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INTRODUCTION |
AE1, also called Band 3, is the plasma membrane chloride
bicarbonate anion exchange protein of the erythrocyte (1) and kidney
(2). AE1 is a member of a multigene family (3); AE2 is most broadly
expressed in tissues such as gastric parietal cells (4) and choroid
plexus (5); AE3 is expressed in excitable tissues: heart (6), brain
(7), and retina (8). All of the anion exchangers have a two-domain
structure: a 55-kDa membrane domain responsible for anion exchange and
a 43-77-kDa cytoplasmic domain involved in cytoskeletal interactions.
Membrane domains are highly conserved (80% homology in the whole
domain and close to 90% in the transmembrane segments). All anion
exchangers function by an electroneutral "Ping-Pong" mechanism of
Cl
/HCO3
exchange. The
high abundance of AE1 in the erythrocyte membrane (50% of integral
protein (9)) has made the protein a model for the study of transport
protein structure and function (10, 11).
Several residues have been implicated as part of the anion exchange
mechanism of AE1. On the basis of indirect evidence, Passow has
proposed a model for anion translocation that involves residues from
TMs1 5, 8, 10, 12, and 13, including mouse residues Glu472, Glu535,
Lys539, Glu681, His703,
Arg731, His735, His816, and
His834 (12). Jennings and co-workers (13-15) have strong
evidence to implicate the TM8 residue, Glu681, in the
transport process, since labeling this residue with Woodward's reagent
K and reduction with sodium borohydride resulted in altered anion
exchange kinetics. The functional role of Glu681 in AE1 was
confirmed in mutagenesis experiments of mouse AE1 (16) and extended to
the homologous position of mouse AE2, suggesting that the mechanistic
role of Glu681 is conserved among anion exchange proteins
(17). Woodward's reagent K chemical modification of AE1 abolishes
chloride transport, yet relieves the requirement for proton cotransport
during sulfate transport. During sulfate transport in unmodified AE1, a
proton, supplied by Glu681 is cotransported. Sulfate/proton
cotransport takes place in both inward and outward directions, which
implies that Glu681 has access to both the intracellular
and extracellular sides of the membrane. Taken together,
Glu681 is functionally involved in anion exchange events
and may reside at the permeability barrier of AE1.
Glu681 of TM8 is the best characterized residue that
interacts with anions during translocation event. We have therefore
focused on TM8 in efforts to identify residues involved in anion
translocation. In a previous report we analyzed a panel of introduced
cysteine residues spanning the TM8 region from Ser643 to
Ser690. Using accessibility to chemical modification by
3-(N-maleimidylpropionyl)biocytin and lucifer yellow
iodoacetamide we identified the bilayer spanning residues as the
sequence Met664-Gln683 (18).
Substituted cysteine mutagenesis and sulfhydryl chemistry have proved a
fruitful approach to identify residues of the transport pathway. The
method is to mutate all native cysteine residues in a protein and to
systematically re-introduce unique cysteine residues into the
cysteineless background. Inhibition of anion transport by
sulfhydryl-specific reagents is then assessed for each mutant. The
approach has been useful for studies of the bacterial transport
proteins, lactose permease (19-21) and UhpT (22). Among mammalian
membrane proteins, this approach has proved very successful for ion
channels. Derivatization of a cysteine with a methanethiosulfonate will
impair ion conductance through steric blockage of a confined ion
translocation pore. Pore-lining residues in the cystic fibrosis chloride channel, CFTR (23-25),
-aminobutyric acid receptor
chloride channel (26) and acetylcholine receptor sodium channel (27, 28) have been identified in this way. Differential sensitivity to
inhibition by methanethiosulfonate compounds has also identified residues of the transport pathway in the glutamate transport protein GLT-1 (29), sodium glucose cotransporter (30), and sodium/calcium exchanger (31).
