Mutations of Arg440 and Gly455/Gly456 Oppositely Change pH Sensing of Na+/H+ Exchanger 1*

Shigeo WakabayashiDagger, 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, December 30, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To identify important amino acid residues involved in intracellular pH (pHi) sensing of Na+/H+ exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of Arg440 in IL5 with other residues except positively charged Lys caused a large shift in pHi dependence of 22Na+ uptake to an acidic side, whereas substitution of Gly455 or Gly456 within the highly conserved glycine-rich sequence of TM11 shifted pHi dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly455 with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg440 (R440D) abolished the ATP depletion-induced acidic shift in pHi dependence of 22Na+ uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly455 (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na+ and H+ and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg440 is required for normal pHi sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg440 in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pHi sensor" of Na+/H+ exchanger 1.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Na+/H+ exchangers (NHEs)1 are plasma membrane transporters that regulate pH homeostasis, cell volume, and transepithelial Na+ absorption (1-4). The activity of ubiquitous exchanger isoform NHE1 is controlled by various extrinsic factors including hormones, growth factors, pharmacological agents, and mechanical stimuli (1-4). These stimuli have been shown to enhance NHE1 activity by shifting its intracellular pH (pHi) dependence to an alkaline side. This phenomenon is usually explained by assuming that there exists an allosteric regulatory site for intracellular protons in NHE1 that is distinct from the Na+ or H+ transport site and that external stimuli increase the affinity of such H+ modifier site (Ref. 5; see Refs. 1-4 for reviews). Regulation of NHE1 by these stimuli has been reported to occur through a variety of signaling molecules, i.e. calcinuerin B homologous protein (6, 7), Ca2+/calmodulin (8, 9), low molecular mass GTPases Ras and Rho (10-12), p42/44 mitogen-activated protein kinases (13), p90 ribosomal S6 kinase (14), 14-3-3 protein (15), Nck-interacting kinase (16), and phosphatidylinositol 4,5-bisphosphate (17). However, the interrelationship of these signaling molecules and the mechanism by which they modulate the interaction of the putative H+ modifier site with intracellular protons is still unclear.

The NHE family members have similar overall structures, consisting of an N-terminal transmembrane domain (~500 amino acids) containing 12 membrane spanning segments and a C-terminal cytoplasmic regulatory domain (~300 amino acids) (1-3). We previously showed that deletion of the cytoplasmic domain of NHE1 greatly shifted the pHi dependence of exchange to an acidic side, with the steep pHi dependence of exchange being maintained (18). Furthermore, we have presented evidence that the cytoplasmic domain of NHE1 contains several subdomains, which increase or decrease the pHi sensitivity (19). These data may be consistent with the idea that the putative H+ modifier site exists within the N-terminal transmembrane domain and that the cytoplasmic domain regulates the accessibility of regulatory protons to the site. However, the structural basis for such an idea is largely unknown.

Based on the accessibility of introduced cysteine residues to sulfhydryl reagents, we have recently presented a new membrane topology model of NHE1 in which the exchanger consists of 12 transmembrane segments with N- and C-tails located in the cytosol (20), although a previous study reported the N-tail to be cleaved as a signal peptide during the exchanger biogenesis (21). In a subsequent cysteine-scanning mutagenesis study, we found that mutation of Tyr454 or Arg458 in a new transmembrane domain 11 (TM11) impaired the plasma membrane expression of NHE1 (22), suggesting the importance of these residues for proper folding of NHE1. Furthermore, our preliminary experiments suggested that some residues in TM11 regulate pHi sensitivity. In addition, it was noted that TM11 contains a phylogenetically conserved Gly-rich segment (see Fig. 1B). These findings prompted us to study the functions of amino acid residues within TM11 and adjacent intracellular (IL5) and extracellular loops (EL6).

