Electrical potential differences across salt transporting membranes

The electrical potential differences across membranes where active transport of ions occurs has been examined using the formalism of linear non-equilibrium thermodynamics, and can be represented as the arithmetic sum of a resistive term, a term directly dependent on metabolism (i.e. electrogenic) and terms appropriate for describing a diffusion potential. The Hittorf transport number for each ion in the latter terms is the ratio of the partial conductances of the membrane to that ion to the total membrane conductance, and the conductance to an ion consists of the arithmetic sum of conductance of active and passive pathways providing these are independent. The conductances of active transport mechanisms arise from variation of the rate of transport with the electrochemical potentials against which they operate. The electrogenic term arises from imbalance between anion and cation transport. If an ion is transported by an obligatorily electrically neutral exchange for some other ion such transport gives rise to no electrogenic effect. A membrane will transport salt most efficiently if there is no imbalance between anion and cation transport, when it will not be electrogenic, but modest deviations from this condition will not degrade the efficiency of active transport markedly.     

A theory for the membrane potential of living cells

We give an explicit formula for the membrane potential of cells in terms of the intracellular and extracellular ionic concentrations, and derive equations for the ionic currents that flow through channels, exchangers and electrogenic pumps. We demonstrate that the work done by the pumps equals the change in potential energy of the cell, plus the energy lost in downhill ionic fluxes through the channels and exchangers. The theory is illustrated in a simple model of spontaneously active cells in the cardiac pacemaker. The model predicts the experimentally observed intracellular ionic concentration of potassium, calcium and sodium. Likewise, the shapes of the simulated action potential and five membrane currents are in good agreement with experiment. We do not see any drift in the values of the concentrations in a long time simulation, and we obtain the same asymptotic values when starting from the full equilibrium situation with equal intracellular and extracellular ionic concentrations. Received: 9 December 1998 / Revised version: 30 August 1999 / Accepted: 15 October 1999     

A model study of the contribution of active Na-K transport to membrane repolarization in cardiac cells

A biochemical model of active Na-K transport in cardiac cells was studied in conjunction with a representation of the passive membrane currents and ion concentration changes. The active transport model is based on the thermodynamic and kinetic properties of a six-step reaction scheme for the Na,K-ATPase. It has a fixed Na:K stoechiometry of 3:2, and its activation is governed by three parameters: membrane potential intracellular Na+ concentration, and interstitial K+ concentration. The Na-K pump current is directly proportional to the density of Na,K-ATPase molecules. The passive membrane currents and ion concentration changes involve only Na+ and K+ ions, and no attempt was made to provide a precise representation of Ca2+ currents or Ca2+ concentration changes. The surface-to-volume ratio of the interstitial compartment is 55 times larger than that of the intracellular compartment. The flux balance conditions are such that the original equilibrium concentration values are re-established at each stimulation cycle. The underlying assumptions of the model were checked against experimental measurements on Na-K pump activity in a variety of preparations. In addition, the qualitative validation of the model was carried out by comparing its behavior following sudden frequency shifts to corresponding experimental observations. The overall behavior of the model is quite satisfactory and it is used to provide the following indications: (1) when the intracellular and interstitial volumes are relatively large, the ion concentration transients are small and the pumping rate depends essentially on average concentration levels. (2) An increase in internal Na+ concentration potentiates the response of the Na-K pump to rapid membrane depolarizations. (3) When the internal Na+ concentration is large enough, the Na-K pump current transient plays an important role in shaping the plateau and repolarization phase of the action potential. (4) A rapid increase in external K+ concentration during voltage clamp in multicellular preparations could saturate the Na-K pump response and lead to a fairly linear dependence of the pump activity on the internal Na+ concentration.     

