Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8
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ABSTRACT |
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Na+/H+ exchange is a passive process not requiring expenditure of metabolic energy. Nevertheless, depletion of cellular ATP produces a marked inhibition of the antiport. No evidence has been found for direct binding of nucleotide to exchangers or alteration in their state of phosphorylation, suggesting ancillary factors may be involved. This possibility was tested by comparing the activity of dog red blood cells (RBC) and their resealed ghosts. Immunoblotting experiments using isoform-specific polyclonal and monoclonal antibodies indicated RBC membranes express Na+/H+ exchanger isoform 1 (NHE1). In intact RBC, uptake of Na+ was greatly stimulated when the cytosol was acidified. The stimulated uptake was largely eliminated by amiloride and by submicromolar concentrations of the benzoyl guanidinium compound HOE-694, consistent with mediation by NHE1. Although exchange activity could also be elicited by acidification in resealed ghosts containing ATP, the absolute rate of transport was markedly diminished at comparable pH. Dissipation of the pH gradient was ruled out as the cause of diminished transport rate in ghosts. This was accomplished by a "pH clamping" procedure based on continued export of base equivalents by the endogenous anion exchanger. These observations suggest a critical factor required to maintain optimal Na+/H+ exchange activity is lost or inactivated during preparation of ghosts. Depletion of ATP, achieved by incubation with 2-deoxy-D-glucose, inhibited Na+/H+ exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate of Na+/H+ exchange in ATP-depleted but otherwise intact cells was similar to the transport rate of ghosts. Therefore, we tentatively conclude that full activation of NHE1 requires both ATP and an additional regulatory factor, which may mediate the action of the nucleotide. Ancillary phosphoproteins or phospholipids or the kinases that mediate their phosphorylation are likely candidates for the regulatory factor(s) that is inactivated or missing in ghosts.
pH regulation; amiloride; red blood cells; ghosts
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INTRODUCTION |
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SODIUM/HYDROGEN EXCHANGE is detectable in the plasma membrane of virtually all mammalian cells, where it plays an important role in the regulation of intracellular pH (pHi) and cell volume. In addition, Na+/H+ exchange is essential for transepithelial movement of Na+ and acid equivalents in certain regions of the renal and gastrointestinal tracts. These specialized functions are mediated by distinct isoforms of the Na+/H+ exchanger (NHE), which have recently been identified and studied in isolation by heterologous expression in mutant cells that are devoid of endogenous exchangers (25, 28, 33, 41). To date, six different NHE isoforms have been described, and at least four of these appear to function at the plasma membrane (for review, see Ref. 34).
In all cases studied to date, the exchange reaction appears to be reversible and driven solely by the transmembrane chemical gradients of Na+ and H+. Transport is generally believed to be passive, not requiring expenditure of metabolic energy. Indeed, NHE activity was first detected in isolated membrane vesicles derived from epithelial brush borders, a system presumably devoid of energy sources (26, 32). Nevertheless, it was subsequently established that in intact cells depletion of ATP induces a marked depression of the rate of exchange (3, 4). The inhibitory effect was not caused by alteration of the ionic gradients but instead reflects a form of allosteric regulation of the exchanger by the nucleotide. All the plasmalemmal isoforms that have been studied by heterologous transfection are inhibited by depletion of ATP, with varying degrees of sensitivity (25, 28).
The mechanism whereby ATP modulates NHE remains obscure. The isoforms studied to date exist as phosphoproteins in untreated cells. In the case of NHE1, which has been studied most extensively, the extent of phosphorylation was not detectably altered when the cells were ATP depleted for the short periods required to induce profound inhibition of ion transport (15). Furthermore, the inhibitory effect of metabolic depletion persisted in truncated NHE1 mutants lacking all the putative phosphorylation sites (15, 40). Thus changes in the phosphorylation state of the exchanger itself are unlikely to mediate the effect of ATP.
