1 Cell Biology Program, Hospital for Sick Children, Toronto M5G 1X8, and 2 Department of Surgery, Toronto Hospital and University of Toronto, Toronto, Ontario M5G 1L7; and 3 Department of Physiology, McGill University, Montreal, Quebec, Canada H3G 1Y6
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ABSTRACT |
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Na+/H+ exchanger (NHE)
activity is exquisitely dependent on the intra- and extracellular
concentrations of Na+ and H+. In addition,
Cl ions have been suggested to modulate NHE activity, but
little is known about the underlying mechanism, and the
Cl
sensitivity of the individual isoforms has not been
established. To explore their Cl
sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3)
were heterologously expressed in antiport-deficient cells. Bilateral
replacement of Cl
with nitrate or thiocyanate inhibited
the activity of all isoforms. Cl
depletion did not affect
cell volume or the cellular ATP content, which could have indirectly
altered NHE activity. The number of plasmalemmal exchangers was
unaffected by Cl
removal, implying that inhibition was
due to a decrease in the intrinsic activity of individual exchangers.
Analysis of truncated mutants of NHE1 revealed that the anion
sensitivity resides, at least in part, in the COOH-terminal domain of
the exchanger. Moreover, readdition of Cl
into the
extracellular medium failed to restore normal transport, suggesting
that intracellular Cl
is critical for activity. Thus
interaction of intracellular Cl
with the COOH terminus of
NHE1 or with an associated protein is essential for optimal activity.
antiport; type 1 Na+/H+ exchanger; anion dependence; osmotic activation; volume regulation
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INTRODUCTION |
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SODIUM/HYDROGEN EXCHANGERS (NHEs) are integral membrane proteins found in all eukaryotic cells studied to date. Several isoforms have been identified, and some reside in endomembranes (e.g., type 6 Na+/H+ exchanger), while others are exclusively or predominantly plasmalemmal [e.g., types 1-3 Na+/H+ exchanger (NHE1-3)] (for review, see Refs. 28 and 39). The latter transporters promote the electroneutral 1:1 exchange of intracellular H+ for extracellular Na+ across the surface membrane, thereby eliminating excess acid generated by actively metabolizing cells. Exchange of Na+ for H+ is also crucial for the regulation of cellular volume and for the (re)absorption of NaCl across renal, intestinal, and other epithelia. The hydropathy profiles of all members of the NHE family suggest that they share a similar transmembrane organization: an NH2-terminal hydrophobic domain composed of 12 membrane-spanning helices and a COOH-terminal hydrophilic cytoplasmic domain. Recent structural studies support this topological paradigm. The membrane-embedded region is highly homologous among the various isoforms and was shown to be both necessary and sufficient to catalyze ion exchange (28, 39). In contrast, greater divergence is observed between the cytosolic tails, which are thought to confer to the individual isoforms their characteristic pattern of regulation by hormones and growth factors (11, 28, 39).
Because NHE plays a central role in salt and water homeostasis,
feedback regulation would ensure accurate control of these parameters.
Accordingly, NHE activity is not only dictated by the concentration of
its physiological substrates, Na+ and H+, but
is also exquisitely sensitive to the cell volume (4). In
addition, evidence exists that anions are also involved in NHE
regulation. The first definitive indication that Cl
affects the activation of the antiporter was provided by Parker (29), who observed that in dog erythrocytes, NHE failed to
respond to cell shrinkage when Cl
was replaced with
either thiocyanate or nitrate. A similar effect of anionic replacement
on osmotic activation of NHE was subsequently reported in rabbit red
blood cells (18) and in barnacle muscle fibers
(9). More recently, a Cl
-dependent
Na+/H+ exchange, presumably catalyzed by an
amiloride-resistant form of NHE, was also detected in the apical
membrane of colonic crypt cells (31). In the latter case,
however, the basal activity, as opposed to the osmotic activation, was
affected by Cl
. In contrast, in salivary acinar cells,
the presence of Cl
was reported to inhibit
Na+/H+ exchange (32). In this
tissue, carbachol induces the opening of Cl
channels,
leading to Cl
efflux and cell shrinkage. Both the
decreased volume and the reduced intracellular Cl
concentration were shown to contribute to the carbachol-evoked activation of the Na+/H+ exchange (12,
32).
