From the * Division of Cell Biology, Hospital for Sick Children, Toronto, M5G 1X8; and Department of Physiology, McGill University,
Montreal, H3G 1Y6, Canada
We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using
the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na+-driven pHi recovery
from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably
transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP
perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of
the antiporter was obtained at ~5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange
even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also
known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger,
this requirement has been attributed to an aminophospholipid translocase, or "flippase." The involvement of this
enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated
by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as
ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that
the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPS
were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted
for ATP. In contrast, GTP
S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal
activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis
and is likely an energy-dependent event. The second process does not involve hydrolysis of the
-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.
Na+/H+ exchangers (NHEs),1 or antiporters, are ubiquitous membrane transport proteins that play a major
role in the regulation of intracellular pH (pHi) and of
cellular volume (Grinstein et al., 1985; Moolenaar,
1986
; Demaurex and Grinstein, 1994
). Under physiological conditions, NHEs catalyze the electroneutral exchange of extracellular Na+ for intracellular H+, a process which is competitively inhibited by amiloride and its analogues (see Grinstein et al., 1989
; Wakabayashi et
al., 1992b
). The rate of exchange is dictated largely by
the state of protonation of an allosteric "modifier" site
on the cytosolic side of the antiporter. Protonation of
the modifier stimulates Na+/H+ exchange, defending
the cell from excessive acidification, while the occurrence of deprotonation near the physiological pHi deactivates the antiporter, precluding a potentially deleterious alkalinization of the cytosol (Aronson et al., 1982
;
Grinstein et al., 1984
; Aronson, 1985
).
Na+/H+ exchange activity has been observed in virtually all eukaryotic cells studied thus far. Yet, the kinetic and pharmacological properties of the exchange
process vary widely between individual cell types and
even between different domains of the plasma membrane in the case of asymmetric cells, e.g., the apical
and basolateral membranes of epithelial cells (Haggerty et al., 1988). The structural basis of this functional diversity is attributed, at least in part, to the existence of distinct isoforms of the NHEs. In mammalian tissues, five separate isoforms have been identified to
date (Sardet et al., 1989
; Orlowski et al., 1992
; Tse et
al., 1992
; Wang et al., 1993
; Klanke et al., 1995
). All
NHE isoforms are integral plasma membrane proteins
of ~80-110 kD, with 10-12 predicted transmembrane domains and a long COOH-terminal cytosolic tail. The
transmembrane-spanning segments are highly conserved between isoforms and contain the ion transport
and inhibitor-binding sites. The cytosolic tail, which is
more variable in sequence, is involved in the regulation of antiporter activity, as it contains numerous potential
sites for phosphorylation by protein kinases and, in the
case of NHE-1, a calcium/calmodulin-binding site.
NHE-1, the "housekeeping" isoform, is present in most
mammalian cells and is localized to the basolateral membrane of epithelial cells. The other isoforms have
a more restricted tissue distribution: NHE-2, 3, and 4 are expressed predominantly in the kidney, intestine,
and stomach, whereas NHE-5 is found mostly in the
brain, spleen, and testis.
Fluxes through the antiporter are driven by the combined chemical gradients of Na+ and H+ and hence do
not directly consume metabolic energy. The lack of requirement for ATP hydrolysis during ion transport is
demonstrated by the ability to measure Na+/H+ exchange in plasma membrane vesicles prepared in the
absence of ATP (Murer et al., 1976; Kinsella and Aronson, 1980
). In intact cells, however, ATP appears to be
required for optimal function of the antiporter. Procedures that reduce intracellular ATP levels drastically inhibit Na+/H+ exchange in a variety of native systems
(Grinstein et al., 1985
; Cassel et al., 1986
; Little et al.,
1988
; Burns et al., 1991
) and in antiport-deficient cells
transfected with either NHE-1, 2, or 3, the only isoforms thus far expressed in heterologous systems (Wakabayashi et al., 1992a
; Levine et al., 1993
; Demaurex and Grinstein, 1994
; Kapus et al., 1994
). The effect of
ATP depletion cannot be explained by dissipation of
the Na+ and H+ gradients, as in most studies a defined
intracellular pH was maintained and changes in the
transmembrane Na+ gradient were minimized. Thus,
the effect of ATP is not attributable to changes in the
concentration of the substrate or regulatory ions. Kinetic analysis showed that the predominant effect of
ATP depletion is to alter the sensitivity of the antiporter
to intracellular [H+]; much more acidic levels must be
reached to stimulate exchange, resulting in virtual inactivity of Na+/H+ exchange at pHi levels higher than
6.5. In most studies, a parallel reduction in the maximal rate of transport (Vmax) has also been observed, although estimates of Vmax are limited by the range of
pHi values that can be imposed without affecting cellular integrity.
