Microelectrode studies in toad urinary bladder epithelium. effects of Na concentration changes in the mucosal solution on equivalent electromotive forces

Microelectrode techniques were employed to measure membrane potentials, the electrical resistance of the cell membranes, and the shunt pathway, and to compute the equivalent electromotive forces (EMF) at both cell borders in toad urinary bladder epithelium before and after reductions in mucosal sodium concentration. Basal electrical parameters were not significantly different from those obtained with impalements from the serosal side, indicating that mucosal impalements do not produce significant leaks in the apical membrane. A decrease in mucosal Na concentration caused the cellular resistance to increase and both apical and basolateral EMF to depolarize. When Na was reduced from 112 to 2.4 mM in bladders with spontaneously different baseline values of transepithelial potential difference (Vms), a direct relationship was found between the change in Vms brought about by the Na reduction and the base-line Vms before the change. A direct relationship was also found by plotting the change in EMF at the apical or basolateral border caused by a mucosal Na reduction with the corresponding base-line EMF before the change. These results indicate that resting apical membrane EMF (and, therefore, resting apical membrane potential) is determined by the Na selectivity of the apical membrane, whereas basolateral EMF is at least in part the result of rheogenic Na transport. These results are consistent with data of others that suggested a link between the activity of the basolateral Na pump and apical Na conductance.     

Effect of pentachlorophenol (PCP) on frog cornea epithelium

Pentachlorophenol (PCP) is a toxic substance that affects many tissues adversely. Present experiments, using an in vitro preparation, were designed to study whether PCP affected the electrophysiological parameters of the bullfrog cornea epithelium, specifically, the Na+/K+ ATPase pump and the K+ conductance located in the basolateral membrane and the Cl- conductance located in the apical membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (Isc) conditions. Addition of PCP to a concentration of 5 x 10-5 M to the tear solution gave a marked decrease in Isc; a marked depolarization of the intracellular potential, Vo; and minimal but significant decreases in the apical membrane fractional resistance, fRo, and in the transepithelial conductance, gt. Isc experiments in Cl--free solutions with amphotericin B in the tear solution confirm results indicating that PCP inhibits the active transepithelial transport mechanism and produces a small increase in the basolateral membrane resistance due to a decrease in the K+ conductance.     

Amphotericin B enhanced anomalous potential difference response to changes in aqueous K+ in frog cornea

An increase in aqueous K+ from 0 to 4 mM increased the potential difference (anomalous response of electrogenic (Na+ + K+)-ATPase antiport) by 1.1 mV in Cl(-)-free solutions compared to 6.8 mV in Cl- solutions. With amphotericin B added to the tear solution in Cl(-)-free solutions, the anomalous PD response for the addition of 4 mM K+ to the aqueous solution was about 20 mV, significantly greater than in Cl- solutions. This anomalous response was inhibited by ouabain. These data support the electrogenicity of the (Na+ + K+)-ATPase pump. It is also evident that, for the pump to respond, Na+ should readily enter the cell. This may be accomplished experimentally, either across the basolateral membrane in Cl- solutions or across the apical membrane in Cl(-)-free solutions with amphotericin B present in the tear solution.     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.     

Pseudo-streaming potentials in Necturus gallbladder epithelium. I. Paracellular origin of the transepithelial voltage changes

Apparent streaming potentials were elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution. In NaCl Ringer's solution, the transepithelial voltage (Vms) change (reference, basolateral solution) was positive with sucrose addition and negative with sucrose removal. Bilateral Cl- removal (cyclamate replacement) had no effect on the polarity or magnitude of the Vms change elicited by addition of 100 mM sucrose. In contrast, bilateral Na+ removal (tetramethylammonium [TMA+] replacement) inverted the Vms change (from 2.7 +/- 0.3 to -3.2 +/- 0.2 mV). Replacement of Na+ and Cl- with TMA+ and cyclamate, respectively, abolished the change in Vms. Measurements of cell membrane voltages and relative resistances during osmotic challenges indicate that changes in cell membrane parameters do not explain the transepithelial voltage changes. The initial changes in Vms were slower than expected from concomitant estimates of the time course of sucrose concentration (and hence osmolality) at the membrane surface. Paired recordings of the time courses of paracellular bi-ionic potentials (partial substitution of apical Na+ with tetrabutylammonium [TBA+]) revealed much faster time courses than those produced by sucrose addition, although the diffusion coefficients of sucrose and TBACl are similar. Hyperosmotic and hypoosmotic challenges yielded initial Vms changes at the same rate; thereafter, the voltage increased with hypoosmotic solution and decreased with hyperosmotic solution. These late voltage changes appear to result from changes in width of the lateral intercellular spaces. The early time courses of the Vms changes produced by osmotic challenge are inconsistent with the expectations for water-ion flux coupling in the junctions. We propose that they are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow.     

