INTRODUCTION

The plasma membrane Na⁺/H⁺ exchanger (NHE)¹ participates in the control of intracellular pH (pH_i) and maintenance of cellular volume, transepithelial Na⁺ reabsorption, and the cell proliferation response to growth factors (1). To date, five distinct isoforms (NHE1 to NHE5) have been isolated (2, 3, 4, 5, 6, 7, 8). They share ~40-70% amino acid identity (M_r ranging from ~81-93 kDa) and exhibit similar membrane topologies, with 10-12 predicted N-terminal membrane-spanning domains and a large C-terminal cytoplasmic region. They also show differences in their patterns of tissue expression, membrane localization, biochemical and pharmacological characteristics, and physiological functions (reviewed in Refs. 9 and 10).

The NHE is a known target for inhibition by the diuretic compound amiloride and its analogues (11). Amiloride analogues containing hydrophobic substituents on the 5-amino group of the pyrazine ring have higher affinity and specificity for NHE relative to other ion transporters. Using a heterologous expression system, the NHE isoforms exhibit a wide range of affinities for amiloride and its analogues that span over 2 orders of magnitude and show the following order of sensitivity: NHE1 > NHE2  NHE3 (12, 13). Recently, new benzoyl guanidinium antagonists of NHE activity, (3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE694) and its related compound HOE642, have also been found to inhibit the isoforms with a similar rank order as the amiloride compounds but over a larger concentration range (3-4 orders of magnitude) (14, 15). The more selective binding properties of these compounds have been exploited therapeutically as more effective agents in the treatment of cardiac ischemia and reperfusion injuries (15, 16, 17). Other pharmacological agents such as cimetidine, clonidine, and harmaline also exhibit differential affinities for the NHE isoforms (12, 13). While these compounds are chemically unrelated to amiloride or HOE694, they possess either an imidazoline or guanidinium moiety and hence bear some structural similarity to these compounds.

Biochemical analyses indicate that inhibition by amiloride compounds (18, 19, 20, 21), cimetidine (22), and HOE694 (14) is reduced by high external Na⁺. This competitive inhibition indicates they bind near the external Na⁺ transport site and may also share a common site. However, under different anionic buffer conditions, amiloride and its derivatives also inhibit transport noncompetitively, suggesting that the external Na⁺ and amiloride binding sites may not be identical (23, 24). Furthermore, the extracellular Na⁺- and amiloride-binding sites can be altered independently of each other using genetic selection techniques (25). Overall, these data indicate that amiloride and possibly other antagonists may bind to multiple regions of the exchanger.

Recent molecular studies have shown that predicted membrane-associated domains of NHE1 are targets for interaction with amiloride and its analogues. Residues in the fourth (Phe¹⁶⁵ and Leu¹⁶⁷) and the ninth (His³⁴⁹) transmembrane domains appear to contribute to amiloride sensitivity without affecting Na⁺ affinity (26, 27). However, mutations at these sites produce only modest changes in drug sensitivity and do not confer the full degree of resistance observed for NHE3, implicating additional residues in amiloride binding. Subsequent analysis of chimeric proteins of NHE1 and NHE3 has confirmed that all the molecular determinants for isoform-specific drug sensitivities reside within the N-terminal transmembranous regions, although their precise locations have yet to be defined (28).

The objective of this study was to conduct a more systematic analysis of putative membrane-spanning segments of the NHE that may contribute to drug recognition. To accomplish this, we exploited the striking pharmacological differences and extensive sequence homology in the N-terminal transmembranous regions between NHE1 and NHE3 to create chimeric exchangers. Unique restriction endonuclease sites were engineered into the N-terminal transmembranous region of each cDNA by site-directed mutagenesis, and chimeric cDNAs were constructed by exchanging homologous segments. The specific drug sensitivity profiles of each chimera were then determined in stably transfected cells and compared with their parental exchangers.

EXPERIMENTAL PROCEDURES

Carrier-free ²²NaCl (radioactivity, 5 mCi/ml) was obtained from DuPont NEN. Amiloride, cimetidine, and ouabain were purchased from Sigma, and the amiloride derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was obtained from Molecular Probes (Eugene, OR). HOE694 was generously provided by Dr. Hans J. Lang (Hoechst AG, Frankfurt am Main, Germany). -Minimal essential medium, fetal bovine serum, kanamycin sulfate, and trypsin-EDTA were purchased from Life Technologies, Inc. Cell culture dishes and flasks were purchased from Becton Dickinson and Co. (Montréal, Québec). All other chemicals and reagents used in these experiments were purchased from British Drug House (St. Laurent, Québec) or Fisher Scientific and were of the highest grade available.