In this study, we found that mutation of Arg440 within IL5 decreases the pHi sensitivity, whereas mutation of Gly residues within the Gly-rich segment of TM11 increases it, suggesting that the region encompassing IL5 and TM11 is critical for the regulatory function of NHE1. This is the first identification of important residues involved in pHi sensing within the transmembrane domain of NHE1.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Biotin maleimide was purchased from Molecular Probes Inc. MTSET and streptavidin-conjugated agarose were purchased from Toronto Research Chemicals Inc. and Pierce, respectively. Amiloride derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was a gift from the New Drug Research Laboratories of Kanebo, Ltd. (Osaka, Japan). 22NaCl was purchased from PerkinElmer Life Sciences. An NHE1-specific polyclonal antibody (RP-cd) was raised as described previously (8). All other chemicals were of the highest purity available.

Cells, Culture Conditions, and Stable Expression-- An Na+/H+ exchanger-deficient cell line (PS120) (23) and corresponding transfectants were maintained in Dulbecco' s modified Eagle' s medium (Invitrogen) containing 25 mM NaHCO3 and supplemented with 7.5% (v/v) fetal calf serum, penicillin (50 units/ml), and streptomycin (50 µg/ml). The cells were maintained at 37 °C in the presence of 5% CO2. PS120 cells (5 × 105 cells/100-mm dish) were transfected with each plasmid construct (20 µg) by the calcium phosphate co-precipitation technique. Cell populations that stably express mutant NHE1 were selected by the "H+ killing" procedure as described (18).

Construction of Na+/H+ Exchanger Mutants-- A plasmid carrying cDNA coding for the Na+/H+ exchanger (NHE1 human isoform) containing some unique restriction sites cloned into mammalian expression vector pECE was described previously (18). A cDNA construct for Cys-less NHE1 devoid of all endogenous cysteine residues was also described previously (20). For construction of point mutant cDNAs, we used a PCR-based strategy as described previously (20), using two template plasmids coding for the wild-type or Cys-less NHE1. Briefly, using synthesized sense and antisense primers containing appropriate mutations, DNA fragments were generated by PCR. These fragments were digested and inserted into appropriate sites on the wild-type or Cys-less NHE1 plasmid. The DNA sequences of the PCR fragments were confirmed with a PerkinElmer Life Sciences model 373S autosequencer. We used the prefix "cl-" for point mutants produced from Cys-less NHE1 as a template.

Labeling with Biotin Maleimide-- Biotin maleimide labeling of NHE1 mutant molecules was carried out as described previously (20). Briefly, confluent cells cultured on 60-mm dishes were washed twice with 5 ml of PBSCM (phosphate-buffered saline containing 0.1 mM CaCl2 and 1 mM MgCl2, pH 7.2) and then incubated in 1 ml of PBSCM containing 0 or 5 mM MTSET for 30 min at room temperature. The cells were washed twice with 5 ml of PBSCM and then incubated in 1 ml of PBSCM containing 0.5 mM biotin maleimide (100 mM stock in Me2SO) for 30 min at room temperature. The cells were washed once with PBSCM containing 1% 2-mercaptoethanol, once with PBSCM, and then collected in the tube by centrifugation. The cells were solubilized with 1 ml of lysis buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Hepes/Tris (pH 7.4), 1 mM phosphate-buffered saline, and 1 mM benzamidine. After centrifugation for 10 min at 15,000 rpm, the supernatant was mixed with streptavidin-agarose beads (30 µl of resin), followed by incubation for 1 h at 4 °C with mild agitation. The agarose beads were washed more than five times with 1 ml of lysis buffer, mixed with 50 µl of SDS-PAGE sample buffer containing 3% SDS, and then boiled for 10 min at 100 °C. The proteins were separated on an 8.5% gel by SDS-PAGE and analyzed by immunoblotting with the NHE1 antibody as described previously (20). The blots were developed with an ECL detection system (Amersham Biosciences). When indicated, the cells were permeabilized with digitonin (50 µg/ml) or streptolysine O (Sigma) immediately before biotinylation, as described previously (20).