We used substituted cysteine mutagenesis and sulfhydryl chemistry to
identify pore-lining residues in the TM8 region of human AE1
chloride/bicarbonate exchange protein. Previously we constructed a
cysteineless form of human AE1, called AE1C
, and
characterized the protein as fully functional (32). We have
systematically replaced the residues of AE1 from the glycosylation site
at Asn642 through TM8 into the cytoplasmic region and
identified the sequence Met664-Gln683 as
spanning the bilayer (18). In this report we measure the effect of two
sulfhydryl reagents upon AE1 anion transport function, using BCECF
fluorescence to monitor intracellular pH shifts associated with
Cl
/HCO3
exchange in
transfected HEK293 cells. Of the two sulfhydryl reagents, pCMBS is
membrane-impermeant but slowly transported by AE1, while MTSEA is
membrane-permeant in its unprotonated form (33). We have identified a
sequence of leucine, isoleucine, and alanine residues that lie on one
face of an
-helix and, when mutated to cysteine, are susceptible to
inhibition by pCMBS and MTSEA.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction endonucleases were from New England
Biolabs. ECL chemiluminescent reagent, horseradish peroxidase
conjugated to sheep anti-mouse IgG, Hyperfilm, and Immobilon-P membrane
were from Amersham Pharmacia Biotech. BCECF-AM was from Molecular
Probes. Poly-L-lysine, pCMBS, and nigericin were from
Sigma. Coverslips were from Fisher. MTSEA was from Toronto Research Chemicals.
Construction of Mutant Anion Exchangers--
A human AE1
cDNA construct, called AE1C
, in which all five
cysteine codons were mutated to serine was constructed previously (32)
in the expression vector pRBG4 (34). Individual cysteine codons were
introduced into AE1C
, to yield mutants, each with a
unique cysteine codon (18). Introduced cysteine mutants at amino acids
645-647 were not constructed because their codons overlap with the
SmaI site (nucleotides 2048-2053) used to clone introduced
cysteine mutants into AE1C
. Mutagenesis was performed
using a polymerase chain reaction megaprimer mutagenesis strategy (35,
36). Polymerase chain reaction primers were designed using the Primers
program (Whitehead Institute for Medical Research). Polymerase chain
reaction was performed using an ERICOMP thermal cycler and either Vent
DNA polymerase (New England Biolabs) or Pwo polymerase (Boehringer Mannheim). Mutants were verified by DNA sequencing.
Protein Expression--
Anion exchangers were expressed by
transient transfection of human embryonic kidney (HEK) 293 cells (37),
as described previously (38), except that calcium
phosphate-precipitated plasmid was added at 2.8 µg of anion exchanger
plasmid with 4.2 µg of pRBG4 carrier/100-mm tissue culture dish.
Cells were grown at 37 °C, in a 5% CO2 environment in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 5% (v/v) fetal bovine serum (Life Technologies, Inc.) and
5% (v/v) calf serum (Life Technologies, Inc.) and harvested 48 h
post-transfection.
Anion Exchange Assays--
HEK293 cells were grown on top of
7 × 11-mm glass coverslips in 60-mm tissue culture dishes and
transfected as described. Two days post-transfection, coverslips were
rinsed with serum-free Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) and loaded with BCECF-AM by incubation in 4 ml of
serum-free Dulbecco's modified Eagle's medium, containing 2 µM BCECF-AM, for 20-30 min, at 37 °C. Coverslips were
mounted in a fluorescence cuvette, with perfusion capabilities.
Intracellular pH was monitored by measuring fluorescence at excitation
wavelengths 440 and 502 nm and emission wavelength 529 nm, in a Photon
Technologies International RCR spectrofluorometer. The cuvette was
perfused at 3.5 ml/min alternately with Ringer's buffer (5 mM glucose, 5 mM potassium gluconate, 1 mM calcium gluconate, 1 mM MgSO4,
2.5 mM NaH2PO4, 25 mM
NaHCO3, 10 mM Hepes, pH 7.4) containing 140 mM sodium chloride (chloride buffer) or 140 mM
sodium gluconate (chloride-free buffer). Both buffers were bubbled
continuously with air containing 5% carbon dioxide. Intracellular pH
was calibrated by the nigericin-high potassium method (39), using three
pH values from pH 6.5 to 7.5. Transport rates were determined by linear
regression of the initial linear rate of change of pH, using the
Kaleidagraph program (Synergy Software).
