Proc. Natl. Acad. Sci.

USA
Vol. 90, pp. 4508-4512, May 1993
Biochemistry

A point mutation of the Na+/H+ exchanger gene (NHEJ) and

amplification of the mutated allele confer amiloride
resistance upon chronic acidosis
(amiloride-binding site/Na+/H+ exchanger isoforms/pH regulation/gene amplification)
LAURENT COUNILLON, ARLETTE FRANCHI, AND JACQUES POUYSStGUR
Centre de Biochimie-Centre National de la Recherche Scientifique, Universitd de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice, France
Communicated by Edward A. Adelberg, December 18, 1992

ABSTRACT The diuretic drug amiloride and its 5-amino impaired in their capacity to bind MPA, Chinese hamster lung
substitute N5-methyl-N5-propylamiloride (MPA) are potent fibroblasts (CCL39 diploid cell line) were submitted repeat-
inhibitors of the growth factor-activatable Na+/H+ exchanger edly to lethal intracellular acidifications in the presence of
isoform 1 (NHE1). This inhibitor competes with Na+, presum- MPA. During this selection procedure, a functional Na+/H+
ably by interacting with the ion-transport site of the NHE exchanger is absolutely required to protect cells against lethal
molecule. As an approach to identify this site, we previously acidosis. The MPA concentration was then steadily increased
reported the use of a specific H+-killing selection technique for over a 6-mo selection period from 0.3 AM (half-lethal doses
isolating amiloride-resistant variants of Chinese hamster lung for CCL39 cells) up to 40 AM and 300 ,M at which,
fibroblasts. After long-term selection, two variants, AR40 and respectively, AR40 and AR300 stable variants were isolated
AR300, 100- and 1000-fold, respectively, resistant to MPA, (13). Here we provide molecular evidence that this 1000-fold
were isolated. By comparing NHE1 cDNA sequences of paren- acquired MPA resistance to chronic lethal acidosis results
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tal and two variant cell lines, we show that the 1000-fold from a single mutation within the structural NHEl-encoding
resistance to MPA results from two sequential genetic events. gene that decreases 30-fold the affinity for MPA; the mutant
(i) In one AR40 allele a point mutation, Phe-167 -+ Leu, occurs allele was then amplified in AR300 cells.
in the middle of the fourth putative transmembrane segment of
NHEL. Producing this mutant protein from human NHE1
cDNA by site-directed mutagenesis increased the K, for MPA by MATERIALS AND METHODS
30-fold, as seen in AR300 cells. (ii) An ==10-fold amplification Cell Culture. Chinese hamster lung fibroblast (CCL39 cell
of the mutated allele, which contributes to the acquired MPA line), the CCL39-derived PS120 variant, and PS120 cells
resistance, accounts for the V.IX increase. Mutating a close transfected with the Na+/H+ antiporter cDNA constructs
residue, Phe-165 -* Tyr, increased by 40-fold the K, for were maintained in Dulbecco's modified Eagle's medium/
amiloride and reduced Na+ transport rate 3- to 4-fold, indi- 7.5% fetal calf serum/penicillin at 50 units/ml/streptomycin
cating that we have identified a critical domain of the NHE at 50 ,ug/ml in a humidified atmosphere of 5% C02/95% air at
molecule that controls amiloride binding and Na+ transport. 370C.
Interestingly, the epithelial amiloride-resistant NHE isoforms cDNA Cloning. Total RNA was extracted from AR300 cells
that occurred naturally possess some of the amino acid sub- by using the LiCl precipitation method. Poly(A)+ RNAs were
stitutions described here. selected on oligo(dT) columns (Pharmacia, type 7) and were
reverse transcribed by using oligo(dT) priming (Amersham
Amiloride is an antihypertensive diuretic agent possessing and Boehringer Mannheim cDNA synthesis kits). Double-
saliuretic and antikaliuretic properties (1) that inhibits the stranded cDNAs over 2 kb were size-selected on an agarose
eukaryotic Na+/H+ exchanger and epithelial Na+ channel in gel and cloned into AgtlO bacteriophage (Amersham AgtlO
the range of micromolar concentrations (2, 3). Amiloride and cDNA cloning kit), yielding a library containing 4 x 105
its derivatives have provided the pharmacological means to independent clones. After three rounds of screening with the
discriminate between various Na+ transport systems that 32P-labeled human NHE1 cDNA as a probe (Amersham
include several isoforms of Na+/H+ exchangers (NHE1- random-priming labeling kit), followed by sequencing and
NHE4) (4-6), Na+/Ca2+ exchanger (7), and the epithelial restriction map analysis, isolated cDNA clones were found to
Na+ channel (8). As far as NHE1 is concerned, amiloride be highly homologous to the human cDNA. The largest clone
inhibits Na+ transport competitively, and substitution of was missing 750 bp at the 5' extremity of the coding region.
alkyl groups at the 5-amino position of the molecule increases A second library was built into AgtlO from AR300 poly(A)+
up to 100-fold the potency of inhibition (9, 10). In spite of this RNA, with random primers, to increase the probability of
specificity and potency of inhibition, amiloride derivatives reverse-transcribing 5' regions of the mRNA. Clones from
have not permitted isolation of the receptor or characteriza- this library bearing 2 x i05 independent recombinants of
tion of the corresponding site by standard biochemical tech- >700-bp length provided sequences containing the transla-
niques. Our approach to this question exploited the toxicity tion initiation region (14), which allowed the design of oligo-
of intracellular protons and the selectivity of the amiloride nucleotides for PCR amplification of the missing fragment.
analog N5-methyl-N5-propylamiloride (MPA) to isolate mu- The 5' oligonucleotide contained an EcoRI restriction site
tants impaired in their capacity to exchange Na+ for H+ and followed by a translation initiation sequence, whereas the 3'
be inhibited by MPA (11). One class, devoid of exchanger oligonucleotide was situated immediately after the first Sac I
activity, allowed us to clone a NHE isoform by gene com- restriction site of the AR26 cDNA (Fig. 1). PCR was done on
plementation (4, 12). To isolate the second class of mutants ""10 ng of randomly primed first-stranded cDNA (Cetus Taq
polymerase). Complete cDNAs could be reconstituted by
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" Abbreviations: NHE1, Na+/H+ exchanger isoform 1; MPA, N5-
in accordance with 18 U.S.C. §1734 solely to indicate this fact. methyl-N5-propylamiloride.