As the NHE-1 isoform is phosphorylated in unstimulated cells (Sardet et al., 1990, 1991
), it was proposed
that the inhibition caused by ATP depletion was due to
the loss of constitutive phosphate groups. However, recent studies failed to demonstrate a difference in the
phosphorylation state of control and ATP-depleted
NHE-1 transformants. One-dimensional phosphopeptide maps of control and depleted cells were indistinguishable (Goss et al., 1994
). Furthermore, normal exchange activity was observed in a deletion mutant lacking all major phosphorylation sites (Wakabayashi et al.,
1994
). Thus, changes in the phosphorylation state of
the antiporter itself do not appear to mediate the effect
of ATP. Since no consensus nucleotide-binding sequences such as Walker motifs are apparent in the primary structure of the NHE isoforms (Sardet et al.,
1989
; Orlowski et al., 1992
; Wang et al., 1993
), direct
ATP binding to the antiporter appears equally unlikely. The effect of ATP thus appears to involve separate, as
yet unidentified components.
Like the NHEs, the Na+/Ca2+ antiporter is equally
susceptible to depletion of ATP (Baker and McNaughton, 1976; Hilgemann, 1990
; Collins et al., 1992
; Hilgemann et al., 1992
). Though the Na+/Ca2+ exchanger
bears no structural analogy to the NHE family, the two
systems share some functional features. In particular,
both transporters are activated allosterically by their intracellular substrate ion (Ca2+ and H+, respectively)
and depletion of ATP results in altered allosteric behavior in both systems. Thus, lessons learned from the
Na+/Ca2+ exchanger could help clarify the mode of action of ATP on the NHE. Specifically, the ATP sensitivity of the Na+/Ca2+ exchanger has been reported to involve changes in the lipid distribution of the plasma
membrane (Hilgemann and Collins, 1992
). The asymmetric composition of the plasma membrane is actively
maintained by phospholipid flippases, which preferentially distribute negatively charged lipids to the inner
(cytosolic) leaflet (for review, see Zachowski, 1993
).
The negative surface charge, or a particular lipid composition of the membrane, has been postulated to stabilize the Na+/Ca2+ protein in its functional state. Inhibition of the flippase upon ATP depletion is expected
to cause a redistribution of the phospholipids across
the bilayer (e.g., Dumaswala et al., 1996
), resulting in an inhibition of exchange. Accordingly, in studies using excised macropatches, adding exogenous phosphatidylserine (PS) to the inner (cytosolic) leaflet increased Na+/Ca2+ exchange, whereas transport was inhibited by screening PS head groups or by inhibiting
the flippase (Hilgemann and Collins, 1992
; Collins and
Hilgemann, 1993
). Given the similarities between the two cation exchangers, a similar mechanism might also
underlie the ATP dependence of Na+/H+ exchange.
To gain insight into the mechanism of ATP dependence of Na+/H+ exchange, we used two approaches. Glass micropipettes were used to perfuse various nucleotides into cells while measuring NHE activity using a pH-sensitive dye. In addition, the involvement of the flippase was assessed by measuring phospholipid translocation, using single cell fluorescence imaging. To simplify the analysis, cells expressing only a single, well defined isoform of the exchanger were employed. For this purpose, mutagenized Chinese hamster ovary (CHO) cells devoid of a functional antiporter (AP-1) were stably transfected with the rat NHE-1, the ubiquitous isoform of the exchanger.
Materials and Solutions
Dioleoylphosphatidylcholine and 1-C6-2-C12-NBD-phosphatidylserine
(NBD-PS) were from Avanti Polar Lipids (Alabaster, AL). Nigericin,
27
bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF)
free acid and acetoxy methyl ester were purchased from Molecular Probes (Eugene, OR). Mes and medium RPMI-1640 were
from Sigma Chemical Co. (St. Louis, MO). All other chemicals
were of analytical grade and were obtained from Aldrich Chemical Co. (Milwaukee, WI). Bath solutions contained 135 mM aspartate and 100 mM Tris, titrated to pH = 8.0 with 85 mM of either NaOH (Na+-medium) or CsOH (Na+-free medium). Pipette
solutions contained 150 mM Cs-aspartate and 5 mM Mes, pH 6.0-
7.0, as indicated. Unless otherwise specified, all solutions contained 1 mM MgCl2 and 1 mM ethylene glycol-bis(amino-ethyl ether)N,N,N
,N
-tetra acetic acid (EGTA); pipette solutions also contained 200 µM BCECF (free acid). The osmolarity was set to 280 ± 5 mosM (pipette) and to 300 ± 5 mosM (bath).