Some electrophysiological consequences of electrogenic sodium and potassium transport in cardiac muscle: a theoretical study

A previous paper described a kinetic model for electrogenic sodium-potassium transport in cardiac muscle, combining a thermodynamically-constrained transport model with simple passive permeabilities for sodium and potassium to generate a cardiac action potential (Chapman, Kootsey & Johnson, 1979). The present paper explores the extent to which this simplest of active-passive transport models can account (without further modification) for the electrophysiological behavior of cardiac muscle. The long term (several minutes) changes in the duration of the action potential observed following a change in stimulation rate are predicted by the model through a shift in the steady-state current-voltage relationship caused by small changes in inside ion concentrations. The diastolic hyperpolarization observed following an increase in rate is also predicted, including the linear relationship between the maximum diastolic depolarization and the rate of stimulation. Varying the outside potassium concentration in the model produces changes in the rest potential and current-voltage relationship similar to published data. Deviations from ideal potassium electrode behavior occur at both high and low concentrations because of effects on the pump. The model not only predicts the observed shift of the current-voltage curve in the depolarizing direction with increasing [K⁺]₀, but also the crossing of the curve in normal [K ⁺]₀ without having to assume a variation in g_K. Anoxia was introduced into the model by changing the concentrations of ATP and ADP, thereby enabling the model to account for the rapid diastolic depolarization observed in myocardial ischemia.     

Electrogenic property of Na +, K+ -ATPase through computer simulation

A computer simulation of the electrogenic nature of the membrane-boundNa⁺, K⁺-ATPase is presented. The model involves coupling twosimulation systems for passive and active transports, usinga minimum of empirical parameters, and studies the contributionof the pump to the membrane potential. The simulation resultsindicate that electrogenic active transport accelerates therestoration of the resting electrochemical gradients and contributes0.44–1.1 mV to the resting potential of the membrane,depending on the Na:K coupling ratio. The effect of membranepotential and the physical positioning of the enzyme from thepassive transporting channel on the enzyme function is alsopresented. The validity of the model is checked by comparingour results with reported literature values.     

Physiological implications of K accumulation in heart muscle

K+-selective microelectrodes in conjugation with the voltage clamp technique were used to examine the voltage and time dependence of K+ efflux and accumulation in cardiac muscle. K+ efflux per action potential is about 10 to 30 pmoles/cm2 per sec. Accumulation of K+ in the paracellular space plays an important role in regulation of action potential duration, so that the [K+]o prior to generation of an action potential determines the duration of following action potential. This regulation is brought about by the shift of inward rectifying K+ current along the voltage axis, so at higher [K+]o there is more outward current at plateau potentials. Monitoring [K+]o after a period of rapid beating provides quantitative data regarding Na-pump activity. The data suggest the Na-pump is electrogenic, making it difficult to assess the extent of K+ accumulation from the measurements of resting potential alone. These studies indicate that changes in [K+]o not only reflect outward membrane currents and Na-pump activity, but also play an important physiological regulatory role in determining the duration of the action potential.     

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.     

Direct effects on the membrane potential due to "pumps" that transfer no net charge

The effects of active ionic transport are included in the derivation of a general expression for the zero current membrane potential. It is demonstrated that an active transport system that transfers no net charge (nonrheogenic) may, nevertheless, directly alter the membrane potential. This effect depends upon the exchange of matter within the membrane between the active and passive diffusion regimes. Furthermore, in the presence of such exchange, the transmembrane active fluxes measured by the usual techniques and the local pumped fluxes are not identical. Several common uses of the term “electrogenic pump” are thus shown to be inconsistent with each other. These inconsistencies persist when the derivation is extended to produce a Goldman equation modified to account for active transport; however, that equation is shown to be limited by less narrow constraints on membrane heterogeneity and internal electric field than those previously required. In particular, it is applicable to idealized mosaic membranes limited by these requirements.     

Evidence for an electrogenic ion transport pump in cells of higher plants

Summary Cyanide (CN) and dinitrophenol (DNP) rapidly depolarize the cells of oat coleoptiles (Avena sativa L., cultivar Victory) and of pea epicotyls (Pisum sativum L., cultivar Alaska); the effect is reversible. This indicates that electrogenesis is metabolic in origin, and, since active transport is blocked in the presence of CN and DNP, perhaps caused by interference with ATP synthesis, that development of cell potential may be associated with active ion transport. Additional evidence for an electrogenic pump is as follows. (1) Cell electropotentials are higher than can be accounted for by ionic diffusion. (2) Inhibition of potential, respiration, andactive ion transport is nearly maximal, but a potential of –40 to –80 mV remains. This is probably a passive diffusion potential since, under these conditions, a fairly close fit to the Goldman constant-field equation is found in oat coleoptile cells.     