Transport Inhibition Assays--
Transport experiments were
performed as described above. Uninhibited rates of anion exchange were
determined by perfusion with chloride Ringer's followed by
chloride-free Ringer's, chloride Ringer's, and chloride-free
Ringer's. The cuvette was then perfused with 10 ml of chloride-free
Ringer's containing either: 5 mM MTSEA or 0.2 mM pCMBS. Cells were incubated with the inhibitor for a total of 8 min, followed by washing with chloride-free Ringer's until
the fluorescence base line was stable (350-550 s). Cells were then
perfused with chloride Ringer's, chloride-free Ringer's, and chloride
Ringer's. Inhibition of anion exchange was determined from the initial
rates of alkalinization and acidification observed as buffers were
changed, before and after inhibitor addition. Rates were determined by
linear regression, using Kaleidagraph software. Percent residual
activity was calculated as: % residual activity = rate of
fluorescence change after sulfhydryl reagent/rate of fluorescence
change without treatment × 100.
Electrophoresis and Immunoblotting--
Samples were
electrophoresed on 8% acrylamide gels (40) and transferred to
Immobilon membrane (41). AE1 was detected by incubation of the blot
with 10 ml of TBSTM (TBST buffer (0.1% (v/v) Tween 20, 137 mM NaCl, 20 mM Tris, pH 7.5), containing 5% (w/v) non-fat dry milk powder (Carnation) and 3 µl of anti-human C-terminal peptide antibody 1657 (18). After washing blots were probed
with 10 ml of 1:3000 diluted horseradish peroxidase conjugated to goat
anti-rabbit IgG. After 1.5-h incubation, blots were washed with TBST
and visualized using ECL reagent and Hyperfilm.
Molecular Biological Methods--
Plasmid DNA for transfections
were prepared using Qiagen columns (Qiagen Inc.). DNA sequencing was
performed by the Core Facility in the Department of Biochemistry,
University of Alberta, with an Applied Biosystems 373A DNA sequencer.
All other procedures followed standard protocols (42).
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RESULTS |
Expression of Introduced Cysteine Mutants--
A form of human AE1
in which all cysteine codons were mutated to serine codons was
constructed previously and called AE1C
(32). Each codon
of the AE1 sequence from Asn643 to Ser690
(except Ser645-Arg647) was individually
mutated to a cysteine codon and cloned into AE1C
(18).
This sequence begins immediately after the extracellular site of
glycosylation (Asn642) and proceeds through TM8
(Met664-Gln683), into an intracellular region
(18). This array of introduced cysteine mutants provides a system in
which to search systematically for residues that line the transmembrane
anion translocation channel.
Fig. 1 is an immunoblot of HEK293 cells
expressing wild-type AE1 and a subset of AE1 mutants. The immunoblot
shows that AE1 mutants are expressed to similar levels as wild-type
AE1. HEK293 cells do not express AE1 that is detectable
immunologically, as shown by the vector control lane. Previously we
measured the degree of cell surface processing of the array of
introduced cysteine mutants (18). Only W648C and E681C had reduced
processing to the cell surface, but in both cases it was only a partial
impairment.

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Fig. 1.
Expression of AE1 and introduced cysteine
mutants in HEK293 cells. HEK293 cells were transfected with AE1
cDNA, solubilized in SDS-PAGE sample buffer, and subjected to
electrophoresis on a 7.5% acrylamide gel. Proteins were transferred to
Immobilon-P membrane, and the immunoblot was incubated with anti-AE1
antibody, 1657 (18). Numbers indicate the position of the
introduced cysteine mutation. WT is wild-type; V
is vector, pRBG4-transfected cells.
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Measurement of Anion Exchange Activity--
Anion exchange
activity of each introduced cysteine mutant was measured using a
fluorescence assay. Transfected HEK293 cells, grown on the surface of
coverslips, were loaded with the pH-sensitive dye BCECF-AM and
suspended in a fluorescence cuvette. The cells were alternately
perfused with Ringer's buffer containing chloride, followed by
chloride-free Ringer's buffer. The result of these manipulations is to
change the direction of the transmembrane chloride gradient (34). In
cells expressing functional anion exchangers chloride will move in one
direction across the membrane, in exchange for bicarbonate in the other
direction. As bicarbonate leaves the cell, the cell acidifies; when
bicarbonate enters the cells, it alkalinizes. At the end of each
experiment, fluorescence levels were calibrated with pH standards, to
allow conversion of fluorescence data to absolute intracellular pH
values. Transport activity is determined by measuring the rate of
alkalinization and acidification after switching buffer solutions.