Cells
AP-1 cells were stably transfected with the full-length cDNAs encoding the NHE-1 isoform of the rat antiporter, as previously described (Orlowski, 1993; Wang et al., 1993
). The transfected cells
were grown in
-minimal essential medium (Ontario Cancer Institute, Toronto, Canada) supplemented with 10% FCS and 1%
antibiotic solution (penicillin and streptomycin, GIBCO BRL,
Gaithersburg, MD) and passaged twice a week. Cultures were reestablished from frozen stocks, and cells from passages 3 to 12 were grown on glass coverslips for 24-48 h before the experiments.
Fluorescence Microscopy
Ratio fluorescence imaging was performed on a Zeiss Axiovert 100 TV inverted microscope (Zeiss, Obeskochen, Germany) equipped with a 75 W Xenon epifluorescence lamp (XB0 75; Zeiss), a shutter/filter-wheel assembly (Lambda 10; Sutter Instrument Co.,
Novato, CA), a NeoFluar 63×/1.25 objective and a cooled, digital
CCD camera (TEA/CCD 1317; Princeton Instruments, Trenton,
NJ) with a high resolution detector (1,317 × 1,025 pixels, KAF
1400; Eastman Kodak Co., Rochester, NY), interfaced to a Pentium 90 computer (Dell Inc., Canada) via a 12 bit, 1 MHz camera
controller board (ST-38; Princeton Instruments). Image acquisition
and excitation filter selection was controlled by the Metafluor
software (Universal Imaging Corp.). Dynamic ratio fluorescence
measurements were performed on a Nikon Diaphot TMD microscope (Nikon Canada, Toronto, Canada) equipped with a 100 W
Xenon lamp, a shutter/rotating mirror/fiber optic assembly (RatioMaster; Photon Technologies Inc., South Brunswick, NJ), a
Fluor 40×/1.3 oil-immersion objective, and a high-sensitivity
photometer (D-104; Photo Technologies Inc.) interfaced to a
386 computer (NEC, Canada) via a 12 bit A/D board (Labmaster; Scientific Solutions Inc., Solon, OH). Photometric data were
acquired at 10 Hz using the Oscar software (PTI). Both the Nikon and Zeiss microscopes were equipped with a separate light
source for long-wavelength transillumination ( > 620 nm), a
580-nm emission dichroic mirror, and a separate video camera
(MTI 72; Dage-MTI, Michigan City, IN) to allow continuous differential interference contrast (DIC) or Hoffmann-enhanced visualization of the cells during the fluorescence measurements.
Electrophysiology
Cells were voltage-clamped at 60 mV in the whole-cell configuration of the patch clamp technique using an Axopatch-1D amplifier (Axon Instruments Inc., Foster City, CA), as described
(Demaurex et al., 1995
). Electrodes were made from borosilicate
glass (World Precision Instruments, Sarasota, FL) using a horizontal
puller (P-87; Sutter Instrument Co., Novato, CA) and a microforge (MF-9; Narishige USA, Greenvale, NY). Pipette resistance
ranged from 2 to 10 M
and seal resistance from 10 to 50 G
.
Series resistance varied between 5 and 30 M
and cell capacitance between 12 and 34 pF.
Measurement of Phospholipid "Flipping"
The ability of cells to translocate lipids was assessed either directly by measuring the internalization of fluorescent phosphatidylserine (NBD-PS), or indirectly by quantifying the binding of
extracellular rhodamine-heptalysine (the kind gift of Dr. D. Hilgemann, University of Texas Southwestern Medical Center,
Dallas, TX) to cell membranes. Unilamellar vesicles containing
NBD-PS/DOPC (1:2, mol/mol) were prepared by ethanol injection, according to Hanada and Pagano (1995). Briefly, aliquots of
the lipids in solution were mixed, dried under N2, redissolved in
50 µl ethanol, and injected into 5 ml of deionized water using a
Hamilton syringe while vortex mixing. Then, 5 ml of twofold concentrated PBS was added, yielding a final concentration of 20 µM
NBD-PS. To measure lipid uptake, cells adhered to coverslips
were rinsed twice with PBS, incubated for 10 min in the experimental buffer and then for 10 min with PBS supplemented with 1 ml of the suspension of NBD-PS-containing donor vesicles. NBD-PS was removed from the cell surface by a 3 × 10 min incubation
with 2 ml of a solution containing 2% defatted albumin at 4°C
(back-exchange), and membrane-associated fluorescence was
measured on the imaging system using 450-nm excitation and 542-nm emission filters. For Rhod-Lys7 binding measurements,
cells were incubated for 10 min in the indicated buffer and subsequently for 10 min with PBS supplemented with 2 µM Rhod-Lys7,
rinsed twice with PBS and fluorescence was measured on the imaging system using 535-nm excitation and 565-nm emission filters.