The source of the differential Cl responsiveness of the
individual biological systems tested to date remains unclear. In view of the divergent sensitivity of the individual isoforms to other regulatory factors, it is conceivable that the reported differences toward Cl
are attributable to differential expression of
distinct isoforms of the NHE in the tissues examined. The analysis may
have been confounded further by the simultaneous expression of multiple isoforms in a single cell type, particularly in the case of epithelia. Finally, there is evidence that the same isoform may behave differently depending on the cellular context in which it is expressed (23, 27).
The primary aim of the present study, therefore, was to explore the
effect of Cl on the activity of the three major
plasmalemmal isoforms of NHE (NHE1, NHE2, and NHE3) and to further our
understanding of the underlying mechanism(s). To eliminate the
ambiguity introduced by the coexistence of multiple isoforms and to
circumvent differences induced by cellular context, individual isoforms
from a defined species were expressed singly in the same cell type. Rat
NHE1-3 were transfected into an antiport-deficient line of Chinese
hamster ovary (CHO) cells termed AP-1 (27). The
effect of Cl
and its sidedness and the responsiveness to
osmotic challenge in the presence and absence of the anion and
structure-function analysis were performed in such heterologous transfectants.
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MATERIALS AND METHODS |
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Materials and solutions. Nigericin and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) were obtained from Molecular Probes (Eugene, OR). Chymotrypsin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and cytochalasin B were from Sigma Chemical (St. Louis, MO). Mouse monoclonal antibodies to influenza virus hemagglutinin (HA) were obtained from BAbCo (Berkeley, CA). Horseradish peroxidase-coupled goat anti-mouse antibodies were from Jackson ImmunoResearch Laboratories (Mississauga, ON). Alexa 488-conjugated goat anti-mouse antibody was from Molecular Probes. Enhanced chemiluminescence reagents were from Amersham (Buckinghamshire, England). 125I-labeled goat anti-mouse IgG was from ICN Pharmaceuticals (Irvine, CA). 3-O-[3H]methyl-D-glucose was from NEN (Boston, MA). Trypsin-EDTA was purchased from Life Technologies (Burlington, ON). The ATP assay kit was from Calbiochem (San Diego, CA). All other chemicals were of analytical grade and were obtained from Aldrich Chemical (Milwaukee, WI).
The isotonic Na+-rich medium contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4. PBS contained (in mM) 150 NaCl, 10 KCl, 8 sodium phosphate, and 2 potassium phosphate, pH 7.4. Isotonic Na+-free medium contained (in mM) 140 KCl, 1 CaCl2, 1 MgSO4, 5.5 glucose, and 25 N-methyl-D-glucammonium-HEPES, pH 7.4. Isotonic ClCells. WT5 is a subline of wild-type CHO cells. AP-1 cells, which are devoid of endogenous Na+/H+ exchange activity, were derived from mutagenized WT5 cells as previously described (34). AP-1/NHE1, AP-1/NHE2, and AP-1/NHE3 cells were obtained by stable transfection of AP-1 cells with the complete coding region of the rat NHE1, NHE2, or NHE3, respectively, as described in detail elsewhere (27). For immunological detection of the proteins, epitope-tagged versions were used. In AP-1/NHE1HA cells, one copy of an influenza virus HA peptide (YPYDVPDYA) was appended to the COOH cytoplasmic tail of the coding region of the NHE1 (35). In AP-1/NHE2'813HA, the HA epitope, preceded by a single glycine linker, was inserted at the COOH terminus of NHE2 (after amino acid 813) (6). In AP-1/NHE3'38HA3 cells, a triple HA epitope was inserted into the coding region within the first predicted extracellular loop of NHE3 (21).
