(Received for publication, August 1, 1995; and in revised form, September 26, 1995)
From the
The erythrocyte anion exchanger AE1 (band 3) serves as an important model for the study of the mechanism of ion transport. Chemical modification of human erythrocyte AE1 has previously suggested that glutamic acid residue 681 lies within the transport pathway and can cross the permeability barrier. This glutamate is conserved in all anion exchangers sequenced to date. We examined the effect on divalent (sulfate) and monovalent (chloride and bicarbonate) anion transport of mutating the corresponding glutamates in mouse AE1 and the closely related anion exchanger, AE2. Substitution of this conserved glutamate with uncharged or basic amino acids had a negligible effect on the maximal rate of sulfate-sulfate exchange in AE-reconstituted proteoliposomes, but largely abolished the steep pH dependence of sulfate transport observed in wild-type AE1 and AE2. In contrast, exchange of monovalent anions was undetectable in cells expressing these mutants. Replacement of the conserved glutamate with aspartate abolished both monovalent and divalent anion transport. These data suggest that the conserved glutamate residue plays a dual role in determining anion selectivity and in proton coupling to sulfate transport. A model explaining the role of the conserved glutamate in promoting ion selectivity and pH regulation is discussed.
Plasma membrane anion exchangers (AE) ()constitute a
diverse family of transporters, which, in mammalian cells, contribute
to the control of transmembrane anion gradients, cell volume, and
acid-base homeostasis. Three members of this family, designated
AE1-AE3, have been cloned and demonstrated by functional
expression to mediate transmembrane exchange of the physiological
substrates, chloride and bicarbonate (reviewed in Kopito(1990) and
Alper(1991)). In mammals, AE1-AE3 are expressed in a tissue- and
cell-specific manner. The best characterized among these is AE1, which
was first identified as the major integral membrane protein of the
erythrocyte, known as ``band 3''. AE1-mediated anion exchange
is rapid, electrically silent, and tightly coupled (see Passow(1986),
Knauf(1986), and Macara and Cantley(1983) for reviews). The latter two
features are best explained by a ``ping-pong'' kinetic model
in which anion translocation in one direction is temporally distinct
from, but dependent upon, anion translocation in the opposing
direction. This tight substrate coupling ensures that the flow of one
substrate is driven by the electrochemical potential of the other.
Thus, anion exchangers can be used by cells to establish Cl
gradients at the expense of pH
(HCO
) (Vaughan-Jones, 1979, 1982) or pH
gradients at the expense of Cl
(Paradiso et al., 1987).
Anion exchange by AE1 is highly selective, both for
anions over cations and among different anions (see Passow(1986),
Knauf(1986), and Macara and Cantley(1983) for reviews). AE1 displays
strong selectivity for its physiological substrates, transporting
chloride 10-250 times more rapidly than other halides.
Remarkably, despite their dissimilar size, charge distribution, and
atomic structures, chloride and bicarbonate are transported by AE1 at
nearly the same rate (Lowe and Lambert, 1982). This contrasts sharply
with the transport of the divalent sulfate anion, which, although
structurally similar to bicarbonate, is transported by AE1 at a rate
nearly 10,000-fold slower than chloride or bicarbonate (Brahm, 1988;
Schnell et al., 1977). Protons have opposing effects on the
transport by AE1 of monovalent anions such as Cl and
HCO
and divalent anions like sulfate.
These dramatic differences in transport rates and proton dependence
between the transport of monovalent and divalent anions have been
reconciled by Gunn(1972) who proposed that protonation of a titratable
group with pK
5 could convert AE1
from a monovalent to a divalent anion carrier. Milanick and Gunn(1984)
further suggested that the transport mechanism requires at least two
distinct ion binding sites: one for anions and one for protons. They
proposed that proton binding could serve to alter the net charge of the
anion binding pocket that crosses the permeability barrier during
translocation (Milanick and Gunn, 1984). This model is consistent with
the observation of a stoichiometric flux of protons accompanying
sulfate during sulfate-Cl
countertransport (Jennings,
1976). The AE2 exchanger, which is expressed in a wide variety of cell
types (Kopito, 1990), shares key features with AE1, including a
preference for Cl
and HCO
as substrates, sensitivity to stilbene disulfonate inhibitors
(Lee et al., 1991), and proton-stimulated sulfate transport
(Sekler et al., 1995). These conserved features, therefore,
may reflect fundamental aspects of the underlying transport mechanism.
