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PubMed1986

Membrane potential dependency of glutamic acid transport in rabbit jejunal brush-border membrane vesicles: K+ and H+ effects.

We have applied our recently developed approach for quantitative generation and estimation of membrane potential differences (Berteloot, A. (1986) Biochim. Biophys. Acta 857, 180-188) to the reevaluation of glutamic acid transport rheogenicity in rabbit jejunal brush-border membrane vesicles. Membrane diffusion-potentials were created by altering iodide concentrations in the intra- and extravesicular compartments while keeping isosmolarity, isotonicity and ionic strength constant by chloride replacement. The known value of ion permeabilities relative to sodium in this preparation also allows calculation of membrane potential differences using the Goldman-Hodgkin-Katz equation. This strategy appears superior to more classical methods involving ionophore-induced membrane diffusion-potentials of protons or potassium as both cations have been shown to participate in the transport mechanism. In this paper, we demonstrate that this approach is perfectly suitable for the investigation of membrane potential dependency of glutamic acid transport as our results showed that chloride replacement by iodide did not affect uptake in vesicles with membrane potential clamped to zero by gramicidin D (sodium conditions) or by gramicidin D plus valimonycin (sodium + potassium conditions). The method thus allows to dissociate membrane potential effects from possible effects that might be introduced by altering the anion species. In these conditions, our studies clearly demonstrate that glutamic acid uptake, whether analyzed over a 1 min time scale or under initial rate conditions, was sensitive to membrane potential differences. However, our results also show that the electrogenicity of the transport system varied depending upon the intravesicular presence or absence of potassium, its presence stimulating the membrane potential dependency of uptake. This effect is modulated by the internal pH and it is concluded that inside H+ and K+ are not equivalent as countertransported cations. The external pH also seems to modulate the response to potential by acting on the fully loaded form(s) of the transporter. The possibility that outside H+ competes for (an) external Na+ binding site(s) and/or precludes the attachment of (an) extra sodium ion(s) should be considered.

Animals↗
PubMed1982

Membrane potential in a potassium transport-negative mutant of Escherichia coli K-12. The distribution of rubidium in the presence of valinomycin indicates a higher potential than that of the tetraphenylphosphonium cation.

The membrane potential across the cytoplasmic membrane of EDTA-treated cells of a K+ transport-negative mutant of Escherichia coli K-12 was estimated from the equilibrium distribution of different lipid-soluble cations. With glucose as a substrate and at low K+ out, the membrane potential calculated from the distribution ratio of 86Rb+ in the presence of valinomycin (delta psi rb+) was considerably higher than that indicated by the [3H]tetraphenylphosphonium cation (delta psi TPP+). The lipid-soluble anion phenyldicarbaundecaborane (PCB-) increased delta psi TPP+ close to delta psi Rb+. To investigate whether these results were due to different binding of the cations to cellular components, residual Rb+ and TPP+ uptake was measured in cells permeabilized with 5% n-butanol (by volume). In those cells the distribution ratios or Rb+, K+ and Na+ approached a value of 4, indicating that the uptake of all three ions was driven by a residual negative surface potential or transmembrane Donnan potential (internally negative). The distribution ratio of TPP+ was 3--4-times higher than that of other cations and up to 10 mM TPP+ out was almost independent of the added TPP+ concentration. This extra uptake presumably represents binding of TPP+ to the cellular membranes. Thus, at pH 7.5, delta psi Rb+ was about 180--200 mV, whereas after correction for binding delta psi TPP+ was 110--150 and 150--170 mV in the absence and presence of PCB-, respectively. It is proposed that TPP+ indicates too low a potential, because by its strong binding it decreases the negative surface potential of the cytoplasmic membrane, and thereby inhibits its own further uptake. This is taken to mean that TPP+ distribution can be used as a qualitative probe only for the bacterial membrane potential.

Cell Membrane↗
PubMed1995

Xenopus laevis oocyte: using living cells to teach the theory of cell membrane potential.