Intracellular Cl depletion and
determination.
For depletion of Cl
, cells were incubated for the
indicated time at 37°C in isotonic Na+-rich
Cl
-free medium. Cellular Cl
content was
determined according to the method of Zall et al. (40)
with slight modifications. Briefly, after the appropriate preincubations, cells were washed three times with large volumes of
ice-cold Cl
-free medium. Next, the cells were lysed with
1 mM nitric acid, scraped off the wells with a rubber policeman, and
sedimented. The supernatant was collected and mixed with an equal
volume of the photometric determination mixture (1 part of mercuric
thiocyanate in 0.417% methanol solution, 1 part of 20.2% ferric
nitrate, and 13 parts of water). The absorbance of the test samples and
Cl
standards was then determined at 480 nm using a
Hitachi U-2000 spectrophotometer.
Cell volume measurement. Cells grown on multiwell plates were incubated in isotonic Na+-rich medium in the presence of tritiated 3-O-[3H]methyl-D-glucose (1 mCi/ml) for 20 min. This time is sufficed for equilibration of 3-O-[3H]methyl-D-glucose across the membrane, as revealed by previous determinations. The cells were then washed three times with ice-cold Na+-rich medium containing 2 µM cytochalasin B, and finally 0.5 ml of 1% SDS was added. The solubilized cells were collected from the wells and counted in an LKB 1217 beta counter. Intracellular volume was calculated from the specific activity of the loading solution, measured in parallel. Sizing of cells was also performed electronically using a model ZM Coulter counter and C1000 Channelyzer, as described earlier (14).
Measurement of NHE activity.
Stably transfected cells grown to 60-70% confluence on glass
coverslips were incubated for 30 min at 37°C in Na+-rich
medium with or without Cl. BCECF-AM (2 µg/ml) was added
during the last 15 min of this incubation. In experiments in which the
Na+-dependent recovery of intracellular pH
(pHi) was measured, an acute acid load was imposed by the
NH
to depleted cells was made at this stage, together with
Na+. To measure the fluorescence of BCECF, the coverslip
was placed in a thermostatted Leiden holder on the stage of a Nikon
TMD-Diaphot microscope equipped with a Nikon Fluor oil-immersion
objective. A chopping mirror was used to direct the excitation light
alternately to two excitation filters (500 ± 10 nm and 440 ± 10 nm) in front of a xenon lamp. To minimize dye bleaching and
photodynamic damage, neutral density filters were used to reduce the
intensity of the excitation light reaching the cells. The excitation
light was directed to the cells via a 510-nm dichroic mirror, and
fluorescence emission was collected through a 535 ± 25 nm
band-pass filter. Photometric data were continuously acquired at 1 Hz
using Felix software (Photon Technologies, South Brunswick, NJ).
Quantification of surface NHE1,
NHE2, and NHE3.
AP-1/NHE1HA, AP-1/NHE2'813HA3, or
AP-1/NHE3'38HA3 cells grown on six-well plates were
preequilibrated for 30 min at 37°C with Na+-rich medium
containing Cl, nitrate, or thiocyanate. Surface NHE1 was
quantified using a previously established procedure (35).
Briefly, AP-1/NHE1HA cells were incubated for 5 min at
37°C in the presence or absence of chymotrypsin (100 U/ml). The cells
were then scraped off with a rubber policeman, washed twice using
Na+-rich medium containing 100 µM TPCK, resuspended in
twice-concentrated Laemmli sample buffer, and boiled for 5 min. Samples
were subjected to electrophoresis in 7.5% polyacrylamide gels and
transferred to nitrocellulose. Blots were blocked with 5% nonfat dried
milk and exposed to a 1:5,000 dilution of mouse monoclonal antibodies against HA. The secondary antibody, goat anti-mouse coupled to horseradish peroxidase, was used at 1:5,000 dilution. Immunoreactive bands were visualized using enhanced chemiluminescence.
