Association with {beta}-COP regulates the trafficking of the newly synthesized Na,K-ATPase.

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and ß-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, ß-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase ß-subunit, the Na,K-ATPase α-subunit interacts with ß-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase ß-subunit, the α-subunit does not interact with ß-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and ß-subunits, newly synthesized α-subunit associates with ß-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and ß-subunits to form the pump holoenzyme. The interaction with ß-COP was reduced by mutating a dibasic motif at Lys(54) in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase ß-subunit expression. Although the Lys(54) α-subunit reaches the cell surface without need for ß-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the ß-subunit.

Mouse GLUT9: evidences for a urate uniporter.

GLUT9 (SLC2A9) is a newly described urate transporter whose function, characteristics, and localization have just started to be elucidated. Some transport properties of human GLUT9 have been studied in the Xenopus laevis oocyte expression system, but the type of transport (uniport, coupled transport system, stoichiometry ... .) is still largely unknown. We used the same experimental system to characterize in more detail the transport properties of mouse GLUT9, its sensitivity to several uricosuric drugs, and the specificities of two splice variants, mGLUT9a and mGLUT9b. [(14)C]urate uptake measurements show that both splice variants are high-capacity urate transporters and have a K(m) of approximately 650 microM. The well-known uricosuric agents benzbromarone (500 microM) and losartan (1 mM) inhibit GLUT9-mediated urate uptake by 90 and 50%, respectively. Surprisingly, phloretin, a glucose-transporter blocker, inhibits [(14)C]urate uptake by approximately 50% at 1 mM. Electrophysiological measurements suggest that urate transport by mouse GLUT9 is electrogenic and voltage dependent, but independent of the Na(+) and Cl(-) transmembrane gradients. Taken together, our results suggest that GLUT9 works as a urate (anion) uniporter. Finally, we show by RT-PCR performed on RNA from mouse kidney microdissected tubules that GLUT9a is expressed at low levels in proximal tubules, while GLUT9b is specifically expressed in distal convoluted and connecting tubules. Expression of mouse GLUT9 in the kidney differs from that of human GLUT9, which could account for species differences in urate handling.

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases.

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.

Cysteine-scanning mutagenesis study of the sixth transmembrane segment of the Na,K-ATPase alpha subunit.

The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,K-ATPase alpha subunit was explored by cysteine scanning mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K-pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin-induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites.

Functional effects of Na+,K+-ATPase gene mutations linked to familial hemiplegic migraine.

Familial hemiplegic migraine type 2, an autosomal dominant form of migraine with aura, has been associated with four distinct mutations in the alpha2-subunit of the Na+,K+-ATPase. We have introduced these mutations in the alpha2-subunit of the human Na+,K+-ATPase and the corresponding mutations in the Bufo marinus alpha1-subunit and studied these mutants by expression in Xenopus oocyte. Metabolic labeling studies showed that the mutants were synthesized and associated with the beta-subunit, except for the alpha2HW887R mutant, which was poorly synthesized, and the alpha1BW890R, which was partially retained in the endoplasmic reticulum. [3H]ouabain binding showed the presence of the alpha2HR689Q and alpha2HM731T at the membrane, whereas the alpha2HL764P and alpha2HW887R could not be detected. Functional studies with the mutants of the B. marinus Na+,K+-ATPase showed a reduced or abolished electrogenic activity and a low K+ affinity for the alpha1BW890R mutant. Through different mechanisms, all these mutations result in a strong decrease of the functional expression of the Na+,K+-pump. The decreased activity in alpha2 isoform of the Na+,K+-pump expressed in astrocytes seems an essential component of hemiplegic migraine pathogenesis and may be responsible for the cortical spreading depression, which is one of the first events in migraine attacks.

Cation stoichiometry and cation pathway in the Na,K-ATPase and nongastric H,K-ATPase.

