From the * Molecular Medicine and Renal Units, Beth Israel Hospital; Critical Care Research Laboratories, Departments of Medicine
(Nephrology) and Anesthesia, The Children's Hospital; § Department of Medicine and || Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02215; and ¶ Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique, Marseille,
France
Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino
acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO42 cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl
influx
and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO42
. E699Q-mediated Cl
influx was activated by elevation of intracellular SO42
, but E699Q-mediated Cl
efflux was undetectable. The DNDS (4,4
-dinitrostilbene-2,2
-disulfonic acid) sensitivity of E699Q-mediated SO42
efflux was indistinguishable from that of wt AE1-mediated Cl
efflux. The extracellular anion selectivity of E699Q-mediated SO42
efflux was similar to that of wt AE1-mediated Cl
efflux. The stoichiometry of E699Q-mediated exchange of
extracellular Cl
with intracellular SO42
was 1:1. Whereas SO42
injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO42
, but not of Cl
or gluconate, into oocytes expressing E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl
with isethionate caused a 28-mV hyperpolarization in SO42
-loaded oocytes expressing
E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl
-dependent depolarization of SO42
-preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl
was of magnitude sufficient to account for measured 35SO42
efflux. Thus, AE1 E699Q-mediated SO42
/
Cl
exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data
confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of
AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange
without alteration of SO42
/Cl
exchange stoichiometry.
The anion exchanger 1 (AE1)1 (band 3) erythroid anion exchanger is one of the most extensively studied
membrane transport proteins (Passow, 1986; Tanner,
1993
; Alper, 1994
). Erythroid AE1-mediated exchange
of Cl
for HCO3
serves to increase the total CO2 carrying capacity of blood. Intracellular HCO3
generated
by red cell carbonic anhydrase from CO2 released from respiring tissues is exchanged for extracellular Cl
in
the capillaries of the peripheral circulation. In the pulmonary capillaries the reverse sequence occurs, and extracellular HCO3
enters the cell in exchange for intracellular Cl
. The newly entered HCO3
is converted to
CO2 by carbonic anhydrase, and the CO2 can diffuse from the blood cell across endothelium, interstitial
space, and alveolar epithelium into the alveolar airspace for expiration.
Despite many years of study, the molecular mechanism by which AE1 mediates anion exchange remains
unclear. Only recently has data emerged identifying
amino acid residues that may participate directly in
binding and translocating anions. Chemical modification studies of red cells have contributed to these identifications. The requirement for liganding anions focused initial attention on cationic amino acid residues.
Indeed, chemical modification and pH titration of AE1
in red cells led to the hypothesis that one or more arginine residues play important roles in AE1 transport function (Wieth et al., 1982; Julien and Zaki, 1988
). Reductive methylation of AE1 lysines inhibited transport
(Jennings, 1982
), whereas dansylation of red cells produced reciprocal changes in Cl
and SO42
transport
(Lepke and Passow, 1982
; Berghout et al., 1988
). Mutation of the lysine residues that provide covalent attachment sites to the isothiocyanate moieties of cyanostilbene
disulfonates reduced inhibition by these site-directed
reagents, but did not change the K1/2 for extracellular
Cl
of AE1-mediated transport (Wood et al., 1992
). A
different lysine residue has also been identified as exposed to both intracellular and extracellular aqueous
media by fluorescence quench of pyridoxal phosphate
covalently bound to red cell AE1 (Bar-Noy and Cabantchik, 1990
).
The mildly acidic pK of inhibition by protons of red
cell Cl/Cl
exchange led to the implication of histidine residues in AE1 transport function by study of the
effect of diethylpyrocarbonate modification of red cells
(Izuhara et al., 1989
) and by site-directed mutagenesis
(Muller-Berger et al., 1995b
). SO42
transport in red
cells was found to be stimulated by acidification, with a
pKa of 5.5, and was accompanied by proton uptake (Milanick and Gunn, 1982
, 1984
). The demonstration of
H+-SO42
cotransport led to the proposal that the residue responsible for proton binding is a residue of
acidic pKa. Both titration experiments and chemical
modification studies have implicated carboxylate residues in AE1-mediated H+-SO42
/Cl
exchange.