Despite the wealth of kinetic and biophysical data on anion exchanger-mediated transport, and despite the availability of cloned cDNAs for three members of the AE family, the mechanistic details of how anion exchangers maintain a remarkably robust ability to catalyze extremely fast transport of the structurally dissimilar chloride and bicarbonate anions are known. Moreover, little is known about the amino acids specifically involved in ion binding, transport, or pH regulation. Chemical modification studies of anion transport in erythrocytes have provided some support for the involvement of certain amino acids in the anion exchange reaction cycle. These studies (Julien and Zaki, 1988; Wieth et al., 1982) have suggested that at least one unidentified arginine is essential for anion translocation. While modification of lysines by stilbene disulfonate and other lysine-specific reagents inhibits transport activity (Cabantchik and Rothstein, 1974; Jennings and Nicknish, 1985), site-directed mutagenesis of the lysines which constitute the covalent binding site for stilbene disulfonates fails to alter the ion affinity or the rate of ion transport, suggesting that they are not directly involved in the transport process (Garcia and Lodish, 1989; Wood et al., 1992). The participation of other AE1 lysines in anion translocation has not been resolved.
Carboxyl residues have also been implicated
in AE-mediated anion transport. Treatment of red blood cells with
carbodiimide compounds inhibits monovalent anion transport (Craik and
Reithmeier, 1985; Bjerrum et al., 1989). More recently,
treatment of red blood cells with carboxyl-specific Woodward's
reagent K (WRK), followed by borohydride reduction, inhibited chloride
transport by 75-80% while stimulating
SO
/Cl
exchange
(Jennings and Al-Rhaiyel, 1988). Significantly, WRK treatment
eliminates both the pH dependence of sulfate transport and the proton
flux which normally accompanies
SO
/Cl
exchange
(Jennings and Al-Rhaiyel, 1988), suggesting that the WRK modified
carboxyl may be the same as the titratable group originally proposed by
Gunn(1972). The WRK-modified carboxyl residue in human AE1 was recently
identified by Edman degradation to be Glu-681, predicted to be located
near the cytoplasmic interface of transmembrane helix 8 and is
conserved among all anion exchangers known to date (Jennings and Smith,
1992).
The present work examines the functional and structural consequences of mutating the glutamate in mouse AE1 (Glu-699) and AE2 (Glu-1007), which correspond to the WRK-reactive residue (Glu-681) in human erythrocyte AE1. The data support the hypothesis that this glutamate in mouse AE1 and AE2 plays a key role in determining the ion selectivity and kinetics of anion transport by these closely related anion exchangers.
Figure 1:
Expression level and
sulfate transport by wild-type and mutant anion exchangers. Crude
microsomes prepared from cells transfected with wild-type and mutant
AE1 (A) or AE2 (B) were analyzed by immunoblotting (top) or for sulfate-sulfate exchange (bottom). Amino
acid at position 699 (AE1) or 1007 (AE2) is indicated across the bottom; Glu is wild-type. Efflux of vesicular
[S]SO
into
SO
containing medium was determined at
pH 6 in the presence (shaded bars) or absence (open
bars) of the anion exchange inhibitor DIDS as described under
``Experimental Procedures.'' Values indicated represent the
mean ± S.E. of triplicate
measurements.
To exclude the possibility that the observed differences were attributable to variation in the expression levels of the wild-type and mutant proteins, the AE content of the proteoliposomes used in this experiment was assessed by immunoblotting using an antibody that recognizes the C-terminal domain of AE1 and is cross-reactive with AE2 (Thomas et al., 1989) (Fig. 1). The data indicate that all anion exchangers and mutants within each class (AE1 and AE2) were expressed at similar levels. The doublet of AE2 bands at 160 and 140 kDa correspond to mature (bearing complex oligosaccharides) and immature (high-mannose oligosaccharides) AE2 (Ruetz et al., 1993), suggesting that wild-type and mutant AE2 proteins were similarly distributed in pre- and post-Golgi compartments. Together, these data suggest that the capacity to transport divalent anions by AE1 is not explained simply by the charge or protonation state of the side chain at position 699, as suggested previously by chemical modification studies. Moreover, these data suggest that the structural requirements at this site are largely conserved between the related anion exchangers AE1 and AE2.