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.     

Intracellular K+ activities in Aplysia gut: effects of transport inhibitors on the Na+ pump

Na+ absorption by the Aplysia californica foregut is affected through an active Na+ transport mechanism located in the basolateral membrane of the epithelial absorptive cells. Since Cl- absorption by the Aplysia gut has been shown to be very different from that demonstrated in vertebrate gut, the present study was undertaken to discern if Na+ transport was also different from that observed in vertebrate preparations. Utilizing microelectrode technique, it was demonstrated that intracellular K+ activity is above electrochemical equilibrium in the Aplysia absorptive cells and that serosal ouabain, Ba2+ or Cd2+ abolished this asymmetry in K+ electrochemical potential. Neither bumetanide nor furosemide had any effect on intracellular K+ activities, mucosal membrane potentials or transepithelial potentials in the Aplysia gut preparation. These results are consistent with the operation of a basolateral Na+/K+ pump.     

Rheogenic transport in the renal proximal tubule

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.     

Voltage dependent membrane conductances in cultured renal distal cells.

Cultured Na(+)-transporting epithelia from amphibian renal distal tubule (A6) were impaled with microelectrodes and analyzed at short-circuit and after transepithelial voltage perturbation to evaluate the influence of voltage on apical and basolateral membrane conductances. For equivalent circuit analysis, amiloride was applied at each setting of transepithelial potential. At short-circuit, apical and basolateral membrane conductances averaged 88 and 497 microS/cm2, respectively (n = 10). Apical membrane conductance, essentially due to Na(+)-specific pathways, decreased after depolarization of the apical membrane. The drop was considerably larger than predicted by the Goldman-Hodgkin-Katz (GHK) constant-field equation. This suggests decrease in permeability of the apical Na+ channels upon depolarization. Basolateral membrane conductance, preferentially determined by K+ channels, increased after hyperpolarization of the basolateral membrane. This behavior is contrary to the prediction of the GHK constant field equation and reflects inward rectification of the K+ channels. The observed rectification patterns can be valuable for maintenance of cellular homeostasis.     

The cellular mechanism of active chloride secretion in vertebrate epithelia: studies in intestine and trachea

The cellular mechanism of active chloride secretion, as it is manifested in the intestine and trachea, appears to possess the following elements: (1)NaCl cl-transport across the basolateral membrane; (2) Cl- accumulation in the cell above electrochemical equilibrium due to the Na+ gradient; (3) a basolateral Na+-K+ pump that maintains the Na+ gradient; (4) a hormone-regulated Cl- permeability in the apical membrane; (5) passive Na/ secretion through a paracellular route, driven by the transepithelial potential difference; and (6) an increase in basolateral membrane K+ permeability occurring in conjunction with an increase in Na+-K+ pump rate. Electrophysiological studies in canine trachea support this model. Adrenalin, a potent secretory stimulus in that tissue, increases apical membrane conductance through a selective increase in Cl- permeability. Adrenalin also appears to increase basolateral membrane K+ permeability. Whether or not adrenalin also increases paracellular Na+ permeability is unclear. Some of the testable implications of the above secretion model are discussed.     