The mechanism of cation translocation by the Na,K-ATPase was investigated by cysteine scanning mutagenesis and measurements of accessibility through exposure to cysteine reagents. In the native protein, accessible residues were found only at the most extracellular residues of the 5th and 6th transmembrane segments (TMS) and the short loop between them. However, after modification by palytoxin a number of residues became accessible along the whole length of the 5th TMS and in the outer half of the 6th TMS, showing the contribution of each of these segments to the "channel" formed by the palytoxin-transformed Na,K-pump. Assuming that this structure is similar in the native and the palytoxin-transformed pump, our data allow us to determine the residues lining the cation pathway from the extracellular solution to their binding sites. A critical position in the 5th TMS contains a lysine conserved in all known nonelectrogenic H,K-ATPases, and a serine in all known electrogenic Na,K-ATPase sequences. Wild-type or mutant Na,K-or H,K-ATPase a subunits were coinjected with the Bufo beta2 subunit in Xenopus oocytes and Rb(86) uptake and electrophysiological measurements were performed. An electrogenic activity was recorded for the H,K-ATPase mutants in which the positively charged lysine had been replaced by neutral or negatively charged residues, while nonelectrogenic transport was observed with the S(782)R mutant of the Na,K-ATPase. The presence or the absence of a positively charged residue at the S(782) position appears to be critical for the stoichiometry of cation exchange.

FXYD proteins: new tissue- and isoform-specific regulators of Na,K-ATPase.

The recently defined FXYD protein family contains seven members that are small, single-span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the gamma subunit of Na,K-ATPase, which was shown to be a specific regulator of renal alpha1-beta1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the gamma subunit and have found that CHIF (corticosteroid hormone-induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K-ATPase and preferentially with alpha1-beta isozymes in native tissues, and produce distinct effects on the transport properties of Na,K-ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K-ATPase by FXYD proteins that involves a tissue-specific expression of an auxiliary subunit of distinct Na,K-ATPase isozymes.

FXYD7, the first brain- and isoform-specific regulator of Na,K-ATPase: biosynthesis and function of its posttranslational modifications.

The FXYD protein family has recently been defined as a result of the search for homologues of the Na,K-ATPase gamma subunit, CHIF, and phospholemman in EST and gene data banks. FXYD7 has been seen to have a role as a brain- and isozyme-specific regulator of Na/K-ATPase. In this study, the biosynthesis, membrane topology, nature, and role of the processing of FXYD7 are investigated.

Selected contribution: limiting Na(+) transport rate in airway epithelia from alpha-ENaC transgenic mice: a model for pulmonary edema.

The amiloride-sensitive epithelial Na(+) channel (ENaC) is essential for fluid clearance from the airways. An experimental animal model with a reduced expression of ENaC, the alpha-ENaC transgenic rescue mouse, is prone to develop edema under hypoxia exposure. This strongly suggests an involvement of ENaC in the pathogenesis of pulmonary edema. To investigate the pathogenesis of this type of edema, primary cultures of tracheal cells from these mice were studied in vitro. An ~60% reduction in baseline amiloride-sensitive Na(+) transport was observed, but the pharmacological characteristics and physiological regulation of the channel were similar to those observed in cells from wild-type mice. Aprotinin, an inhibitor of serine proteases, blocked 50-60% of the basal transepithelial current, hypoxia induced downregulation of Na(+) transport, and beta-adrenergic stimulation was effective to stimulate Na(+) transport after the hypoxia-induced decrease. When downregulation of ENaC activity (such as observed under hypoxia) is added to a low "constitutive" ENaC expression, the resulting reduced Na(+) transport rate may be insufficient for airway fluid clearance and favor pulmonary edema.

Epithelial sodium channel: a ligand-gated channel?

