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J. Biol. Chem., Vol. 278, Issue 44, 43580-43585, October 31, 2003

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Kinetic Dissection of Two Distinct Proton Binding Sites in Na⁺/H⁺ Exchangers by Measurement of Reverse Mode Reaction^*

Shigeo Wakabayashi, Takashi Hisamitsu, Tianxiang Pang, and Munekazu Shigekawa

From the Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565 Japan

Received for publication, June 24, 2003 , and in revised form, July 24, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We examined the effect of intracellular acidification on the reverse mode of Na⁺/H⁺ exchange by measuring ²²Na⁺ efflux from ²²Na⁺-loaded PS120 cells expressing the Na⁺/H⁺ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of ²²Na⁺ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H⁺ transport site. Intracellular pH (pH_i) dependence of ²² Na⁺ efflux roughly exhibited a bell-shaped profile; mild acidification from pH_i 7.5 to 7 dramatically accelerated ²²Na⁺ efflux, whereas acidification from pH_i 6.6 gradually decreased it. Alkalinization above pH_i 7.5 completely suppressed EIPA-sensitive ²²Na⁺ efflux. Cell ATP depletion and mutation of NHE1 at Arg⁴⁴⁰ (R440D) caused a large acidic shift of the pH_i profile for ²²Na⁺ efflux, whereas mutation at Gly⁴⁵⁵ (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na⁺/H⁺ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Na⁺/H⁺ exchangers (NHEs)¹ belong to one of the secondary active transporter families that catalyze the transport of ions or solutes using a driving force generated by active ion pumps. NHEs are involved in various cellular functions such as pH homeostasis, cell volume regulation, and transepithelial Na⁺ absorption (1–4), and at least eight isoforms differing in tissue and subcellular localization have been identified. The activities of NHEs are controlled by various extrinsic factors including hormones, growth factors, pharmacological agents, and mechanical stimuli (1–4). Many of these stimuli modulate the activity by changing the apparent affinity for intracellular H⁺ (Refs. 5–8; see Refs. 1–4 for reviews).

Physiologically, NHE catalyzes an electroneutral exchange of extracellular Na⁺ for intracellular H⁺, i.e. a forward mode of exchange with the aid of a constant driving force provided by a Na⁺ pump. However, NHE is also able to catalyze the exchange of intracellular Na⁺ for extracellular H⁺, i.e. a reverse mode of exchange under certain conditions. In cells, NHE is inactivated usually at an intracellular pH (pH_i) of 7.2, a value much lower than that (>8) predicted from the thermodynamic equilibrium between intracellular and extracellular Na⁺ and H⁺ ions. This "set point" behavior has been attributed to the existence of an allosteric regulatory site(s) called the "H⁺ modifier" site or "pH sensor" in NHE. Twenty years ago, Aronson et al. (9, 10) elegantly presented evidence for such a site based on analysis of the kinetics of ion exchange in renal brush border membrane vesicles; they found that ²²Na⁺ uptake into Na⁺-loaded vesicles (Na⁺/Na⁺ exchange) is stimulated by intravesicular H⁺ and that ²²Na⁺ efflux is stimulated by intravesicular H⁺, a finding opposite to the expected competitive interaction of H⁺ with Na⁺. These findings led to the idea that NHE becomes active only when the internal H⁺ modifier site is occupied by a proton. In many subsequent studies using native or NHE-transfected cells, exchange activity was reported to exhibit a complex, cooperative dependence on the internal H⁺ concentration despite its hyperbolic dependence on external Na⁺ or H⁺ concentration (11–18), suggesting the involvement of at least two binding sites for internal H⁺. However, because only the forward mode of exchange was measured in these studies and because very accurate measurement of pH_i is sometimes difficult, it still remains difficult to clearly distinguish the H⁺ modifier and H⁺ transport sites. Furthermore, it is not clear that the H⁺ modifier site exists in various NHE isoforms, because detailed analysis has not yet been performed using cells expressing each NHE isoform.