Measurement of 22Na+ Uptake-- 22Na+ uptake activity and its pHi dependence were measured by the K+/nigericin pHi clamp method (19). Serum-depleted cells in 24-well dishes were preincubated for 30 min at 37 °C in Na+-free choline chloride/KCl medium containing 20 mM Hepes/Tris (pH 7.4), 1.2-140 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 5 µM nigericin. In some experiments (see Fig. 6), the preincubation solution contained 2 µg/ml oligomycin and 5 mM 2-deoxyglucose instead of glucose. 22Na+ uptake was started by adding the same choline chloride/KCl solution containing 22NaCl (37 kBq/ml) (final concentration, 1 mM), 1 mM ouabain, and 100 µM bumetanide. In some wells, the uptake solution contained 0.1 mM EIPA. After 1 min, the cells were rapidly washed four times with ice-cold phosphate-buffered saline to terminate 22Na+ uptake. pHi was calculated from the imposed [K+] gradient by assuming the equilibrium [K+]i/[K+]o = [H+]i/[H+]o and an intracellular [K+] of 120 mM. The data were normalized as to protein concentration, which was measured by bicinchoninic assay system (Pierce) using bovine serum albumin as a standard.

Measurement of Changes in pHi-- The changes in pHi induced by various extracellular agents were measured by the [14C]benzoic acid equilibration method, as described previously (18).

Statistics-- The data for the pH dependence of 22Na+ uptake were simulated by fitting the values to the sigmoidal dose-response equation, the rate of EIPA-sensitive 22Na+ uptake = Vmax/(1 + 10^(log(pK - pHi)n)) (Vmax, the maximal rate of 22Na+ uptake; pK, pHi giving half-maximal 22Na+ uptake; n, Hill coefficient), with the aid of a simulation program included in the graphing software Graphpad Prizm (Microsoft Corp.). The concentration dependences of extracellular Na+, pH, and EIPA for 22Na+ uptake were also fitted to steady-state kinetic equations with the aid of the same simulation program. Kinetic parameters are expressed as the best fitted values with standard errors, whereas other data are expressed as the means ± S.D. for at least three determinations.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Precise Topology of the Putative 11th Transmembrane Domain and Neighboring Loops of NHE1-- Based on the results of cysteine accessibility analysis, we previously presented a novel topology model of NHE1, as shown in Fig. 1A (20). Here, to obtain more precise topological information of the regions surrounding TM11, we introduced single Cys residues into these regions of Cys-less NHE1 and expressed mutants in the exchanger-deficient cells PS120. All of the mutant exchangers except cl-K438C, cl-R440C, cl-I451C, cl-G456C, and cl-R458C were well expressed in PS120 cells (Fig. 2A; also see Fig. 7 of Ref. 20). On SDS-PAGE, we observed two forms of exchanger protein with high (~110 kDa) and low (~90 kDa) molecular masses, which are thought to be a mature protein with N- and O-linked glycosylation and an immature protein containing only N-linked high mannose oligosaccharide, respectively (24). The cells were then incubated with 0.5 mM biotin maleimide after preincubation with or without a membrane-impermeable MTSET, and biotinylated proteins were subsequently recovered with streptavidin-agarose, followed by immunoblot analysis with an anti-NHE1 to visualize biotinylated exchangers (Fig. 2B).


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Fig. 1.   Topology of NHE1 and sequence alignment of TM11. A, left side, a topological model of NHE1 based on our previous cysteine accessibility analysis (20). Right side, precise topologies of IL5, TMs 11 and 12, and EL6 revealed in this study. The positions examined for cysteine accessibility are represented by a single-letter amino acid code. Black and gray circles represent residues accessible to external and internal SH probes, respectively, whereas white circles represent residues not or only faintly labeled by biotin maleimide (see Fig. 2). White squares represent residues (Lys438, Arg440, Ile451, Gly456, and Arg458) not studied here because cells expressing mutants could not be obtained. B, sequence alignment of TM11 and its neighboring regions in the exchangers from different species. GenBankTM accession numbers for these proteins are as follows: human NHE1, P19634; rat NHE2, NP_036785; rat NHE3, NP_036786; rat NHE4, P26434; human NHE5, NP_004585; human NHE6, NP_006350; human NHE7, NP_115980; mouse NHE8, NP_683731; trout beta NHE, A46188; green crab NHE, AAC26968; Caenorhabditis elegans NHE, NP_509830; Saccharomyces cerevisiae NHX1, NP_010744; cynechobacterium (Synechocystis sp.) Syn-NhaP, NP_441245; Pseudomonas aeruginosa NhaP, NP_252576; and Bacillus subtilis NhaG, BAA89487.