Jennings and colleagues have investigated further the
involvement of glutamate residues in human AE1-mediated anion exchange. They reported that reduction of
the E681 carboxylate to the corresponding alcohol by
treatment with Woodward's reagent K (WRK) followed by reduction with borohydride (BH4) produced a complex pattern of changes in anion transport (Jennings
and Anderson, 1987; Jennings and Smith, 1992
). Cl
/
Cl
exchange was inhibited, whereas SO42
i/SO42
o
and SO42
i/Cl
o exchange were stimulated 5- to 10-fold
and 80-fold, respectively. Protons were no longer
cotransported with SO42
, and the activation of SO42
transport by acidic pH was abolished. Moreover, in
WRK-BH4 modified cells, trans Cl
-dependent efflux of
35SO42
was accelerated by cationophores, and ionophore-mediated 86Rb efflux was activated by extracellular Cl
. (Jennings and Al-Rhaiyel, 1988
; Jennings,
1995
). Residue E681 of AE1 was defined as the principal target for WRK-BH4 modification of intact human
erythrocytes (Jennings and Smith, 1992
). These workers proposed that E681 is exposed to both intracellular
and extracellular aqueous spaces during the anion exchange cycle and is a proton binding site for H+/SO42
cotransport. The data further suggested that modification of the AE1 residue E681 to the corresponding alcohol conferred on modified AE1 the ability to mediate electrogenic exchange of intracellular SO42
for extracellular Cl
(Jennings, 1995
).
We were prompted by these findings to examine the
functional role of the corresponding glutamate residue
in mouse AE1, E699, as expressed in the Xenopus oocyte
from cRNA. The Xenopus oocyte was the first heterologous system used for functional expression of recombinant AE1 (Bartel et al., 1989; Garcia and Lodish, 1989
) and has been developed by subsequent investigators for
examination of structure-function relationships in the
AE1 polypeptide (Kietz et al., 1991
; Groves and Tanner,
1993
; Chernova et al., 1995
; Muller-Berger, 1995a, b). The
Xenopus oocyte has been used to confirm the electroneutrality of wild-type (wt) AE1-mediated Cl
/anion
exchange, but also to demonstrate the potential-dependence of transport rates (Grygorczyk et al., 1987
).
We have found decreased Cl transport in oocytes
expressing mouse AE1 mutated at residue E699. Only
one among the several amino acid substitutions tested,
E699Q, displayed increased SO42
transport. Efflux of
intracellular SO42
was easily detected and required the
presence of extracellular Cl
or other appropriate anions. In contrast efflux of intracellular Cl
was undetectable regardless of the extracellular anion present. Whereas AE1 E699Q mediated 1:1 electroneutral
SO42
/SO42
exchange, exchange of intracellular
SO42
for extracellular Cl
by AE1 E699Q was accompanied by inward currents, consistent with electrogenic
outflow of anions. E699Q-mediated anion fluxes and currents were inhibited by the stilbene disulfonate,
DNDS (4,4
-dinitrostilbene-2,2
-disulfonic acid). The
stoichiometry of SO42
efflux to Cl
influx to inward
current was consistent with 1:1 electrogenic anion exchange.
Our results with recombinant mutant AE1 confirm
and extend several earlier conclusions from the studies
of Jennings and colleagues on red cells: (a) E699 serves
as the proton binding site during H+SO42/Cl
exchange by wt AE1; (b) charge neutralization at E699
leads to electrogenic exchange of SO42
for Cl
with a
1:1 stoichiometry; and (c) such electrogenic exchange is asymmetric. The results further show that electroneutrality and 1:1 stoichiometry are properties of the AE1
protein that can be uncoupled. Recent results from two
additional groups address the functional role of E699.
Muller-Berger et al. (1995a)
showed that AE1 E699D
expressed in Xenopus oocytes exhibits increased pK for
Cl
/Cl
exchange. Sekler et al. (1995)
found in microsomes prepared from transfected HEK293 cells that
whereas AE1 E699D exhibited complete loss of SO42
/
SO42
exchange, AE1 E699Q exhibited increased
SO42
/ SO42
exchange accompanied by loss of the
pH-dependence characteristic of wt AE1.