To obtain insight into the modifier role of intracellular H⁺,in this study we measured pH_i dependence of ²²Na⁺ efflux from cells expressing NHE1, NHE2, or NHE3. We found that ²²Na⁺ efflux is dramatically stimulated by intracellular acidification but almost completely inhibited by modest alkalinization (pH_i 7.5), which provides a strong piece of evidence for the existence of an intracellular H⁺ modifier site in these NHE isoforms. We show that pH_i dependence of the Na⁺/H⁺ exchange can be explained by assuming the interaction of multiple protons with the regulatory site(s) and the interaction of a single proton with the transport site.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The amiloride derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was a gift from New Drug Research Laboratories of Kanebo, Ltd. (Osaka, Japan). ²²NaCl was purchased from PerkinElmer Life Sciences. All other chemicals were of the highest purity available.

Cell Culture and Stable Expression—A Na⁺/H⁺ exchanger-deficient cell line (PS120) (19) and corresponding transfectants were maintained in Dulbecco' s modified Eagle' s medium (Invitrogen) as described (16). Plasmid transfection and selection of cell populations expressing NHE variants were performed as described (16).

Construction of Na⁺/H⁺ Exchanger Mutants—A plasmid carrying cDNA coding for the Na⁺/H⁺ exchanger isoforms NHE1–3 cloned into the mammalian expression vector pECE was described previously (16). The construction of two mutants, G455Q and R440D, was also described previously (20). Gly⁴⁵⁵ and Arg⁴⁴⁰ were reported to be located within the putative transmembrane-spanning domain 11 (TM11) and the intracellular loop 5 (IL5) connecting transmembrane domains 10 and 11 (20).

Measurement of ²²Na⁺ Efflux—Serum-depleted cells in 24-well dishes were loaded with ²²Na⁺ by preincubating them for 30 min at 37 °C in chloride/KCl medium comprising 20 mM Hepes/Tris (pH 7.4), 0.2–1.2 mM ²²NaCl (37 kBq/ml), 1.9–140 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, and 5 µM nigericin. Choline chloride was added to the medium to maintain the total concentration of KCl plus choline chloride at 140 mM. After removal of the radioactive preincubation solution, ²²Na⁺ efflux was started by adding the same choline chloride/KCl medium except that it additionally contained 2 mM ouabain and 100 µM bumetanide but not ²²Na⁺. For the data at zero time, this efflux solution was not added. In some wells, the efflux solution contained 0.1 mM EIPA or 5 mM amiloride. At the times indicated in Figs. 1, 3, and 5, cells were rapidly washed four times with ice-cold PBS to terminate ²²Na⁺ efflux. pH_i was calculated from the imposed [K⁺] gradient by assuming the equilibrium [K⁺]_i /[K⁺]_o and an intracellular [K⁺] of 120 mM. Data were normalized as to the protein concentration, which was measured with a bicinchoninic assay system (Pierce) using bovine serum albumin as a standard.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Time courses of 22Na+ efflux from PS120 cells and their NHE1 transfectants. PS120 cells or NHE1 transfectants were pHi-clamped at 7 or 7.5 by incubating with 5 µM nigericin plus 48 or 140 mM KCl, respectively, as described under "Experimental Procedures." At the same time, 22Na+ was loaded into the cells in the presence of 1 mM 22NaCl. After removal of the radioactive pHi clamp solution, the cells were incubated in the Na+-free non-radioactive efflux solution. A, illustration representing the intra- and extracellular ionic conditions in the 22Na+ efflux experiment. pHo, extracellular pH. B, time courses of 22Na+ loading in PS120 cells expressing NHE1 measured at pHi 7.5 () or 7 (•). C, time courses of 22Na+ efflux in PS120 cells were measured in the presence (•) and absence () of 0.1 mM EIPA at pHi 7. D and E, time courses of 22Na+ efflux in cells expressing NHE1 were measured in the presence (•) and absence () of 0.1 mM EIPA at a pHi of 7 and 7.5, respectively. Data represent means ± S.D. of three determinations.