ATP is also unlikely to regulate exchange by directly binding to the NHE. Analysis of the primary sequence of the known isoforms does not reveal the presence of consensus nucleotide binding sites, and direct association has not been documented experimentally. Therefore, it is more likely that regulation involves ancillary molecules, which could themselves be the primary targets of ATP. Such a mechanism has been invoked in the case of the Na+/Ca2+ exchanger, which is similarly activated by ATP (23). As in the case of NHE, neither direct phosphorylation nor nucleotide binding is likely to be responsible for the regulation of the Na+/Ca2+ exchanger (7). Instead, two types of associated molecules have been proposed to mediate regulation by ATP. In giant cardiac membrane patches, the level of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been suggested to be an important determinant of exchange activity. ATP is envisaged to modulate the Na+/Ca2+ exchanger by dictating the extent to which phosphatidylinositol becomes phosphorylated to yield PtdIns(4,5)P2 (21). In squid nerve fibers, modulation of Na+/Ca2+ exchange by ATP was also reported to depend on an ancillary factor (11). In this case, however, the regulatory component was found to be cytosolic.
There is precedent for the regulation of NHE activity by independent, associated factors. The apical isoform of the exchanger, NHE3, is inhibited by elevation of cAMP. Although some authors believe that direct phosphorylation of the antiporter by protein kinase A is involved (27), others have reported that an additional protein cofactor is required for the manifestation of the effect (42). Indeed, two such regulatory molecules, termed NHE regulatory factor (NHERF) and NHERF-2 (also known as E3KARP and TKA-1), have recently been identified and sequenced (43, 44). We therefore considered the possibility that ancillary factors could also be important for the ATP dependence of NHE. Red blood cells (RBC) were found to be particularly useful for this purpose because they can be lysed in mildly hypotonic solutions and then resealed upon restoration of the original osmolarity. By varying the extent of the initial dilution, different concentrations of cytosolic factors can be trapped in the resulting ghosts. For this study, we used canine RBC, which were shown by the pioneering work of Parker (36) to display considerable antiport activity. Herein we report that these RBC express NHE1 that is sensitive to ATP. The stimulatory effect of the nucleotide requires the presence of an additional regulatory factor that is lost or inactivated during preparation of RBC ghosts.
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MATERIALS AND METHODS |
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Reagents and solutions. Amiloride and 2-deoxy-D-glucose were obtained from Sigma Chemical (St. Louis, MO). DIDS (disodium salt) was obtained from Molecular Probes (Eugene, OR). Enhanced chemiluminescence reagents were from Amersham International (Buckinghamshire, UK). The ATP assay kit was purchased from Calbiochem (San Diego, CA). 22Na+ was obtained from New England Nuclear Life Science Products (Boston, MA). All other chemicals were of analytical grade and were obtained from Aldrich Chemical (Milwaukee, WI). HOE-694 [(3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate] was kindly provided by Dr. A. Durckheimer (Hoechst, Frankfurt, Germany).
Polyclonal antibodies to NHE1 were raised by injecting rabbits with a fusion protein constructed withPreparation of RBC, ghosts, and membranes. Ghosts were prepared by a modification of the method of Parker (35). Briefly, RBC were obtained from heparinized blood of healthy dogs. After centrifugation, the plasma and buffy coat were discarded, and the RBC were washed three times with 2-3 volumes of 165 mM NaCl plus 1 mM HEPES (pH 7.3). The RBC were then diluted with 1 volume of this medium and chilled for 5 min on ice. This suspension was added to 10 volumes of an ice-cold lysing medium containing (in mM) 20 KCl, 4 MgSO4, 0.3 CaCl2, and 1.2 acetic acid. The suspension was brought to pH 5.8 while being stirred in an ice bath and incubated for 5 min. This suspension was then mixed with 1 volume (equal to the volume of the original cell suspension) of resealing solution containing (in mM) 1,458 NaCl, 162 KCl, 12 MgSO4, 30 Tris base, and 12 ATP. The mixture was titrated to pH 7.0 and, after 5 min on ice, incubated for 60 min at 37°C. The resealed ghosts were separated from the hemolysate by centrifugation at 13,000 g for 15 min and suspended in storage solution containing (in mM) 126 NaCl, 15 KCl, 1 MgCl2, 20 Tris-MES, 10 glucose, and 0.5 EGTA (pH 7.4). To prepare sodium gluconate or potassium gluconate-loaded ghosts, these salts were used to replace NaCl and KCl in the washing, lysing, resealing, and storage solutions. For cell volume and Na+ content measurements, ghosts were loaded with potassium gluconate. Counting and sizing of RBC and resealed ghosts were performed electronically using a model ZM Coulter counter and C1000 Channelyzer, as described earlier (16).