Complementary DNA fragments of rat NHE1 (NotI-NsiI fragment, nucleotides 761 to +3820) and NHE3 (KpnI-SmaI fragment, nucleotides 44 to +4288) were initially subcloned into the mammalian expression plasmid pCMV and called pCMV/NHE1 and pCMV/NHE3, respectively, as described previously (12). To facilitate construction of NHE chimeras, it was necessary to remove parts of the 5- and/or 3-untranslated regions of NHE1 and NHE3 in order to create a number of useful single restriction endonuclease sites. For NHE1, nucleotides 761 to 42 in the 5-untranslated region were removed by polymerase chain reaction mutagenesis. As well, nucleotides +3454 to +3820 in the 3-untranslated region and part of the pCMV polylinker region were eliminated by removing an XbaI-XbaI fragment. For NHE3, nucleotides +2950 to +4288 in the 3-untranslated region and part of the polylinker were eliminated by removing an NsiI-NsiI fragment. Unique SpeI and BstBI sites were also eliminated from pCMV and NHE1, respectively, by site-directed mutagenesis (29). The removal of a unique BstBI site in NHE1 (nucleotide position +266 relative to the ATG start codon) did not change the amino acid sequence. The modifications resulted in both cDNAs being flanked by single HindIII and XbaI sites at the 5- and 3-ends, respectively. In addition, several unique restriction endonuclease sites (AgeI, BstBI, SpeI, NheI, and EagI) were engineered simultaneously into each cDNA at analogous positions in the N-terminal transmembranous regions by site-directed mutagenesis. In NHE1, the sites for AgeI, BstBI, SpeI, NheI, and EagI were created at amino acid positions 208, 281, 327, 391, and 504, respectively, whereas in NHE3, these sites were inserted at positions 155, 228, 276, 340, and 454, respectively. For the most part, generation of each site either preserved the native amino acid sequence or resulted in substitution of an amino acid present in one isoform with the homologous residue found in the other isoform (i.e. for NHE1, L208T (AgeI) and S391A (NheI); for NHE3, K277S (SpeI) and K454R (EagI)). The modified cDNAs were completely sequenced to ensure that other random mutations were not introduced during the mutagenesis procedure. These modified plasmids were labeled pNHE1 (E1) and pNHE3 (E3), respectively. Chimeric NHEs were then constructed by exchanging homologous domains within the entire N-terminal transmembranous regions of E1 and E3. The isoform composition of each chimera was further verified by either polymerase chain reaction analysis and Southern blotting using isoform-specific oligonucleotides or DNA sequencing.

Chemically mutagenized Chinese hamster ovary cells (AP-1) devoid of endogenous NHE activity (30) were transfected with plasmids containing the various NHE constructs by the calcium phosphate-DNA coprecipitation technique of Chen and Okayama (31). Starting 48 h after transfection, the AP-1 cells were selected for survival in response to repeated (5-6 times over a 2-week period) acute NH₄Cl-induced acid loads (i.e. H⁺-suicide technique) (12, 32) in order to discriminate between Na⁺/H⁺ exchanger positive and negative transfectants.