Measurement of Na+/H+ Exchange in Patch-clamped Cells
NHE activity in voltage-clamped cells was measured microfluorimetrically on the photometric system described above (see also Demaurex et al., 1995). Cells were incubated for 15 min with 2 µM BCECF-AM, then patch-clamped in the whole-cell configuration and used to measure BCECF fluorescence using alternate
495/440-nm illumination. The pipettes contained acidic solutions of low buffering power (5 mM Mes) to facilitate detection
of NHE-induced changes of intracellular pH. When the cells
were perfused in Na+-free media, the cytosolic pH had equilibrated with the pipette pH after 5 min. NHE was initiated by
switching the perfusate to a Na+-containing medium. Cells were
perfused at 0.5 ml/min and bath exchange was complete within
1 min. Calibration of fluorescence ratio vs. pH was performed on
intact cells (not patched), using nigericin (5 µM final) according
to Thomas et al. (1982)
. One calibration curve was obtained every day by averaging data from three to six cells sequentially perfused with KCl media buffered at four different pH values ranging from 6.0 to 7.5. All measurements were carried out at 37°C.
Data Analysis and Statistics
Quantification of cell-associated fluorescence was performed using the Metamorph/Metafluor package (Universal Imaging, Inc.). Data were graphed using the Origin software (MicroCal Software Inc., Northampton, MA) and are shown as means ± one standard error (SE) of the number of experiments indicated.
To date, the ATP dependence of the NHE has been
studied only in intact cells, which were subjected to
metabolic depletion by the combined use of mitochondrial and glycolytic inhibitors. This approach precludes
detailed study of the ATP concentration dependence
and of the reversibility of the process. Moreover, the
nucleotide specificity of the phenomenon cannot be
explored in intact cells. To circumvent these limitations, we studied the activity of the antiport in cells
where the cytosol could be perfused with the solution
of choice via a patch pipette in the "whole-cell" configuration (i.e., after applying suction to rupture the
membrane patch). Cytosolic pH was measured microfluorimetrically using the excitation ratio of BCECF. We
found earlier that, when using cells with robust NHE
activity and pipettes containing solutions of low buffering power, the extrusion of H+ by the antiport can outstrip the diffusion of buffers to and from the pipette,
resulting in measurable pHi changes (Demaurex et al.,
1995). In these conditions, the plateau pHi reached after recovery is not necessarily the "set-point" of the antiporter, but an equilibrium between acid extrusion and
acid diffusion from the pipette into the cell.
ATP perfusion restores Na+/H+ exchange in depleted cells. Intact, untreated NHE-1 transfectants displayed robust Na+/H+ exchange activity, readily detectable as a Na+-dependent pHi recovery from an acid
load (Fig. 1 A, top trace). In agreement with earlier findings (Goss et al., 1994; Kapus et al., 1994
), the antiport
activity in these cells was markedly inhibited when intracellular ATP levels were reduced by a short (10 min)
incubation with 5 mM 2-deoxy-D-glucose and 5 µg/ml
oligomycin (Fig. 1 A, lower trace). To test whether the effect of metabolic inhibition was reversible, the nucleotide was re-introduced to depleted cells using a patch
pipette. After metabolic depletion, the cells were patch-clamped in the whole-cell configuration using a pipette
containing 10 mM ATP-Mg, and the cytosol was allowed to equilibrate with the perfusing solution for 5 min
prior to recording. Acidic pipette solutions (pH = 6.0)
of low buffering power (5 mM Mes) were used to maximize NHE activation and facilitate the detection of the
pHi changes. With ATP-Mg present in the patch pipette, Na+/H+ exchange could be readily restored in
cells that had been metabolically depleted (Fig. 1 B, top
trace), although the lag time between Na+ addition and
the onset of the alkalinization was consistently prolonged (see below). In contrast, no exchange activity
was observed when the depleted cells were perfused
with a pipette solution devoid of ATP (Fig. 1 C, bottom
trace). Thus, ATP-Mg is sufficient to restore Na+/H+ exchange in metabolically poisoned cells.
ATP Removal Inhibits NHE
In addition to depleting intracellular ATP, mitochondrial and/or glycolysis inhibitors increase the intracellular concentrations of ADP and AMP. As all previous studies of the ATP dependence of NHE have relied on metabolic inhibitors, increased ADP and AMP, instead of decreased ATP, could possibly account for the inhibition of Na+/H+ exchange. To test this possibility, we used the patch pipette to deplete the cells of all adenine nucleotides. A 5-min equilibration of otherwise untreated cells with a pipette devoid of nucleotides resulted in full inhibition of Na+/H+ exchange (Fig. 1 C, bottom trace), suggesting that this procedure lowered cytosolic ATP levels as efficiently as a 10-min incubation with metabolic inhibitors. Since intracellular ADP and AMP levels are expected to decrease in parallel with ATP, the inhibition observed under these conditions could not have been caused by an increase in the mono- or diphospho-nucleosides.