Materials
Female Xenopus laevis were purchased from NASCO (Madison, WI), maintained at room temperature in running distilled water, and fed with frog brittle (NASCO). DNDS was from Pfaltz & Bauer (Waterbury, CT). Bumetanide was obtained from Dr. P. Feit (Leo Pharmaceuticals Ballerup, Denmark). All salts were analytical grade and were purchased from Fluka Chemical Corp. (Ronkonkoma, NY) or Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Na36Cl was from Amersham Corp. (Arlington Heights, IL) or NEN-Dupont (Boston, MA); Na235SO4 was from ICN Biomedicals Inc. (Costa Mesa, CA).
Solutions
ND-96 contained (in mM): 96 NaCl, 2 KCl, 1.8 MgCl2, 1 CaCl2,
and 5 HEPES hemisodium, pH 7.40. In some experiments 96 mM NaCl was replaced mole for mole with the sodium salt of either gluconate, isethionate, nitrate, bromide, or iodide. Alternatively, 96 mM NaCl was replaced with 64 mM Na2SO4 or with 70 mM sodium phosphate, pH 7.4. All Cl-free solutions included
the above mentioned concentrations of K+, Mg2+, and Ca2+ as
the gluconate salts.
Construction of AE1 Mutants
The 563 nt Sma1/Sph1 fragment encoding murine erythroid
AE1 nt 2117-2679 (Kopito and Lodish, 1985) was excised from
the murine kidney AE1 plasmid pBL (Brosius et al., 1989
) and
subcloned into M13mp19. Mutations in AE1 E699 were constructed in this phage subclone by the dut
/ung
method
(Kunkel et al., 1991
) using the degenerate oligonucleotide 5
-CAT
TTTCCTT[C,G,T,A][C,G,T,A]GTCT-3
and the Muta-Gene T7
Kit (Bio-Rad Laboratories, Richmond, CA). Mutant phage were
detected by sequencing across the mutation site. Double-stranded DNAs from phage in which E699 had been mutated to
R, K, G, T, and Q were used to reconstruct full-length mutant
AE1 cDNAs. Mutant plasmid cDNAs were again sequenced across
the mutation site.
Expression of cRNA in Xenopus Oocytes
Transcription template was made by linearizing plasmids with
HindIII. cRNA transcription with T7 RNA polymerase was performed with the Megascript Kit (Ambion Inc., Austin, TX). Manually defolliculated oocytes prepared as previously described
(Humphreys et al., 1994) were microinjected with 20 ng cRNA
and incubated in ND-96 at 19°C for 1-14 d.
Immunoprecipitation of Total and Surface AE Proteins from Xenopus Oocytes
Groups of 10-12 oocytes previously injected with water or with 20 ng cRNA were incubated in ND-96 containing 1-1.5 mCi/ml of
35S-methionine (20-30 µM) for 48-72 h. Metabolically labelled
oocytes were washed in modified ND-96, pH 8.0, then incubated
for 1 h at 4°C in the same medium in the presence or absence of
5 mg/ml chymotrypsin. Oocytes were then washed three times in
10 ml ND-96, pH 7.4 containing 2 mM PMSF and 1 mg/ml BSA
Fraction V, and once more in the same medium containing 100 µg/ml chymostatin and 1 mg/ml BSA. Groups of washed oocytes
were manually homogenized at 4°C with a fitted Teflon® pestle
(Kontes, Vineland, NJ) in microfuge tubes with 100 µl oocyte immunoprecipitation (IP) buffer containing (in mM) 50 Tris-HCl, 1 EDTA, 1 PMSF, and 0.04 each of leupeptin, pepstatin, and antipain, pH 7.6. The extract was incubated with shaking for 30 min
at 4°C, then centrifuged 10 min in a microfuge. The resultant supernatants were brought to 500 mM NaCl and precleared with
5% normal rabbit serum. Precleared supernatants were incubated 1 h with rabbit polyclonal antiserum raised against mouse
AE1 aa 214-228 (Alper et al., 1989), followed by precipitation
with protein A-agarose. The protein A-agarose pellets were
washed six times in 1 ml IP buffer containing 500 ml NaCl, six
more times in 1 ml IP buffer without NaCl, then analyzed by SDS-PAGE fluorography.
