tested. Motoneurons ipsilateral to the walking front leg exhibited phasic membrane potential modulations coupled to steps in accordance with previously published results. Coupling patterns were typical for a given motoneuron pool. Local nonspiking mesothoracic interneurons that provide synaptic drive to tibial motoneurons also contribute to the modulation of membrane potential of tibial motoneurons during front-leg walking. We hypothesize that the tonic depolarization of motoneurons during walking is a cellular correlate of arousal that usually increases effectiveness of phasic excitation in supporting motoneuron firing.

Animals↗
PubMed1989

Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.

The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development.

Cell Membrane↗
PubMed1993

A photocross-linking fluorescent indicator of mitochondrial membrane potential.

Ionic dyes that distribute across membranes according to electrical potential have proven valuable as fluorescent indicators of mitochondrial energetics in living cells. Applications have been limited, however, as potential-dependent staining is lost during cell fixation. We have produced a membrane potential indicator whose potential-dependent distribution can be made permanent, to enable correlation of membrane potential with cytochemical information from immunofluorescence. A carbocyanine dye was derivatized with a photoreactive nitrophenylazide moiety so that irradiation would induce nonspecific, covalent attachment to nearby molecules. Photo-induced cross-linking was observed in paper chromatography, when irradiation caused immobilization of the dye. The new dye, named PhoCy (photofixable cyanine), showed specific staining of mitochondria in living Swiss 3T3 fibroblasts. When living cells were stained, irradiated, and fixed with formaldehyde, mitochondrial staining was retained owing to cross-linking with mitochondrial components. Omission of irradiation eliminated mitochondrial staining in fixed cells. Labeling, irradiation, and fixation procedures were optimized to produce bright specific staining with minimal background. The indicator's sensitivity to mitochondrial potential was demonstrated by treating cells with 2,4-dinitrophenol, an uncoupler of mitochondrial electron transport, which decreased mitochondrial staining in living cells and in the corresponding fixed specimens.

3T3 Cells↗
PubMed1999

Calcium channels, potassium channels and membrane potential of smooth muscle cells of human allantochorial placental vessels.

The membrane potential (Um), the main factor of the excitation-contraction coupling, of human allantochorial placental vascular smooth muscle cells (VSMCs) has been previously shown to depend on voltage-sensitive K+ channels. These channels were blocked by high external K+. To characterize other channels which regulated Um, various constrictor or/and vasodilators and channel blockers were used. Serotonin depolarized VSMCs, in normal medium, but induced a more marked depolarization in VSMCs predepolarized by high external K+. This depolarization was inhibited by nifedipine, a blocker of voltage-gated Ca2+ channels. Acetylcholine, sodium nitroprusside (without effect on Um in normal medium), hyperpolarized the predepolarized-high K+ medium VSMCs. This hyperpolarization was inhibited after addition of charybotoxin (a blocker of Ca2+-activated K+ channels) or/and glibenclamide (a blocker of ATP-sensitive K+ channels). A similar effect was obtained with isoproterenol. These results indicated that membrane potential of human placental allantochorial VSMCs was regulated by voltage-gated, Ca2+- and ATP-sensitive K+ channels and by voltage-dependent Ca2+ channels.

Acetylcholine↗
PubMed1983

Comparison of indirect probes of membrane potential utilized in studies of human neutrophils.

Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3'dipentyloxacarbocyanine [di-O-C5(3)], 3,3'dipentylindocarbocyanine [di-I-C5(3)], and 3,3'dipropylthiodicarbocyanine [di-S-C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di-S-C3(5) not only exhibits a different fluorescence response mechanism from di-O-C5(3) [and di-I-C5(3)] but also that the fluorescence of di-S-C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB-, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB- are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di-O-C5(3) and di-I-C5(3) but cannot be avoided with di-S-C3(5) or TPMP+/TPB-. Using di-O-C5(3) with the fluorescence-activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe-specific problems or response heterogeneity.

Carbocyanines↗
PubMed1991

Role of membrane potential in Ba2+ induced automaticity in guinea pig cardiac myocytes.