View larger version (28K): [in this window] [in a new window]   FIG. 3. 22Na+ efflux in cells expressing G455Q or R440D. A and B, time courses of 22Na+ efflux from cells expressing G455Q were measured in the presence (•) and absence () of 0.1 mM EIPA at pHi 7 or 7.5, respectively, as described under "Experimental Procedures." C and D, time courses of 22Na+ efflux from cells expressing R440D were measured in the presence (•) and absence (•) of 0.1 mM EIPA at pHi 7 or 7.5, respectively. Data represent means ± S.D. of three determinations.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Time courses of 22Na+ efflux in cells expressing NHE2 or NHE3. Cells expressing NHE2 or NHE3 were pHi-clamped and 22Na+-loaded as described under "Experimental Procedures". A and B, time courses of 22Na+ efflux in cells expressing NHE2 were measured in the absence () and presence (•) of 5 mM amiloride at pHi 7.5 and 7, respectively. C and D, time courses of 22Na+ efflux in cells expressing NHE3 were measured in the absence () and presence (•) of 5 mM amiloride at pHi 7.5 and 7, respectively. Data represent means ± S.D. of three determinations.

Measurement of ²²Na⁺ Uptake—²²Na⁺ uptake activity was measured using serum-depleted cells grown in 24-well dishes in the presence or absence of EIPA as described previously (20).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To measure ²²Na⁺ efflux, we first preincubated PS120 cells or their NHE transfectants for 30 min in a radioactive solution containing 1 mM ²²NaCl, the K⁺/H⁺ ionophore nigericin, and either 48 or 140 mM KCl. This preincubation allowed cells to be pH_i-clamped at 7 or 7.5 and, at the same time, to be loaded with ²²Na⁺. ²²Na⁺ was rapidly taken up by cells and reached nearly to the plateau after the preincubation for 20 min under both conditions (Fig. 1B), suggesting that the radioactivity of ²²Na⁺ was equilibrated in the extra- and intracellular medium. The intracellular ²²Na⁺ concentration at 30 min was 1–2 mM by assuming a value of 5 µl of cell water per milligram of protein. After removal of the preincubation solution, cells were placed in a Na⁺-free, non-radioactive solution (pH 7.4) to start ²²Na⁺ efflux. By this procedure, we were able to measure ²²Na⁺ efflux in the presence of an outwardly directed H⁺ gradient (at pH_i 7) or in almost the absence of the H⁺ gradient (at pH_i 7.5) (Fig. 1A). We expected that NHE-independent background ²²Na⁺ leakage would be minimal, because the efflux medium contained the Na⁺-pump inhibitor ouabain and the Na⁺/K⁺/Cl^– cotransporter inhibitor bumetanide but not , a substrate for both cotransporter and Na⁺-dependent exchanger. In fact, we observed only slow ²²Na⁺ efflux in exchanger-deficient PS120 cells and their NHE1 transfectants in the presence of the NHE-specific inhibitor EIPA (Fig. 1, C–E).

As expected, EIPA-sensitive ²²Na⁺ efflux was not detectable in exchanger-deficient PS120 cells whose pH_i was maintained at 7 (Fig. 1C) or 7.5 (not shown). In NHE1 transfectants, however, rapid EIPA-sensitive ²²Na⁺ efflux was observed at pH_i 7 (Fig. 1D), although little EIPA-sensitive ²²Na⁺ efflux was observed at pH_i 7.5 (Fig. 1E). Thus, ²²Na⁺ efflux via NHE1 is dramatically stimulated by intracellular acidification, whereas it is completely inhibited by alkalinization. If we assume that NHE1 catalyzes a counter transport reaction only involving the transport site, the outwardly directed H⁺ gradient should lead to inhibition of ²²Na⁺ efflux. The data support the view that activity of NHE1 is regulated by protonation/deprotonation of the H⁺ modifier site(s), which is different from the H⁺ transport site.