The cells were grown to confluence in 24-well plates. NHE activity was determined by preloading the cells with H⁺ using the NH₄Cl technique (33) and then measuring the initial rates of ²²Na⁺ influx essentially as described (12). Briefly, the cell culture medium was aspirated and replaced by isotonic NH₄Cl medium (50 mM NH₄Cl, 70 mM choline chloride, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, 20 mM HEPES-Tris, pH 7.4). Cells were incubated in this medium for 30 min at 37 °C in a nominally CO₂-free atmosphere. After acid loading, the monolayers were rapidly washed twice with isotonic choline chloride solution (125 mM choline chloride, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, 20 mM HEPES-Tris, pH 7.4). ²²Na⁺ influx assays were initiated by incubating the cells in isotonic choline chloride solution containing 1 mM ouabain and 1 µCi of ²²NaCl (carrier-free)/ml. The assay medium was K⁺-free and included ouabain to prevent the transport of ²²Na⁺ by Na⁺-K⁺-Cl cotransporter and Na⁺,K⁺-ATPase, respectively. The influx of ²²Na⁺ was terminated by rapidly washing the cell monolayers four times with 4 volumes of ice-cold isotonic saline solution (130 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, 20 mM HEPES-NaOH, pH 7.4). The washed cell monolayers were solubilized in 0.5 ml of 0.5 N NaOH, and the wells were washed with 0.5 ml of 0.5 N HCl. Both the solubilized cell extract and wash solutions were added to vials, and radioactivity was assayed using a liquid scintillation counter. Under the conditions of H⁺ loading used in this study, uptake of ²²Na⁺ was linear with time for 8-10 min (at low Na⁺ concentrations at 22 °C). Therefore, ²²Na⁺ uptakes were measured after 5 min except when examining the kinetics of NHE activity as a function of extracellular Na⁺ concentration. We found that ²²Na⁺ influx was linear with time for only 4 min when [Na⁺]_o was increased to 40 mM. Hence, an uptake time of 1 min was chosen for this set of experiments (i.e. when the extracellular Na⁺ concentration ranged from 1.25 to 40 mM). Measurements of ²²Na⁺ influx specific to the Na⁺/H⁺ exchanger were determined as the difference between the initial rates of H⁺-activated ²²Na⁺ influx in the absence and presence of 1 mM amiloride (a concentration sufficient to inhibit NHE1 or NHE3 under these experimental conditions) and expressed as amiloride-inhibitable ²²Na⁺ influx. Protein content was determined using the Bio-Rad DC protein assay procedure. All results represent the average of at least two or three experiments.

RESULTS

To systematically evaluate structural domains responsible for drug sensitivity, we took advantage of the strong sequence homology and large difference in affinities (2-3 orders of magnitude) between NHE1 and NHE3 for various pharmacological antagonists (summarized in Fig. 1) (12, 14). Nucleotides at homologous positions in the N-terminal transmembranous regions of the NHE1 and NHE3 cDNAs were substituted by site-directed mutagenesis to create several unique restriction endonuclease sites. These changes either preserved the native amino acid sequence or resulted in substitution of an existing amino acid with the corresponding residue found at the homologous position of the other isoform. These modified exchangers were named E1 and E3 (illustrated in Fig. 2) to distinguish them from their respective NHE1 and NHE3 wild types.

Most E1 and E3 chimeras were constructed by substituting segments of E3 with homologous regions of E1 (Fig. 2), with the expectation that one or more of the resulting chimeras would exhibit increased drug sensitivity relative to E3. The acquisition of sensitivity by E3-dominant chimeras was considered more informative since a ``gain of function'' would more likely result from the transfer of structural components that directly contribute to the drug binding site than would a loss of drug sensitivity by E1, which could result from nonspecific alterations in protein structure. These chimeras were then transfected into Chinese hamster ovary cells devoid of endogenous transporter activity (AP-1) and stably selected for their ability to survive repeated intracellular acid loads. Of the 12 chimeras examined, only five (E3-1HA, E3-1BS, E3-1SN, E3-1BN and E1-3SN) were active. The other chimeras did not confer cell survival in response to repeated intracellular acidifications. The nonfunctional chimeras all contained foreign putative transmembrane (M) domains M6-M7 and/or M10-M12. Although speculative, these regions may be essential for homodimer assembly and targeting to the cell surface (34).

To characterize the drug sensitivity of the functional chimeras, concentration-response experiments were conducted with amiloride, EIPA, HOE694, and cimetidine. The inhibitor concentration profiles for E3, E3-1HA, E3-1BS, E3-1SN, and E1 are shown in Fig. 3, and the values for apparent half-maximal inhibition (K_0.5) of H⁺_i-activated ²²Na⁺ influx are summarized in Table I. All four antagonists inhibited ²²Na⁺ influx into cells expressing E1 to a significantly greater extent than those expressing E3, with the difference in potency being 90-, 445-, 7553-, and 170-fold for amiloride, EIPA, HOE694, and cimetidine, respectively. These results are similar to those previously reported for the unmodified wild type exchangers (see Fig. 1). Comparison of the chimeras revealed that only E3-1SN, which contained a 66-amino acid segment of E1 (composed of the putative fourth cytoplasmic loop, M9 domain, and fifth exoplasmic loop), consistently showed an increase in its sensitivity to inhibition by these compounds. EIPA and HOE694 sensitivities were increased most (57- and 37-fold, respectively), whereas amiloride and cimetidine sensitivities were both increased about 2-fold. The E3-1BS chimera (containing the M8 domain of E1) also showed a 2-fold increase in sensitivity to EIPA but not to the other compounds. The E3-1BN chimera, which contained the M8 and M9 domains of E1, gave similar results to E3-1SN (data not shown). In contrast, E3-1HA showed a small decrease (2.5-fold) in its affinity for cimetidine, suggesting that the region encompassing membrane-spanning domains M1-M5 may also influence the sensitivity to certain drugs.

