Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane.

When human red cells are incubated at 37 degrees C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, upcouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change.     

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane

When human red cells are incubated at 37°C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb⁺ (K⁺) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb⁺ (K⁺) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca²⁺ (Gardos effect). The electron donors require the presence of intracellular Ca²⁺ to be effective, but at levels that do not stimulate by themselves the fluxes of K⁺. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca²⁺ alone; antimycin, uncouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect.The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca²⁺ more effective to elicit rapid K⁺ movements. Plasma membrane oxidoreductase activities could be engaged in this change.     

Caveolins bind to (Na+, K+)/H+ exchanger NHE7 by a novel binding module

NHE7 was identified as the first mammalian organelle-membrane type (Na+, K+)/H+ exchanger that may contribute to the ion homeostasis in the trans-Golgi network (TGN) and endosomes. Here we show that caveolins directly bind to the C-terminal extension of NHE7 by an unconventional binding-module. NHE7 is partly associated with caveolae/lipid raft fractions, and heterologous expression of caveolin dominant-negative mutants as well as cholesterol depriving drugs diminished such associations. In contrast to the wild type NHE7, a deletion mutant lacking the C-terminal extension was predominantly detected in non-caveolae/lipid rafts. We further show that a small fraction of NHE7 is targeted to the cell surface and subsequently internalized. Endocytosis of NHE7 was efficiently inhibited by pharmacological maneuvers that block clathrin-dependent endocytosis, whereas dominant-negative caveolin mutants or methyl beta-cyclodextrin did not affect NHE7-internalization. Thus, NHE7 associates with both caveolae/lipid rafts and non-caveolae/lipid raft, and the two pools likely exhibit separate dynamics.     

Molecular cloning and characterization of a novel (Na+,K+)/H+ exchanger localized to the trans-Golgi network

The luminal pH of organelles along the secretory and endocytic pathways of mammalian cells is acidic and tightly regulated, with the [H+] varying up to 100-fold between compartments. Steady-state organellar pH is thought to reflect a balance between the rates of H+ pumping by the vacuolar-type H+-ATPase and H+ efflux through ill-defined pathways. Here, we describe the cloning of a novel gene (NHE7) in humans that is homologous to Na+/H+ exchangers, is ubiquitously expressed, and localizes predominantly to the trans-Golgi network. Significantly, NHE7 mediates the influx of Na+ or K+ in exchange for H+. The activity of NHE7 was also found to be relatively insensitive to inhibition by amiloride but could be antagonized by the analogue benzamil and the unrelated compound quinine. Thus, NHE7 displays unique functional and pharmacological properties and may play an important role in maintaining cation homeostasis of this important organelle.     

Non-selective voltage-activated cation channel in the human red blood cell membrane

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.     

Differential effect of HOE642 on two separate monovalent cation transporters in the human red cell membrane.

Residual K(+) fluxes in red blood cells can be stimulated in conditions of low ionic strength. Previous studies have identified both the non-selective, voltage-dependent cation (NSVDC) channel and the K(+)(Na(+))/H(+) exchanger as candidate pathways mediating this effect, although it is possible that these pathways represent different modes of operation of a single system. In the present study the effects of HOE642, recently characterised as an inhibitor of the K(+)(Na(+))/H(+) exchanger, on NSVDC has been determined to clarify this question. Radioisotope flux measurements and conductance determinations showed that HOE642 exerted differential effects on the NSVDC channel and the K(+)(Na(+))/H(+) exchanger, confirming that the salt loss observed in low ionic strength solutions represents contributions from at least two independent ion transport pathways. The findings are discussed in the context of red blood cell apoptosis (eryptosis) and disease.     

The arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes.

In saline environments, plants accumulate Na(+) in vacuoles through the activity of tonoplast Na(+)/H(+) antiporters. The first gene for a putative plant vacuolar Na(+)/H(+) antiporter, AtNHX1, was isolated from Arabidopsis and shown to increase plant tolerance to NaCl. However, AtNHX1 mRNA was up-regulated by Na(+) or K(+) salts in plants and substituted for the homologous protein of yeast to restore tolerance to several toxic cations. To study the ion selectivity of the AtNHX1 protein, we have purified a histidine-tagged version of the protein from yeast microsomes by Ni(2+) affinity chromatography, reconstituted the protein into lipid vesicles, and measured cation-dependent H(+) exchange with the fluorescent pH indicator pyranine. The protein catalyzed Na(+) and K(+) transport with similar affinity in the presence of a pH gradient. Li(+) and Cs(+) ions were also transported with lower affinity. Ion exchange by AtNHX1 was inhibited 70% by the amiloride analog ethylisopropyl-amiloride. Our data indicate a role for intracellular antiporters in organelle pH control and osmoregulation.     

Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.     

Pressure and temperature effects on human red cell cation transport

Summary The effects of hydrostatic pressure and temperature on the three components of K⁺ uptake in human red cells have been investigated, using ouabain and bumetanide to distinguish between the pump, passive diffusion and cotransport. The pressure sensitivity for passive diffusion has been shown to depend on the counter-ion present. The order of this effect, Cl^–>Br^–>NO ₃ ^– >I^–, is the same as for the ionic partial modal volumes and the Hofmeister series. We have analyzed our experimental results thermodynamically, and propose a model for the activated transition-state complex of the potassium ion which involves the loss of water molecules from the secondary hydration shell, cosphere II.     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Ouabain-resistant human lymphoblastoid lines altered in the (Na+ + K+)-dependent ATPase membrane transport system.

Diploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)-dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 muM ouabain. Ouabain-resistant (OUA(R)) cells with normal growth at 50 times this dose were recovered at a frequency 1 X 10(-6). This frequency was increased 9-fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR-191, under conditions where both increased the frequency of 8-azaguanine-resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUA(R) clones show showed 30-50% of the wild type amount of 3H-ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 muM at 0.5 mM K+, as observed in wild-type cells. Both the initial rate of uptake of 86Rb+ in OUA(R) cells and the (Na+ + K+)-dependent ATPase activity of OUA(R) plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUA(R) cells in the absence of ouabain were unchanged compared with wild type cells.     

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.     

Effect of cisplatin on H+ transport by H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane

The effect of the potent anticancer drug cisplatin, cis-diamminedichloroplatinum (II) (CDDP), on H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane was examined. To measure H+ transport by vacuolar H+ -ATPase in renal brush-border membrane vesicles, we employed a detergent-dilution procedure, which can reorientate the catalytic domain of H+ -ATPase from an inward-facing configuration to outward-facing one. ATP-driven H+ pump activity decreased markedly in brush-border membrane prepared from rats two days after CDDP administration (5 mg/kg, i.p.). In addition, N-ethylmaleimide and bafilomycin A1 (inhibitors of vacuolar H+ -ATPase)-sensitive ATPase activity also decreased in these rats. The decrease in ATP-driven H+ pump activity was observed even at day 7 after the administration of CDDP. Suppression of ATP-driven H+ pump activity was also observed when brush-border membrane vesicles prepared from normal rats were pretreated with CDDP in vitro. In contrast with H+ -ATPase, the activity of Na+/H+ exchanger, which was determined by measuring acridine orange fluorescence quenching, was not affected by the administration of CDDP. These results provide new insights into CDDP-induced renal tubular dysfunctions, especially such as proximal tubular acidosis and proteinuria.     

Anion-coupled Na efflux mediated by the human red blood cell Na/K pump

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity. Although it can be concluded that Na efflux in the uncoupled mode occurs by means of a cotransport with cellular anions, the molecular basis for this change in the internal charge structure of the pump and its change in ion selectivity is at present unknown.     

Characterization of the Na+/H+ exchanger in human epidermoid carcinoma A431 vesicles

A previous report from this laboratory (Rothenberg et al., 1983a) demonstrated the presence of an Na+/H+ exchanger in human epidermoid carcinoma A431 cells. We now characterize surface-derived membrane vesicles from this cell line which contain a functional Na+/H+ exchanger. The Na+/H+ exchanger in A431 vesicles shares a number of characteristics in common with previously described Na+/H+ exchangers including the following: (1) Na+ uptake is stimulated by an outward-directed pH gradient and inhibited by an inward-directed pH gradient. (2) Na+ uptake is inhibited by amiloride and its analogs and their relative effectiveness is similar in vesicles and A431 cells. (3) The Na+/H+ exchanger uses Na+ or Li+ as a substrate but not K+ or Cs+. (4) H+ efflux is stimulated by an inward-directed Na+ gradient and inhibited by the amiloride analog 5-N-dimethylamiloride. The Na+/H+ exchanger in these membrane vesicles is activated allosterically by low intravesicular pH. The apparent pKa of the activating site is 6.4-6.6, characteristic of the NA+/H+ exchanger before activation by mitogens.     

Effect of peroxynitrite on passive K+ transport in human red blood cells.

Peroxynitrite is generated in vivo by the reaction between nitric oxide, from endothelial and other cells, and the superoxide anion. It is therefore pertinent to examine its effects on the membrane permeability of red blood cells. Treatment of human red blood cells with peroxynitrite (nominally 1 mM) markedly stimulated passive K+ permeability. The main effect was on a Cl(-)-independent K+ pathway, which remains unidentified. Although K+-Cl- cotransport (KCC) was stimulated, this was dependent on saline composition, being inhibited by physiological levels of glucose (IC50 4 mM), and also by sucrose and MOPS. Effects on the Cl(-)-independent K+ pathway were less dependent on saline composition, and were not inhibited by amiloride, ethylisopropylamiloride, dimethylamiloride or gadolinium. Na+-K+-2Cl- cotransporter was inhibited whilst there was little effect on the Gardos channel (Ca2+-activated K+ channel). Peroxynitrite was markedly more effective in oxygenated cells than deoxygenated ones. Treatment with peroxynitrite per se did not affect initial cell volume. Anisotonic swelling modestly increased the Cl(-)-independent K+ influx, but did not affect peroxynitrite-stimulated KCC. Decreasing extracellular pH from 7.4 to 7.2 or 7.0 increased KCC stimulation, whilst the Cl(-)-independent component of K+ transport was lowest at pH 7.2. Finally, protein phosphatase inhibition with calyculin A (100 nM) inhibited KCC, implying that, as with other KCC stimuli, peroxynitrite acts via decreased protein phosphorylation; pre-treatment with calyculin A also inhibited the Cl(-)-independent component of K+ transport. These findings are relevant to the actions of peroxynitrite in vivo.