To examine pH_i dependence of ²²Na⁺ efflux, we clamped pH_i at various values by incubating NHE1-expressing cells for 30 min in solutions containing nigericin and different concentrations of KCl. Because ²²Na⁺ was taken up by cells via the exchanger during pH_i clamping, ²²Na⁺ loading was greater in cells clamped at lower pH_i than in cells clamped at higher pH_i. To minimize such differences in ²²Na⁺ loading, we used a pH_i clamp solution containing different concentrations (0.2–1.2 mM) of ²²NaCl. As shown in Fig. 2A, the level of ²²Na⁺ loading varied from 10 to 16 nmol/mg (corresponding to intracellular concentrations of 2–3 mM), indicating that the outwardly directed Na⁺ gradient produced under the conditions used was not very different. Fig. 2A also shows levels of ²²Na⁺ remaining in cells 3 min after the addition of the efflux solution with or without EIPA. We plotted the EIPA-sensitive fraction of ²²Na⁺ efflux (Fig. 2B, open circles), and the values normalized to the initial level of ²²Na⁺ loading as a function of pH_i (Fig. 2C). The ²²Na⁺ efflux increased steeply with decreasing pH_i from 7.5 to 7.2, reached the maximum at pH_i 6.6, and then decreased with decreasing pH_i from 6.6 to 5.6. The bell-shaped pH_i profile of ²²Na⁺ efflux suggests that there are at least two intracellular proton-binding sites, each involved in the stimulation and inhibition of NHE1 function, respectively.

View larger version (36K): [in this window] [in a new window]   FIG. 2. The pHi profile of 22Na+ efflux. Cells expressing the wild-type NHE1 were pHi-clamped with K+ /nigericin and, at the same time, loaded with 22Na+ under control or ATP-depletion conditions as described under "Experimental Procedures." This preincubation solution contained 140, 76, 48, 19, 7.6, and 1.9 mM KCl and 0.2, 0.3, 0.5, 0.7, 0.9 and 1.2 mM 22NaCl for pHi values of 7.5, 7.2, 7, 6.6, 6.2 and 5.6, respectively. After removal of the radioactive preincubation solution, the cells were incubated in the Na+-free non-radioactive efflux solution in the presence or absence of 0.1 mM EIPA. A, the intracellular contents of 22Na+ present at the start of efflux and 22Na+ remaining in cells at 3 min after the switch to the EIPA-free and EIPA-containing medium were plotted against pHi. Data represent means ± S.D. of three determinations. B, absolute values of EIPA-sensitive 22Na+ efflux (3 min) under control ( and ) or ATP depletion conditions (•) were plotted against pHi. For control, results from two independent experiments were represented, and one () was calculated from the data in panel A. C, EIPA-sensitive 22Na+ efflux was normalized to the initial 22Na+ content and then plotted against pHi. Data represent means ± S.D. of three determinations. D, for comparison, pHi dependence of 22Na+ uptake is presented under control () or ATP-depletion conditions (•).

We examined the effect of cell ATP depletion on the pH_i dependence of ²²Na⁺ efflux. We treated cells for 30 min with metabolic inhibitors 2-deoxyglucose (5 mM) and oligomycin (2 µg/ml) during pH_i clamping. Such treatment decreased cell ATP to less than 5% of that in normal cells (16). In these cells, the initial ²²Na⁺ loading was 6–9 nmol/mg (corresponding to intracellular concentrations of 1.2–1.8 mM) at pH_i values from 6.6 to 7.5, although ²²Na⁺ was loaded to high levels (20–30 nmol/mg) at pH_i 6.2 or 5.6. EIPA-sensitive ²²Na⁺ efflux and its normalized values were plotted against pH_i. As shown in Fig. 2, B and C, ²²Na⁺ efflux was almost completely inhibited by ATP depletion at least in the pH_i range from 6.6 to 7.5, consistent with the previous finding (16, 21–23) that ATP depletion greatly shifts the pH_i dependence of the forward mode of Na⁺/H⁺ exchange to an acidic side, as shown in Fig. 2D. It is also noted that, similar to the forward mode, the inhibitory effect of ATP-depletion on ²²Na⁺ efflux is alleviated by acidification.