4508
 Biochemistry: Counfllon et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4509
5 E B_I AcI0 Sa Ac B 3' was stably transfected into NHE-deficient PS120 cells (21), it
.
i could restore Na+-dependent intracellular pH regulation.
Human C28 cDNA Clone Most important, 22Na+-uptake kinetic studies revealed that
M nooding this cDNA conferred an identical MPA pharmacological
E B Ac Sa region profile as that seen in the AR300 parental cell line: Ki for MPA
I Inonooding = 1.5 ,uM instead of 0.05 ,M observed in CCL39 cells or in
Human probe used to screen region PS120 cells transfected with the human NHE1 cDNA (Fig. 2).
the cDNA library 250 bp This result validates the hypothesis that the MPA-resistant
Ac Sa Ac B Sa3n phenotype seen in AR300 cells was due to a mutation(s)
l I I 1. within the gene for the Na+/H+ exchanger and demonstrates
44- 70b bpT
-,- , w-m
-
AR26 cDNA clone
that the cDNA coding for this mutated antiporter has been
E B Ac Sa cloned.
To identify the mutation(s) by sequence comparison, we
cloned the corresponding cDNA of the parent Chinese ham-
PCR enginered fragment ster cell line CCL39 (22). Like the human counterpart, it
E B Ac Sa Ac contains 12 putative transmembrane segments and two con-
5
I I . II l
B Sa 31 served glycosylation sites, and it displays 92.5% homology at
the amino acid level with the human NHE1 (4, 22).
Expressed MPA resistant cDNA Identification of the Mutation Responsible for MPA Resis-
tance. Before sequencing the complete AR300-derived
FIG. 1. Human probe and organization of the cDNA fragments cDNA, we first constructed chimeric antiporters by using
isolated during cloning of AR300 NHE1 cDNA. Restriction en- corresponding wild-type and mutant cDNA restriction frag-
zymes: Ac, Acc I; B, BamHI; E, EcoRI; Sa, Sac I. ments (see Fig. 3). By comparing the MPA-sensitivity in the
using the biggest clone obtained from the oligo(dT)-primed H+-killing test, as well as the amiloride sensitivity of Na+
library and the fragment synthesized by PCR. uptake, the area containing the mutation(s) has clearly been
Site-Directed Mutagenesis. An HindIII-Sac I restriction narrowed to a 700-bp cDNA fragment encoding transmem-
brane segments 2-7 in the putative topological model de-
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fragment isolated from plasmid pEAP-A5' (15) cloned into duced from the NHE1 primary sequence (4, 23).
pECE mammalian expression vector (16) was subcloned into The comparative nucleotide sequence of this 700-bp frag-
the pTZ18 vector. Site-directed mutagenesis was done on ment revealed only a single point mutation (C -- T) that
single-stranded plasmid, according to the method described generated a Leu-167 -- Phe substitution in the middle of the
by Kunkel (17) (Bio-Rad kit). Mutagenized cDNA fragments fourth putative transmembrane segment (Fig. 4, Upper).
were reintroduced in pEAP-A5' cloned in pECE by enzy- Subsequent analysis of the entire sequence revealed that this
matic restriction cutting and subsequent ligation. Leu-167 -* Phe substitution was the only mutation seen.
Expression of Na+/H+ Antiporters. NHE1 cDNAs from
AR300, CCL39, and human were subcloned between EcoRI 10
NH2
and Sac I restriction sites of the pECE expression vector
polylinker. The cDNA coding for the NHE2 isoform was
provided by M. Donowitz (Johns Hopkins University, Bal- 8