Isotopic Flux Studies
Measurement of 35SO42 influx was carried out as follows: Oocytes were injected 10 min before initiation of influx measurements with 50 nl of a solution containing (in mM) 130 Na2SO4,
50 HEPES, pH 7.4. The flux assay was initiated by transfer of
groups of 6-10 water-injected or 8-12 cRNA-injected oocytes into
microtiter wells containing 149 µl of Na isethionate influx medium containing 2 mM Na2SO4 and 1 µl (5 µCi) Na235SO4. Influx
assays were carried out at room temperature for 15 min, then terminated by rapid transfer of groups of oocytes through three 25-ml room temperature washes in isotonic Na gluconate medium. In experiments designed to measure the stoichiometry of SO42
/
SO42
exchange, influx medium contained 64 mM Na2SO4 instead of 96 mM Na isethionate.
Assay of 35SO42 efflux was carried out as follows: Oocytes were
injected with 50 nl of a solution containing Na235SO4 (0.25-0.5
µCi, 3-6 µM) in 50 mM HEPES, pH 7.4, with or without 130 mM
Na2SO4, and maintained for 10 min post-injection in SO4-free, Cl-free medium containing 96 mM Na isethionate. Efflux was initiated by transfer of individual oocytes into 1 ml of efflux medium. At regular intervals, 950 µl of this medium was removed for scintillation counting and replaced with fresh medium. All efflux experiments ended with a final period of efflux into medium containing 100 µM DIDS or DNDS before solubilization of the
washed oocyte in 100 µl 1% SDS. The sum of efflux fractions and
residual cpm in the oocyte was >98% of originally injected cpm.
Data were plotted as ln (% cpm remaining) vs. time. Efflux rate
constants were measured from linear least squares regressions
calculated from the last three time points for each experimental
condition, and corrected by subtraction of the efflux rate constant of water-injected oocytes studied in the same experiment.
Effluxes were calculated as products of the measured rate constants and intracellular ion concentration (determined as described below). Oocyte water space was assumed to be 450 nl
(500 nl acutely following 50 nl microinjection of isotope). As described in RESULTS, endogenous oocyte SO42
concentration
was assumed to be 1.1 mM (1.0 mM following microinjection of
50 nl fluid).
Measurement of 36Cl influx was carried out as described by
Humphreys et al. (1994)
in the presence of 10 µM bumetanide.
Influx periods were 15-30 min, during which uptake was linear.
For some experiments, individual oocytes kept in isethionate medium were injected with 50 nl of 50 mM HEPES, pH 7.40, with or
without 130 mM Na2SO4, then maintained 10 min in isethionate
medium before initiation of the influx experiment in ND-96 or
other media as indicated.
Measurement of 36Cl efflux was carried out as described previously (Humphreys et al., 1994
), modified only in that oocytes
were placed into isethionate medium for 10 min after isotope injection. Efflux was initiated by transfer of individual oocytes into
medium containing ND-96 or other media as indicated.
Peak anion transport activities were exhibited by wt AE1 at 3-4 d
post-injection of cRNA, as noted previously (Brosius et al., 1989;
Humphreys et al., 1995
). However, anion transport activities of
oocytes expressing AE1 E699Q required up to 7 d to reach peak values. Wt AE1-expressing oocytes began to die as soon as 4 d post-injection and were unusable after 10 d. In contrast, AE1 E699Q-expressing oocytes maintained in ND-96 remained
healthy and functional for as long as 15 d or more post-injection
with cRNA.
Determination of Oocyte (Ovary) Intracellular
SO42 Concentration
Samples for sulfate determination were prepared as follows. Resected fragments of Xenopus ovary were centrifuged in a microcentrifuge at 18,000 rpm for 1 h. 130 µl of the supernatant fraction were diluted with water to 500 µl and filtered through a Centricon 3 filter (Amicon Corp., Danvers, MA) in a Sorvall RT
6000B at 3,000 rpm for 4 h. This crude cytosol fraction was subjected to sulfate analysis by a barium precipitation method
(Greenberg et al., 1980). Costa et al. (1989)
have shown that intracellular Na and Cl activities and contents determined in ovary
extracts and in pooled, isolated oocytes did not differ.