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Fig. 2.   Biotinylation of neighboring regions of TM11. Cells expressing single cysteine mutants of Cys-less NHE1 were treated with or without 5 mM MTSET. Then cells were incubated with 0.5 mM biotin maleimide, solubilized with lysis buffer, and treated with streptavidin-agarose as described under "Experimental Procedures." A total cell lysate (20 µg) (A) or the proteins recovered with streptavidin-agarose (B and C) were separated by SDS-PAGE, and the proteins were visualized by immunoblot analysis with an NHE1 antibody. In C, cells were permeabilized with digitonin before biotinylation also as described under "Experimental Procedures."

Mutant exchangers cl-L468C, cl-L469C, cl-D470C, cl-K471C, cl-K472C, cl-H473C, cl-F474C, cl-P475C, and cl-M476C with high molecular mass were efficiently labeled with biotin maleimide (Fig. 2B; not shown for cl-K471C, but see Ref. 20). cl-E151C, used as a positive control, was also labeled, whereas Cys-less NHE1 was not (data not shown; see Ref. 20). The biotinylation of mutant exchangers was significantly inhibited by pretreatment with membrane-impermeable MTSET (Fig. 2B), suggesting that these residues face the outside. On the other hand, cl-G466C, cl-D478C, and Cys477* (native cysteine) were not labeled with biotin maleimide, and cl-Y467C was only faintly labeled (Fig. 2B). In contrast, cl-N437C, cl-I441C, cl-V442C, cl-K443C, cl-K447C, and cl-D448C were labeled with biotin maleimide only when the cells were permeabilized with digitonin (Fig. 2C; data not shown for some mutants) or streptolysine O (see Ref. 20), suggesting that these positions face the inside. Some Cys mutants, i.e. cl-F439C, cl-L444C cl-T445C, cl-P446C, and cl-Q449C, were only faintly labeled even when cells were permeabilized with digitonin (Fig. 2C; data not shown for cl-T445C and cl-P446C). We found that introduced cysteines at Phe450, Ile452, Ala453, Tyr454, Gly455, Leu457, Gly459, Ala460, Ile461, Ala462, Phe463, Ser464, and Leu465 were not labeled with biotin maleimide in either intact or permeabilized cells (data not shown; see Ref. 20). Based on these results, we propose that: (i) the putative TM11 consists of 18 residues from Gln449 to Tyr467, (ii) the putative TM12 starts from Cys477, (iii) the region from Leu468 to Met476 forms the extracellular loop (EL6), (iv) the region from Asn437 to Asp448 forms part of the intracellular loop (IL5), and (v) some residues in IL5 may not be completely exposed to the cytosol. These topological data would be useful for the functional study of the exchanger in which residues in TM11 and its surrounding regions are mutated (see below).