iN -" 9 '~NH2
timore). The cDNAs coding for NHE3 and NHE4 isoforms
were provided by J. Orlowski (McGill University, Montreal).
PS120 cells were transfected with the calcium phosphate
precipitation method (18), and positive transfectants were
selected by their ability to survive a 1-hr acidification induced
by NH+ prepulse acid loading, as described (4). R2
Other Methods. 22Na+-uptake studies, Southern blot, and
immunoblot analyses were done as described (12, 15, 19).
Protein concentrations were determined by the method of ._04)
Lowry et al. (20). ._b

RESULTS
+
Cloning the cDNA of the "MPA-Resistant" Exchanger. 0
m
Genomic DNA, from the MPA-resistant variant AR300, ._
EZ

transfected into NHE-deficient mouse fibroblasts, restored 0

Na+/H+ exchange activity with the corresponding lower OA
affinity for MPA (12). This finding was important because it 0.1 1 100
provided strong evidence for the existence of a mutated gM MPA
NHEl-encoding gene in AR300 cells. We therefore decided
to clone the MPA-resistant exchanger by screening cDNA FIG. 2. Amiloride structure and MPA pharmacological profile of
libraries prepared from the hamster AR300 cell line with the CCL39 and AR300 cells. (Upper) Structure of protonated form of
human NHE1 cDNA as probe. amiloride and of analog MPA. (Lower) Concentration-response
A cDNA containing the entire AR300 NHE-coding se- curves for inhibition of 22Na+ influx by MPA in CCL39 cells (o) and
quence was cloned, as judged by restriction enzyme pattern AR300 cells (o). NH4+-loaded cells were incubated for 6 min in a
medium (pH 7.4) containing 22Na+ (carrier-free), 1 mM ouabain, and
analysis, partial DNA sequencing, and hybridization with 5' various MPA concentrations. Initial rates of 22Na+ uptake were
and 3' probes of the corresponding human NHE1 cDNA. Fig. determined, as described (12, 13). Dose-response curves clearly
1 outlines the two-step cloning procedure used, and details show that PS120 cells expressing NHE1 cDNAs cloned from CCL39
are described in Materials and Methods. When this cDNA, (s) and from AR300 cells (e) exhibit the same pharmacological profile
deleted of most of its 5'- and 3'-noncoding regions (Fig. 1), as the "parental" CCL39 and AR300 cell lines, respectively.
 4510 Biochemistry: Counillon et al. Proc. Natl. Acad. Sci. USA 90 (1993)
PARENTAL MPA
s b ~~~~Phenotype
01.5pM)
(AR300) <

m [ e OSENSITIVE
(CCL39)