Other methods. 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. Unless otherwise specified, all experiments were carried out at 37°C. Data are presented as means ± SE of the number of experiments specified.
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RESULTS |
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Depletion of intracellular Cl.
To examine the impact of Cl
on NHE activity, CHO cells
expressing individual isoforms of the antiporter were depleted of
Cl
. Reduction of the intracellular Cl
concentration was carried out by incubation of the cells in a solution
in which this anion was substituted by equimolar
NO
. As illustrated in Fig.
1, this procedure resulted in a
rapid decrease in intracellular Cl
content. Nearly 50%
of the intracellular Cl
was lost within 5 min, while
longer incubation time resulted in further loss of Cl
,
reaching ~70% after 30 min. The residual component may represent a
slowly exchangeable pool of intracellular Cl
.
Alternatively, cellular components other than Cl
may
produce a spurious reading in the photometric assay. In either case,
depletion of the cytosolic Cl
pool is in all likelihood
nearly complete after 30 min. This time was chosen for all subsequent
experiments.
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Effect of Cl depletion on
Na+/H+
exchange mediated by NHE1, NHE2, and
NHE3.
Though Cl
is not transported by the NHEs
(20), depletion of this anion was reported to reduce the
rate of cation exchange in a variety of cells, while stimulation was
reported in one instance. Expression of different isoforms in these
systems may account for the apparent discrepancies. We therefore
compared the effect of Cl
depletion on NHE activity in
cells transfected with the individual isoforms. To assess the effect of
anions over a wide pHi range, control or
Cl
-depleted cells were initially acid loaded by the
NH
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The sidedness of the Cl requirement.
In the above experiments, Cl
was depleted bilaterally. To
establish whether intra- and/or extracellular Cl
is
required for the maintenance of optimal NHE activity, we depleted intracellular Cl
and reintroduced this anion to the
bathing medium at the time of Na+ readdition. Figure
3A shows that when measured at pHi 6.6, inhibition of NHE1 activity in Cl
-depleted cells was
similar in the absence (NO
. The fact that readdition of Cl
to the
extracellular space during the recovery phase failed to restore NHE
activity suggests that intracellular Cl
is the critical
determinant of NHE1 activity. Similar results were obtained with NHE3
(Fig. 3B).
Effect of Cl depletion on
ATP content.
Though ATP is not hydrolyzed during the Na+/H+
exchange cycle, depletion of this nucleotide drastically reduces the
rate of transport in a variety of cells (3, 5). In cells
transfected with individual isoforms, NHE1, NHE2, and NHE3 all proved
to be sensitive to metabolic depletion (19, 23). The
reduced NHE activity of Cl
-depleted cells superficially
resembles the inhibition evoked by ATP depletion. It was therefore
important to establish whether Cl
depletion alters the
cellular ATP content, since such an effect could contribute to the
observed inhibition. Direct measurements using the luciferin-luciferase
assay indicated that the ATP content of Cl
-depleted cells
was identical to that of Cl
-containing controls,
irrespective of the anion used for depletion (99 ± 6% and
90 ± 9% of the control level for depletion in
NO
medium, respectively; data
are means ± SE of 6 determinations). Thus the inhibition of NHE
induced by anion substitution is not mediated by alteration in the
content of ATP.
Quantitation of plasmalemmal NHE1,
NHE2, and NHE3.
The observed decrease in the rate of transport throughout the
pHi range studied is consistent with a reduction in the
number of exchangers exposed on the cell surface. In this respect, it is noteworthy that NHE3 has been shown to be present not only in the
plasmalemma but also in an intracellular vesicular compartment (10) and that a net reduction in transport can result from
redistribution of antiporters between these pools (21). It
was, therefore, important to test whether the density of plasmalemmal
antiporters is altered upon depletion of Cl. To this end,
we assessed the number of exchangers present at the cell surface both
before and after anion substitution. Quantitation of NHE1 was based on
the chymotrypsin sensitivity of the surface-exposed exchangers, whereas
antibodies were used to estimate the surface density of NHE2 and NHE3,
which were tagged with the HA epitope.