Table I. Comparison of the inhibition constants of parental and chimeric rat Na+/H+ exchangers for various pharmacological antagonists Values for half-maximal inhibition (K0.5) were determined from the logit transformation of the sigmoidal inhibition data presented in Figs. 3 and 4. The transformation involved plotting the ln(P/(100  P)) (where P represents the percentage of inhibition) as a function of the log(inhibitor). The K0.5 is the concentration when logit = 0. Values represent the mean ± S.D. Chimeras Inhibition constants (K0.5) Amiloride EIPA HOE694 Cimetidine E3 1.3  ± 0.1 × 104 8.5  ± 1.0 × 106 6.4  ± 0.3 × 104 5.6  ± 0.5 × 103 E3-1HA 1.5  ± 0.1 × 104 8.8  ± 0.3 × 106 8.5  ± 0.7 × 104 1.4  ± 0.1 × 102 E3-1BS 1.4  ± 0.1 × 104 3.7  ± 0.2 × 106 8.9  ± 0.4 × 104 3.5  ± 0.2 × 103 E3-1SN 7.5  ± 0.7 × 105 1.5  ± 0.1 × 107 1.8  ± 0.3 × 105 2.7  ± 0.2 × 103 E1-3SN 1.7  ± 0.1 × 104 3.4  ± 0.3 × 106 2.8  ± 0.2 × 104 1.4  ± 0.1 × 103 E1 1.6  ± 0.1 × 106 1.9  ± 0.1 × 108 8.5  ± 0.2 × 108 3.3  ± 0.4 × 105

To verify the importance of the SN region containing M9 and adjacent loops to drug sensitivity, the reciprocal chimera was analyzed (E1-3SN). Substitution of the SN segment of E1 with the homologous region of E3 drastically reduced the sensitivity of the chimera to inhibition by amiloride, EIPA, HOE694, and cimetidine by 108-, 179-, 3341-, and 42-fold, respectively, relative to E1 (Fig. 4 and Table I).

Concentration-response profiles for inhibition of parental and E1-dominant chimeric Na

The rat NHE1 isoform has a 2-fold lower affinity for Na⁺ than does NHE3 (10.0 ± 1.4 and 4.7 ± 0.6 mM, respectively) (12). Since the binding of these compounds can exhibit competitive or mixed competition for Na⁺ under particular conditions, we assessed whether the chimeras exhibited the appropriate kinetic behaviors. To this end, the initial rates of H⁺_i-activated ²²Na⁺ influx were examined in cells expressing functional chimeras at varying extracellular Na⁺ (Na⁺_o) concentrations. As illustrated in Fig. 5, A and B, the velocity of amiloride-inhibitable ²²Na⁺ influx gradually approached saturation with increasing Na⁺_o concentration for parental and chimeric exchangers, consistent with simple Michaelis-Menten kinetics. Analysis of the data using the algorithm of Eadie-Hofstee (V versus V/[S]) (Fig. 5, C and D) yielded straight lines, consistent with preservation of a single Na⁺ binding site in each case. Apparent Na⁺ affinity constants (K_Na) and maximum velocities (V_max) were estimated from the negative slope and y-intercept of these fits, respectively, and are given in Table II. These data show that the modified parental E1 and E3 exchangers retained a 2-fold difference in their apparent affinities for Na⁺ (27.0 ± 1.7 and 12.3 ± 0.9 mM, respectively), although the affinities are somewhat lower than those for unmodified NHE1 and NHE3. By contrast, all the chimeras exhibited a further 2-fold reduction in their Na⁺ affinity. In particular, the reciprocal chimeras E1-3SN and E3-1SN retained their normal Na⁺ transport properties; i.e. they did not show a reversal of their Na⁺ affinities compared with the parental exchangers. The E3-1SN chimera maintained a 2-3-fold higher Na⁺ affinity than the E1-3SN chimera, although both affinities were reduced.