The epithelial sodium channel (ENaC) is a key component of the transepithelial Na+ transport. In epithelia, it is responsible for the maintenance of Na+ balance (which in turn controls extracellular fluid volume and arterial blood pressure) and the regulation of airway surface fluid. While the regulation of channel synthesis and surface density have been well described, the control of channel opening is still poorly understood. The channel has a large extracellular domain of as yet unknown function; a number of extracellular factors have been shown to modulate ENaC activity, including extracellular Na+ itself (through a phenomenon called 'self-inhibition'), several other organic or inorganic cations, which seem to interfere with self-inhibition, and serine proteases. Although a direct interaction with the extracellular domain of ENaC has not yet been demonstrated for each of these modulators, the available data strongly suggest that ENaC behaves as a ligand-gated channel similar to several other members of the ENaC/degenerin family.

A link between FXYD3 (Mat-8)-mediated Na,K-ATPase regulation and differentiation of Caco-2 intestinal epithelial cells.

FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na+ and K+ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na+ and K+ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.

Effects of mineralocorticoid and K+ concentration on K+ secretion and ROMK channel expression in a mouse cortical collecting duct cell line.

The cortical collecting duct (CCD) plays a key role in regulated K(+) secretion, which is mediated mainly through renal outer medullary K(+) (ROMK) channels located in the apical membrane. However, the mechanisms of the regulation of urinary K(+) excretion with regard to K(+) balance are not well known. We took advantage of a recently established mouse CCD cell line (mCCD(cl1)) to investigate the regulation of K(+) secretion by mineralocorticoid and K(+) concentration. We show that this cell line expresses ROMK mRNA and a barium-sensitive K(+) conductance in its apical membrane. As this conductance is sensitive to tertiapin-Q, with an apparent affinity of 6 nM, and to intracellular acidification, it is probably mediated by ROMK. Overnight exposure to 100 nM aldosterone did not significantly change the K(+) conductance, while it increased the amiloride-sensitive Na(+) transport. Overnight exposure to a high K(+) (7 mM) concentration produced a small but significant increase in the apical membrane barium-sensitive K(+) conductance. The mRNA levels of all ROMK isoforms measured by qRT-PCR were not changed by altering the basolateral K(+) concentration but were decreased by 15-45% upon treatment with aldosterone (0.3 or 300 nM for 1 and 3 h). The paradoxical response of ROMK expression to aldosterone could possibly work as a preventative mechanism to avoid excessive K(+) loss which would otherwise result from the increased electrogenic Na(+) transport and associated depolarization of the apical membrane in the CCD. In conclusion, mCCD(cl1) cells demonstrate a significant K(+) secretion, probably mediated by ROMK, which is not stimulated by aldosterone but increased by overnight exposure to a high K(+) concentration.

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes.

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.

Sodium self-inhibition of human epithelial sodium channel: selectivity and affinity of the extracellular sodium sensing site.

The epithelial Na(+) channel (ENaC) is present in the apical membrane of "tight" epithelia in the distal nephron, distal colon, and airways. Its activity controls the rate of transepithelial sodium transport. Among other regulatory factors, ENaC activity is controlled by the concentration of extracellular Na(+), a phenomenon named self-inhibition. The molecular mechanism by which extracellular Na(+) concentration is detected is not known. To investigate the properties of the extracellular Na(+) sensing site, we studied the effects of extracellular cations on steady-state amiloride-sensitive outward currents in Na(+)-loaded oocytes expressing human ENaC and compared them with self-inhibition of inward current after fast solution changes. About half of the inhibition of outward Na(+) currents was due to self-inhibition itself and the rest might be attributed to conduction site saturation. Self-inhibition by extracellular Li(+) was similar to that of Na(+) except for slightly slower kinetics. Ionic selectivity of the inhibition for steady-state outward current was Na(+) > or = Li(+) > K(+). We estimated an apparent inhibitory constant (K(I)) of approximately 40 mM for extracellular Na(+) and Li(+) and found no evidence for a voltage dependence of the K(I). Protease treatment induced the expected increase of the amiloride-sensitive current measured in high-Na(+) concentrations which was due, at least in part, to abolition of self-inhibition. These results demonstrate that both self-inhibition and saturation play a significant role in the inhibition of ENaC by extracellular Na(+) and that Na(+) and Li(+) interact in a similar way with the extracellular cation sensing site.