RBC membranes were prepared for immunoblot analysis as follows. RBC were washed three times with 5 volumes of 150 mM NaCl plus 5 mM sodium phosphate (pH 8.0). After centrifugation, packed RBC (1 ml) were lysed by mixing with 40 ml of ice-cold 5 mM sodium phosphate (pH 8.0). The resulting membranes were sedimented at 30,000 g for 15 min. The clear red supernatant was removed by aspiration, and the pellet was washed twice with the lysis solution. Each 1-ml pellet of freshly prepared ghosts was resuspended in 40 ml of prewarmed 0.5 mM sodium phosphate (pH 9.0). The suspension was incubated at 37°C for 20 min. The resulting stripped membranes were sedimented at 30,000 g for 30 min and used for immunoblotting as described below. The pH 9.0 stripping procedure was also used to prepare membranes from resealed ghosts.pHi manipulation and determination.
In RBC, which are rich in
Cl/HCO
3
exchangers, the relationship between
pHi and the extracellular pH is
dictated by the transmembrane
Cl
gradient. Because
HCO
3 is a base equivalent, rapid anion
exchange ensures that the ratio of internal
H+ concentration to external
H+ concentration equals the ratio
of external Cl
concentration to internal
Cl
concentration. We took
advantage of this unique feature of RBC to set their
pHi to the desired level by
incubating them for 10 min at 37°C, in medium containing (in mM)
140 KCl, 40 sucrose, 0.15 MgCl2,
10 glucose, and 20 Tris-MES (pH 6.0-7.0). After this initial
incubation, DIDS was added to a final concentration of 200 µM, and
the cells were incubated for a further 30 min. Treatment with this
anion exchange inhibitor enabled us to subsequently suspend the cells
in media of different pH, without dissipation of the transmembrane pH
gradient. The cells were finally sedimented and resuspended in the
medium of choice.
ATP depletion and determination. For depletion of ATP, cells were incubated for 2 h at 37°C in PBS (pH 7.8) containing 10 mM 2-deoxy-D-glucose. Cellular ATP content was determined using the Calbiochem assay kit. Cells (~5 × 105) were extracted with 0.4 ml of 8% perchloric acid and placed on ice. The extract was then neutralized with 1 M K2CO3, debris were sedimented, and aliquots of the supernatant (10 µl) were mixed with the buffer and luciferin-luciferase mixture provided by the kit manufacturer. Sample luminescence was determined using a Beckman LS7000 counter and compared with ATP standards.
Isotope fluxes.
NHE activity was measured as the rate of amiloride-inhibitable
22Na+
influx at 37°C. Uptake of
22Na+
was initiated by resuspension of a pellet of
~108 acid-loaded RBC or ghosts
in 0.5 ml of medium consisting of (in mM) 135 NMDG-Cl, 2 NaCl, 2 MgCl2, 1 KCl, 0.5 EGTA, and 10 glucose with or without 1 mM amiloride, plus 0.5 µCi of
22Na+.
Isotope uptake was terminated after 10 min by aspirating the radiolabeled medium and rapidly washing the cells or ghosts twice with
ice-cold medium containing 128 mM NMDG-Cl and 13 mM potassium gluconate. Pellets were lysed with 1 ml of distilled water and counted
in a LKB 1282 gamma counter. As necessary, e.g., when ghosts were acid
loaded by imposing various inward
Cl gradients, the
compositions of the radiolabeled and wash solutions were adjusted
appropriately.
22Na+
uptake was linear for at least 15 min under all the experimental conditions used.
NHERF expression and purification. Hexahistidine-tagged full-length NHERF was produced by insertion of rabbit NHERF cDNA into pET30A (Novagen, Madison, WI) as described (19, 20). Fusion proteins were expressed according to the manufacturer's instructions and purified using nickel beads (Novagen).
Immunoblotting. For immunoblotting, samples were mixed with 0.25 volume of 5× concentrated Laemmli sample buffer and boiled for 5 min. The samples were subjected to SDS-PAGE and transferred to nitrocellulose. Blots were blocked with 5% nonfat dried milk and exposed to a 1:5,000 dilution of affinity-purified anti-NHE1, NHE3, or NHE4 polyclonal antibodies or to a 1:1,000 dilution of affinity-purified anti-NHE1 monoclonal antibody. The secondary antibodies, goat anti-rabbit or anti-mouse coupled to horseradish peroxidase, were used at a 1:5,000 dilution. Immunoreactive bands were visualized using enhanced chemiluminescence.