, CHIMERA & A C G T A C G T
c
SENSITIVE
c C
(0.05GM) T F CT F
L T

CT T F
m~
IRESISTANT
(1.59M) c F
CTV
TG
CT G V
G
Ac D GA D
SENSITIVE
(0.05gMl) Wild-tyrpe MPA-resistant
NHE1 NHE1
AMILORIDE BINDING
SITE LOCATION a
% of Amiloride -sensitive
22 Na+ uptake

FIG. 3. MPA phenotype of different NHE1 cDNA chimera

constructs. The chimeras used to map a piece of DNA containing the
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mutation(s) responsible for the MPA-resistance phenotype were

constructed by enzymatic restriction cutting and subsequent religa-
tion of wild-type and mutant AR300 cDNAs. Resulting chimeric
cDNAs have been inserted in the pECE expression vector and
expressed in PS120 cells. The black areas of the putative topological
model of the Na+/H+ exchanger correspond to regions of the 0.01 0.1 1 100
molecule encoded by the AR300 mutant cDNA. The corresponding
phenotype is indicated beside each chimera with the inhibition UMMMPA
constants in parenthesis.
FIG. 4. Identification of NHE1 mutation responsible for MPA-
Accordingly, this mutation introduced by site-directed mu- resistant phenotype of AR300 variant by nucleotide sequence anal-
tagenesis in the human NHE1 cDNA shifted the correspond- ysis and MPA pharmacological profiles. (Upper) Portion of DNA
sequencing gel showing single nucleotide change (C -+ T) in codon
ing MPA-sensitive phenotype to an MPA-resistant one with 167 resulting in Leu -+ Phe. (Lower) Dose-response curves for
the corresponding 30-fold increase in K, value for MPA (Fig. inhibition of 22Na+ influx by MPA in PS120 cells transfected with
4, Lower). This result shows that this sole mutation is human NHE1 cDNA (m) and with human NHE1 cDNA containing
responsible for the observed altered pharmacological profile the Leu-167 -- Phe mutation (o). The human NHE1 cDNA was
of the antiporter expressed by AR300 cells. Also, this result mutagenized according to the method of Kunkel (17).
strongly suggests that Leu-167 interacts with amiloride, pre-
sumably by hydrophobic interaction with the pyrazine ring of NHEI alleles, and therefore we conclude that the observed
the molecule and, to a greater extent, with the 5-amino pharmacological profile results from the coexpression in
substituents because affinity for MPA is much more affected equal amounts of both MPA-resistant and -sensitive NHE1
than affinity for amiloride when this leucine is changed to a molecules.
phenylalanine. Investigation of Other Amino Acids Involved in Amiloride
Elucidation of the Molecular Mechanisms Responsible for Binding. In contrast to NHE1, Na+/H+ exchanger isoforms
Acquisition of Resistance. During the continuous selection expressed in epithelial cells have been reported to display
that led to the emergence of the MPA-resistant phenotype, relative resistance to amiloride and its analogs (24, 25). With
AR40 cells, which display an "intermediate phenotype," the recent availability of the corresponding sequences for
emerged quickly (-1 mo), whereas an additional 5 mo were
required to yield the highly resistant AR300 cells. It was,
therefore, of interest to elucidate the mechanism of evolution kb
,4