When 10 mM ATP was present in the pipette, the response observed in untreated cells was similar to that reported above for cells that had been metabolically depleted prior to patching (cf. Fig. 1, B and C). This implies that exposure to the metabolic inhibitors had no effects on NHE-1 activity other than those associated with ATP depletion. Thus, ATP removal mimicked the effects of metabolic inhibitors, whereas ATP re-perfusion restored Na+/H+ exchange, demonstrating that intracellular ATP-Mg is both necessary and sufficient for normal NHE-1 function.
Kinetics of the pHi Changes Observed in Perfused Cells
The pHi changes observed in patch-clamped NHE-1
transfectants differed somewhat from the response observed in intact cells, in that the lag-time between Na+
re-addition and the onset of the alkalinization was
markedly increased (Fig. 1). As the cytosol had been
extensively dialyzed during the 5-min pre-equilibration
with the pipette solution, this long lag-time could be
due to the loss of a soluble component(s) required for
optimal Na+/H+ exchange. However, analysis of the
pH dependence of the effect suggested a simpler explanation. As shown in Fig. 2 A, the lag time was much reduced when the pH of the pipette was more alkaline (e.g., 6.5). Systematic study of this relationship in multiple experiments showed that the lag was progressively
decreased as the pipette pH was elevated (Fig. 2 B, inset). We therefore believe that the lag period reflects, at
least in part, diffusional delay of H+ (equivalents) from
the membrane to the area where fluorescence is recorded, which includes out-of-focus fluorescence from
the pipette shank. Because patch-perfused cells are an
open system, H+ (equivalents) extruded from the cells
by NHE can be replaced not only by cellular buffers,
but also by incoming pipette buffer. It therefore requires larger amounts of H+ and longer times to detect
pHi changes in perfused cells than in intact ones. This
delay is more noticeable at low pH because the buffering power is greater. pH-dependent changes in the diffusional properties of the cells may also contribute to
this effect. For instance, the state of actin polymerization and cross-linking is known to increase at lower pH
(Yuli and Oplatka, 1987; Edmonds et al., 1995
), likely
retarding diffusion across the cytosol.
pH Dependence of NHE in Perfused Cells
Despite the variable lag period, the effect of pHi on the
rate of NHE could be compared in ATP-depleted and
ATP-replete cells. Consistent with the allosteric activation of the antiporter by intracellular H+ ions, the pH
recovery rates of ATP-containing cells were high at
acidic pHi and were reduced at more alkaline levels, becoming virtually undetectable at values higher than pH
6.75 (Fig. 2 B). By comparison, NHE activity was insignificant in the depleted cells down to pH 6.0. If, as proposed earlier, the effect of ATP depletion is merely a
shift in the pH dependence of the antiporter (Cassel et
al., 1986; Wakabayashi et al., 1992a), the shift in the
transfected cells is very large (>0.75 pH U), since the
threshold of activation was not reached even at the lowest pH values tested in our experiments. It must be
borne in mind, however, that diffusion of acidic solution from the pipette might blunt small NHE responses, shifting the apparent threshold of activation
to lower values.
The Antiporter Requires Millimolar Intracellular ATP Concentrations
Since the patch pipette could both deplete and restore
intracellular ATP levels without need for metabolic inhibitors, we used this approach to establish the ATP
concentration dependence of Na+/H+ exchange. In
cells that had not been exposed to the metabolic inhibitors, intracellular perfusion of submillimolar ATP concentrations failed to sustain Na+/H+ exchange, whereas
a maximal response was obtained with doses above 10 mM (Fig. 3). A similar dose-response was observed in cells exposed to metabolic inhibitors (not shown).
As the perfused cells likely consume some ATP, conceivably approaching the rate of nucleotide diffusion from the pipette, our assay may overestimate the ATP concentration required for optimal antiport function. To ensure that the concentrations in the pipette truly reflected the prevailing concentration of the nucleotide in the cytosol, we compared the effects of perfusion in the presence and absence of an ATP-regenerating system. For these experiments, an ATP dose near the activation threshold was chosen, so that small increases in the effective concentration would result in sizable changes in activity. The presence of creatine-phosphate (10 mM) and creatine kinase (80 U/ml) did not enhance the response to 1 mM ATP (Fig. 3 B, open circle), suggesting that the rate of delivery of ATP through the pipette exceeded the intracellular consumption of the nucleotide. Thus, we believe that the dose-response shown in Fig. 3 B accurately reflects the ATP dependence of NHE-1.