Measurements of Membrane Potential as a Function of Intracellular Anion
Defolliculated oocytes were incubated for 2-4 d at 19°C after injection of cRNA or water. Oocytes were placed in a superfusion chamber, impaled with a 3 M KCl-filled microelectrode, and allowed to recover for several minutes until a stable membrane potential was observed. Pulses of 10 nA were injected at 3-s intervals to monitor input resistance. Monitoring of membrane potential and current injection were performed with a CA 100 voltage clamp amplifier (Biologic, Echirolles, France). A second microelectrode was then introduced into the oocyte, through which the designated K+ salts (40-60 nl, titrated to pH 7.4) were injected into the oocytes with 10 s pressure pulses from a pneumatic picopump (WPI, Hertfordshire, UK). Membrane potential and input resistance were monitored for at least 10 min after pressure injection. Data were printed on a thermal array recorder (Graphtec, Japan) or processed through an analog-digital converter at 5 kHz, then transferred to computer for further analysis with custom-written software.
Measurements of Membrane Potential and Current as a Function of Extracellular Anion
After injection of cRNA or water, oocytes were incubated in
ND96 for 4-14 d at 19°C. Oocytes were then injected with 50 nl of
solution containing (in mM) 130 Na2SO4, 50 HEPES, pH 7.40, and placed in medium in which 96 mM Na isethionate replaced
NaCl. Between 1 and 6 h afterward, single oocytes were placed in
a 5-ml bath chamber (Model RC-11, Warner Instrument Corp.,
Hamden, CT) on the stage of a dissecting microscope and impaled with microelectrodes under direct view. Current and potential-sensing electrodes were pulled from borosilicate glass
(1.2-mm outer diameter, 0.94-mm inner diameter; Sutter Instrument Co., Novato, CA) on a Flaming/Brown Model P-97 micropipette puller (Sutter Instrument Co.). The electrodes were filled
with 3 M KCl and had resistances of 2-3 M.
After impalement, oocyte membrane potential was monitored
until it had stabilized (typically 3-7 min). Oocytes were voltage clamped using a Geneclamp 500 amplifier (Axon Instruments,
Foster City, CA) interfaced to an 80486 50 MHz IBM-compatible
computer (Dell 450/ME, Austin, TX) via a DigiData 1200 AD/D
board (Axon Instruments). Electrical connections between the
electrodes and the amplifier were made using Ag/AgCl pellets or
wires and 3 M KCl/3% agarose bridges. Current was measured as
that flowing to ground via the bath reference electrode. Errors
induced by voltage drops across the bath ground were eliminated
by use of a second reference electrode and a virtual-ground circuit. Current signals were filtered at 200 Hz before digitization.
Oocytes were maintained in open-circuit conditions except during measurement of whole oocyte currents, for which membrane
potential was clamped at a holding potential of 50 mV, and test
potential steps at 20-mV intervals between
100 or
80 mV to
+80 mV were imposed for 800-ms time periods. Whole oocyte
currents recorded at each test potential were acquired to hard
disk for later analysis with pClamp software (Axon Instruments).
Oocytes clamped to test potentials of +80 mV or higher displayed activation of endogenous outward currents.
Estimate of Proton Flux Accompanying Sulfate Efflux
Oocytes previously injected with 50 nl of 130 mM Na2SO4 were
placed on coverslips in 1 µl droplets of modified ND-96 containing 5 µM BCECF free acid and 0.5 mM MOPS, pH 7.4, as buffer
(Jaisser et al., 1993). The coverslips were mounted in a customized chamber on an inverted microscope stage, and the oocytes
in their droplets were alternately irradiated at 440 and 495 nm.
BCECF fluorescence excitation ratios were acquired at 530 nm
from the extracellular fluid and recorded to optical disk with an
Image 1 digital ratio imaging system (Universal Imaging, West
Chester, PA) as previously described (Humphreys et al., 1994
,
1995
). Calibration of the BCECF free acid fluorescence ratio was
performed as described (Thomas et al., 1979
). JH+ from oocyte
into the surrounding droplet was calculated by multiplying the
measured dpHi/dt of the droplet by the calculated buffer capacity of the extracellular medium. AE1 E699Q-mediated proton efflux was estimated by subtracting outward JH+ of water-injected
oocytes from that of E699Q-expressing oocytes.