Sulfate transport by AE1 has been reported previously
to be steeply pH-dependent, suggesting the participation of protonation
of a specific acidic residue in the transport process (Schnell et
al., 1977). Because we have previously observed a similar pH
dependence of sulfate transport by AE2 (Sekler et al., 1995),
we examined the effects of the above-described mutants on the pH
dependence of sulfate transport (Fig. 2). Initial rates of
sulfate transport were determined under pH-clamp conditions between pH
5.5 and 7.5, such that intravesicular and extravesicular pH were
matched. As observed previously, the pH dependence of wild-type AE1 and
AE2 was characteristically steep. At pH 7.5, sulfate transport rates by
wild-type AE1 and AE2 at pH 7.5 were reduced by 75-80% of the
rate at pH 5.5. By contrast, the pH dependence of AE1 (Glu-699) and AE2
(Glu-1007) mutants was significantly diminished. The initial rates of
sulfate efflux of AE1E699Q and AE1E699A mutants were only 20%
lower at pH 7.5 than at pH 5.5. This pH insensitivity of sulfate
transport was even more dramatic for the AE21007Q and AE21007K mutants;
we could detect no significant difference in sulfate transport rate
between pH 5.5 and 7.5. We conclude that this conserved glutamate plays
a key role in determining the pH dependence of sulfate exchange
mediated by AE1 and AE2.
Figure 2:
pH dependence of sulfate/sulfate exchange
mediated by wild-type and mutant anion exchangers. Tracer
[S]SO
efflux from
proteoliposomes reconstituted with AE1 (left panel) or AE2 (right panel). Amino acids at AE1 position 699 were glutamate
(wild type, squares); glutamine (circles), or alanine (diamonds). Amino acids at AE2 position 1007 were glutamate
(wild type) (squares), glutamine (circles), or lysine (diamonds). Intravesicular and extravesicular pH were clamped
with nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the values indicated on
the abscissa, and sulfate transport rates were determined as
described in the legend to Fig. 1and under ``Experimental
Procedures.'' Values indicated represent the means of triplicate
measurements. The specific activity of sulfate efflux for AE1 and AE2
at pH 6 were 213 ± 27 and 264 ± 35 cpm of
[
S]SO
/mg of
protein
min, respectively.
Figure 3: Expression of AE2 at the cell surface is unaffected by mutations at position Glu-1007. HEK cells expressing wild-type (Glu) AE2 or AE2 substituted at position 1007 with the indicated residues were surface labeled by biocytin hydrazine, lysed, and immunoprecipitated with AE antibody. Immunocomplexes containing labeled AE2 were separated on SDS-polyacrylamide gel electrophoresis, transferred to nitrocellullose, and detected using streptavidin peroxidase as described under ``Experimental Procedures.''
Expression of
wild-type AE2 resulted in a marked acceleration of the initial rate of Cl efflux from HEK cells compared with vector transfected
control cells (Fig. 4A). Moreover, the transport was
completely dependent on the presence of trans-Cl
, was inhibited by the impermeable
anion gluconate, and was completely inhibited by the anion exchange
inhibitor, DIDS. Increasing external Cl
from 30 to 50
mM stimulated a significant increase in the rate of
Cl
efflux from 1.54 ± 0.22 to 2.2
± 0.23
10
cpm
Cl
mg protein
min
. These experiments show that
AE2-mediated Cl
efflux from HEK cells is inhibited by
disulfonic stilbenes and is stimulated by the presence of a trans-anion, suggesting that AE2 in these cells is
functionally expressed at the cell surface.