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Effect of Cl depletion on the osmotic
responsiveness of the NHE isoforms.
Activation of Na+/H+ exchange is an important
mechanism in the restoration of cell volume in osmotically shrunken
cells (4). NHE1 and NHE2 can fulfill this role, since both
of these isoforms are activated upon cell shrinkage (2, 19; but see
Ref. 25 for discrepant view). In contrast, the epithelial
brush border isoform NHE3 is inhibited when the cell shrinks
(19), perhaps guarding the organism against solute
(re)absorption under abnormally hypertonic conditions.
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Role of the cytosolic tail in conferring
Cl sensitivity to the exchanger.
The cytosolic COOH-terminal domain of the antiporters is thought to be
essential for regulation of activity by hormones, growth factors, and
cell volume (see Refs. 28 and 39 for review). On
the other hand, the transport and (part of) the allosteric H+-sensitive sites are thought to reside in the
transmembrane moiety. To define the region(s) of the protein
responsible for the sensitivity to anions, we compared the
Cl
sensitivity of the full-length exchanger with that of
a truncated mutant (NHE1
582) lacking a large portion of the
cytoplasmic tail. At pHi 6.4-6.8, Cl
depletion resulted in a 62 ± 4% decrease in the activity of the wild-type NHE1 (n = 40), whereas it caused only a
31 ± 3% drop in the activity of
582 (n = 15).
The effect of further truncations could not be assessed accurately,
since their basal activity was marginal.
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DISCUSSION |
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Cl was initially shown to be involved in the
regulation of NHE in red blood cells. Using dog erythrocytes, Parker
(29) reported that replacement of Cl
with
NO
abolished the
shrinkage-induced activation of the antiporter. If the cells were first
shrunken in the presence of Cl
and then briefly treated
with low concentrations of the mild fixative, glutaraldehyde, the
exchanger remained in the activated state, in spite of the subsequent
removal of Cl
or the restoration of isotonic cell volume
(30). These findings were interpreted to mean that the
activation, rather than the function, of the exchanger was sensitive to
the anion composition. Similarly, Motais et al. (26) found
that Cl
was required for the cAMP-induced stimulation of
the exchanger of trout red blood cells. As in the previous case,
however, once the trout antiport was activated, the anion sensitivity
was lost.
The results in these studies differ from those reported here in two
significant aspects. First, mammalian NHE1-3 were found to be
anion dependent in unstimulated cells, i.e., in isotonic media devoid
of hormonal or other agonists. Second, in the cases of NHE1 and NHE2,
the volume-induced response persisted in the absence of
Cl, in contrast to the results in erythrocytes. The
explanation for this divergent behavior may lie in the pHi
dependence of the different systems, which in turn reflects their
primary function. In unstimulated red blood cells, NHE activity is
negligible at or near physiological pH. In these cells, stabilization
of the pHi is accomplished through the exchange of
Cl
for bicarbonate, by far the predominant transport
system in their membrane. Therefore, the primary role of
Na+/H+ exchange in red blood cells is thought
to be in the regulation of cellular volume (4). This
contrasts with the exchangers of nucleated mammalian cells, where NHE
activity is mainly required for pH regulation or transepithelial
electrolyte transport. For these functions, NHE must be active at or
very near the physiological pH. The factor(s) responsible for this
basal activity, which may be related to those stimulated by shrinkage
or cAMP in red blood cells, could be the target of Cl
ions.
This explanation is consistent with the reported effects of
Cl in barnacle muscle fibers. Like red blood cells,
barnacle fibers have negligible NHE activity near the resting
pHi but show a significant activation by hyperosmolarity
(9). This activation was similarly found to depend on
Cl
, an effect that was attributed to coupling of NHE with
a heterotrimeric GTP-binding protein (8, 9). Subsequently,
Hogan and colleagues (15) found that the barnacle
exchanger could also be activated by a profound acidification. Because
this acid-induced effect was also sensitive to anion replacement, it
was suggested that regulation by the GTP-binding protein may be exerted
tonically. A similar mechanism may operate in mammalian cells. G
protein control of NHE1 in salivary cells has been reported
(17), although no such evidence exists at present for the
other isoforms of the exchanger.