A K<Superscript>+</Superscript>/H<Superscript>+</Superscript> P-ATPase transport in the accessory cell membrane of the blowfly taste chemosensilla sustains the transepithelial potential

An electrogenic K(+) transport in the tormogen cell of insect chemosensilla is involved in the generation and maintenance of the transepithelial potential (TEP). To gain more information about the K(+) transport system underlying the TEP generation and the location of its components in the plasma membrane of the tormogen cell, we studied the effects of inhibitors of K(+)/H(+) P-ATPase (bafilomycin A1, omeprazole and Na-orthovanadate), of K(+)/Cl(-) co-transport (bumetanide), of Cl(-) channels (NPPB) and of a K(+) channel blocker (BaCl(2)). The relationship between TEP amplitude and spike firing activity was also studied. Experiments were performed on the labellar chemosensilla of the blowfly Protophormia terraenovae using a modified tip-recording technique. Results show that: (a) K(+)/H(+) P-ATPase inhibitors significantly decrease the TEP, when properly applied to the labellum for 20 min, so as to reach the basolateral side of the plasma membrane, while no effect was detected when applied to the apical side, (b) bumetanide, NPPB and BaCl(2) decrease the TEP value only when administered to the apical side, (c) spike activity is positively correlated with the TEP. A model is proposed of the active and passive K(+) transports sustaining the TEP associated with the blowfly chemosensilla.     

Electrogenic ion transport by Na+,K+-ATPase

Experimental data on the ion electrogenic transport by Na+,K+-ATPase available in the literature are analyzed. Special attention is paid to the measurements of unsteady-state electric currents initiated by alternating voltage or rapid introduction of the substrate. In the final part, a physical model of the Na+,K+-ATPase functioning is discussed. According to this model, active transport is carried out by opening and closing of the access channels used for the sodium and potassium exchange between solutions on either side of the membrane. The model explains most of the experimental data, although some details (the channel size, rates of individual transport steps) need further refinement.     

Zebrafish heart as a model for human cardiac electrophysiology

The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.     

Ion transport by rabbit colon. I. Active and passive components.

Descending rabbit colon, stripped of muscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO3 secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit current ISC is determined solely by the rate of active Na transport and that the net movements of Cl and HCO3 are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO3 secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxes Jism of Na, K and Cl suggest that JNasm,JIsm and one-third of JCl-sm may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such that Pk:PNa:PCl= 1.0:0.07:0.11. Electrolyte transport by in vitro rabbit colon closely resembles that reported from in vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Electrical signals in higher plants: Mechanisms of generation and propagation

Local stimulation induces generation and propagation of electric signals in higher plants. Noninvasive stimulus induces an action potential and damaging influences lead to the variation potential. The mechanism of the generation of an action potential is rather complex in nature and is associated with both activation of ion channels (Ca²⁺, Cl^–, and K⁺) and transient change in the activity of the plasma membrane H⁺-ATPase. Generation of the variation potential, the duration of which is considerably longer than that of the action potential, is based on transient inactivation of the electrogenic pump; however, passive ion fluxes also contribute to such process, which causes qualitative similarity of the mechanisms of action potential and variation potential generation. Propagation of electrical signals mainly occurs in conducting bundles; thus, transfer of an action potential is associated with vascular parenchyma and sieve elements, while the variation potential is connected to the xylem vessels. The mechanism of the distribution the action potential is similar to nerve impulse transmission, while generation of the variation potential is induced by transfer of a chemical substance, whose propagation is accelerated by a hydraulic wave.     