Figure 4:
AE2 glutamate 1007 is essential for
Cl/Cl
exchange. A,
characterization of the Cl
efflux assay. HEK cells
expressing wild-type AE2 were loaded with
Cl
and the rate of isotope efflux was determined in the presence of
media containing 20 mM (squares) or 50 mM chloride (triangles). Control efflux determinations on
AE2-transfected (circles) or mock-transfected cells (diamonds) were performed in the presence of 30 mM Cl
alone (diamonds), 30 mM gluconate (open circles), or 30 mM
Cl
containing 300 µM DIDS (closed
circles). The specific activities for the
[
Cl
] efflux measurement for
wild-type AE2 in 30 and 50 mM Cl
,
respectively, were 1.54
10
± 0.22 and
2.2
10
± 0.23 cpm
[
Cl
]/mg of protein
min. B, effect of substitution of Glu-1007 (squares)
with aspartate (triangles), lysine (circles), or
glutamine (diamonds) on tracer
[
Cl
] efflux into media
containing 20 mM Cl
. The specific activity
for the chloride efflux measurement for wild-type AE2 was 1.4
10
± 0.017 cpm of
[
Cl
]/mg of protein
min.
To determine the effect
of Glu-1007 substitution on Cl transport mediated by
surface-expressed AE2, we measured the rate of
Cl
efflux from cells expressing
wild-type AE2 or Glu-1007 mutants thereof (Fig. 4B).
The data indicate that substitution of Glu-1007 with Gln, Lys, or Asp
decreased the capacity of AE2 to stimulate
Cl
efflux above background levels. These data suggest that, in
contrast to transport of the divalent anion sulfate, chloride transport
by AE2 is extremely sensitive to the nature of amino acid side chain at
position 1007.
The physiological substrates of the AE class of anion
exchangers are chloride and bicarbonate, which bind to AE1 with similar
affinities and are transported at similar rates. Because of the marked
structural and electrostatic differences between Cl,
a halide, and HCO
, a planar oxyanion, it
was important to investigate the effect of Glu-1007 mutations on
Cl
/HCO
exchange, using
an assay for intracellular pH which exploits the pH-sensitive
intracellular fluorescent indicator BCECF. Using this assay, cells
expressing wild-type AE2 responded to the application of a large
outward Cl
gradient by a rapid (
50 s) increase
in intracellular pH of
0.6 pH unit (Fig. 5). We have
demonstrated previously that this pH increase is the result of the
AE-mediated influx of bicarbonate (Lee et al., 1991). This pH
change is similar in magnitude to previously observed values (Lindsey et al., 1990) and was reversed upon restoring extracellular
[Cl
]. There was no significant response in
cells transfected with vector plasmid. None of the cells expressing
mutant AE2, substituted with either Asp, Lys, or Gln at position 1007,
showed detectable HCO
transport
stimulated by either inward or outward directed Cl
gradients. Moreover, we were unable to detect significant
HCO
transport driven by outward or inward
gradients of nitrate, a rapidly transported substrate for wild-type AE1
and AE2 (data not shown). These findings confirm that Glu-1007
contributes a critical role to the transport of monovalent halides or
oxyanion substrates in either direction across the membrane. Because
the level of expression of AE2 and AE2 mutants at the cell surface of
these cells was similar (Fig. 3), we conclude that the specific
amino acid side chain at position 1007 of AE2 contributes critically to
the transport in either direction of structurally unrelated monovalent
anion substrates.
Figure 5:
AE2
glutamate 1007 is essential for
Cl/HCO
exchange.
Intracellular pH was monitored in HEK cells expressing the indicated
AE2 constructs (or vector) and loaded with the intracellular pH
indicator dye BCECF. Cells were equilibrated in normal Ringer's
solution buffered with
HCO
/CO
, pH 7.4. At the time
indicated the normal Ringer's solution was replaced with
isoosmotic solution lacking Cl
(replaced with
gluconate). Intracellular pH and time scales are
indicated.
The above data show that a conserved glutamate at position 699 of mouse AE1 and position 1007 of mouse AE2 plays a key role in determining the ion selectivity and kinetics of anion transport by these closely related anion exchangers. Substitution of this residue with amino acids containing basic or neutral side chains effectively eliminated the capacity for exchange of the physiological monovalent anions chloride and bicarbonate. By contrast, although the same mutations had no measurable effect on the rate of transport of the divalent anion sulfate at acidic pH, these mutations sharply attenuated the steep pH dependence of sulfate transport. The different consequences of these mutations for monovalent and divalent anion transport suggest a critical role for this glutamate in distinct steps of the chloride and sulfate transport cycles and provide some insight into the basic mechanism by which these exchangers bind and translocate anions.