ATP Depletion Inhibits the Phospholipid Flippase
Direct phosphorylation of the antiporter had been suggested to account for its ATP dependence (Wakabayashi et al., 1992b). However, most protein kinases have
affinities for ATP in the micromolar range. The unusually high ATP dependence of the Na+/H+ exchanger is
incompatible with this model and instead resembles that reported for the cardiac Na+/Ca2+ exchanger
(Collins et al., 1992). In the latter case, high concentrations of ATP are thought to be required to support
"flipping" of membrane phospholipids (Hilgemann
and Collins, 1992
). The asymmetric distribution of
membrane phospholipids, which is maintained actively
by the ATP-dependent "flippase," has been shown to be
essential for optimal Na+/Ca2+ exchange activity
(Hilgemann and Collins, 1992
; Collins and Hilgemann, 1993
; see INTRODUCTION).
To investigate whether phospholipid flipping underlies the ATP sensitivity of the antiporter, we estimated
flippase activity in control and ATP-depleted cells. To
obtain a direct measure of the activity of the enzyme,
which translocates lipids from the outer to the inner
monolayer, we measured the incorporation of fluorescent PS into the cells. Liposomes containing NBD-labeled PS were added to adherent cells, which were allowed to
incorporate the fluorescent lipid. A 10-min incubation
at 37°C ensued, during which the lipid was allowed to
translocate to the inner monolayer and subsequently to
endomembrane compartments. Next, fluorescent lipid
bound to the extracellular face of the plasma membrane was removed (back-exchange) by addition of defatted albumin, and the intracellular NBD-PS was finally measured by fluorescence imaging. After a 10-min
incubation with 2 µM NBD-PS, bright fluorescence was
observed in control cells (Fig. 4 A, left), whereas no signal was discernible in either ATP-depleted or in cells
treated with diamide, a known inhibitor of the phospholipid flippase (Hilgemann and Collins, 1992) (Fig.
4 A, middle and right). Quantitation of the fluorescence
images confirmed that ATP depletion and diamide inhibited the incorporation of fluorescent phosphatidylserine by 96 and 84%, respectively (Fig. 4 B). Thus,
the ATP depletion protocol used for assessment of
NHE activity was indeed sufficient to inhibit the plasma
membrane phospholipid flippase.
If inhibition of the flippase is responsible for the effects of ATP depletion on NHE-1, diamide would be
predicted to be an equally effective inhibitor of Na+/
H+ exchange. In contradiction with this hypothesis, a
10-min incubation with 5 mM diamide, which inhibits
the flippase by >80% (Fig. 4 B), did not alter Na+/H+
exchange (Fig. 5, top trace; compare with Fig. 1 A).
Higher diamide concentrations and/or longer incubation times were similarly ineffective (not shown). More
importantly, NHE-1 activity in diamide-treated cells was
still sensitive to ATP depletion (Fig. 5, lower trace), implying that the step that confers ATP dependence to
NHE-1 is insensitive to diamide. Thus, it appears most
unlikely that the ATP dependence of NHE-1 is mediated by the phospholipid flippase.
Analysis of the divalent cation dependence of the exchanger also suggests that the flippase does not account
for the effects of ATP on NHE. The lipid transferase is
characterized by a strict requirement for magnesium
(Auland et al., 1994). In contrast, in ATP-depleted cells
Na+/H+ exchange could be restored if the potassium
salt of ATP was used instead of Mg-ATP (Fig. 6, A and
B). These experiments were performed adding 2 mM
EDTA to the perfusion pipette, to ensure complete chelation of endogenous (cellular) Mg2+, and 5 mM
EDTA to the external medium, to exclude influx and the
possibility of submembraneous Mg2+ microdomains. As
shown in Fig. 6 C, the ATP dose-dependence obtained with K-ATP and Mg-ATP was indistinguishable. Together,
these findings indicate that the biological process underlying the ATP-sensitivity of NHE-1 is Mg2+-insensitive and is
therefore unlikely to be the lipid flippase.
ATP Hydrolysis Is Not Absolutely Required for Stimulation of Na+/H+ Exchange
The complex of ATP with Mg2+ (or Ca2+) is generally
the substrate used by hydrolases. The finding that divalent cation chelation by EDTA did not impair the stimulatory effect of ATP on NHE suggested that hydrolysis
of the nucleotide may not be required. This notion was
tested by comparing the effects of ATP with those of
poorly hydrolyzable or nonhydrolyzable analogues.