We describe a student course in membrane physiology that includes demonstration of the cell membrane potential of a living cell, the Xenopus laevis oocyte. By use of K+ macroelectrodes and a computer simulation program, students are introduced to the membrane concept on the basis of ion gradients, diffusion potentials, and membrane conductances. Subsequently, some basic principles are demonstrated in living cells by measuring the cell membrane potential of a X. laevis oocyte. The dependence of the cell membrane potential on ion gradients is shown by stepwise elevation of the extracellular K+ concentration. The clinical relevance of disturbances of the K+ homeostasis is discussed in this context. The effect of changing the membrane conductance for particular ions on the cell membrane potential is demonstrated by applying a K+ channel blocker. The experiments are discussed in the context of the action of pharmaceuticals and toxins. This student course on membrane physiology has now been held for 2 yr for medical and dental students, and their response to the oocyte demonstration, as evaluated by a student questionnaire, was mainly positive.

Animals↗
PubMed2004

A voltage-dependent K+ current contributes to membrane potential of acutely isolated canine articular chondrocytes.

The electrophysiological properties of acutely isolated canine articular chondrocytes have been characterized using patch-clamp methods. The 'steady-state' current-voltage relationship (I-V) of single chondrocytes over the range of potentials from -100 to +40 mV was highly non-linear, showing strong outward rectification positive to the zero-current potential. Currents activated at membrane potentials negative to -50 mV were time independent, and the I-V from -100 to -60 mV was linear, corresponding to an apparent input resistance of 9.3 +/- 1.4 G Omega (n= 23). The outwardly rectifying current was sensitive to the K(+) channel blocking ion tetraethylammonium (TEA), which had a 50% blocking concentration of 0.66 mM (at +50 mV). The 'TEA-sensitive' component of the outwardly rectifying current had time- and membrane potential-dependent properties, activated near -45 mV and was half-activated at -25 mV. The reversal potential of the 'TEA-sensitive' current with external K(+) concentration of 5 mm and internal concentration of 145 mM, was -84 mV, indicating that the current was primarily carried by K(+) ions. The resting membrane potential of isolated chondrocytes (-38.1 +/- 1.4 mV; n= 19) was depolarized by 14.8 +/- 0.9 mV by 25 mM TEA, which completely blocked the K(+) current of these cells. These data suggest that this voltage-sensitive K(+) channel has an important role in regulating the membrane potential of canine articular chondrocytes.

Animals↗
PubMed1985

[Does non-quantum secretion of acetylcholine from motor neuron endings participate in the neurotrophic control of the membrane potential of muscle fibers in the rat?].

The resting membrane potential of synaptic zone of muscle fibres within 2-3 mm (near) and 9-11 mm (far) of the nerve section of the rat diaphragm muscle was measured after 3 hours or on the 5th day after the motor nerve section. The membrane potential of "near" fibres was lower than that of "far" fibres. The presence of carbamylcholine or cGMP in the culture medium maintained the membrane potential of "near" fibres close to that of "far" fibres. Ouabain prevented these effects. On the 5th day after the nerve section the presence of carbamylcholine or cGMP had no effect on the membrane potential. In the in vitro experiments the nonquantal acetycholine release was equal in "near" and "far" fibres and just the same as in the control preparations. A conclusion is made that synaptic acetylcholine from motor nerve endings in nonquantal form does not play a significant role in neurotrophic control of the resting membrane potential of muscle fibres membrane in mammals.

Acetylcholine↗
PubMed1977

The effect of Na, K and Cl ions on the resting membrane potential of sino-atrial node cell of the rabbit.

The resting membrane potential of S-A node cell was investigated by observing the response of the membrane potential to change in [K+]O or [Cl-]O under the presence or absence of Na ion. The slope of the change in membrane potential per decade change in [K+]O increased from 12.3 to 44 mV by removal of Na ion from the external medium, suggesting an extensive contribution of Na ion to the resting membrane potential. To determine the relative conductance between Na and K ions, Cl ion in Tyrode solution was substituted with SCN ion, which is 2.1 times more permeable than Cl ion, in order to eliminate the contribution of Cl concentration cell to the resting membrane potential. The contribution of the Cl ion to the resting membrane potential could be examined only in Na-free medium. Acetate ion has been reported to be 0.5 times less permeable than Cl ion. The replacement of Cl ion by acetate ion in various proportions caused a transient depolarization. The slope of this transient depolarization per decade change of [Cl-]O between 10 to 100 mM was determined to be -6.8mV. Under the assumption that the resting membrane potential could be determined by the parallel concnetration cells for Na, K and Cl, relative conductance ratio between K, Na and Cl was calculated to be 1:0.58:0.15.