Fig. 2 shows an example of an anion
exchange assay, in this case showing the activity of wild-type AE1,
AE1C
, and vector-transfected cells. Fig. 2 shows that the
transport activity of AE1, AE1C
, and pRBG4 are 0.49, 0.20, and 0.014 pH/min, respectively, during the alkalinization phase.
Vector-transfected HEK293 cells have a background anion exchange
activity around 10% of AE1C
. Table
I shows the transport activity determined
for each of the introduced cysteine mutants, relative to
AE1C
. Extracellular mutants W648C, I650C, P652C, G654C,
L655C, F659C, P660C, and W662C and transmembrane mutants A671C, F679C,
and E681C all have greatly impaired anion exchange function. These
mutants therefore were not subjected to inhibition assays with
sulfhydryl reagents.

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Fig. 2.
Assay of anion exchange activity. HEK293
cells transfected with human AE1 cDNA (A),
AE1C cDNA (B) or pRBG4 vector alone
(C) were grown on glass coverslips, then loaded with
BCECF-AM, pH-sensitive dye. Cells were suspended in a fluorescence
cuvette and intracellular pH was monitored, as described under
"Experimental Procedures." Cells were perfused with either
Ringer's buffer containing 140 mM NaCl, or Ringer's
buffer, with NaCl replaced by 140 mM sodium gluconate. The
bar at the top of each panel represents the time
period when the cuvette was perfused with chloride containing
(solid bar) or chloride-free buffer (open
bar).
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Table I
Transport activity of AE1 introduced cysteine mutants
Introduced cysteine residues with low or no functional activity are
marked by an asterisk and were not characterized for sulfhydryl
inhibition. ND refers to data not determined. Data are shown ± S.E.
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Inhibition of Anion Exchange Activity by Sulfhydryl
Reagents--
To begin to identify the residues that line the
hydrophilic transmembrane anion translocation channel of AE1, we
attempted to inhibit anion exchange activity of introduced cysteine
mutants with sulfhydryl reagents. The protocol we used was to mount
coverslips containing transfected HEK293 cells in the fluorimeter and
to assay anion exchange activity as described above. However, during one incubation with chloride-free Ringer's buffer, the buffer was
modified with either 5 mM MTSEA or 0.2 mM
pCMBS. The anion exchange assay was then repeated after washing away
the sulfhydryl compound so that only the effect of covalent sulfhydryl
modification was measured.
Fig. 3 is an example of a sulfhydryl
inhibition assay. In this example, I684C AE1 was treated with MTSEA.
The inhibition assay proceeded as seen in the anion exchange assays,
but the data were not calibrated for intracellular pH, so that the
y axis represents raw fluorescence data. Since transport
inhibition is calculated as the ratio of transport in the presence and
absence of sulfhydryl compound, absolute pH values are not required.
The reduced slope of the curves that follow MTSEA incubation shows that
anion exchange is inhibited. As perfusion was switched from one
solution to another, we frequently observed a transient rise in
fluorescence ratio followed by a fall, as seen in this figure. We
attribute this artifact to changes of fluid pressure. Slopes were
calculated in the region following this artifact when it was
observed.

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Fig. 3.
Inhibition of I684C AE1 by MTSEA. HEK293
cells transfected with I684C AE1 cDNA were subjected to the anion
exchange assay protocol. Cells were perfused alternately with
chloride-containing (open bar, across the top)
and chloride-free Ringer's buffer (black bar).
A, during each experiment, with the same cells on a
coverslip, cells were subjected to perfusion with the two buffers. The
cells were then incubated with 5 mM MTSEA, in chloride-free
Ringer's buffer, and washed for approximately 500 s with
chloride-free Ringer's buffer (not shown). B, after MTSEA
treatment, the cells were perfused again with the two buffers. Residual
activity was calculated as the transport activity remaining after
treatment with MTSEA.