°
of the fibroblast population exposed to this chronic acidosis. 6.5 :- e,,
'MTgg,,;
(i) A marked difference was noticed between AR40 and
AR300 at the NHEI gene copy level. Fig. 5 shows that AR300
has amplified the NHEI gene -10-fold with no sign of 4.3 -

rearrangement. This genetic event could easily account for

overexpression of the Na+/H+ exchange activity reported
(13). We then analyzed by PCR the corresponding FIG. 5. Genomic DNA analysis of Chinese hamster fibroblasts
"amiloride-binding" region of the AR40 NHE1-encoding and derived null and MPA-resistant variants. Southern blots of the
gene. Sequence analysis of 11 cDNA clones, prepared by
CCL39, PS120, AR40, and AR300 cell lines. Twenty micrograms of
EcoRI-cut genomic DNA was electrophoresed, blotted onto Nylon
using AR40 mRNA, revealed that 5 of them were wild type, filter, and hybridized with 32P-labeled human NHE1 probe [H18-Pst
whereas the others possessed the same mutation as the I; (4)]. The arrow indicates the NHE1-specific EcoRI fragment
AR300 cells. This distribution of cDNA sequences clearly detected by the probe. Note the marked amplification in AR300 cells
indicates that AR40 cells possess both wild-type and mutant over wild-type (CCL39) and AR40 cells.
 Biochemistry: Counfllon et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4511
NHE2, NHE3, and NHE4 isoforms (5, 6), it was of particular c 40
interest to examine the amino acid region proposed as the ~A \~~, ((N
"amiloride-binding site." The sequence of amino acids E
164VFFLFLLPPI173 is rather well conserved among the four 0.- 30
isoforms; boldface letters correspond to complete conserva- (U
Mutated NHE1 -8
tion among these forms (Table 1). Some differences, how- 20
ever, are noteworthy. The pharmacological sensitivity of the u 0
0.
NHE isoforms was tested after expression in PS120 cells. The
NHE2 and NHE3 genes could be expressed, whereas we , 10
were unable to express NHE4. NHE2 expressed in PS120 z
(Xi2 low qu Mo
cells displays a decreased affinity for MPA but does not show cOJ0
CM
......