Other methods. The concentration of Na+ in the hemolysates was determined by flame photometry using Li+ as an internal standard. All experiments were performed on at least three separate occasions, each with triplicate determinations. Data are presented as means ± SE of the number of experiments specified.
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RESULTS |
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Identification of NHE1 in dog RBC.
Isoform-specific antibodies, directed to unique sequences in the
cytosolic domains of NHE1, NHE3, or NHE4, were used to establish which
isoform(s) of the antiporter is present in canine RBC. No antibodies to
the remaining isoforms were available to us at the time of this study.
Anti-NHE3 and NHE4 antibodies failed to react with NHE in dog RBC (data
not shown). In contrast, a polypeptide of ~98 kDa reacted positively
with both the monoclonal and polyclonal anti-NHE1 antibodies (Fig.
1). The immunoreactive band
was somewhat smaller and migrated more sharply than the
human platelet NHE1, which, as shown earlier (1), migrates as a wide
band of ~110 kDa in human platelets. The width of the NHE1 band in
SDS-PAGE has been attributed to carbohydrate heterogeneity, since
impairment of glycosylation yields a sharp band of 85 kDa (8).
Therefore, our results suggest that dog RBC express a form of NHE1 with
reduced and more homogeneous glycosylation.
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ATP depletion inhibits
Na+/H+
exchange in dog RBC.
In native systems, as well as when it is heterologously expressed by
transfection, NHE1 is sensitive to the concentration of cytosolic ATP
(3, 4, 25, 28). To test whether NHE1 is similarly sensitive in dog RBC,
ATP was depleted by omission of glucose from the medium and addition of
2-deoxy-D-glucose. Under these
conditions, >70% of the ATP was depleted after 2 h, as determined
using luciferase [from (6.83 ± 0.22) × 1011
mol/106 cells to (2.00 ± 0.18) × 10
11
mol/106 cells]. We undertook
a detailed analysis of the pHi
dependence of the activity of the exchanger in ATP-depleted and
ATP-replete cells (Fig. 3). As shown
earlier for other cells (41), NHE1 activity increases progressively
with decreasing cytosolic pH. It is noteworthy that depletion of ATP
inhibited the amiloride-sensitive flux at all
pHi levels studied. In the range
studied, the fractional inhibition was greater at more alkaline
pHi values. The
pHi was measured individually in
each experiment at the end of the transport determination. Therefore,
the effect of ATP depletion is due to inhibition of the exchanger and
not to a spurious effect on pHi.
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Na+/H+
exchange in ghosts.
Before this report, NHE activity in dog RBC had been elicited primarily
by osmotic shrinkage (12, 36). This response was lost when the cells
were lysed and the ghosts resealed by conventional means (i.e.,
restoring the surface-to-volume ratio of the original cells; see Ref.
6). It was therefore of interest to determine whether acid-induced NHE
activity would persist in the ghosts. Ghosts were prepared as described
under Preparation of RBC, ghosts, and
membranes and were acid loaded by equilibration with an
external acidic medium, followed by treatment with DIDS to preserve the pH gradient. A comparison of the rates of
22Na+
uptake in acid-loaded RBC and ghosts is presented in Fig.
4, which summarizes results of seven
individual preparations. As in Fig. 3, which illustrates a different
set of experiments over a narrower pH range, the RBC displayed a
robust, pH-dependent uptake. The ghosts, which were obtained from the
same cells illustrated in Fig. 4, also displayed amiloride-sensitive
NHE activity. However, at comparable
pHi values, the rates of
22Na+
uptake were lower than those of RBC. When the
pHi approached neutrality, the
activity of the ghosts became insignificant. The reduced activity of
the ghosts was not due to proteolytic cleavage of NHE1 during the
ghosting and resealing procedures. This was confirmed by immunoblotting
the resealed ghost membranes, as shown in Fig. 4,
inset.
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A method for the imposition of a sustained acid load in ghosts.
To establish definitively whether the process of lysis and resealing
reduces the intrinsic activity of NHE1, we required a procedure whereby
a sustained acidification could be imposed. We took advantage of the
large anion exchange activity of the cells and ghosts to generate and
maintain an acid load of predictable magnitude. Ghosts were resealed in
medium containing gluconate as the predominant anion. Subsequent
resuspension of such ghosts in
Cl-rich media promoted the
exchange of external Cl
for
intracellular HCO
3, generated by
diffusion and hydration of CO2 and
subsequent dissociation of
H2CO3.