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The methodology shown in Fig. 3 was applied to each of the 36 functional introduced cysteine mutants in the
Ser643-Ser690 region of human AE1. Fig.
4 summarizes the results of inhibition with the positively charged methanethiosulfonate compound, MTSEA. MTSEA
is membrane-permeant in its unprotonated form (33) and therefore should
be able to access the entire anion translocation channel, including
sites on the intracellular side of the permeability barrier. Among the
36 introduced cysteine mutants characterized, L673C, A677C, I684C, and
I688C had anion exchange activity that was significantly inhibited by
MTSEA treatment. Maximum inhibition was observed for mutant I684C
(59 ± 3% residual activity). Neither wild-type AE1 nor
AE1C
were inhibited by MTSEA, since they had respective
residual anion exchange rates of 100 and 104% after MTSEA treatment.
The transmembrane permeability barrier has previously been mapped to
Glu681 of human AE1, so that Ile684 and
Ile688 reside beyond the permeability barrier, relative to
the outside of the cell.

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Fig. 4.
Treatment of AE1 introduced cysteine mutants
with MTSEA. HEK293 cells transfected with cDNA for AE1
introduced cysteine mutants were assayed for anion exchange activity
before and after 8-min incubation with 5 mM MTSEA. Residual
activity was calculated as the transport activity remaining after
treatment with MTSEA. Data are plotted (S.E. (n = 2-5). Mutants marked by an asterisk were not analyzed. The
black bar marks the control AE1C , cysteineless
mutant.
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Fig. 5 shows the effect of pCMBS
treatment of AE1 introduced cysteine mutants. Mutants A666C, S667C,
L669C, L673C, L677C, and L680C had impaired anion exchange activity
after treatment with 0.2 mM pCMBS, while all other mutants
were not impaired by pCMBS. There is a graded effect of pCMBS upon the
introduced cysteine residues; the two outermost sites of inhibition
(A666C and S667C) had a lower level of inhibition than the inner sites.
Two mutants, F679C and E681C, whose function is greatly impaired by
cysteine introduction flank the most pCMBS-sensitive mutant, L680C.
L680C is also adjacent to Glu681, which has been proposed
to define the transmembrane permeability barrier. L669C and F680C could
be inhibited by pCMBS, but not by MTSEA. AE1C
was
insensitive to pCMBS, since it had 110% residual activity after pCMBS
treatment. However, wild-type AE1 had modest inhibition with 86 ± 16% residual activity. Previous investigators found that AE1 in red
cells could be covalently inhibited by pCMBS, but only low levels of
inhibition would be expected at the low concentration and incubation
time used in the present study (43). Inhibition of wild-type AE1 by
pCMBS is attributed to the endogenous Cys479.

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Fig. 5.
Treatment of AE1 introduced cysteine mutants
with pCMBS. HEK293 cells transfected with cDNA for AE1
introduced cysteine mutants were assayed for anion exchange activity
before and after 8-min incubation with 0.2 mM pCMBS.
Residual activity was calculated as the transport activity remaining
after treatment with pCMBS. Mutants marked by an asterisk
were not analyzed. Data are plotted (S.E). Mutants without error bars
were analyzed only once; mutants with error bars had n = 2-5. The black bar marks the control AE1C ,
cysteineless mutant.
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Failure to observe complete transport inhibition with both MTSEA and
pCMBS reflects (i) that the assay measures only covalent inhibition,
(ii) chemical reaction only occurred for 8 min, and (iii) only moderate
concentrations of sulfhydryl reagents were used to prevent nonspecific
effects on the cell. In studies to identify pore-lining residues of
channels, a maximum inhibition of 25-40% was also observed (25, 26).
Introduced cysteine mutants were defined as those inhibited by
sulfhydryl reagents if residual activity after inhibition was less than
100%. That is, those mutants whose error bars did not overlap with
100% activity. Because of the large number of mutants analyzed,
experiments were repeated until statistically significant only at those
sites that were inhibited by sulfhydryl reagents.