Identification of Residues Modifying pHi Sensitivity of NHE1-- We introduced single Cys residues into the region covering most of IL5 and TM11 in the wild-type NHE1. In the wild-type background, R440C was well expressed in PS120 cells, but K438C, I451C, Y454C, and R458C were not. All Cys mutants expressed well in cells exhibited high EIPA-sensitive 22Na+ uptake activity when cells were acidified. We found that substitution of Arg440 with Cys greatly shifted the pHi dependence of 22Na+ uptake to an acidic side with the pK of less than 6.2 (Fig. 3A). We could not determine the precise pK value for this mutant because of incomplete attainment of the maximal activity. On the other hand, mutations at other neighboring residues in IL5 did not significantly affect the pHi dependence (Fig. 3B). Intriguingly, unlike the Arg440 mutation, mutations of Gly455 and Gly456 in TM11 significantly shifted the pHi dependence of 22Na+ uptake to an alkaline side, respectively (Fig. 3C). The pK values increased by ~0.3 pH units with these mutations. When these experiments were repeated more than three times, the pK values of mutant exchangers other than R440C, G455C, and G456C were in the range of 6.5-6.8, which is not very different from that of the wild-type NHE1 (Fig. 3D). Thus, we identified Arg440 in IL5 and Gly455/Gly456 in TM11 as residues greatly influencing the pHi sensitivity of NHE1.


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Fig. 3.   pHi dependence of exchange activity in cells expressing various NHE1 cysteine mutants. A-C, pHi dependence of 22Na+ uptake was measured in cells expressing the wild-type or cysteine mutant exchangers in the presence or absence of 0.1 mM EIPA. pHi was clamped at various values with K+/nigericin. The wild-type or mutant exchangers exhibited high 22Na+ uptake activities (15-50 nmol/mg/min at pHi 5.4). The data were fitted to the sigmoidal dose dependence equation as described under "Experimental Procedures." In B and C, the data were normalized to the maximal uptake activity. D, pK values obtained by curve fitting. The bars represent the standard errors given by the curve fitting program, whereas the numbers represent Hill coefficients.

We substituted Arg440 and Gly455 with other residues and analyzed the functional consequences of these mutations. Fig. 4 shows the pHi dependence of 22Na+ uptake in cells expressing R440D or R440K. Amino acid substitution of Arg440 with Asp greatly shifted the pHi dependence to an acidic side. In the inset to Fig. 4, we present the ratios between the uptake activities of various substitution mutants of Arg440 at pHi 6.8 and 5.4, in place of the respective pHi dependences. The ratio decreased significantly when Arg440 was substituted with Lys, His, Asp, Glu, or Leu, indicating the acidic shift in the pHi dependence. It is notable that the effect of the Arg440 to Lys substitution on the pHi dependence was modest (pK = ~6.4) compared with the substitution with other residues (Fig. 4). Thus, the positive charge of Arg440 appears to be important for the normal pHi sensitivity.


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Fig. 4.   Effect of amino acid substitution at Arg440 on pHi dependence of exchange activity. pHi dependences of EIPA-sensitive 22Na+ uptake were compared among cells expressing the wild-type NHE1, R440K, and R440D. Inset, ratios between activities of various substitution mutants of Arg440 at pHi 6.8 and 5.4 are presented. The cells expressing these exchangers exhibited high 22Na+ uptake activities (20-45 nmol/mg/min at pHi = 5.4). The data are the means ± S.D. of three determinations.

Fig. 5 (A and B) shows the results of amino acid substitution at Gly455. Substitution of Gly455 to Gln, Thr, or Val significantly shifted the pHi dependence of 22Na+ uptake to an alkaline side, whereas substitution to Ala, Asp, Asn, or Ser did not shift or only modestly shifted. We plotted the pK values against the surface area or volume of substituted amino acid side chains. The data suggest that the pK increases when Gly455 is substituted with bulky residues (Fig. 5C).


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Fig. 5.   Effect of amino acid substitution at Gly455 on pHi dependence of exchange activity. A, pHi dependence of 22Na+ uptake was measured in cells expressing the wild-type or cysteine mutant exchangers in the presence or absence of 0.1 mM EIPA. The wild-type or mutant exchangers exhibited high 22Na+ uptake activities (20-45 nmol/mg/min at pHi = 5.4). B, pK values were obtained by curve fitting as described under "Experimental Procedures." The bars represent the standard errors given by the curve fitting program, whereas the numbers represent Hill coefficients. C, pK values were plotted against the surface area (43) or volume (44) of substituted amino acid side chains.

