The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Regulation of cytosolic sodium ion activity in frog sartorius

To explore the regulation of cytosolic sodium ion activity in the frog sartorius, we used Na(+)-selective microelectrodes to monitor intracellular sodium ion activity in situations of lowering external sodium concentration and elevating external potassium concentration. Reductions of 20%, 40%, 60% and 80% in extracellular sodium concentration produced slight but statistically insignificant changes in the membrane potential of the muscle. However, cytosolic sodium ion activity decreased significantly from 10.0 +/- 1.1 mM to 7.8 +/- 1.1 mM, 7.1 +/- 1.4 mM, 6.5 +/- 1.2 mM and 5.9 +/- 1.1 mM, respectively. In addition, elevation of the external potassium concentration from 2 mM to 12 mM, 32 mM and 62 mM caused respective stepwise depolarization of membrane potential from -87.2 +/- 1.6 mV to -62.4 +/- 3.6 mV, -45.4 +/- 3.0 mV, -27.2 +/- 1.8 mV. Under these conditions, the cytosolic sodium ion activity decreased from 10.5 +/- 1.4 mM to 7.3 +/- 1.6 mM, 6.4 +/- 1.1 mM and 5.2 +/- 0.8 mM, respectively. The results illustrate that the net sodium flux is out of cell either in the reduction of sodium chemical gradient or in the potassium depolarization across the cell membrane.     

Membrane potential of primitive red cells from chick embryo is a proton potential

The membrane potential of primitive red cells from 4- and 6-day old chick embryos has been determined using the fluorescent dye Dis-C3-(5). At day 4 the membrane potential Em was -44 mV for pH 7.4 and 20 degrees C and -36 mV at day 6. Both values are far removed from the equilibrium potential for chloride, which is about -14 mV at day 6. Changes in the external potassium, sodium or chloride concentration were without effect on the membrane potential, except at very high potassium concentrations, where a small but significant depolarization was observed at day 6. The measurements gave the same results in the absence or presence of the anion exchange blocking agent DIDS. Three pieces of evidence indicate that the membrane potential of primitive red cells is primarily caused by an electrogenic H+ conductance: 1) The measured membrane potential of -36 mV at day 6 is close to the previously determined proton equilibrium potential (Baumann and Haller, 1983) EH + of -36 mV. 2) Addition of the electrosilent Cl-/OH- exchanger tributyltin causes a significant depolarization of about 20 mV at day 4 and about 14 mV at day 6. 3) Measurement of hydrogen ion fluxes demonstrate a potential dependent proton conductance, which increases with depolarization. These results indicate that large qualitative differences exist with regard to the mechanisms involved in the generation of membrane potential and hydrogen distribution between red cell and plasma of embryonic and adult chicken.     

Explanation for constancy of intracellular chloride concentration with respect to membrane potential in guinea pig mammary gland.

1. The relative permeabilities for sodium, potassium and chloride in guinea pig mammary gland slices are determined by means of ion flux studies with radioisotopes. 2. Assuming that there are no significant electrogenic potential components, we calculate permeability ratios PNa/PK = 0.97 and Pc1/PK = 1.25. 3. Substitution of these values in the Goldman equation yields membrane potentials of--15 mV before and--13 mV after ouabain treatment. 4. This small change in membrane potential explains the absence of a significant change in chloride content upon ouabain application, which leads to large changes in intracellular sodium and potassium concentrations.     

Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of Ambystoma oocytes

A voltage-gated hydrogen ion-selective conductance has been previously described in the immature oocyte of the urodele amphibian Ambystoma. The present study was prompted by reports that changes in membrane voltage and internal pH, as well as in internal sodium ion concentration, occur during the hormone-induced maturation of oocytes from other amphibians. As activation of membrane currents might mediate changes in internal ion concentrations in addition to altering the membrane voltage, microelectrode recording techniques have been employed to examine changes in membrane conductances which occur during maturation of Ambystoma oocytes. It was observed that during the first 5 hr of maturation the magnitude of the hydrogen ion conductance gradually decreased, and that subsequently there was an increase in the amplitude of a voltage-dependent noninactivating sodium conductance. After 6 to 7 hr, after the loss of the hydrogen conductance and at about the time of germinal vesicle breakdown, the resting potential of the oocyte spontaneously shifted from approximately -10 mV to approximately +30 mV, where it remained until at least 24 hr after the initiation of maturation. This voltage transition was due to the appearance of mechanisms generating inward current in the oocyte membrane; part of this inward current was due to the tonic activation of the sodium conductance. Changes in internal pH and internal sodium ion concentration occurred during maturation, as judged from shifts in the reversal potentials of both hydrogen and sodium currents. A gradual decrease in internal hydrogen ion concentration was observed up until the time of disappearance of the hydrogen conductance (change in internal pH from about 7.15 in immature oocytes to about 7.40 by 3 hr after application of progesterone). This was followed, as sodium conductance increased, by an apparent rise in the internal sodium ion concentration (from about 6 mM to about 17 mM by 10 hr postprogesterone).     