The electrogenic sodium pump activity in Aplysia neurons is not potential dependent

We have investigated the potential dependence of the electrogenic sodium pump in Aplysia neurons by recording the potential and current induced by sudden change of the artificial sea water from one containing K+ at various concentrations to K+ -free sea water in the presence or absence of ouabain. Both K+ free sea water and ouabain block sodium transport and result in a significant depolarization due to removal of a maintained outward current that is a result of transport of more Na+ out of the cell than K+ into the cell during pump operation. In the presence of ouabain there is, however, an inward current induced by changing external K+ concentration from zero to some value between 1 and 20 mM, and this current is greater with a greater K+ concentration gradient. The current induced by change from zero to 1 mM K+ does not show any potential dependence, although those currents induced by higher K+ concentrations are potential dependent. We conclude that the activity of the electrogenic sodium pump is not potential dependent, but that the potential independence is obscured if higher concentrations of K+ are used to activate the electrogenic sodium pump.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

Energy coupling for membrane hyperpolarization in Lemna: Evidence against an ATP-fueled electrogenic pump as the exclusive mechanism

The vacuolar electrical potential of Lemna paucicostata 6746 has an active component of about-130 mV. This hyperpolarization above the diffusion potential was maintained when dicyclohexyl carbodiimide (DCCD) or arsenate (0.1 mM or 5 mM final concentrations, respectively) were added in the light or after the plants had been kept in darkness for 1 h. The ATP level was reduced to 11±3% by DCCD and to 56±6% by arsenate under conditions identical to those during the potential measurements. In this report, it is discussed whether these results could be interpreted in terms of a putative electrogenic ATPase in the plasma membrane of Lemna. Rb⁺-influx in illuminated plants was 12.5% or 52% of the control when ATP generation was inhibited by DCCD or arsenate. This finding is regarded as justifying the assumption that the availability of ATP at plasmalemma-located transport sites is drastically decreased by these inhibitors.A passive proton-permeability in the cell membrane was induced with different concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The potential decrease, caused by the current through this shunt, was not affected by DCCD. It therefore seems less conceivable that the cell membrane remains hyperpolarized because of an increase of membrane resistance concomitant to the inhibition of the pump.The significance of respiratory processes for membrane hyperpolarization is displayed by the depolarizing action of anoxia or KCN. As ATP was found to be non-limiting under these conditions, the inhibition of the electrogenic pump is regarded as being in discord with the concept of an electrogenic ATPase, which is solely responsible for membrane hyperpolarization.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N, N-dicyclohexyl carbodiimide - DES diethylstilbestro - DNP 2,4-dinitrophenol - POPOP 1,4-bis (2-(5-phenyloxazolyl))-benzene - PPO 2,5-diphenyloxazole     

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.     

Time-dependent extracellular ion transport

The theory of the time-dependent behaviour of the extracellular ion transport processes associated with electric currents of biologic origin is developed. It is shown that the time course of the ratio of extracellular electric potential gradient to current density, following initiation or shutdown of electrogenic membrane transport, should provide a sensitive test for the occurrence of proton or hydroxyl transport.     

The Effect of Intracellular Current Pulses in Smooth Muscle Cells of the Guinea Pig Vas Deferens at Rest and during Transmission

The effect of intracellular current pulses on the membrane of smooth muscle cells of the guinea pig vas deferens at rest and during transmission was studied. Two main response types were identified: active response cells, in which a spike was initiated in response to depolarizing currents of sufficient strength and duration; passive response cells, in which depolarizing currents gave only electrotonic potential changes. These cells were three times more numerous than the active response cells. During the crest of the active response the input resistance fell by about 25% of the resting value. Comparison of the active response with the action potential due to stimulating the hypogastric nerve showed that the former was smaller in amplitude and had a slower rate of rise and higher threshold. Electrical coupling occurred between the smooth muscle cells during the propagation of the action potential. Depolarizing current pulses had no effect on the amplitude of the excitatory junction potential (E.J.P.) in passive response cells, but in general did decrease its amplitude in active response cells. These results are discussed with respect to the mechanism of autonomic neuroeffector transmission.