decreased affinity for amiloride. Its Ki value for MPA is 10

times higher than it is for NHE1, and its amino acid sequence min
in the 164-173 region varies by only one conservative sub- FIG. 6. Na+/H+ exchange activity of various mutated NHE1
stitution (Phe-168 -* Tyr) (Table 1). Introduction of this cDNAs expressed in hamster lung fibroblasts. (Left) Comparison of
substitution into NHE1 does not affect the K, for either MPA initial rates of amiloride-sensitive 22Na+ influx in CCL39 cells (-) and
or amiloride. This result clearly indicates that the pharma- in PS120 cells transfected with human NHE1-mutagenized cDNAs
cology for 5-amino substitutes of amiloride is also dictated by F168Y (A), L167F-F168Y (o), and F165Y-F168Y (o). The CCL39
additional residues, outside the 164-173 amino acid stretch. cells exhibit a lower antiporter activity (i) because of the lower
NHE3, however, possesses the same Leu-167 -* Phe sub- transcriptional activity of the endogenous promoter when compared
stitution as that found in AR40/AR300 mutants. In addition, with the simian virus 40 promoter of the pECE vector used to express
NHE3 has also the "neutral" conservative Phe-168 -- Tyr the mutated cDNAs and (ii) because deletion of the 5' untranslated
region of the mutated cDNAs enhances their expression (15). (Right)
change found in NHE2. When both substitutions, Leu-167 -+ Immunoblot analysis of the expression level of the 3 site-directed
Phe and Phe-168 -- Tyr, are introduced into NHE1 and the mutated NHE1 cDNAs. About 50 ,ug of crude membrane proteins
mutated molecule is expressed in PS120 cells, we observed was separated on SDS/PAGE and blotted onto a nitrocellulose
exactly the pharmacology of AR300 but did not observe that membrane. The blot was incubated with RP1-C28 anti-NHEl anti-
of NHE3 (Table 1). Indeed, NHE3 expressed in PS120 cells body (19) and revealed by enhanced chemiluminescence technique,
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displays a notable amiloride-resistant phenotype with a 50- as described (15). Protein concentration in each lane is normalized by
fold and 200-fold increase in the Ki values for amiloride and quantitation of Gja2 subunit (a12) protein using a Gja2 antibody on
the same blot.
MPA, respectively (Table 1). Here also we must conclude
that besides the key region where the Leu-167 -* Phe very amiloride-resistant isoforms. In addition, the NHE1
mutation defines an "amiloride hot spot," other residues molecule bearing the Phe-165 -+ Tyr and Phe-168 -- Tyr
outside the 164- to 173-amino acid stretch contribute to the substitutions of NHE4 showed a 3- to 4-fold decrease in Na+
recognition of amiloride and its derivatives. NHE4 possesses transport rate when compared with NHE1 and all the previ-
the "neutral" substitution Phe-168 -- Tyr and another con- ously characterized mutated antiporters (Fig. 6 Left). As the
servative change, Phe-165 -- Tyr. Introduction of these affinity for external Na+ is apparently not modified (results
mutations into NHE1 profoundly altered the amiloride phar- not shown) and the quantity of antiport protein expressed in
macology: it increased Ki values for amiloride and MPA 30- membranes is not decreased (Fig. 6 Right), we conclude that
to 40-fold. This result is particularly important, as it inde- the Phe-165 -* Tyr substitution affects both amiloride binding
pendently shows that the hydrophobic pocket 164VFFLFLl69 and the Vma,, for Na+ transport.
contributes to the recognition of amiloride and of its 5-amino
substitutes. Unfortunately, at this stage it was not possible to
express the NHE-4 isoform in PS120 cells, and therefore its DISCUSSION
pharmacological profile with the corresponding NHE1 site- To gain some insight into residues involved in the Na+
directed mutated cDNA could not be compared. Neverthe- transport site or nearby the NHE1 transporter, we exploited
less, we could easily predict that NHE4 will be one of the the competitive interaction of the amiloride analog with this
Table 1. IC5o of MPA and amiloride for the various NHE site. Previously, we showed that a long-term adaptation of
isoforms and the mutated NHE1 cDNAs cells in culture to chronic acidosis with increased MPA
concentrations led to the isolation of a highly MPA-resistant
IC50 for IC50 for variant, AR300 (13). Here we showed that this 1000-fold
Amiloride MPA, amiloride, acquired resistance resulted from two genetic events: (i) A
cDNA binding site Mutation ,uM AM point mutation that occurred spontaneously in AR40 and was
NHE1 164VFFLFLLPPI173 0.05 3 selected because it reduced the affinity of MPA by 30-fold.
AR300 VFFFFLLPPI 1.5 15 However, in AR40 the coexistence of the wild-type and
NHE1 L167F 1.5 15 mutated NHEI alleles gave only an intermediate MPA-
NHE2 VFFLYLLPPI 0.5 3 resistant phenotype. (ii) At this stage, increased stringency of
NHE1 F168Y 0.05 3 selection for an additional 5 mo could have generated either
NHE3 VFFFYLLPPI 10 150 the emergence of secondary mutations or amplification of the
L167F mutated NHE gene. Molecular analysis of AR300 has clearly
NHE1 F168Y 1 15 shown that gene amplification of the mutated allele prevailed,
NHE4 VYFLYLLPPI Unknown Unknown as seen for other examples of drug resistance in eukaryotic
cells (26).