Previous studies using the carboxyl-specific WRK followed by
reductive cleavage permitted ``chemical mutagenesis'' of
human erythrocyte AE1 in which one or more glutamic acid carboxyls on
the protein were converted to the corresponding alcohol,
5-hydroxynorvaline (Jennings and Anderson, 1987). This effect was later
shown to be due to modification of a single glutamate, Glu-681,
corresponding to position 699 in mouse AE1 (Jennings and Smith, 1992).
WRK/NaBH modification of AE1 inhibited
Cl
/Cl
exchange while stimulating
SO
/SO
exchange (Jennings and Al-Rhaiyel, 1988). Moreover, this
modification, which is structurally similar to permanent protonation of
the carboxyl, abolished the pH dependence of sulfate transport and
eliminated the proton flux normally associated with divalent anion
movement (Jennings and Al-Rhaiyel, 1988; Jennings and Smith, 1992).
These findings led Jennings to propose that Glu-681 is the titratable
site on AE1 the protonation state of which appears to switch AE1
between monovalent and divalent anion selectivity (Jennings and Smith,
1992). However elegant, these data left unresolved several key issues.
First, the presence of
20% of unmodified AE1 significantly
complicated the interpretation of the chemical modification data. Is
the residual Cl
/Cl
exchange
following WRK modification entirely explained by the presence of
unmodified protein, or is there residual
Cl
/Cl
exchange following
modification? Second, the borohydride reduction reaction is difficult
to control; it is apparently not possible to ensure cleavage of all of
the arylsulfonate adduct without risking reduction of other groups on
the protein (Jennings and Smith, 1992). Hence it is difficult to
attribute all the properties of WRK-BH4 modified AE1 to the effects
exclusively on Glu-681. Third, although the conversion of E681 to an
alcohol would indeed be expected to render the residue uncharged, such
chemical modification does not permit the substitution of other amino
acids at that position. Consequently, it is not possible to determine
whether the WRK effect is to ``protonate'' Glu-681 or to
introduce detrimental steric consequences by conversion to
5-hydroxynorvaline. These concerns have all been addressed in the
present work using site-directed mutagenesis, which ensures that all
copies of the expressed mutant protein will have the mutant phenotype
and that any amino acid substitution may be made at will. Moreover, in
this paper we have extended the analysis to include the non-erythroid
anion exchanger AE2, in which the residue corresponding to human AE1
Glu-681 is highly conserved by Glu-1007. This issue is of particular
interest, since it has been suggested that AE2 transport proceeds by a
fundamentally different, non-ping-pong mechanism from AE1 (Restrepo et al., 1991). Our data suggest a fundamental mechanistic
conservation not only among anion exchangers of different species (i.e. human and mouse) but also of different members (AE1 and
AE2). Indeed, functionally reconstituted mouse AE3 displays a
strikingly similar pH dependence of sulfate transport to that of AE1
and AE2, (
)suggesting that the conserved glutamate at
position 998 is also the major titratable acidic group in AE3.
This steric constraint suggests a major role for
glutamate 699/1007 in the precise coordination of anions in the binding
pocket. It is possible that the side chain of glutamate 699/1007
participates in accepting hydrogen bonds from residues that either
donate or accept hydrogen bonds to or from chloride. It is also
possible that the side chain of glutamate 699/1007 salt bridges with a
guanidino group of an arginine which would in turn bind the negative
charge of chloride. These indirect roles could allow glutamate 699/1007
to influence the proper geometry of the peptide backbone(s) and or side
chain(s) that binds chloride. Networks of hydrogen bonds, primarily
main chain peptide NH and CO groups in addition to hydroxyl side
chains, are capable of dissipating the charge(s) on a substrate. For
instance, the ligand-bound crystal structures of sulfate- and
phosphate-binding proteins, which are involved in active transport in Salmonella typhimurium, revealed that single OH groups from
side chains of residues such as serines and threonines, together with
peptide backbone NH hydrogen donor groups and CO hydrogen acceptor
groups form multiple hydrogen bonds which give rise to an extensive and
stable network (Pflugrath and Quiocho, 1988; Luecke and Quiocho, 1990;
He and Quiocho, 1991). Such an array of interactions can potentially
moderate opposite charge attractions and facilitate ion movement. That
aspartate cannot replace glutamate to support chloride transport may be
explained by the fact that aspartate and glutamate have markedly
different effects on the conformation and chemical reactivity of
neighboring peptide backbone(s) (Creighton, 1992) and emphasizes that
simple net charge considerations cannot account solely for the observed
tight Cl binding.