Isotopic Chloride Transport by AE1 Mutants
Site-directed mutagenesis was used to modify the wt
glutamate residue at position 699 in recombinant murine
AE1. Two mutations reversed the wt negative charge with
arginine and lysine substitutions. One mutation neutralized the charge with glycine. Two more mutations attenuated the negative charge with the negative dipoles
of threonine and glutamine. The threonine hydroxyl resembled the hydroxyl group of the hydroxynorvaline
product of WRK-BH4 modification, whereas the gluta-mine side chain more closely imitated the steric bulk of
the hydroxynorvaline. cRNAs encoding the full-length
mutant AE1 polypeptides were expressed in Xenopus
oocytes. The ability of the mutant AE1 polypeptides to
transport Cl was compared with that of wt AE1 48 h after cRNA injection. Fig. 1 A shows a 64-fold activation
of unidirectional Cl
influx by wt AE1, from 0.1 to 6.4 nmol Cl
per oocyte · 15 min. In contrast, none of the
mutants mediated Cl
uptake under these conditions.
36Cl efflux mediated by these AE polypeptides is shown
in Fig. 1 B. Cl
efflux was evident in wt AE1-expressing
oocytes but not in oocytes expressing the AE1 mutants.
Exchange of intracellular Cl
for extracellular sulfate
was not observed with either wt AE1 or any of the mutants (Fig. 1 B). This is consistent with a relative rate of
red cell Cl
/sulfate exchange >103 slower than Cl
/
Cl
exchange, and with a detection threshold of the oocyte 36Cl
efflux assay of >1% of the rate of Cl
/Cl
exchange (Humphreys et al., 1994
).
Isotopic Sulfate Transport by AE1 Mutants
AE1 E699Q-mediated 35SO42 efflux was not reproducibly detectable 48 h after cRNA injection. However, by
5 d after cRNA injection, E699Q-expressing oocytes exhibited sulfate/sulfate exchange (Fig. 2 A). In contrast,
neither oocytes expressing wt AE1 nor those expressing
E699 mutants in which K, G, R, or T was substituted showed detectable sulfate/sulfate exchange. AE1
E699Q-mediated sulfate/sulfate exchange was DIDS-sensitive. Replacement of extracellular 64 mM Na2SO4
with 96 mM NaCl accelerated AE1 E699Q-mediated
35SO42
efflux 1.7-fold (Fig. 2 C) but did not produce
detectable wt AE1-mediated 35SO42
efflux, unlike the
acceleration of wt AE1-mediated 36Cl
efflux from oocytes (Fig. 1 B) or from red cells (Hanke-Baier et al.,
1988
; Passow et al., 1992
) produced by the same extracellular substitution.
35SO42 influx into wt AE1-expressing oocytes was undetectable in seven experiments with extracellular
[SO42
] ranging from 2 to 64 mM, consistent with the
low SO42
/Cl
exchange rate of unmodified intact red
cells. 35SO42
influx into AE1 E699Q-expressing oocytes
was detectable at low level in about half of experiments
performed 48 h after cRNA injection.
Biosynthesis and Surface Expression of AE1 E699Q
Since the only AE1 E699 mutant which transported sulfate in the Xenopus oocyte 48 h after cRNA injection was
E699Q, further studies focused on this mutant. Total
accumulation of AE1 E699Q polypeptide was at least
half that of the wt AE1 polypeptide (Fig. 3, lanes 1 and
3). In Fig. 3, 19% of wt AE1 was cleaved by extracellular chymotrypsin (lane 2), as was 15% of AE1 E699Q (lane
4). These figures represent biochemical estimates of
the steady-state proportion of total oocyte AE polypeptide at the oocyte surface. In four similar experiments
using oocytes from two frogs, 32 ± 7% of wt AE1 was
exposed to chymotrypsin digestion at the oocyte surface. The comparable figure for AE1 E699Q was 29 ± 9%. Therefore, surface accumulation of wt and of
E699Q AE1 polypeptides in Xenopus oocytes was of
comparable efficiency.
Stilbene Sensitivity of AE1 E699Q
Wt AE1-mediated 36Cl efflux into chloride medium
was reversibly inhibited in a dose-dependent manner
by the stilbene disulfonate inhibitor, DNDS, with a concentration for half-maximal inhibition of 13 + 8 µM
(Fig. 4 B). This value is similar to the value of 3-5 µM
reported by Passow et al. (1992)
. Half-maximal inhibition by DNDS of AE1 E699Q-mediated 35SO42
efflux
into chloride medium was 8 ± 2 µM (Fig. 4, A and B),
a value not significantly different from that of wt AE1
(P > 0.1). Thus, the apparent affinity of AE1 for an impermeant reversible antagonist applied outside the oocyte was not changed by the single E699Q mutation.