Exchange of Cl
for
cytosolic OH
may have also
contributed to the acidification. Figure
5A
illustrates the effectiveness of this approach: gluconate-loaded ghosts
suspended in high-Cl
medium
have a pHi that is 0.4-0.5 pH
units more acidic than their counterparts suspended in
low-Cl
medium. We found
that the change in pH (
pH) imposed using this method was more stable
than that created using stilbene derivatives (a change of 0.014 ± 0.001 vs. 0.024 ± 0.001 pH units/min, respectively, at a comparable
starting pHi). Over the course
of 10 min, the duration of the transport assay, such a rate of
dissipation altered the pHi only
marginally (from 6.53 ± 0.01 to 6.67 ± 0.01).
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Effect of ATP depletion on
Na+/H+
exchange in ghosts.
The reduced NHE activity of the ghosts superficially resembles the
inhibition induced in the intact cells by ATP depletion. It was
therefore conceivable that the content of ATP of the ghosts was reduced
compared with that of cells. However, direct measurements using
luciferin-luciferase indicated that the ATP content of RBC and resealed
ghosts is similar [(6.83 ± 0.22) × 1011
mol/106 cells and (6.56 ± 0.20) × 10
11
mol/106 ghosts,
respectively]. It therefore appears that the transport rate of
ghosts is reduced, despite the presence of normal ATP levels.
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DISCUSSION |
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NHE1 in RBC.
Our results indicate that NHE1 is present in canine RBC and that it
mediates most or all of the
Na+/H+
exchange in these cells. Briefly, we found that RBC membranes contain
an ~95-kDa polypeptide that is immunoreactive with both polyclonal
and monoclonal anti-NHE1 antibodies (Fig. 1) but not with antibodies to
other isoforms. In addition, RT-PCR analysis of RNA isolated from dog
reticulocytes failed to detect NHE2, NHE3, or NHE4 (unpublished
observations). Although suggestive, the latter observations are not
conclusive because oligonucleotides based on the rat sequences were
used, since the sequences of the canine exchangers are not known. On
the other hand, the exclusive presence of NHE1 was also indicated by
pharmacological experiments in which >95% of the exchange activity
of RBC was inhibited by 5 µM HOE-694. At this dose of the drug,
only NHE1 is significantly inhibited (9).
ATP dependence of intact cells and ghosts. Inhibition of Na+/H+ exchange activity by depletion of ATP has been consistently found in a variety of cellular systems naturally expressing NHE1, as well as in antiport-deficient cells stably transfected with this isoform. A similar observation is reported here in canine RBC. The universality of the phenomenon would seem to suggest that ATP dependence is an intrinsic property of the exchanger. However, we also found that, whereas ghosts display considerable exchange activity, they were unaffected by the presence or absence of the nucleotide. Interestingly, the residual NHE activity observed in ATP-depleted but otherwise intact cells is similar in absolute magnitude to the activity recorded in ghosts. We therefore surmise that a cooperative effect of ATP and a second factor is required for optimal activity of NHE1. Omission of either component results in suboptimal exchange activity. Because the second factor is lost or inactivated during the course of RBC lysis or resealing, the basal transport rate of ghosts is lower and depletion of ATP has little additional effect.