Fig. 3 shows the results of similar ²²Na⁺ efflux measurements using cells expressing G455Q or R440D in which the pH_i dependence of the forward mode of exchange markedly shifts to alkaline and acidic sides, respectively (Fig. 4B; see also Ref. 20). As in the case with the wild type NHE1, ²²Na⁺ efflux in G455Q-expressing cells was much faster at pH_i 7 than at pH_i 7.5 (Fig. 3, A and B). Interestingly, a significant level of EIPA-sensitive ²²Na⁺ efflux was still observed in these cells even at pH_i 7.5 (Fig. 3B), unlike the case with the wild-type NHE1. In contrast, a significant level of EIPA-sensitive ²²Na⁺ efflux was not observed in R440D-expressing cells at pH_i 7 (Fig. 3C) or 7.5 (Fig. 3D), although low levels of ²²Na⁺ efflux were observed in a more acidic range of pH_i (Fig. 4A). Fig. 4 shows pH_i profiles of ²²Na⁺ efflux in cells expressing G455Q or R440D. The pH_i profile was significantly shifted to an alkaline side in G455Q-expressing cells compared with cells expressing the wild-type NHE1 (Fig. 4, A and B). In contrast, the pH_i profile was markedly shifted to an acidic side in cells expressing R440D.

View larger version (27K): [in this window] [in a new window]   FIG. 4. The pHi profile of 22Na+ efflux and 22Na+ uptake in cells expressing G455Q or R440D. A, cells expressing G455Q or R440D were pHi-clamped with K+/nigericin and, at the same time, loaded with 22Na+ as described under "Experimental Procedures." The concentrations of KCl and 22NaCl in preincubation solutions were the same as those given in the legend to Fig. 2. Absolute values of EIPA-sensitive 22Na+ efflux (3 min) were plotted against pHi. B, EIPA-sensitive 22Na+ efflux was normalized to the initial 22Na+ content and then plotted against pHi. Data represent means ± S.D. of three determinations. C, for comparison, 22Na+ uptake data were represented.

Finally, we examined whether intracellular acidification activates ²²Na⁺ efflux in cells expressing NHE2 or NHE3 (Fig. 5). In these experiments, we used a high concentration (5 mM) of amiloride in place of EIPA as an inhibitor, because these isoforms are relatively less sensitive to the amiloride analogue. As in the case of NHE1, modest intracellular acidification (pH_i 7) significantly accelerated amiloride-sensitive ²²Na⁺ efflux from cells expressing NHE2 or NHE3, although ²²Na⁺ efflux from NHE2 transfectants was relatively slow (Fig. 5). The slow efflux in NHE2 transfectants may be due to lower expression of the exchanger in the plasma membrane, because exchange activity of these transfectants was 20–30% of that of NHE1 or NHE3 transfectants.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this work we tried to kinetically dissect proton-binding sites in the Na⁺/H⁺ exchanger by measuring EIPA-sensitive ²²Na⁺ efflux from cells expressing different NHE isoforms. We loaded cells with ²²Na⁺ while clamping pH_i at various values using a K⁺/nigericin technique and measured ²²Na⁺ efflux at a constant extracellular pH (7.4) in the nominal absence of extracellular Na⁺. To avoid possible interaction of cytosolic Na⁺ with the H⁺ modifier site as suggested previously (14, 25), we limited the extent of ²²Na⁺ loading to low levels (2–3 mM). To our knowledge, ²²Na⁺ efflux has never been measured using cultured cells under such defined conditions.

²²Na⁺ efflux from cells expressing NHE1, NHE2, or NHE3 was markedly stimulated by cytoplasmic acidification, contrary to the prediction based on reduced H⁺ concentration gradient. As proposed previously (9, 10), the results can be interpreted as indicating that these NHE isoforms possess the H⁺ modifier site and that occupancy of the latter by a proton(s) results in activation of exchange activity. We observed that the NHE1-mediated ²²Na⁺ efflux exhibited roughly bell-shaped pH_i dependence. Modest cytosolic alkalinization (pH_i 7.5) abolished ²²Na⁺ efflux (Figs. 1D, 2B, and 4), whereas modest cytosolic acidification from pH_i 7.5 to 7–7.2 dramatically enhanced it (Figs. 1C, 2B, and 4). The increase in ²²Na⁺ efflux in the latter pH_i range was very steep, suggesting the binding of two or more protons to the modifier site. In a near-neutral pH_i range, cell ATP depletion or mutations of NHE1 (R440D and G455Q) caused marked shifts in the pH_i dependence of ²²Na⁺ efflux (Figs. 2B, 3B, and 4). Because they also caused similar large shifts in pH_i dependence of the forward mode of Na⁺/H⁺ exchange under corresponding conditions (20), it is most likely that modulation of Na⁺/H⁺ exchange by these procedures is attributable to altered interaction of the H⁺ modifier site with activating protons. On the other hand, acidosis (<pH_i 6.2) caused an extensive inhibition of ²²Na⁺ efflux (Figs. 2B and 4). This inhibition appears to result from competition between Na⁺ and H⁺ for the intracellular transport site. Our measurement of the descending and ascending slopes of pH_i dependence of ²²Na⁺ efflux thus allowed us to observe the binding of protons to the H⁺ modifier and H⁺ transport sites, respectively. The effect of pH_i on ²²Na⁺ efflux from cells was measured previously using thymic lymphocytes (13) in which a significant difference in ²²Na⁺ efflux was not observed at the two pH_i values tested (7.2 and 6.3). A possible explanation for such data is that the two pH_i values used for thymic lymphocytes may not be optimal for observing a large pH_i-dependent change in ²²Na⁺ efflux.