STUDY OBJECTIVE: The aim was to study in isolated myocardial cells the role of membrane potential in barium induced spontaneous activity and the ionic mechanism of the underlying pacemaker current. DESIGN: The membrane potential and resistance of single myocytes were studied at different voltage levels by means of current and voltage clamp steps in the absence and presence of barium (Ba). EXPERIMENTAL MATERIAL: The membrane potentials and currents of single guinea pig ventricular myocytes were recorded by means of an intracellular microelectrode through which current could also be passed. MEASUREMENTS AND MAIN RESULTS: In the presence of Ba (0.1-0.2 mM), stepwise depolarisations induced a transient overshoot and initiated action potentials followed by an undershoot, diastolic depolarisation and spontaneous discharge. During progressive depolarisations, membrane resistance (Rm) increased, decreased transiently at the end of the action potential, and reincreased during diastole. Stepwise repolarisations had opposite effects. Hyperpolarisations reversed diastolic depolarisation and could unmask oscillatory potentials (Vos). Voltage clamp steps to +20 mV were followed by outward tail currents during which Rm increased. Larger or longer depolarisations were followed by larger outward tail currents at resting potential level. The outward tail current reversed at potentials negative to EK. CONCLUSIONS: In the presence of Ba, applied depolarisation facilitates the induction of spontaneous activity through an interplay between voltage dependent and time dependent Ba block and unblock of gK1, voltage dependent increase in Rm, increased potassium driving force, and negative shift in the slow inward current threshold and sometimes Vos. The pacemaker potential underlying spontaneous activity is due to the slow re-establishment of Ba block of IK1 during diastole.

Action Potentials↗
PubMed1985

Effects of intracellular sodium and potassium iontophoresis on membrane potentials and resistances in toad urinary bladder.

Glass microelectrodes were used to measure membrane potentials and the ratio of apical to basolateral membrane resistances before and after the passage of current from the potential-recording microelectrode to ground, in toad urinary bladder epithelium, in order to iontophorese cations into the cell. After application of the current, there was a transient change in the tip potential of the microelectrode. This artifact was measured with the microelectrode in the mucosal medium and was subtracted from the potential recorded in the cell. The serosal medium was bathed by Ringer's solution containing 51.5 mM K+ to minimize any current-induced increase of K+ in the unstirred layer. Under those conditions, both Na+ and K+ iontophoresis caused a significant hyperpolarization of basolateral membrane potential (Vcs) and a significant increase in the ratio of apical to basolateral membrane resistances (Ra/Rb). When bladders were exposed to amiloride in the mucosal solution, Na+ iontophoresis caused the basolateral membrane to hyperpolarize, but no significant changes were observed in Ra/Rb. When Na+ was injected in the presence of serosal ouabain, Vcs depolarized and Ra/Rb increased. K+ iontophoresis caused the basolateral membrane potential to hyperpolarize in the presence of ouabain but Ra/Rb did not change significantly. These results indicate that the Na+ pump in toad bladder is rheogenic, that apical Na+ conductance is sensitive to the cell levels of Na+ and K+ and that the basolateral membrane is K+ permeable.

Amiloride↗
PubMed1974

Membrane potential and input resistance in acinar cells from cat and rabbit submaxillary glands in vivo: effects of autonomic nerve stimulation.

1. Membrane potential and input resistance measurements were made from acinar cells of cat and rabbit submaxillary glands in vivo, using intracellular glass micro-electrodes.2. The mean resting cell membrane potential was higher than previously reported, but ranged widely from -15 to -80 mV.3. Single shock electrical stimulation of the parasympathetic nerve fibres evoked characteristic potential changes. In some cases monophasic hyperpolarizations, in others biphasic responses (depolarization - hyperpolarization) were observed.4. The latency of the hyperpolarizing response was considerably longer (300-550 msec) than the latency of the biphasic response (about 150 msec).5. Hyperpolarizing and biphasic responses could be observed in the same cell at different levels of membrane potential. The initial depolarization of the biphasic response was dependent on the magnitude of the resting potential in such a manner that it was very small or absent at the lowest potentials and increased gradually with increasing level of the resting potential.6. Single-shock stimulation of the sympathetic nerve fibres to the gland did not evoke any response. In the cat, repetitive stimulation evoked hyperpolarizing or biphasic responses similar to those seen after repetitive stimulation of the parasympathetic nerve fibres. In the rabbit small hyperpolarizations were seen in a few cells only; mostly there was no response. Repetitive stimulation of the parasympathetic nerve fibres to the rabbit submaxillary gland evoked complex potential changes mostly of the depolarization-hyperpolarization type.7. Both single shock and repetitive stimulation of the parasympathetic nerve fibres evoked marked reductions in cell input resistance. In the hyperpolarizing cell type the conductance change sometimes preceded the potential change whereas they always occurred simultaneously in the biphasic cell type.8. It is concluded that both the hyperpolarizing and the biphasic secretory potentials are derived from the same type of acinar cells. The neurotransmitter released by the parasympathetic nerve endings (ACh) acts on the acinar cell membrane by increasing the ion permeability.