Earlier work using nucleated cells patched in the whole cell configuration (i.e., under conditions in which the cytosol is continuous with the filling solution of the pipette) found that the ATP sensitivity of NHE1 was preserved for many minutes (10). This observation is not necessarily in conflict with the present findings in RBC. First, dialysis of cellular components through the comparatively small opening of the pipette is slow, limiting the loss of macromolecules. Second, lysis of the ghosts at low ionic strength may have dislodged loosely bound proteins, which would otherwise remain attached and would not diffuse in the case of patch-clamped cells. The nature of the putative cofactor required for ATP to exert its stimulatory action remains unknown. We attempted to regenerate the ATP sensitivity of the ghosts by resealing within them concentrated cytosol. (The supernatant of a lysed suspension was freeze dried, resuspended in a volume of water equal to the original volume of the cells, and used as the resealing solution.) These attempts were unsuccessful, suggesting that the factor is labile. In this regard, our results differ from those of Colclasure and Parker (6), who could restore the volume-induced activation of NHE by resealing RBC cytosol or concentrated protein solutions into ghosts with decreased volume-to-surface ratios. Therefore, although macromolecular crowding can appropriately explain the osmotic sensitivity of the exchanger, it is unlikely to contribute to the metabolic dependence of NHE1. Accordingly, the inhibitory effects of ATP depletion occur without discernible volume changes. Although the detailed mechanism of regulation of ATP remains unresolved, several possibilities can be contemplated. First, it is conceivable that the lysis or resealing procedures may have altered the conformation of NHE1 itself. Second, an ATP-dependent reversible association with a soluble or integral membrane (phospho)protein may regulate the exchanger. An analogous mechanism operates in the case of the neuronal Na+/Ca2+ exchanger, which is similarly ATP sensitive. In this case, a soluble cytoplasmic protein isolated from squid axoplasm or brain reconstitutes the ATP stimulation of the exchanger (11). At least three distinct proteins have been reported thus far to interact with NHE1: calmodulin, 70-kDa heat shock protein (HSP-70), and a calcineurin homologue protein (CHP) (2, 29, 38). A 24-kDa polypeptide also shown to bind to NHE1 in vivo (14) may be identical or related to CHP. Of these, only HSP-70 interacts with the exchanger in an ATP-dependent manner: the nucleotide induces dissociation of the chaperone with NHE1 (38). The possible role of HSP-70 in the regulation of NHE1 activity, however, remains to be demonstrated directly. As has been reported for a variety of other transporters and channels (30, 31, 39), it is alternatively possible that NHE1 may be associated with and regulated by the cytoskeleton. This interaction could, in turn, be modulated by ATP. Association with the cytoskeleton has been suggested by the modification of NHE activity by changes in cell size and shape (17, 25) and by the uneven distribution of NHE1 on the surfaces of some cells (18, 38). Moreover, the actin cytoskeleton rearranges upon ATP depletion (13). Yet, to our knowledge, direct evidence of an interaction between the skeleton and NHE1 is still lacking. The proteins reported thus far to interact with NHE1 (see above) are not known cytoskeletal components. Finally, it is possible that the regulatory component is not a protein but a (phospho)lipid. In this regard, a possible role of acidic phospholipid asymmetry in the control of NHE1 was considered earlier (10). This hypothesis was entertained in view of results suggesting control of Na+/Ca2+ exchange by phosphatidylserine and ethanolamine (22). However, this notion was not borne out by direct experimental analysis (10). Instead, it is possible that, as also suggested for a variety of K+ channels and for the cardiac Na+/Ca2+ exchanger (21, 24), NHE1 may be modulated by polyphosphoinositides. Depletion of ATP is accompanied by a concomitant reduction of the polyphosphoinositide pool, due to the dynamic equilibrium between inositide kinases and phosphatases. If, as is the case for other transporters (21, 24), PtdIns(4,5)P2 is essential for optimal NHE1 function, the inhibitory effect of ATP depletion may be mediated by depletion of this lipid. Any of the above hypotheses could be easily reconciled with the requirement for both ATP and a second, soluble and/or labile cofactor. The inhibition noted in ghosts may have resulted from loss or inactivation of a regulatory protein that associates directly with NHE1. Such a protein could be a soluble or cytoskeletal polypeptide. Alternatively, a kinase responsible for protein or lipid phosphorylation may be the elusive factor. In any event, loss of such a component would clearly result in inhibition of the exchanger despite the presence of ATP. In conclusion, NHE1 can apparently exist in two distinct functional states: an optimal conformation that transports cations effectively and requires both ATP and an as yet unidentified cofactor and a suboptimal conformation that predominates when either ATP or the cofactor is lacking. The factor, which likely mediates the action of the nucleotide on the exchanger, confers on NHE1 a higher sensitivity to pHi. ![]() |
ACKNOWLEDGEMENTS |
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We thank Marcella Prasad for help during the initial phases of this study.
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FOOTNOTES |
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This research was supported by the Canadian Cystic Fibrosis Foundation (CCFF) and the Medical Research Council of Canada. O. Aharonovitz is the recipient of a CCFF Postdoctoral Fellowship.
S. Grinstein is cross-appointed to the Department of Biochemistry of the University of Toronto, is an International Scholar of the Howard Hughes Medical Institute, and is the current holder of the Pitblado Chair in Cell Biology.
Present address of N. Demaurex: Dept. of Physiology, University of Geneva Medical Center 1, Michel-Servet, CH-1211 Geneva 4, Switzerland.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Grinstein, Div. of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8 (E-mail: sga{at}sickkids.on.ca).
Received 30 September 1998; accepted in final form 24 February 1999.
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