Potassium secretion by the cortical collecting tubule

The isolated perfused rabbit cortical collecting tubule has been shown to actively transport K from bath to lumen. The first step in this process is active uptake of K across the basolateral membrane via and Na:K exchange pump as evidenced by: 1) basolateral localization and Na:K exchange properties of the ouabain-sensitive Na,K-ATPase, 2) ouabain sensitivity of the Na and K fluxes, 3) interdependence of the Na and K fluxes, and 4) ouabain-sensitivity of 42K uptake into the cell across the basolateral membrane. At the luminal border, a significant K permeability of the apical cell membrane has been identified using electrophysiological techniques. This K permeability is insensitive to the diuretic amiloride, and, thus, differs from the pathway for Na entry, which is highly amiloride sensitive. A significant K permeability of the paracellular pathway is not apparent. It is concluded that K secretion by the rabbit cortical collecting tubule occurs via a two-step process: active uptake of K across the basolateral membrane via the Na:K exchange pump, followed by passive efflux of K across the apical membrane via an amiloride-insensitive K conductive pathway.     

Electrophysiology of flounder intestinal mucosa. II. Relation of the electrical potential profile to coupled NaCl absorption

We characterized the hyperpolarization of the electrical potential profile of flounder intestinal cells that accompanies inhibition of NaCl cotransport. Several observations indicate that hyperpolarization of psi a and psi b (delta psi a,b) results from inhibition of NaCl entry across the apical membrane: (a) the response was elicited by replacement of mucosal solution Cl or Na by nontransported ions, and (b) mucosal bumetanide or serosal cGMP, inhibitors of NaCl influx, elicited delta psi a,b and decreased the transepithelial potential (psi t) in parallel. Regardless of initial values, psi a and psi b approached the equilibrium potential for K (EK) so that in the steady state following inhibition of NaCl entry, psi a approximately equal to psi b approximately equal to ECl approximately equal to EK. Bumetanide decreased cell Cl activity (aClc) toward equilibrium levels. Bumetanide and cGMP decreased the fractional apical membrane resistance (fRa), increased the slope of the relation of psi a to [K]m, and decreased cellular conductance (Gc) by approximately 85%, which indicates a marked increase in basolateral membrane conductance (Gb). Since the basolateral membrane normally shows a high conductance to Cl, a direct relation between apical salt entry and GClb is suggested by these findings. As judged by the response to bumetanide or ion replacement in the presence of mucosal Ba, inhibition of Na/K/Cl co-transport alone is not sufficient to elicit delta psi a,b. This suggests the presence of a parallel NaCl co-transport mechanism that may be activated when Na/K/Cl co-transport is compromised. The delta psi a,b response to reduced apical NaCl entry would assist in maintaining the driving force for Na-coupled amino acid uptake across the apical membrane as luminal [NaCl] falls during absorption.     

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.     

Cl Secretagogues Reduce Basolateral K Permeability in the Rabbit Corneal Epithelium

The stromal-to-tear transport of Cl by the rabbit corneal epithelium is increased by pharmacological effectors (secretagogues) that raise cAMP. It is well established that such secretagogues increase the apical membrane permeability to Cl and thus facilitate the efflux of the anion. However, we and others have found that cAMP-elevating agents frequently decrease the transepithelial potential difference across the rabbit cornea. The mechanism underlying this latter phenomenon had not been characterized. In this report, transepithelial and microelectrode studies were combined with measurements of unidirectional fluxes of 36Cl, 22Na and 86Rb to show that secretagogues known to act via cAMP also decrease the K permeability of the basolateral membrane, which by cellular depolarization would decrease apical Cl secretion. This effect was increasingly pronounced as a function of concentration when agents (e.g., epinephrine, isoproterenol) were applied to the apical side of the preparations. The addition of these agonists to the basolateral bathing solution, or of forskolin to the apical side, solely elicited inhibitions of basolateral K permeability. It seems that apical Cl and basolateral K conductances are independently and inversely regulated by cAMP. The opposite effects that cAMP could have on fluid secretion and epithelial thickness, by increasing apical Cl permeability but decreasing basolateral K permeability, may serve as a mechanism to maintain epithelial thickness within a narrow range.     

An analytical model of ionic movements in airway epithelial cells.