Fig. 6 places the sulfhydryl reagent
inhibited residues in models of the transmembrane segment 8 region. In
a helical wheel plot of residues Met664-Ile688
(Fig. 6A), it is clear that sulfhydryl-sensitive sites
cluster on one face of the helix. Only a single hydrophilic site
(Ser667) was sensitive to inhibition. The remaining
sensitive sites are all aliphatic residues: leucine, isoleucine, or
alanine residues in wild-type AE1. Interestingly the only two
introduced cysteine mutants (A671C and F679C) that have greatly
impaired transport activity localize on the face of the helix directly
opposite from the sulfhydryl-sensitive residues we have identified. The
helical face containing Ala671 and Phe679 has
been identified as the most hydrophobic surface of the helix and is
predicted to face lipid (10).

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Fig. 6.
Topology and helical wheel models for the
transmembrane segment 8 region of human AE1. Topology model is
based on a structure determined by introduced cysteine methods (18).
Black circles represent introduced cysteine mutant with
transport activity inhibited by both MTSEA and pCMBS; dark
gray residue is inhibited by pCMBS only; light gray
residues are inhibited by MTSEA only. A, helical wheel model
(3.6 amino acids/helical turn) of residues Met664-
Ile688 of human AE1. Asterisks mark introduced
cysteine mutants with greatly impaired function. B, topology
model for transmembrane segment 8 of AE1. Circles filled
gray represent introduced cysteine mutants with transport activity
inhibited by MTSEA and pCMBS compounds. The large branched structure at
Asn642 marks the site of N-linked glycosylation.
The black box represents the proposed transmembrane region,
while the unfilled box represents a proposed extension of
the transmembrane segment 8 helix beyond the membrane surface. The
jagged lines with black ovals on the end
represent lipid molecules.
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The transmembrane topology diagram (Fig. 6B) illustrates the
topological disposition of residues in the region, as determined by
accessibility to labeling by biotin maleimide (18). The residues we
have identified as sensitive to inhibition by MTSEA and pCMBS form a
clear line through the transmembrane region. Interestingly, the
sequence of sulfhydryl reagent-sensitive residues starts beneath the
predicted surface of the bilayer and extends beyond the surface of the
membrane. Since helical periodicity of anion exchange activity is
maintained up to Ile688, Fig. 6B models the
Ile684-Ser690, shown previously to be
extramembraneous (18), as a helical region.
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DISCUSSION |
In this paper we have examined the amino acid sequence from
SER643-SER690 of human AE1, to identify
residues that line a transmembrane anion translocation pore. We have
focused on this region because it contains Glu681, shown
previously to be accessible to membrane impermeant Woodward's reagent
K, from either side of the membrane (13-15). This residue may
therefore reside at the transmembrane permeability barrier. Because
Glu681 is accessible to Woodward's reagent K and has been
implicated as one residue that interacts with anions, at least during
sulfate transport, we decided to examine the region surrounding
Glu681 to find other residues that might form the anion
translocation channel. We recently determined the topology of the
Ser643-Ser690 region and localized
Glu681 to within four residues of the cytosolic surface of
the protein (18).
Our results have identified a sequence of introduced cysteine mutants
(Ala666, Ser667, Leu669,
Leu673, Leu677, Leu680,
Ile684, and Ile688) in the transmembrane
segment 8 region of human AE1 that can be inhibited either by the
sulfhydryl reagents pCMBS or MTSEA. The locations of the mutants have a
helical periodicity and reside on one face of a predicted
-helix,
which extends at least one turn beyond the surface of the membrane.
Seven of the eight identified residues are found within a 100° arc of
a TM8 helical wheel plot, indicating that the region forms an
-helix. The sensitive helical face is directly opposite the most
hydrophobic face of TM8 and is adjacent to Glu681, which
was shown previously to interact with anions during transmembrane translocation. The simplest explanation for our data is a covalent interaction of introduced cysteine residues with the sulfhydryl compounds; at sites that line the pore, these reagents block the pore
and inhibit anion transport. On this basis we propose that Ala666, Ser667, Leu669,
Leu673, Leu677, Leu680,
Ile684, and Ile688 line the transmembrane
translocation channel and interact with substrate anions as they cross
the bilayer. An alternate possibility is that the inhibited residues
form a conformationally active surface of AE1, part of a cleft where
two helices meet and whose movements are blocked by sulfhydryl
modification. However, this model is unlikely since such a cleft would
not be likely to have access to pCMBS and MTSEA, two hydrophilic reagents.