The precise mechanism of action of Cl ions remains
undefined. The data in Fig. 4 imply that the number of transporters
available at the membrane is unaffected. Thus it is most likely that
the intrinsic activity of each exchanger is depressed when
Cl
is removed. NHE1 is known to possess an autoinhibitory
domain that constitutively represses the activity of the transport site (37). The effect exerted by this domain can be modulated
by calcium/calmodulin and may also be sensitive to Cl
ions. However, part of the effect of Cl
ions persisted in
NHE1
582, a truncated form lacking the autoinhibitory domain.
Therefore, other mechanisms must be invoked. For instance, Cl
may alter the sensitivity of the exchangers to
intracellular H+ ions, an explanation consistent with the
leftward shift in pHi dependence noted in Fig. 2. The
latter is dictated primarily by an allosteric "modifier" site that
confers cooperativity to the H+ concentration dependence
(1). The modifier was initially postulated to be a
discrete site located in the transmembrane domain (38), but an important contribution of the cytosolic tail was suggested more
recently (16). Regardless of its precise composition, the modifier site is generally accepted to face the cytoplasmic milieu, compatible with the finding that, at least in the case of NHE1, the
site of action of Cl
is intracellular.
Another important volume-dependent ion transporter, the
Na+-K+-Cl cotransporter, has also
been proposed to be regulated by intracellular Cl
(22, 24; however, see Ref. 13 for alternative view). In this system, it has been suggested that changes in intracellular
Cl
concentration regulate the activity of
serine/threonine kinase(s) that directly phosphorylate and thereby
activate the cotransporter. This, together with the earlier reports in
red blood cells, raised the possibility that cellular effectors,
including NHE, detect volume changes by sensing the associated
alterations in Cl
concentration. However, the
hyperosmotic activation of NHE1 and NHE2 persisted following bilateral
Cl
removal. An important implication of this finding is
that the shrinkage-induced rise in intracellular Cl
concentration cannot itself be the trigger for the osmotic stimulation of NHE. Since the intracellular Cl
content was reduced by
at least 70%, and the addition of 100 mM NaNO3 decreased
the cell volume by not more than 40%, the Cl
concentration in the depleted, shrunken cells would remain well below
(<50%) the resting intracellular level under isotonic conditions. Therefore, other volume sensors must relay the information to activate NHE.
In summary, we found that anion sensitivity is a common feature of the
three major NHE isoforms. In all cases, the presence of
Cl was found to be essential for optimal activity and, at
least in the case of NHE1, the site of action of the anion is
intracellular. Accordingly, deletion of part of the cytosolic tail
reduced the anion dependence of transport. The results can be most
readily explained by direct binding of the anion to the COOH-terminal region of the exchanger or to an ancillary protein that in turn interacts with the cytosolic aspect of the exchanger.
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ACKNOWLEDGEMENTS |
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This work was supported by the Canadian Cystic Fibrosis Foundation and the Canadian Institutes for Health Research (CIHR). O. Aharonovitz was supported by the Arthritis Society of Canada and the Canadian Cystic Fibrosis Foundation.
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FOOTNOTES |
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A. Kapus is a CIHR Scholar. K. Szászi is supported by a CIHR fellowship. J. Orlowski is supported by a Scientist Award from CIHR. S. Grinstein is supported by a Distinguished Scientist Award from the CIHR and is cross-appointed to the Department of Biochemistry of the University of Toronto. S. Grinstein is an International Scholar of the Howard Hughes Medical Institute and is the current holder of the Pitblado Chair in Cell Biology.
Address for reprint requests and other correspondence: S. Grinstein, Cell Biology Program, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: sga{at}sickkids.on.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 May 2000; accepted in final form 13 February 2001.
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