We attempted to reproduce by simulation the pH_i dependence of the forward and reverse modes of Na⁺/H⁺ exchange and their modulation by ATP depletion or the exchanger mutations using a simplified reaction model and the assumption that three protons (n = 3) cooperatively interact with the modifier site and a single proton interacts with the transport site (Fig. 6, A and B). We were able to at least qualitatively reproduce complex pH_i profiles of the forward and reverse modes of exchange and their modulation (Fig. 6, C and D). For example, ATP depletion or mutation of Arg⁴⁴⁰ inhibited the uptake and efflux activities more strongly in the neutral pH_i range (6.6–7.5). In addition, pH_i dependence between 6.6 and 5.6 was very steep when the activity was measured as uptake but not as efflux (Figs. 2 and 4) under ATP depletion or in cells expressing R440D (compare the pH_i profiles in Figs. 2, 4 and 6). Thus our measurement of pH_i dependence of ²²Na⁺ efflux permitted us to analyze kinetic properties of the H⁺ modifier site interacting with activating protons. The observed properties are compatible with the predicted roles of the exchanger in cell pH_i regulation, i.e. protection of cells from excessive alkalosis, acceleration of recovery of cells from acidosis, and modulation of exchange activity due to alteration in H⁺ affinity of the modifier site, in particular in the near-neutral pH_i range.

View larger version (35K): [in this window] [in a new window]   FIG. 6. Schemes explaining the pHi dependence of the Na+/H+ exchange. We assumed that multiple protons (n molecules) cooperatively bind to the H+ modifier sites (circular space), whereas a single proton binds to the transport site (rectangular space) on the cytosolic side of the exchanger. In addition, we assumed that these two types of H+ binding sites are independent, i.e. H+ binding at one site does not affect the H+ affinity for the other. In the forward mode of exchange (22Na+ uptake) (A), only the exchanger occupied by protons at both sites would be able to participate in the exchange reaction, whereas in the reverse mode of exchange (22Na+ efflux) (B), the exchanger having protons at the modifier site but 22Na+ at the transport site would become active. In the reverse mode, H+ would competitively inhibit Na+ binding at the transport site. Based on these assumptions, we simulated the pHi dependences of 22Na+ uptake (C) and 22Na+ efflux reactions (D). We used the following steady-state equations that were developed assuming a rapid equilibrium of H+ and Na+ binding (37): v/Vmax = [H+]/Ka(1 + (Kb/[H+])n) + [H+](1 + (Kb/[H+])n)) for the relative rate of 22Na+ uptake, and v/Vmax = [Na+]/(Ks (1 + [H+/Ka + (kb/[H+]n) + ([H+]/Ka)(Kb/[H+] n) + [Na+](1 + (Kb/[H+]) n))) for the relative rate of 22Na+ efflux, where Ka and Kb (K b' = (Kb)n) are the intrinsic H+ dissociation constants for the transport and modifier sites, respectively, and Ks is the Na+ dissociation constant for the transport site. For simulation of 22Na+ efflux, Ks and the intracellular Na+ concentration were assumed to be 10 and 2 mM, respectively. The values for other parameters obtained by means of manual fitting trials are given in panels C and D.