Our observation that the hydrophobic amino acids leucine, isoleucine,
and alanine line the TM8 portion of the anion translocation channel at
first seems surprising. The alkyl side chains of these amino acids are
not hydrophilic and cannot contribute hydrogen bonds to line an aqueous
channel. However, hydrophobic residues have been found to form part of
the lining of the cystic fibrosis chloride channel, CFTR (23, 24), the
-aminobutyric acid receptor channel (26), and the acetylcholine
receptor Na+ channel (28). The transmembrane carbohydrate
translocation channel of the maltoporin protein is lined with
hydrophobic residues (44). Similarly, the crystal structure of a
bacterial K+ channel illustrated that the ion translocation
pore is lined with hydrophobic amino acids (45). The authors speculated
that the hydrophobic lining provides the most energetically favorable surface for movement of ions. Interactions with the wall of the pore
would only impede ion transit through the pore. A hydrophobic lining of
the AE1 anion translocation pore could be important to facilitate the
high turnover rate (105 anions·s
1 (46)), which is an
order of magnitude slower than ion channel fluxes, but still very fast.
The observation that the pore-lining face of the helix is not centered
on Glu681 may also seem surprising, because
Glu681 interacts with sulfate anions during transport
(13-15). However, Glu681 is on the edge of the pore in our
model (Fig. 6) and could extend its side chain into the pore.
Interestingly, Passow has presented a model of the AE1 anion
translocation pathway in which TM8 interacts with TM9 and TM10, but
E681 is predicted to be on the edge of the pore (12).
Mutant S667C stands apart from the other sulfhydryl reagent-sensitive
sites. This is the only hydrophilic site observed among the pore-lining
residues. All other sites cluster on one face of the helix, while S667C
is 60° removed from the rest of the mutants, on a helical wheel plot.
S667C is close to Pro670, which may kink and twist the TM8
helix. This would result in exposure of a discontinuous helical face to
line the pore. That is the TM8 helical region above and below
Pro680 may not be directly aligned.
Our results may provide insight into the structure of the anion
translocation pore. We observed that AE1 introduced cysteine mutants
are only partially (<40%) inhibited by pCMBS. Similarly, in studies
of the CFTR chloride channel, pCMBS was only able to inhibit to
25-75% (24). In contrast translocation pathway mutants of the
bacterial glucose 6-phosphate transporter, UhpT, were nearly fully
inhibited by pCMBS (22). This difference in sensitivity to pCMBS may
reflect the size of the anion translocation pore, since glucose
6-phosphate is larger than chloride. The CFTR channel is estimated to
have a pore diameter of at least 6 Å (24), which provides at least an
approximation for the size of the AE1 pore.
One prevailing model of AE1 suggests that the anion translocation pore
forms an outward facing funnel and narrows to a permeability barrier at
Glu681 (47). Our data may provide some support for the
model. The first sulfhydryl reagent-sensitive site (Ala666)
is located after the start of the TM8 (Met664) (18). Since
both pCMBS and MTSEA are expected to inhibit anion exchange by steric
blockage of a pore, the observation that the inner portion of TM8 is
more sensitive to sulfhydryl reagents than the outer suggests a more
open outer region and a more closed pore in the inner region. In line
with this model, we found that L669C was sensitive to pCMBS, but not
MTSEA, reflecting the larger steric bulk of pCMBS. Furthermore, A666C
and S667C, the outermost sensitive mutants, both had lower pCMBS
sensitivity than the inner mutants. We found that the sequence of
pCMBS-inhibited residues ended at Leu680, which is
consistent with the identification of Glu681 as the site of
the permeability barrier. Since pCMBS can permeate the membrane only
via AE1, it will not accumulate on the cytosolic surface to any extent.
Conversely, MTSEA is membrane permeant in its deprotonated form (33)
and therefore can access the anion translocation pore from both the
extracellular and intracellular surfaces. Finally, our data show that
the greatest sensitivity to pCMBS is found with mutant L680C, which is
adjacent to Glu681, the presumed permeability barrier. The
high sensitivity at this site may reflect a smaller pore diameter.