Mutations of Arg440 and Gly455 Do Not Change the Apparent Affinities for Extracellular Na+, H+, and EIPA-- We analyzed several other kinetic parameters of Na+/H+ exchange in cells expressing R440D and G455Q that exhibited acidic and alkaline pHi dependence of 22Na+ uptake, respectively. Fig. 6 (A-C) shows the extracellular Na+ concentration dependence of EIPA-sensitive 22Na+ uptake. The Km values for Na+ estimated from double reciprocal plots (Fig. 6, A-C, insets) were similar for the wild-type NHE1 (9.22 ± 1.04 mM), R440D (9.30 ± 0.90 mM), and G455Q (14.08 ± 1.75 mM). The dependences of 22Na+ uptake on extracellular pH (pHo) and on the EIPA concentration were also similar for the wild-type NHE1, R440D, and G455Q (pK for pHo, 7.29 ± 0.03, 7.50 ± 0.08, and 7.38 ± 0.02; IC50 for EIPA, 106.3 ± 7.3, 91.2 ± 0.1, and 85.0 ± 8.0 nM, respectively). The data suggest that mutations of Arg440 and Gly455 only affect the pHi dependence of exchange.


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Fig. 6.   Comparison of kinetic properties of wild-type and mutant exchangers. A-C, EIPA-sensitive 22Na+ uptake in cells expressing the wild-type NHE1, R440D, or G455Q was measured as a function of Na+ concentration. Insets, double reciprocal plots of exchange activity versus Na+ concentration. D and E, EIPA-sensitive 22Na+ uptake in cells expressing the wild-type NHE1, R440D, or G455Q was measured as a function of extracellular pH or EIPA concentration.

Effects of Mutations of Arg440 and Gly455 on Some NHE Regulatory Functions-- Cellular ATP depletion is known to inhibit the exchange activity by reducing the pHi sensitivity and/or maximal activity (18, 25-28). Fig. 7 (A and B) shows the effect of ATP depletion on the pHi dependence of 22Na+ uptake in cells expressing R440D or G455Q. Interestingly, ATP depletion did not change the pHi sensitivity in cells expressing R440D (Fig. 7A), although it caused ~50% reduction of Vmax (data not shown). In contrast, ATP depletion greatly shifted the pHi dependence of cells expressing G455Q to an acidic side without a change in Vmax. However, it is notable that even under ATP depletion G455Q exhibited more alkaline pHi dependence of 22Na+ uptake (pK, ~6.4) compared with the wild type (pK, ~6.0).


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Fig. 7.   Effect of ATP depletion on pHi dependence of exchange activity in cells expressing R440D or G455Q. A and B, pHi dependence of EIPA-sensitive 22Na+ uptake was measured under control (open circles) or ATP-depleted conditions (closed circles) in cells expressing R440D or G455Q, respectively. In each panel, exchange activities are normalized to the ones measured at pHi of 5.4.

Finally, we measured the changes in pHi caused by various extracellular stimuli. In cells expressing the wild-type NHE1 and G455Q, we detected cytoplasmic alkalinization induced by thrombin, platelet-derived growth factor BB, hyperosmotic stress (sucrose), phorbol 12-myristate 13-acetate, lysophosphatidic acid, and the recently described activator Li+ (29), indicating that all of these stimuli are capable of activating exchangers (Fig. 8). Of note, the extent of cytoplasmic alkalinization caused by hyperosmotic stress or Li+ was reduced in cells expressing G455Q. In contrast, such alkalinization by extracellular stimuli was not observed in cells expressing R440D. Lack of alkalinization was presumably due to the acidic shift of the pHi dependence of activity of R440D.