NHE1 F165Y 1 100 This study has identified an amino acid, Leu-167, of the
IC50 values have been measured by inhibition of initial rates of NHE1 protein, which is likely to contact the amiloride
amiloride-sensitive 22Na+ influx. Measurements were made in du- molecule. Indeed, that this substitution induces a much
plicate, as described (12, 13). The variations between duplicate weaker effect on the Ki for amioride than for MPA suggests
experiments did not exceed 20%. Boldface letters indicate amino acid that Leu-167 contacts the 5-amino substituents of the MPA
conservation. Underlined amino acids indicate variations from the molecule. We cannot exclude, however, that Leu-167 is, in
NHE1 sequence. fact, not involved in amiloride binding but, instead, induces
 4512 Biochemistry: Counillon et al. Proc. Natl. Acad. Sci. USA 90 (1993)
a conformational change, thus altering the pharmacological and S. Wakabayashi for technical advice and critical discussion; F.
profile. However, this explanation seems unlikely for at least MacKenzie and R. Reithmeier for critical reading of the manuscript;
three reasons: (i) An important conformational change might and D. Grail for technical assistance on cell culture. We are grateful
be expected to have dramatic effects for function of the to Drs. M. Donowitz and J. Orlowski for providing NHE2, NHE3, and
NHE4 sequences before publication. This work was supported by
antiporter or, at least, should have pleiotropic effects on the grants from the Centre National de la Recherche Scientifique (CNRS,
other kinetic parameters of the Na+/HI exchange; this is not UMR134), the Institut National de la Sante et de la Recherche
the case. (ii) A conservative hydrophobic amino acid substi- Medicale (CRE 910205), the Association Franco-Israelienne pour la
tution (Leu -- Phe) has not been reported to strongly desta- Recherche Scientifique et Technologique (AFIRST), the Fondation
bilize a-helical structures (27) and is, therefore, unlikely to pour la Recherche M6dicale, and the Association pour la Recherche
induce important conformational changes in the protein. (iii) contre le Cancer (ARC).
This nonpolar amino acid presumably involved in the binding 1. Cragoe, E. J. (1979) in Amiloride and Epithelial Sodium Trans-
of amiloride and of its 5-amino-substituted derivatives is port, ed. Cuthbert, A. W., Fanelli, G. M. & Scriabine, A.
situated within a hydrophobic pocket of the Na+/H+ ex- (Urban & Schwarzenberg, Baltimore), pp. 3-20.
changer, a result in good agreement with data from the 2. Benos, D. (1982) Am. J. Physiol. 242, C131-C145.
structure-activity relationship studies in the amiloride series 3. Grinstein, S. (1988) in Na-H Exchange, ed. Grinstein, S. (CRC,
(28). Boca Raton, FL).
Leu-167 -- Phe substitution of AR300 has highlighted a 4. Sardet, C., Franchi, A. & Pouyss6gur, J. (1989) Cell 56,
hydrophobic region of the fourth transmembrane a-helix that 271-280.
we refer to here as a putative amiloride-binding site. This 5. Orlowski, J., Kandasamy, R. A. & Shull, G. E. (1992) J. Biol.
Chem. 267, 9331-9339.
notion is reinforced by two additional findings. (i) We have 6. Tse, C. M., Brant, S. R., Walker, S., Pouyss6gur, J. & Dono-
found that this substitution occurred naturally in the se- witz, M. (1992) J. Biol. Chem. 267, 9340-9346.
quence of NHE3, an epithelial isoform recently cloned that 7. Kaczorowski, G. J., Barrows, G. J., Dethmers, J. K. & Trum-
displays an amiloride-resistant phenotype (5, 6). Although we ble, M. J. (1985) Biochemistry 24, 1394-1403.
have shown that this substitution cannot by itself account for 8. Cuthbert, A. W. (1976) Mol. Pharmacol. 12, 945-957.
the very amiloride-resistant phenotype of NHE3, it is prob- 9. Vigne, P., Frelin, C., Cragoe, E. J. & Lazdunski, M. (1983)
ably involved. (ii) Examination of the corresponding NHE4 Biochem. Biophys. Res. Commun. 116, 86-90.
sequence has outlined a nearby conservative substitution, 10. L'Allemain, G., Paris, S. & Pouyssegur, J. (1984) J. Biol.
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11. Pouyss6gur, J. (1985) Trends Biochem. Sci. 10, 453-455.
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MPA (20-fold) but also that ofamiloride (30- to 40-fold) (Table Proc. Natl. Acad. Sci. USA 83, 9388-9392.
1). Therefore, Phe-165 probably contacts the pyrazine ring 13. Franchi, A., Cragoe, E. & Pouyss6gur, J. (1986) J. Biol. Chem.
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The putative fourth transmembrane domain, where the 14. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148.
above-mentioned interaction sites occur, exhibits, indeed, 15. Wakabayashi, S., Sardet, C., Fafournoux, P. & Pouyss6gur, J.
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'64VFFLFLLPPI'73, previously identified as the amiloride- 16. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A.
binding site, contains a very unusual doublet of prolines. & Rutter, W. J. (1986) Cell 45, 721-732.
17. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492.
Although single proline residues have often been reported to 18. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A.,
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We thank Drs. P. Fafournoux, S. Meloche, C. Sardet, K. Seuwen, Cardiol. 17, 1029-1042.