Animals↗
PubMed1983

On the mechanism by which Mg2+ and adenine nucleotides restore membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate.

The presence of ATP or ADP in the incubation medium prevents the collapse of membrane potential induced by external Ca2+ and phosphate. The same adenine nucleotides are unable to restore collapsed membrane potential unless Mg2+ are also added. Bongkrekate is also able to prevent the effects of external Ca2+ and phosphate and when added after membrane potential has collapsed strongly potentiates the restorative action of ATP or ADP. Atractyloside has an opposite effect.

Adenine Nucleotides↗
PubMed1999

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.

BACKGROUND: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. METHODS: Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent. RESULTS: In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium. CONCLUSIONS: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.

Calibration↗
PubMed2001

Dendritic spatial flicker of local membrane potential due to channel noise and probabilistic firing of hippocampal neurons in culture.

Whole-cell recordings and imaging of dissociated hippocampal neurons stained with voltage sensitive dye provide a new microscopic picture of neuronal excitation. This is the first attempt to combine imaging of active channel clusters on the geometry of live neurons and a theoretical approach. During single somatic action potentials and the back-invasion into the neurites, local mean potentials are generated at sites of active channel clusters which are unevenly distributed in the neuronal membrane. Similar mean membrane potentials are observed in the neurites and at the soma. Identical action potentials produce different spatial patterns of mean membrane potentials from trial to trial. This spatial variability is explained by the stochastic behavior of the channels in the clusters. When hippocampal neurons are excited by synaptic inputs, their evoked responses are probabilistic and generate variable spatial patterns of mean membrane potential trial after trial. Our stochastic model reproduces this random behavior by assuming that the voltage fluctuations generated by channel noise are added to the synaptic potentials reaching the soma. We demonstrate that the probability of action potential initiation depends on the strength of the synaptic input, the diameter of the dendrites and the relative positions of the channel clusters, of the synapse and of the soma.

Action Potentials↗
PubMed1998

Charybdotoxin block of Ca(2+)-activated K+ channels in colonic muscle depends on membrane potential dynamics.

Charybdotoxin (ChTX) is a specific blocker of Ca(2+)-activated K+ channels. The voltage- and time-dependent dynamics of ChTX block were investigated using canine colonic myocytes and the whole cell patch-clamp technique with step and ramp depolarization protocols. During prolonged step depolarizations, K+ current slowly increased in the continued presence of ChTX (100 nM). The rate of increase depended on membrane potential with an e-fold change for every 60 mV. During ramp depolarizations, the effectiveness of ChTX block depended significantly on the rate of the ramp (50% at 0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanism in which ChTX slowly "unbinds" in a voltage-dependent manner. A simple kinetic model was developed in which ChTX binds to both open and closed states. Slow unbinding is consistent with ChTX having little effect on electrical slow waves recorded from circular muscle while causing depolarization and contraction of longitudinal muscle, which displays more rapid "spikes." Resting membrane potential and membrane potential dynamics are important determinants of ChTX action.

Animals↗
PubMed1984

Can a Ca2+ pump in the endoplasmic reticulum of the Lepidium root be the trigger for rapid changes in membrane potential after gravistimulation?

Since gravistimulation is followed by alterations in the external current symmetry (Behrens et al., 1982), the effect of gravistimulation on cellular membrane potential was investigated using conventional glass microelectrode techniques. The resting potential of statocytes in a vertically oriented root is approx. -118 mV. Upon gravistimulation, the membrane potential is temporarily depolarized (lag time = 2 s) to a potential of approx. -93 mV. This depolarization is only observed in statocytes located on the physically lower root flank while those on the corresponding upper flank become weakly hyperpolarized (approx. -13 mV). These results reflect altered ion fluxes across the plasma membrane. The perception of gravistimulus was suggested to result from a pressure of the amyloplasts on the distal endoplasmic reticulum (ER) of the statocytes (Sievers and Volkmann, 1972). A causal relationship between changes in ER-amyloplast interactions and the rapid alterations in plasma membrane potential described above is not known. A candidate for such an intracellular messenger is Ca2+. As a first step in establishing the validity of such an assumption, we have isolated ER membranes from roots. When incubated with micromolar concentrations of Ca2+, the vesicular membrane fraction accumulates Ca2+. The accumulation is ATP-dependent and -specific and is directly coupled to ATP hydrolysis since a protonophore shows no inhibitory effect. Thus, in analogy to the sarcoplasmic reticulum of muscle, regulation of an ER-localized Ca2+ compartment might be an important step in such complex processes as stimulus-transduction in gravitropism.

Adenosine Triphosphate↗