Animals↗
PubMed1986

Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP.

The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Hormone responses of in vitro bone nodule cells: studies on changes of intracellular calcium and membrane potential in response to parathyroid hormone and calcitonin.

We used two techniques to study the responses of individual in vitro bone nodule cells to parathyroid hormone (PTH) and calcitonin (CT). These techniques are laser scanning confocal imaging with a fluorescent indicator to measure intracellular free [Ca2+], and microelectrode impalement to measure the electrical potential difference across the cell membrane. We applied these measurement techniques to cells in the top cellular layer of nodules that form in vitro in cultures of cells obtained from fetal rat calvaria. Our measurements showed a transient increase in intracellular free [Ca2+] following application of PTH or CT. The duration of the increase in fluorescent intensity following PTH application varied from about 100 to more than 300 s, and the duration following CT application was from 30 to 80 s. In some measurements we applied both hormones in sequence, and observed that some cells showed an intracellular [Ca2+] response to both hormones, while other cells apparently responded to only one or the other of the hormones, or to neither. We also observed membrane potential changes in response to PTH and to CT. The membrane potential response to CT was quite small. The time courses of these membrane potential changes consisted of a depolarizing phase lasting about 100 s (with both hormones) followed by a hyperpolarizing phase (with PTH). Control measurements using only the vehicle solutions were carried out with both techniques, producing negligible responses.

Animals↗
PubMed1966

Application of the sucrose-gap method to determine the ionic basis of the membrane potential of smooth muscle.

1. A study has been made of the ionic basis of the smooth muscle membrane potential by changing the ionic environment of the ureter and recording the resultant potential changes with the sucrose gap. The relation of these potential changes to the membrane potential of individual cells has also been studied.2. It is shown that if the ionic environment of all the cells in the tissue is uniformly changed, then the potential change recorded by the sucrose gap is proportional to the magnitude of the short circuit factor, the liquid junction potential change at the sucrose-test solution interface, and the amplitude of the membrane potential change of a single cell.3. A simple method is described for determining the steady value of the short circuiting factor, and the liquid junction potential changes, so that the recorded potentials can be corrected to give membrane potentials.4. The action of isotonic potassium sulphate and isotonic potassium chloride on the membrane potentials of the ureter smooth muscle cells is described.5. When the extracellular potassium concentration is changed reciprocally with the extracellular chloride concentration in order to maintain these ions in a Donnan equilibrium across the muscle cell membrane, the membrane potential is found to decrease by 53 mV for a 10-fold change in external potassium concentration, for concentrations above 10 mM.6. It is concluded that above an external potassium concentration of 10 mM the membrane potential of ureteral smooth muscle cells obeys the prediction of the Nernst equation for a potassium electrode.

Cell Membrane↗
PubMed1991

Does the membrane potential control incorporation of tubulovesicles into the secreting apical membrane of the rat parietal cell?

The present studies were designed to examine the effect of changes in membrane potential by means of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) and variations in the pH of the medium on the secretory response of parietal cells. Studies were performed in vitro using isolated cells from rat stomachs and acid production was indirectly determined by 14C-aminopyrine (AP) accumulation. CCCP affected both basal and histamine-stimulated AP accumulation in a concentration-dependent manner. The AP accumulation ratios depended on pH of the incubation medium; the ratio was lowest at pH 6.6, and increased progressively as the pH of the medium increased to 7.8. Moreover, the decreases in AP accumulation ratios caused by simultaneous addition of CCCP and AP to cell suspensions compared to those in which CCCP was added to incubated cells after achieving the steady-state of AP accumulation were quantitatively similar. These findings suggest that the decrease in AP accumulation due to CCCP treatment is a consequence of an activation of acid secretion rather than an inhibitor of acid production. From the present and previously published data, we propose a working hypothesis: membrane recycling is dependent on changes in apical membrane potential.