A new mathematical model of ion movements in airway epithelia is presented, which allows predictions of ion fluxes, membrane potentials and ion concentrations. The model includes sodium and chloride channels in the apical membrane, a Na/K pump and a cotransport system for Cl- with stoichiometry Na+:K+:2Cl- in the basolateral membrane. Potassium channels in the basolateral membrane are used to regulate cell volume. Membrane potentials, ion fluxes and intracellular ion concentration are calculated as functions of apical ion permeabilities, the maximum pump current and the cotransport parameters. The major predictions of the model are: (1) Cl- concentration in the cell is determined entirely by the intracellular concentration of negatively charged impermeable ions and the osmotic conditions; (2) changes in intracellular Na+ and K+ concentrations are inversely related; (3) cotransport provides the major driving force for Cl- flux, increases intracellular Na+ concentration, decreases intracellular K+ concentration and hyperpolarizes the cell interior; (4) the maximum rate of the Na/K pump, by contrast, has little effect on Na+ or Cl- transepithelial fluxes and a much less pronounced effect on cell membrane polarization; (5) an increase in apical Na+ permeability causes an increase in intracellular Na+ concentration and a significant increase in Na+ flux; (6) an increase in apical Cl- permeability decreases intracellular Na+ concentration and Na+ flux; (7) assuming Na+ and Cl- permeabilities equal to those measured in human nasal epithelia, the model predicts that under short circuit conditions, Na+ absorption is much higher than Cl- secretion, in agreement with experimental measurements.     

Ion transport across the epithelium of the rabbit caecum.

The isolated rabbit caecum was studied in vitro. Under our experimental conditions, the rabbit caecum secreted potassium and chloride and absorbed sodium. To characterize the transport properties of the apical and the basolateral barriers, transepithelial electrical and flux (22Na, 36Cl and 86Rb) measurements and their sensitivity to transport inhibitors (furosemide, DIDS, ouabain and barium) are presented together with intracellular measurements with double-barrelled microelectrodes of intracellular electrical potentials and ionic activities. The fluxes of sodium and chloride were insensitive to DIDS and furosemide. The secretion of potassium and the absorption of sodium were both inhibited by ouabain, indicating that they are coupled through the sodium pump. Ouabain induced a slow fall in the chloride net fluxes, suggesting that these fluxes are also driven by the sodium pump, albeit indirectly. The basolateral to apical fluxes of potassium are insensitive to barium added to the apical side, but are accelerated by the replacement of chloride by gluconate on the apical side, suggesting the presence of a K+/Cl- symport in the apical barrier.     

Effects of ouabain on fluid transport and electrical properties of Necturus gallbladder. Evidence in favor of a neutral basolateral sodium transport mechanism

Net fluid transport (Jv) and electrical properties of the cell membranes and paracellular pathway of Necturus gallbladder epithelium were studied before and after the addition of ouabain (10(-4) M) to the serosal bathing medium. The glycoside inhibited Jv by 70% in 15 min and by 100% in 30 min. In contrast, the potentials across both cell membranes did not decrease significantly until 20 min of exposure to ouabain. At 30 min, the basolateral membrane potential (Vcs) fell only by ca 7 mV. If basolateral Na transport were electrogenic, with a coupling ratio (Na:K) of 3:2, the reductions of Vcs at 15 and 30 min should be 12--15 and 17--21 mV, respectively. Thus, we conclude that the mechanism of Na transport from the cells to the serosal bathing solution is not electrogenic under normal transport conditions. The slow depolarization observed in ouabain is caused by a fall of intracellular K concentration, and by a decrease in basolateral cell membrane K permeability. Prolonged exposure to ouabain results also in an increase in paracellular K selectivity, with no change of P Na/P Cl.     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes ofNecturus small intestine

Intracellular electrical potentials and K activity, (K)c, were determined simultaneously in Necturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (psi mc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of psi mc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine. In the absence of alanine, psi mc averaged -32 mV and (K)c averaged 67 mM. When a steady state was achieved in the presence of alanine these values averaged -24 mV and 50 mM, respectively. The steady-state electrochemical potential differences for K across the basolateral membrane in the absence and presence of alanine did not differ significantly. Inasmuch as the rate of transcellular active Na transport or "pump activity" was increased two- to threefold in the presence of alanine, it follows that, if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease in rs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in "pump activity". This "homocellular" regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and, (ii) repolarize psi mc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.     

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.