Palytoxin acts on Na(+),K (+)-ATPase but not nongastric H(+),K (+)-ATPase.

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase alpha(2)- and Na,K-ATPase beta(2)-subunits; Bufo Na,K-ATPase alpha(1)- and Na,K-ATPase beta(2)-subunits; and Bufo Na,K-ATPase beta(2)-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 microM ouabain. Functional expression was confirmed by measuring (86)Rb uptake. PTX (5 nM: ) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo beta(2)-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase alpha(1)-subunit and Na,K-ATPase beta(1)-subunit; rat Na,K-ATPase alpha(2)-subunit and Na,K-ATPase beta(2)-subunit; and rat Na,K-ATPase beta(1)- or Na,K-ATPase beta(2)-subunit alone. Measurement of increases in (86)Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKalpha(1)/NKbeta(1) and NKalpha(2)/NKbeta(2). Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKalpha(1)/NKbeta(1) exposed to 100 nM PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKbeta(1)- or rat NKbeta(2)-subunit alone. However, in HeLa cells expressing rat NKalpha(2)/NKbeta(2), outward current was observed after pump activation by 20 mM K(+) and a large membrane conductance increase occurred after 100 nM PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.

Role of homologous ASP334 and GLU319 in human non-gastric H,K- and Na,K-ATPases in cardiac glycoside binding.

Cardiac steroids inhibit Na,K-ATPase and the related non-gastric H,K-ATPase, while they do not interact with gastric H,K-ATPase. Introducing an arginine, the residue present in the gastric H,K-ATPase, in the second extracellular loop at the corresponding position 334 in the human non-gastric H,K-ATPase (D334R mutation) rendered it completely resistant to 2mM ouabain. The corresponding mutation (E319R) in alpha1 Na,K-ATPase produced a approximately 2-fold increase of the ouabain IC(50) in the ouabain-resistant rat alpha1 Na,K-ATPase and a large decrease of the ouabain affinity of human alpha1 Na,K-ATPase, on the other hand this mutation had no effect on the affinity for the aglycone ouabagenin. These results provide a strong support for the orientation of ouabain in its biding site with its sugar moiety interacting directly with the second extracellular loop.

The third sodium binding site of Na,K-ATPase is functionally linked to acidic pH-activated inward current.

Sodium- and potassium-activated adenosine triphosphatases (Na,K-ATPase) is the ubiquitous active transport system that maintains the Na(+) and K(+) gradients across the plasma membrane by exchanging three intracellular Na(+) ions against two extracellular K(+) ions. In addition to the two cation binding sites homologous to the calcium site of sarcoplasmic and endoplasmic reticulum calcium ATPase and which are alternatively occupied by Na(+) and K(+) ions, a third Na(+)-specific site is located close to transmembrane domains 5, 6 and 9, and mutations close to this site induce marked alterations of the voltage-dependent release of Na(+) to the extracellular side. In the absence of extracellular Na(+) and K(+), Na,K-ATPase carries an acidic pH-activated, ouabain-sensitive "leak" current. We investigated the relationship between the third Na(+) binding site and the pH-activated current. The decrease (in E961A, T814A and Y778F mutants) or the increase (in G813A mutant) of the voltage-dependent extracellular Na(+) affinity was paralleled by a decrease or an increase in the pH-activated current, respectively. Moreover, replacing E961 with oxygen-containing side chain residues such as glutamine or aspartate had little effect on the voltage-dependent affinity for extracellular Na(+) and produced only small effects on the pH-activated current. Our results suggest that extracellular protons and Na(+) ions share a high field access channel between the extracellular solution and the third Na(+) binding site.