Aminopyrine↗
PubMed1988

Role of sodium pump in membrane potential gradient of canine proximal colon.

A large gradient in membrane potential exists through the thickness of the circular layer in canine colonic muscles. This study tested the effects of several experimental manipulations known to block electrogenic sodium pumping on the resting potentials of colonic muscles. Membrane potentials were recorded with microelectrodes from cells through the circular muscle layer. In cells adjacent to the submucosal surface of the circular layer, application of ouabain (10(-6) to 10(-5) M) caused an average membrane depolarization of 36 mV. Removal of the external K+ resulted in depolarizations similar to the effect of ouabain. Readmission of K+ (5.9 mM) produced repolarization and an additional hyperpolarization that averaged 13 mV beyond the resting potential. When exposed to 15 mM K+, cells hyperpolarized well beyond the estimated potassium equilibrium potential (EK). Ouabain blocked the repolarization in response to reintroduction of external K+. Lowering the bath temperature to 20 degrees C rapidly depolarized membrane potential; rewarming repolarized cells. Ouabain and K+-free solutions blocked the repolarization response to rewarming. Cells also depolarized when exposed to solutions in which the NaCl was replaced with LiCl. Membrane potentials of cells within the bulk of the circular layer decreased as a function of distance from the submucosal border. Cells at the myenteric border of the circular muscle were not significantly affected by ouabain and K+-free solution, but these treatments abolished the gradient in membrane potential across the circular layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

Sensory electrophysiology of bacteria: relationship of the membrane potential to motility and chemotaxis in Bacillus subtilis.

The relationship of membrane potential to motility and chemotaxis of Bacillus subtilis has been tested by using the fluorescence of a cyanine dye as a probe of the potential. The dye fluorescence was found to be an indicator of membrane potential by correlation with triphenylmethylphosphonium ion distribution and with changes due to anaerobicity and ionophore addition. When the potential was sufficient for motility and constant over time, it was found that the absolute level of the potential did not affect the swimming behavior of the bacteria. Transient alteration of the membrane potential did, however, lead to changes in swimming behavior. Attractants were found to alter the swimming behavior of the bacteria without altering the membrane potential. Thus, change of the overall membrane potential of a normal B. subtilis is not required for chemotaxis, but such a change is sensed by the bacteria just as changing levels of attractants and repellents are sensed.

Alanine↗
PubMed1978

The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell.

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.

Amino Acids↗
PubMed1980

Membrane potential changes in lacrimal gland acinar cells elicited by carbachol and epinephrine.

Intracellular microelectrode recordings of acinar cell membrane potentials were made from fragments of the rat lacrimal gland superfused in vitro. The average resting membrane potential was -45 mV. Carbachol and epinephrine produced virtually identical membrane potential changes consisting of an initial hyperpolarization (1 mV), lasting approximately 7 sec, followed by a depolarization of approximately 12 mV. The membrane potential generally returned to prestimulation levels after 2 min of exposure to agonist. The responses to carbachol and epinephrine were blocked by atropine and phentolamine, respectively. Superfusion with media lacking Ca or Cl reduced significantly both the resting membrane potential and the agonist-induced depolarization. The hyperpolarization was increased significantly in the absence of Ca and generally prolonged in the absence of Cl. Superfusion with 10 mM Co had no effect on either the resting membrane potential or the agonist-induced membrane potential changes. The hyperpolarization initiated by agonist was significantly enhanced during superfusion with low K, ouabain or amiloride while the depolarization was significantly reduced during superfusion with low K, amiloride or low Na. Resting membrane potentials during superfusion with low K, amiloride or low Na were not significantly different from control, whereas ouabain caused a small depolarization. It is concluded that muscarinic or alpha adrenergic receptor stimulation initiates a membrane potential change characterized by a hyperpolarization, due to an increased in membrane permeability to K, followed by a depolarization due to an increase in membrane permeability to Na.