Table II. Comparison of the kinetic parameters of parental and chimeric rat Na+/H+ exchangers Values represent the mean ± S.D. Chimeras Kinetic parameters KNa Vmax mm nmol/min/mg E3 12.3  ± 0.9 444  ± 32 E3-1HA 28.8  ± 4.7 208  ± 34 E3-1BS 32.0  ± 4.5 280  ± 39 E3-1SN 22.4  ± 4.3 50  ± 10 E1-3SN 60.8  ± 12.8 415  ± 87 E1 27.0  ± 1.7 621  ± 38

DISCUSSION

The aim of the present study was to identify domains of the Na⁺/H⁺ exchanger that may be important for drug recognition and binding. In particular, we wished to delineate the segments responsible for the distinct drug sensitivity profiles exhibited by two members of the rat NHE family, NHE1 and NHE3. Previous comparisons of their drug sensitivity profiles demonstrated that NHE1 was substantially more sensitive to inhibition by amiloride, EIPA, HOE694, and cimetidine than NHE3 (12, 14). In the present study, we found that the differential potencies of amiloride, EIPA, HOE694, and cimetidine (90-, 445-, 7553-, and 170-fold, respectively) were preserved when unique restriction endonuclease sites were inserted at equivalent positions in the respective cDNAs. These modified parental exchangers (i.e. E1 and E3) served as the starting point when constructing chimeric proteins by exchange of homologous domains within the putative N-terminal transmembranous region, a region that contains all the elements necessary to confer isoform-specific responsiveness to various antagonists.

Twelve chimeras were constructed, of which only five had functional activity. While the reasons for the lack of activity of some chimeras are uncertain, we noted that all of those with a predominantly E3 primary structure contained E1 segments spanning either M6-M7 (i.e. AB fragment) or M10-M12 (i.e. NE fragment). All of our attempts to study the function of chimeras containing various combinations of these segments were unsuccessful. Moreover, the reciprocal chimeras (i.e. those with predominantly E1 primary structure) were also inactive (data not shown). These data suggest that exchanging either one, or both, of these segments disrupts structural elements that are critical for isoform-specific function. An intriguing possibility is that these regions contain structural elements involved in homodimer assembly and function of the exchanger (34). Construction of additional chimeras through creation of additional unique restriction endonuclease sites may help delineate these elements more precisely and restore function.

Analysis of the active chimeras revealed that all of the molecular determinants responsible for the relative drug insensitivity of E3 compared with E1 are confined to the distal portion of the N-terminal transmembranous region (i.e. M6-M12). This was clearly demonstrated by the chimeric transporter E3-1HA, in which the region encompassing M1-M5 of E3 was replaced with the homologous region of E1. Despite the large substitution, the drug sensitivity of E3-1HA was unaltered relative to E3, except for cimetidine, which showed a minor reduction (2.5-fold) in its potency. Further analysis of chimeras constructed by interchanging segments between M6 and M12 revealed that the SN fragment, which spans 66 amino acids and contains the putative fourth cytoplasmic loop, the M9 domain, and the fifth exoplasmic loop, conferred drug sensitivity that was intermediate between the two parental exchangers. More specifically, substitution of the SN fragment of E3 with the homologous domain of E1 increased the chimera's (E3-1SN) affinity for EIPA and HOE694 by 57- and 37-fold and its affinity for amiloride and cimetidine by 2-fold. The more substantial changes for EIPA compared with amiloride suggest that the 5-amino-substituted moiety of EIPA may interact strongly with amino acid determinants within the SN fragment. This would also explain the enhanced sensitivity to HOE694, which shares closest structural similarity to EIPA (see Fig. 1A). In contrast, the affinities of reciprocal chimera (E1-3SN) for amiloride, EIPA, HOE694, and cimetidine were substantially reduced (108-, 179-, 3341, and 42-fold, respectively) when compared with E1. These drastic reductions in drug sensitivity (relative to the -fold increases in drug sensitivity of E3-1SN), particularly for amiloride and cimetidine, are interpreted as evidence for the transfer of several molecular determinants of E3 that confer resistance to these antagonists, and perhaps for partial disruption of the spatial organization of the drug binding pocket of E1. Taken together, these data indicate that the differential drug sensitivity profiles of E1 and E3 are greatly influenced by a specific region (SN), which contains the M9 domain. They also imply a role for other molecular determinants in a region encompassing M6-M12.