The differential effect of Glu-699/1007 substitutions
on monovalent and divalent anion transport suggests two mutually
exclusive pathways for carrier reorientation, which are outlined in Fig. 6. Preferred substrates, e.g. Cl and HCO
(Fig. 6A),
form an initial loose complex with the carrier (step 1), which is
followed by a conformational change (step 2), leading to a tightly
bound (or occluded) form. According to this model, the specificity of
ion transport depends on the ``tightness'' of the tightly
bound state and is, therefore, correlated with the transport rates
rather than with the apparent affinity. We propose that entry into this
tightly bound state requires the participation of the unprotonated WRK
glutamate, perhaps by forming an essential salt bridge or by
contributing to the precise coordination of the substrate anion in the
transition state. There is elegant evidence from the crystal structure
of the S. typhimurium periplasmic sulfate-binding protein that
salt bridges span an opening in the deep cleft wherein a single
dehydrated sulfate is completely engulfed. Removal of these salt
bridges by site-directed mutagenesis reveals that they function to
stabilize the closed liganded form and modulate the kinetics of cleft
opening (Jacobson et al., 1991, 1992). Protonation, or
substitution by mutagenesis, of the WRK Glu in anion exchangers would
interfere with its ability to form salt bridges and hence reduce the
probability of forming the tightly bound occluded state (step 2).
Figure 6: A model for anion exchanger-mediated anion transport. Details are given in the text.
On
the other hand, substrates like sulfate fail to achieve the tightly
bound state necessary for rapid reorientation (Fig. 6B). Binding of sulfate to the protonated form of
the carrier is permissive for a slow conformational reorientation in
which both the substrate and the proton are released to the trans side of the membrane (steps 2a and 2b). Three distinct mechanisms
can be envisioned to account for the effect of protonation at the
conserved glutamate in enhancing sulfate transport. First, protonation
could enhance the loose binding of sulfate (steps 1 and 5), as
suggested by the data of Jennings(1989). However, Milanick and
Gunn(1984) observed only a relatively modest effect of pH on the K for sulfate, while over the same pH range, the
rate of sulfate transport varied by as much as 100-fold (Milanick and
Gunn, 1984; Sekler et al., 1995). Therefore, an effect of
protonation of Glu-699/1007 on sulfate affinity cannot be ruled out.
Second, protonation could allow a tighter occlusion of sulfate for
enhanced stabilization of the transition state (step 2a). However,
substituting the conserved glutamate with alanine, asparagine, and even
lysine had no major effect on the initial rate of sulfate transport,
which reflects the tightness of sulfate occlusion. Residues involved,
either directly or indirectly, in sulfate binding are predicted to be
very sterically sensitive. We propose that protonation could facilitate
the conformational changes associated with sulfate translocation by
reducing the energy barrier to movement (step 2b).
In summary, our
data support a model in which exchange coupling and substrate
specificity are linked to the tightness of substrate binding in the
transition state. The data suggest further that two types of
conformational reorientations, distinguished by their sensitivity to
protonation or mutation at Glu-699/1007, can promote anion exchange.
The first is a rapid pathway (represented in Fig. 6A by
steps 3a and 3b), which requires precise coordination of monovalent
anion binding to stabilize the transition state. This pathway is
extremely sensitive to the side chain and even the protonation state at
position Glu-699/1007. In the second pathway, divalent anion binding is
followed by slow (10
- to 10
-fold slower)
reorientation only if protonation of the glutamate side chain or
mutagenesis reduces the energy barrier to movement.