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Fig. 8.   Extracellular stimuli-induced changes in pHi in cells expressing the wild-type and mutant exchangers. Changes in pHi were measured as described under "Experimental Procedures" using the [14C]benzoic acid equilibration method. The cells expressing the wild type, G455Q, or R440D were stimulated for 15 min at 37 °C with 2 units/ml thrombin, 10 ng/ml platelet-derived growth factor (PDGF) BB, 200 mM sucrose, 1 µM phorbol 12-myristate 13-acetate (PMA), 10 µg/ml lysophosphatidic acid, or 140 mM Li+. For stimulation with Li+, the cells were transferred from Hepes-buffered Dulbecco' s modified Eagle' s medium to medium containing 140 mM LiCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2. and 20 mM Hepes/Tris (pH 7.0).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, after obtaining precise information on the topology of the portion of NHE1 encompassing TM11 and its surrounding regions, we performed scanning mutagenesis analysis of these regions to search for residues possibly involved in the regulation of pHi dependence of Na+/H+ exchange. We found that mutation at Arg440 of IL5 of NHE1 greatly shifted pHi dependence of EIPA-sensitive 22Na+ uptake to an acidic side, whereas mutation at Gly455 or Gly456 of TM11 shifted it to an alkaline side. Mutation of many other residues in IL5 and TM11, in which it did not impair exchanger expression in the plasma membrane, did not alter the pHi dependence. Furthermore, these arginine and glycine mutations did not alter the apparent affinities of NHE1 for external transport substrates Na+ and H+ and the inhibitor EIPA. We observed similar opposite shifts of the pHi dependence with these NHE1 mutants when we measured the reverse mode of Na+/H+ exchange, i.e. EIPA-sensitive 22Na+ efflux from 22Na-loaded cells,2 supporting the possibility that the mutations alter the interaction of intracellular protons with the H+ modifier site rather than the transport site. It thus appears likely that these single mutations exert rather specific effects on the pHi sensor of NHE1.

In addition to the effect on pHi dependence of basal Na+/H+ exchange, mutation of Arg440 abolished the ATP depletion-induced acidic shift of pHi dependence of exchange activity as well as the cytoplasmic alkalinization in response to various extracellular stimuli. We previously reported (18) that deletion of the entire C-terminal cytoplasmic domain of NHE1 causes a large acidic shift of pHi dependence of basal exchange activity and the disappearance of growth factor-induced cytoplasmic alkalinization. The same deletion was also found to abolish the ATP depletion-induced acidic shift of pHi dependence of exchange activity. We subsequently provided evidence that these effects occur because of deletion of the N-terminal portion of the cytoplasmic domain of NHE1 named subdomain I (amino acids 503-600) (19). Thus, the effects caused by deletion of subdomain I and single mutation of Arg440, which is localized in the internal loop (IL5) connecting TMs 10 and 11, are very similar except that in the latter the Vmax of exchange activity is somewhat reduced by ATP depletion.

Recently, we have reported that the calcineurin B homologous protein CHP is an essential co-factor for the expression of high physiological levels of exchange activity in many plasma membrane NHE isoforms (6). We showed that CHP is tightly bound to a short juxtamembrane segment of the exchanger (amino acids 510-530 of subdomain I in the case of NHE1) (6). Interestingly, in addition to the large reduction of Vmax, CHP binding-defective NHE1 mutants exhibited a marked shift of the pHi dependence to an acidic side.3 Thus, the mutation at Arg440, deletion of subdomain I, and the CHP binding-defective mutation all caused the same effect, i.e. a marked shift of the pHi dependence to the acidic side, suggesting that the region of IL5 containing Arg440 and the CHP-subdomain I complex are both involved in the preservation of physiological pHi sensitivity of the exchanger. In this study, we observed that pHi dependence of exchange activity did not change extensively by substitution of Arg440 with similarly charged but somewhat more bulky Lys residue, unlike substitutions with other residues (Cys, His, Asp, Glu, and Leu). This suggests that the positive charge of Arg440 is important. An attractive hypothesis would be that Arg440 interacts with negatively charged residue(s) localized in the CHP-subdomain I complex and that its substitution with other residues or cell ATP depletion results in the disruption of such interaction, thereby altering pHi sensitivity of the exchanger. Consistent with this hypothesis, there is a cluster of acidic amino acid residues on the side opposite to one where CHP binds to NHE1 in a predicted model of CHP structure.2