Cells were ATP depleted, and the ability of different
nucleotides to restore Na+/H+ exchange was evaluated
in internally perfused cells. ATPS, a poorly-hydrolyzable analogue, was nearly as effective as ATP in sustaining Na+/H+ exchange in NHE-1 transfectants at 2 and
10 mM (Fig. 7, A and B). Truly nonhydrolyzable analogues, such as AMP-PNP and AMP-PCP, partially substituted for ATP. The maximal rate of recovery obtained with 10-20 mM of either AMP-PNP or AMP-PCP
averaged ~50% of the maximal response obtained with
ATP (Fig. 7 C). Increasing the concentration of the
nonhydrolyzable analogues up to 40 mM had no further effects (not shown). These nucleotides were
added as the Li+ salt. Because Li+ has been reported to
stimulate antiport activity (Aronson, 1985
), we were
concerned that the observed activation may be due to
the cation, as opposed to the nucleotide. This possibility was ruled out by introduction of an equimolar concentration of Li+ to depleted cells, in the absence of
nucleotides. As shown by Fig. 7 C, Li+ alone had not effect on NHE in ATP-depleted cells.
Finally, we considered that the effects of ATPS or
the nonhydrolyzable analogues might have been due to
formation of poorly hydrolyzable analogues of GTP, via
the nucleoside diphosphokinase (Vu and Wagner,
1993
). GTP analogues are effective stimuli of monomeric and heterotrimeric G proteins, which have been
in turn shown to alter NHE activity in some circumstances. This notion was tested by addition of 100 µM
GTP
S to ATP-depleted cells. This concentration of
the analogue effectively stimulates most G proteins, but
failed to restore Na+/H+ exchange. These observations
suggest that stimulation of G proteins by nonhydrolyzable analogues of GTP, generated by the diphosphokinase, are unlikely to explain the stimulatory effects of
ATP
S and the other analogues of ATP. Thus, binding
of ATP without hydrolysis may suffice for activation of
NHE-1.
Physiological ATP levels are required in order for the
antiporter to catalyze Na+/H+ exchange. This ATP dependence is shared by all the NHE isoforms identified
to date and can be observed both in native tissues and
in cells transfected with defined isoforms of the antiporter (for review, see Demaurex and Grinstein, 1994).
Though the ATP dependence of NHE has been known
for over ten years, the underlying mechanism remains
to be defined. There are no conventional nucleotide-binding motifs identifiable in the sequence of any of
the NHE isoforms. Structure-function analysis has thus
far failed to clarify the mechanism, since results obtained with truncated mutants of NHE-1 and NHE-3
have yielded conflicting results. Progressive deletions of the cytosolic tail abolished the ATP dependence of
NHE-1, but not of NHE-3 (Goss et al., 1994
; Cabado et
al., 1996
). Thus, whereas the transmembrane domain is
sufficient to confer ATP sensitivity to the NHE-3 isoform, the cytosolic tail appears to play a role in the ATP
dependence of NHE-1.
Despite the fact that NHE-1 is constitutively phosphorylated, no changes in the phosphorylation state of this
isoform were detected when cells were metabolically
depleted (Goss et al., 1994). Based on these findings, it
was suggested that direct phosphorylation of the antiporter is not likely to mediate the effects of ATP. In accordance with these findings, we found that analogues
of ATP having either poorly hydrolyzable or nonhydrolyzable
-phosphates substitute at least partially for the
native nucleotide on NHE. The presence and contribution of nucleotide diphosphokinase(s) capable of generating small amounts of ATP from other nucleoside cannot be ruled out. However, metabolic depletion is
expected to deplete GTP and other triphosphates pari
passu with ATP. Moreover, in the patched cells, any remaining nucleoside triphosphates are lost from the
cells as the cytosol is dialyzed against the pipette solution. These observations imply that ATP is virtually negligible and argue not only against direct phosphorylation of the antiporter, but also suggests that protein kinases may not be essential. This conclusion is supported
by the finding that NHE was unaffected by complete removal of Mg2+, an essential cofactor of most protein kinases.
The effect of nonhydrolyzable analogues and the independence of Mg2+ similarly suggest that lipid kinases
or transferases are not responsible for the effects of
ATP. Phospholipid transferases (flippases) presented
an attractive alternative, inasmuch as the ATP dependence of the Na+/Ca2+ exchanger has been attributed
to these enzymes (Hilgemann and Collins, 1992). The
possibility that phospholipid flipping accounted for the
ATP dependence of NHE, however, was not borne out
by our experimental data. Although ATP depletion did
reduce flipping (Fig. 4), causing redistribution of membrane phospholipids (as measured by binding of
rhodamine-heptalysine to the external face of the
membrane; data not shown), extensive inhibition of
the flippase with the oxidizing agent diamide did not
alter Na+/H+ exchange (Fig. 5). These findings indicate that stringent regulation of the lipid composition
of the plasma membrane is not essential for adequate
NHE-1 function, as massive redistribution of membrane phospholipids occurred upon diamide treatment. More importantly, they rule out the notion that
phospholipid flippases are solely responsible for the
ATP dependence of Na+/H+ exchange.