Action of External Divalent Ion Reduction on Sodium Movement in the Squid Giant Axon

Voltage clamp measurements of the sodium potential have been made on the resting squid giant axon to study the effect of variations in external divalent ion concentration upon net sodium flux. From these measurements the intracellular sodium concentration and the net sodium inflow were calculated using the Nernst relation and constant activity coefficients. While an axon bathed in artificial sea water shows a slow increase in internal sodium concentration, the rate of sodium accumulation is increased about two times by reducing external calcium and magnesium concentrations to 0.1 times their normal values. The mean inward net sodium flux increases from a mean control value of 97 pmole/cm² sec. to 186 pmole/cm² sec. in low divalent solution. Associated with these effects of external divalent ion reduction are a marked decrease in action potential amplitude, little or no change in resting potential, and a shift along the voltage axis of the curve relating peak sodium conductance to membrane potential similar to that obtained by Frankenhaeuser and Hodgkin (1957). These results implicate divalent ions in long term (minutes to hours) sodium permeability.     

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.     

Potassium uniport and ATP synthesis in Halobacterium halobium.

Light-driven potassium ion uptake in Halobacterium halobium is mediated by bacteriorhodopsin. This uptake is charge-balanced by sodium ions and not by proton release. Light-induced shifts in concentrations of divalent cations were found to be negligible. The transient changes in extracellular pH (alkaline overshoot) can be understood by the concomitant processes of ATP synthesis, proton/sodium exchange and potassium uptake. The driving force of potassium ion uptake is the membrane potential, no ATP-dependent potassium transport process is found. Fluorescence measurements indicate a high permeability of the membrane to potassium ions compared to sodium ions. Therefore the potassium ion diffusion potential contributes to the membrane potential (about 30 mV/decade) and thereby influences the ATP level. Sudden enhancement of the diffusion potential by the potassium ionophore monactin leads to the expected transient increase in cellular ATP level. Due to the large size (up to 100-fold) of the potassium ion gradient and its high capacity (intracellular concentration up to 3 M) the potassium ion gradient can well serve the cell as a long term storage form of energy.     

Dipole moment changes and voltage dependent membrane capacity of squid axon

Changes in the membrane capacity of squid axons during hyper- and depolarizations are measured between ?160 and +40 mV. After corrections for the series resistance and fringe effect, we found that the membrane capacity increased from 0.68 to 1.2 μF/cm² with depolarization. It was further observed that tetrodotoxin in the external medium eliminated the change in membrane capacity without affecting the conductivity. The voltage-dependent membrane conductivity is, in turn, greatly reduced by the internal cesium ion. These observations clearly indicate that the voltage-dependent membrane capacity and conductivity are closely related to ionic channels. Particularly, the increase in membrane capacity with depolarizations may be due to sodium channels. The change in the dipole moment associated with sodium sites was determined using values of α_{m and}β_m at various depolarizations. We found, based on voltage clamp measurements, that the increase in the dipole moment of the sodium site between ?40 and ?5 mV is 1230 Debye units (D.U.) and 930 D.U. between ?5 and +60 mV, indicating that the depolarization of sodium channels may consist of two different steps.     

Unidirectional sodium and potassium fluxes through the sodium channel of squid giant axons.