Animals↗
PubMed2006

Theoretical evaluation of a possible nature of the outer membrane potential of mitochondria.

A possibility of generation of the outer membrane potential in mitochondria has been suggested earlier in the literature, but the potential has not been directly measured yet. Even its nature, metabolic impact and a possible range of magnitudes are not clear, and require further theoretical and experimental analysis. Here, using simple mathematical model, we evaluated a possible contribution of the Donnan and metabolically derived potentials to the outer membrane potential, concluding that the superposition of both is most probable; exclusively Donnan origin of the potential is doubtful because unrealistically high concentrations of charged macromolecules are needed for maintaining its relatively high levels. Regardless of the mechanism(s) of generation, the maximal possible potential seems to be less than 30 mV because significant osmotic gradients, created at higher values, increase the probability of the outer membrane rupture. New experimental approaches for direct or indirect determination of true value of the outer membrane potential are suggested here to avoid a possible interference of the surface electrical potential of the inner membrane, which may change as a result of the extrusion of matrix protons under energization of mitochondria.

Bacterial Outer Membrane Proteins↗
PubMed1985

Sodium cotransport systems and the membrane potential difference.

Studies with membrane vesicles and with whole cell preparations have shown clearly that the electrochemical gradient of Na+ acting across the cell membrane is closely coupled to the influx and efflux of amino acids or carbohydrates through their cellular pumps. It has been less clear (1) just how tightly solute flow is coupled to that of Na+ in stoichiometrical terms and (2) whether coupling is tight enough to account for the maximum solute gradients that the systems form in vivo. Recent work with ionophores, including nigericin, has revealed circumstances in preparations of mouse ascites-tumor cells where if the sodium gradient hypothesis is correct, electrogenic ion pumping must be supposed to maintain membrane potentials of the order of 80 mV negative. We have used a new fluorescence assay based on an oxonol dye in a search for potentials of that magnitude. Their possible origin is discussed.

Adenosine Triphosphate↗
PubMed1964

INFLUENCE OF SOME IONS ON THE MEMBRANE POTENTIAL OF ASCARIS MUSCLE.

The influence of several ions on the membrane potential of the somatic muscle of Ascaris has been investigated by changing their concentration in the surrounding solution. When [K](o) is increased at the expense of [Na](o) leaving [Cl](o) constant, the membrane potential is first seen to increase. [K](o) higher than 45 mM reduces the membrane potential with a slope of 23 mv for a tenfold change in [K](o). However, when [K](o) is increased keeping [Na](o) and [Cl](o) low and constant, the line relating the membrane potential with log [K](o) has a slope of almost 50 mv. If [Cl](o) is reduced in the absence of external Na, after the [K](o) is increased to 45 mM, the membrane potential decreases with a slope of 59 mv per tenfold change in [Cl](o) in close agreement with the Nernst equation. If Cl(-) is replaced by SO(4) (2-), a depolarization is produced, while chloride replacement by NO(3) (-), Br(-), and I(-) results in a hyperpolarization of the membrane. Removal of the external Na(+) ions increases the average membrane potential by 17 mv.

Animals↗
PubMed1988

Membrane potential and Donnan potential.

The Nernst-Planck-Poisson equations for the potential profile across a membrane are exactly solved without recourse to the assumption of constant field within the membrane. It is assumed that the membrane core of thickness dc is covered by a surface layer of thickness ds in which the membrane-fixed charges are distributed at a uniform density N. The membrane boundary potentials as well as the diffusion potentials contribute to the membrane potential. It is shown that for ds greater or similar 1/k, k being the Debye-Hückel parameter, the potential in the membrane surface layer except in the region very near the membrane/solution boundary is effectively equal to the Donnan potential and that its contribution to the membrane potential becomes dominant as N increases. For low N, on the other hand, the membrane potential arises mostly from the diffusion potential.

Journal Article↗