As opposed to the mutation of Arg440 in IL5, mutation of Gly455 or Gly456 in the glycine-rich region within TM11 shifted the pHi dependence of basal exchange activity to an alkaline side. Furthermore, in the Gly455 mutant, ATP depletion was able to induce a large acidic shift of the pHi dependence without a change in Vmax, a pattern mimicking that of the wild-type NHE1, although the pHi dependence itself was shifted to the alkaline side under these conditions (Fig. 7B). In the same mutant, we observed normal cytoplasmic alkalinization in response to growth factors (Fig. 8). Thus, the underlying mechanisms for the effects of these Gly mutations and that of the Arg440 mutation in IL5 are likely to be different.

The glycine-rich region in TM11 of NHE1 is remarkably conserved in different types of NHEs from bacteria to mammals (Fig. 1B), suggesting its importance for the NHE function. Glycine has been suggested to serve as a molecular hinge permitting the occurrence of conformational changes of intramembrane helices in some proteins (30, 31) or as a notch for orienting multiple helices at the point of closest packing between membrane helices in several polytopic membrane proteins (32). In this study, we observed a clear tendency showing that substitution of Gly455 with more bulky residues are more effective in causing an alkaline shift of pHi dependence of exchange activity (Fig. 5C), which indicates that small residues are required at the position of Gly455 for normal pHi sensitivity. This appears to be in line with the fact that some isoforms such as NHEs 4, 6, 7, and 8 have small residues such as alanine and serine at this position (Fig. 1B). We previously found that single cysteine substitutions at Tyr454 and Arg458, both being close to Gly455 or Gly456 in TM11, cause the retention of NHE1 protein in the endoplasmic reticulum (22), suggesting that these mutations impair proper protein folding. On the other hand, mutation of Gly455 neither prevented the plasma membrane expression of NHE1 protein nor appeared to alter other transport functions of the exchanger significantly. Based on all these pieces of information, we speculate that a relatively mild change in helix packing interaction of TM 11 with other transmembrane helice(s) was induced by the mutation at Gly455 or Gly456, which indirectly modified the conformation and function of the putative H+-regulatory site(s). It is noteworthy that in Escherichia coli antiporter NhaA distantly related to mammalian NHEs, mutation of Gly338 in the glycine-rich region of TM11 was reported to render the antiporter active in the wide range of pH, i.e. practically independent of the pH level between pH 6 and 9 (33).

Although the present and previous results suggest important roles of IL5, TM11, and the subdomain I-CHP complex in the modulation of pHi sensing in NHE1, little is known about structures of the ion transport sites and the putative pH sensor and their interaction. In our previous study of membrane topology of NHE1, we found re-entrant loop-like structures between TMs 4 and 5, between TMs 8 and 9, and between TMs 9 and 10 (20). In addition, both TM4 and TM9 have been shown to be important for the interaction of NHE1 with amiloride analogue inhibitors that compete with the transport substrate Na+ (34-38). Furthermore, other studies identified a few polar amino acid residues required for ion exchange within transmembrane segments in the mammalian NHE1 (38, 39) as well as in the Na+/H+ antiporters from E. coli (40, 41) and Vibrio alginolyticus (42). Much further study is required for the elucidation of the structural aspect of Na+/H+ exchange.

In summary, we have presented evidence that Arg440 in IL5 and Gly455 or Gly456 in TM11 are important residues that oppositely regulate the pHi sensitivity of NHE1. This is the first identification of critical residues involved in pHi sensing within the N-terminal transmembrane domain of NHE1.

    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, by a grant from the Organization of Pharmaceutical Safety and Research of Japan (Promotion of Fundamental Studies in Health Science), and by Health and 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M213243200

2 S. Wakabayashi, unpublished observations.

3 T. Pang, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: NHE, Na+/H+ exchanger; pHi, intracellular pH; TM, transmembrane domain; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; IL, intracellular loop; EL, extracellular loop; CHP, calcineurin B homologous protein; MTSET, 2-trimethylammoniumethyl methanethiosulfonate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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