Unidirectional 22Na-traced sodium influx or 42K-traced potassium efflux across the membranes of voltage-clamped squid giant axons was measured at various membrane potentials under bi-ionic conditions. Tetrodotoxin almost entirely eliminated the extra K+ efflux induced by short repetitive depolarizations in the presence of tetraethylammonium or 3,4-diaminopyridine. A method of determining the voltage dependence of the unidirectional flux through voltage-gated channels is described. This technique was used to obtain the unidirectional flux-voltage relation for the sodium channel in bi-ionic and single-ion conditions. It allows the determination of the unidirectional flux at the zero-current potential which, for influx, was found to be approximately 20% of the value measured 80 mV negative to the zero-current potential. The unidirectional flux ratio under bi-ionic conditions was also measured and the flux ratio exponent found to average 1.15 with an external sodium and an internal potassium solution. A three-barrier, two-site, multi-occupancy model previously obtained for other conditions was found to predict a similar non-unity average for the flux ratio exponent. It is also shown that some single-occupancy models can predict non-unity values for the flux ratio exponent in bi-ionic conditions.     

Membrane potential of the unfertilized sea urchin egg

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm²· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (?70 mV) and specific resistance (420 kΩ · cm²). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were ?75 ± 3 mV and 380 ± kΩ · cm².     

Ionic Blockage of Sodium Channels in Nerve

Increasing the hydrogen ion concentration of the bathing medium reversibly depresses the sodium permeability of voltage-clamped frog nerves. The depression depends on membrane voltage: changing from pH 7 to pH 5 causes a 60% reduction in sodium permeability at +20 mV, but only a 20% reduction at +180 mV. This voltage-dependent block of sodium channels by hydrogen ions is explained by assuming that hydrogen ions enter the open sodium channel and bind there, preventing sodium ion passage. The voltage dependence arises because the binding site is assumed to lie far enough across the membrane for bound ions to be affected by part of the potential difference across the membrane. Equations are derived for the general case where the blocking ion enters the channel from either side of the membrane. For H⁺ ion blockage, a simpler model, in which H⁺ enters the channel only from the bathing medium, is found to be sufficient. The dissociation constant of H⁺ ions from the channel site, 3.9 x 10^-6 M (pK_a 5.4), is like that of a carboxylic acid. From the voltage dependence of the block, this acid site is about one-quarter of the way across the membrane potential from the outside. In addition to blocking as described by the model, hydrogen ions also shift the responses of sodium channel "gates" to voltage, probably by altering the surface potential of the nerve. Evidence for voltage-dependent blockage by calcium ions is also presented.     

Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.     

Conformational studies of Escherichia coli pyruvate oxidase

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3'-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from -51 mV to -36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using 86Rb+ as a radioactive tracer for K+, demonstrated that the depolarization was not caused by changes in (Na+ + K+)-ATPase-mediated K+ influx or in the transmembrane K+ gradient. The permeability ratio (PNa/PK), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of PNa/PK was shown by 24Na+ and 86Rb+ flux measurements to be due to both an increase of the Na+ permeability and a decrease of the K+ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Sodium flux ratio through the amiloride-sensitive entry pathway in frog skin

The sodium flux ratio of the amiloride-sensitive Na+ channel in the apical membrane of in vitro Rana catesbeiana skin has been evaluated at different sodium concentrations and membrane potentials in sulfate Ringer solution. Amiloride-sensitive unidirectional influxes and effluxes were determined as the difference between bidirectional 22Na and 24Na fluxes simultaneously measured in the absence and presence of 10(-4) M amiloride in the external bathing solution. Amiloride- sensitive Na+ effluxes were induced by incorporation of cation- selective ionophores (amphotericin B or nystatin) into the normally Na+- impermeable basolateral membrane. Apical membrane potentials (Va) were measured with intracellular microelectrodes. We conclude that since the flux ratio exponent, n', is very close to 1, sodium movement through this channel can be explained by a free-diffusion model in which ions move independently. This result, however, does not necessarily preclude the possibility that this transport channel may contain one or more ion binding sites.     

Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches

We have studied the effect of N-bromoacetamide (NBA) on the behavior of single sodium channel currents in excised patches of rat myotube membrane at 10 degree C. Inward sodium currents were activated by voltage steps from holding potentials of about -100 mV to test potentials of -40 mV. The cytoplasmic-face solution was isotonic CsF. Application of NBA or pronase to the cytoplasmic face of the membrane irreversibly removed sodium channel inactivation, as determined by averaged single-channel records. Teh lifetime of the open channel at - 40 mV was increased about 10-fold by NBA treatment without affecting the amplitude of single-channel currents. A binomial analysis was used both before and after treatment to determine the number of channels within the excised patch. NBA was shown to have little effect on activation kinetics, as determined by an examination of both the rising phase of averaged currents and measurements f the delay between the start of the pulse and the first channel opening. Our data support a kinetic model of sodium channel activation in which the rate constant leading back from the open state to the last closed state is slower than expected from a strict Hodgkin-Huxley model. The data also suggest that the normal open-channel lifetime is primarily determined by the inactivation process in the voltage range we have examined.     

Brownian dynamics study of flux ratios in sodium channels

Measurements of unidirectional fluxes in ion channels provide one of the experimental methods for studying the steps involved in ion permeation in biological pores. Conventionally, the number of ions in the pore is inferred by fitting the ratio of inward and outward currents to an exponential function with an adjustable parameter known as the flux ratio exponent. Here we investigate the relationship between the number of ions in the pore and the flux ratio exponent in a model sodium channel under a range of conditions. Brownian dynamics simulations enable us to count the precise number of ions in the channel and at the same time measure the currents flowing across the pore in both directions. We show here that the values of the flux ratio exponent n′ ranges between 1 and 3 and is highly dependent on the ionic concentrations in which measurements are made. This is a consequence of the fact that both inward and outward currents are susceptible to saturation with increasing concentration. These results indicate that measurements of the flux ratio exponent cannot be directly related to the number of ions in the pore and that interpretation of such experimental measurements requires careful consideration of the conditions in which the study is made.     

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats (SHR-Okamoto strain adult rats) was measured with conventional 3 mol KCl microelectrodes, in vivo. Peritubular cell membrane potential was not different in SHR (-66.5 ± 0.7 mV) as compared with normotensive control Wistar rats (-67.5 ± 1.2 mV). To test the effects of possible altered sodium membrane transport in SHR on proximal tubule peritubular membrane potential, we allowed SHR and control rats to drink 1% NaCl for two weeks. Again, proximal tubule peritubular membrane potential was not different in SHR on 1% NaCl (-67.0 ± 1.0 mV) as compared with control rats on 1% NaCl (-64.7 ± 1.3 mV). From these results we concluded that peritubular membrane potential in kidney proximal tubular cells of SHR was not different from normotensive Wistar control rats, and if some alteration of sodium transport in kidney proximal tubular cells of SHR could exist, that was not possible to evaluate from the measurements of peritubular membrane potential in kidney proximal tubular cells.     

Acetylcholine-induced receptor-controlled ion flux investigated by flow quench techniques

Using a quench flow technique with membrane vesicles, the acetylcholine receptor-controlled transmembrane ion flux and the inactivation of the receptor with acetylcholine were measured in the msec time region. The ion flux was followed by influx of radioactive tracer ion and the inactivation was followed by an ion flux assay of receptor pre-incubated with ligand. The measurements covered a concentration range to complete saturation of the $\text{active}$ state of the receptor with ligand, and were consistent with a minimal model previously proposed on the basis of experiments with carbamylcholine. The ion translocation rate at saturation with acetylcholine is about twice that at saturation with carbamylcholine and this reflects a more favored channel opening equilibrium for acetylcholine.     

Potassium permeability of Rickettsia prowazekii.

The potassium permeability of Rickettsia prowazekii was characterized by chemical measurement of the intracellular sodium and potassium pools and isotopic flux measurements with 86Rb+ as a tracer. R. prowazekii, in contrast to Escherichia coli, did not maintain a high potassium-to-sodium ratio in their cytoplasm except when the potassium-to-sodium ratio in the extracellular medium was high or when the extracellular concentrations of both cations were low (ca. 1 mM). Both influx and efflux assays with 86Rb+ demonstrated that the rickettsial membrane had limited permeability to potassium and that incorporation of valinomycin into these cells increased these fluxes at least 10-fold. The transport of potassium showed specificity and dependence on rickettsial metabolism. The increased flux of potassium which results from the incorporation of valinomycin into the rickettsial membrane was detrimental to both lysine transport and lysis of erythrocytes by the rickettsiae.