Mutation of Arg⁴⁴⁰ shifts pH_i dependence of the forward mode of Na⁺/H⁺ exchange toward an acidic side, whereas that of Gly⁴⁵⁵ shifts it toward an alkaline side without changing apparent affinities for extracellular substrates Na⁺ and H⁺ and the inhibitor EIPA (20) (see Fig. 4B). Thus, the present data reinforce our previous conclusion that the region encompassing the intracellular loop IL5 and the transmembrane domain TM11 plays a crucial role in the proper functioning of the H⁺ modifier site (20). Although the structure of the H⁺ modifier site is not known, we suggested previously that pH_i sensing of NHE1 may be controlled by a substructure consisting of intracellular loop IL5 and the juxtamembrane subdomain I of the cytoplasmic domain (amino acids 503–595) with a tightly bound calcineurin B homologous protein (20, 26). In general, a histidine residue has been thought to be a good candidate for a residue involved in pH_i sensing, because its imidazole moiety is the only side chain that ionizes in solution within a physiological pH range. Indeed, His²²⁵ and His³⁶⁷ have been identified as important residues for pH sensing of the Na⁺/H⁺ antiporters of Escherichia coli (NhaA) (27, 28) and Schizosaccharomyces pombe (Sod2) (29, 30), respectively. However, little change was observed in pH_i sensitivity when histidine residues at positions 76, 81, 250, 285, 325, 373, 376, 407, 408, and 473 were substituted by cysteine in NHE1,² although a recent study (31) reported that mutations at His⁴⁷⁹ and His⁴⁹⁹ in the juxtamembrane cytoplasmic domain of rabbit NHE3 shifted the pH_i profile to an acidic side. Other histidine residues of NHE1, i.e. His³⁵, His¹²⁰ and His³⁴⁹ in the membrane-spanning segments (32) and those in the histidine cluster (HYGHHH) in the cytoplasmic domain (30), do not appear to directly influence exchange activity.

Although the present results suggest the existence of a H⁺ modifier site in NHEs, it should be noted that complex kinetic effects of protons on exchanger activation have been reported as follows. (i) Transient kinetic studies of the exchanger using kidney brush border membrane vesicles revealed that the exchanger exhibits cooperativity with respect to the external Na⁺ concentration when vesicles are acid-loaded (33), suggesting that protonation of the modifier site may change the oligomeric interaction. (ii) Exchange activity of NHE3 (34, 35) or NHE1 (36) is slowly (35 min) activated by intracellular acidification, suggesting that a slow conformational change or phosphorylation-dependent event may be involved in the expression of exchanger activity. (iii) Intracellular Na⁺ is able to activate the exchanger (14, 25), suggesting a possible interaction of Na⁺ with the H⁺ modifier site. However, the structural basis for these properties of the H⁺ modifier site is not known.

In summary, using cultured cells expressing different NHE isoforms, we obtained evidence for the existence of the H⁺ modifier site(s) distinguishable from the H⁺ transport site. Our results suggest that interaction of multiple protons with the modifier site results in a dramatic activation of Na⁺/H⁺ exchange in response to modest acidification. Clearly, further work is required to clarify the structure and function of the H⁺ modifier site, which is a hallmark of NHE regulation.

	FOOTNOTES

 
^* This work was supported by Grant-in-Aid on Priority Areas 13142210 and Grant-in-Aid for Scientific Research 14580664 from the Ministry of Education, Science, and Culture of Japan, a grant from the Organization of Pharmaceutical Safety and Research (OPSR) of Japan (Promotion of Fundamental Studies in Health Science), Ministry of Health Labour Sciences research grants, and Research on Advanced Medical Technology Grant nano-001.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular Physiology, National Cardiovascular Center Research Inst., Fujishirodai 5-7-1, Suita, Osaka 565-8565, Japan. Tel.: 81-6-6833-5012; Fax: 81-6-6872-7485; E-mail: wak{at}ri.ncvc.go.jp.

¹ The abbreviations used are: NHE, Na⁺/H⁺ exchanger; pH_i, intracellular pH; EIPA, 5-(N-ethyl-N-isopropyl)amiloride.

² S. Wakabayashi, T. Hisamitsu, T. Pang, and M. Shigekawa, unpublished observations.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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