Comparison of the 66 amino acids within the SN segments of E3 and E1 reveals only 50% identity, but the absence of a definitive membrane topology precludes easy identification of candidate residues that mediate differences in drug sensitivity. Precise delineation of these amino acids would require a strategy involving both random and site-specific mutagenesis of NHE1 and the functional selection of mutant exchangers that have increased drug resistance. These studies are currently under way.

Previous molecular studies of NHE mutants demonstrated that drug sensitivity (and possibly drug binding) involves membrane-associated domains that are separated by large distances. Counillon and colleagues (26), using a H⁺-killing selection technique, isolated mutant cells that express an amiloride-resistant variant of the Chinese hamster NHE1 isoform. Sequencing and site-directed mutagenesis of this variant cDNA demonstrated that a single amino acid substitution, Leu¹⁶⁷  Phe, in the putative M4 domain of NHE1 could account for the loss of amiloride sensitivity and probably binding. Likewise, mutagenesis of the equivalent residue in rabbit NHE2 (Leu¹⁴³  Phe) also reduced its sensitivity to amiloride compounds (35). However, mutations at the above mentioned sites did not confer the level of amiloride resistance observed for the NHE3 isoform (12, 14, 26); therefore, it is likely that other residues help determine drug sensitivity. Since phenylalanine is also present at the equivalent position in M4 of native NHE3 (i.e. Phe¹¹⁴ in rat NHE3), this site was presumed to be responsible, at least in part, for its weak sensitivity to amiloride. However, the region around Phe¹¹⁴ did not seem important for isoform-specific drug sensitivity, since exchanging the first five putative transmembrane domains of E3 with the homologous domain of E1 (i.e. E3-1HA) did not affect amiloride, EIPA, or HOE694 sensitivity. These differences can be reconciled by assuming that other molecular determinants within the M1-M5 region of E3 can compensate for any destabilizing effect of Phe¹¹⁴ in M4 for drug binding. Thus, while this site in M4 may participate directly or indirectly in drug sensitivity and possibly binding, it does not appear to be responsible for the observed pharmacological differences between NHE1 and NHE3.

In addition to Leu¹⁶⁷, Wang et al. (27) recently reported that mutation of His³⁴⁹ in the putative M9 domain of human NHE1 to Tyr, Phe, Gly, or Leu produced a modest 2-fold increase (Tyr and Phe) or decrease (Gly and Leu) in amiloride sensitivity, although other amino acid substitutions examined had no effect. These data nicely complement our results with the E3-1SN and E1-3SN chimeras, where interchanging a larger protein segment containing M9 correlated with a substantial reciprocal switch in their drug sensitivities, particularly for EIPA and HOE694.

Biochemical analyses have indicated that the amiloride-based compounds, HOE694 and cimetidine (14, 18, 19, 20, 21, 22), exhibit simple competitive inhibition at the external Na⁺ transport site, implying that they occupy the same binding site. However, mutations at Leu¹⁶⁷ (26) and His³⁴⁸ (27) in NHE1 or Leu¹⁴³ in NHE2 (35) did not influence Na⁺ affinity. This interdependence suggests that other amino acids remote from the Na⁺ transport site confer drug sensitivity. Likewise, none of the functional chimeras showed reciprocal changes in Na⁺ affinity between parental exchangers, although each displayed a 2-fold reduction in Na⁺ affinity. Thus, unlike drug sensitivity, the determinants of cation affinity appear more dependent on the spatial organization of individual isoforms and could not be recreated optimally in the chimeras tested. It is also possible that other regions, possibly within M6-M7 and M10-M12, may contain sites for both Na⁺ affinity and drug binding, but unfortunately they could not be tested because the chimeras were not functionally expressed.

Taken together, these observations suggest that multiple sites in distinct membrane-spanning domains contribute to the drug sensitivity of NHEs. Nevertheless, the region encompassing M9 plays an important role in the differential responses, and possibly binding, between NHE1 and NHE3. Future studies aimed at identifying the precise structural elements involved in sensitivity to these antagonists could enable rational drug design. Compounds that inhibit NHE activity in an isoform-specific manner would permit the selective modulation of the appropriate Na⁺/H⁺ exchanger implicated in diseases such as hypertension (36, 37) and cardiac ischemia and reperfusion injury (15, 16).