GTP-binding proteins have also been invoked as possible regulators of antiporter activity (Davis et al.,
1992). Depletion of GTP might in principle underlie
the ATP dependence of Na+/H+ exchange, as the levels of GTP are expected to decline in parallel with ATP
during metabolic inhibition. However, we found that
GTP
S, when perfused at doses inducing maximal heterotrimeric G-protein activation, did not restore Na+/
H+ exchange activity in nucleotide-depleted cells (Fig.
7). These findings, however, cannot rule out the participation of small, monomeric GTP-binding proteins,
some of which fail to activate their effectors in the presence of GTP
S (Bokoch and Der, 1993
).
Dialysis of otherwise untreated cells with solutions devoid of ATP produced inhibition of the exchanger
(Figs.1, 2, and 3). Conversely, re-introduction of ATP
to depleted cells sufficed to restore transport activity.
Two conclusions can be derived from these findings.
First, that the decreased transport rate caused by metabolic inhibition was reversible and due to the depletion of ATP, rather than to other, unrelated effects of oligomycin and/or deoxyglucose. Second, that intracellular
ATP (or a metabolite thereof) is the only diffusible
component required for optimal antiport activity. It is
noteworthy that near normal Na+/H+ exchange activity was maintained in patch-perfused cells despite extensive dialysis of the cytosol, suggesting that the ATP
sensitivity may be an intrinsic property of the antiporter. Certain ion transporters, such as the cystic fibrosis transmembrane regulator, are directly regulated
by binding of ATP (Anderson et al., 1991; Welsh et al., 1992
). Although consensus ATP-binding motifs such as
Walker lysines are not present in the primary structure
of NHE-1, the binding of the nucleotide to an as yet unidentified motif on the antiporter cannot be ruled out.
Alternatively, the ATP sensitivity of NHE may be conferred by a nondialyzable ancillary component possessing a nucleotide-binding domain.
Whereas the poorly hydrolyzable analogue ATPS could
fully substitute for ATP, analogues where the
-phosphate
is truly nonhydrolyzable, such as AMP-PNP and AMP-PCP, restored exchange activity only partially, even when
high concentrations were used (Fig. 7). Thus, although the direct binding of ATP to target effector proteins
may be sufficient for detectable antiport function, optimal activity also requires the hydrolysis of ATP. This
suggests that two distinct mechanisms may be involved
in the activation of the antiporter: one that needs only
binding of ATP and another that requires hydrolysis of
the nucleotide. As both mechanisms are preserved in
cells dialyzed in the whole-cell configuration, they appear to involve nondiffusible cellular structures. ATP
may be required to preserve the membrane composition or to stabilize cytoskeletal elements needed to
maintain the antiporter in an optimal configuration.
In conclusion, we demonstrated that ATP, at millimolar concentrations, is necessary for adequate activity of the NHE-1 isoform of the antiporter. The antiporter does not require Mg2+ as a cofactor and hydrolysis of ATP is not essential. We ruled out that the effect of ATP is mediated by the membrane phospholipid flippase and suggest instead that optimal antiport activation requires both hydrolysis of ATP and direct binding of the nucleotide to an as yet unidentified effector. Regardless of the detailed mechanism of action of the nucleotide, the fact that the antiporter requires millimolar ATP ensures that Na+/H+ exchange activity is tightly coupled to the metabolic state of the cell. Such coupling would prevent dissipation of the Na+ gradient that would result from excess Na+/H+ exchange in the absence of functional Na+/K+ ATPase. Impairment of Na+/H+ exchange would also protect the cells from osmotic swelling, despite the acidification that accompanies metabolic inhibition. Indeed, it has been noted that cellular edema does not occur during ischemia, but only after reperfusion, when ATP levels are restored.
Original version received 8 July 1996 and accepted version received 13 November 1996.
Address correspondence to Dr. Sergio Grinstein, Division of Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Canada. Fax: 416-813-5028; E-mail: sga{at}sickkids.on.ca
This research was funded by the Medical Research Council of Canada. N. Demaurex is a fellow of the Swiss Foundation for Biological and Medical Research and is supported by grant #31-46859.96 from the Swiss National Science Foundation. S. Grinstein is an International Scholar of the Howard Hughes Medical Institute and is cross-appointed to the Department of Biochemistry of the University of Toronto. J. Orlowski is the recipient of a scholarship from the Fonds de la Recherche en Sante du Quebec (FRSQ).