Extracellular Anion Selectivity of AE1 E699Q
Fig. 5 shows the effect on SO42 efflux of complete replacement of extracellular Cl
with various anions.
SO42
efflux rates relative to those observed in extracellular Cl
were 0.97 ± 0.12 (nitrate), 0.67 ± 0.04 (bromide), 0.20 ± 0.03 (gluconate), 0.20 ± 0.04 (isethionate), 0.16 ± 0.02 (iodide), and 0.04 ± 0.01 (phosphate). The values for nitrate, bromide, and iodide were
close to those reported previously for wt AE1-mediated Cl
efflux from Xenopus oocytes and intact mouse red
cells (Passow et al., 1992
). The relative rates of AE1
E699Q-mediated SO42
exchange with gluconate,
isethionate, and phosphate were higher than expected
for AE1-mediated Cl
exchange with the same anions
in human red cells, but these rates had not been reported for wt AE1 expressed in Xenopus oocytes. Therefore, relative rate constants were also measured for wt
AE1-mediated efflux of 36Cl
into extracellular gluconate and isethionate, and were found to be 0.06 ± 0.02 (n = 13 oocytes, 3 frogs) and 0.11 ± 0.03 (n = 17 oocytes, 4 frogs), respectively. Though slightly lower than the corresponding values for AE1 E699Q, these values
were consistent with the rates of Cl
/gluconate exchange exhibited by trout AE1 (Fievet et al., 1995
) and
by mouse AE2 (Humphreys et al., 1994
).
Stoichiometry of AE1 E699Q-mediated Exchange of
Intracellular SO42 for Extracellular Cl
To estimate the stoichiometry of AE1 E699Q-mediated
SO42/Cl
exchange, it was necessary to measure Cl
influx. Though AE1 E699Q-mediated 36Cl
influx was
minimal or absent two days after cRNA injection (Fig. 1
A), 36Cl
influx and 35SO42
efflux were both detectable
5-7 d after injection of cRNA (Fig. 6). A second requirement was knowledge of the intracellular [SO42
].
Cytosolic fractions isolated from ovarian fragments
were subjected to sulfate analysis by barium precipitation (Greenberg et al., 1980
). This assay indicated an
ovarian cytosolic [SO42
] of 1.1 ± 0.1 mM (n = 5 frogs). On this basis, oocyte cytosol [SO42
] was assumed to be 1 mM after injection of a 50 nl volume into a nominal 450-nl water space.
The stoichiometry of SO42/Cl
exchange by AE1
E699Q was examined
5 d after cRNA injection. AE1
E699Q-mediated SO42
efflux into extracellular Cl
was 0.31 ± 0.05 pmol cell
1 s
1 (n = 59 oocytes, 10 frogs). In 96 oocytes from 10 frogs, AE1 E699Q-mediated Cl
influx was 0.25 ± 0.06 pmol cell
1 s
1. The ratio of the mean SO42
efflux over the mean Cl
influx
was 1.24. In two experiments in which both measurements were performed on oocytes from the same frog,
the mean of the two flux ratios was 0.94. Thus, exchange of intracellular endogenous SO42
for extracellular Cl
by AE1 E699Q was consistent with a stoichiometry of 1:1.
To maximize AE1 E699Q-mediated rates of SO42/
Cl
exchange stoichiometry experiments were performed with oocytes acutely injected with 50 nl of solution containing 130 mM Na2SO4 buffered with Na
HEPES, pH 7.40, to yield a total estimated intracellular
[SO42
] of 14 mM. Such intracellular injection of
SO42
produced a 2.2 ± 0.5-fold increase in AE1
E699Q-mediated 36Cl
influx (Fig. 6 A, P < 0.01). This increase presumably reflected increased saturation of the
intracellular binding site for sulfate on AE1 E699Q. 36Cl
influx assays were performed with AE1 E699Q-expressing
oocytes isolated from the same frog and previously injected on the same day 5-15 d earlier with water or with
cRNA encoding AE1 E699Q. 10 min before initiation
of 36Cl
uptake, these oocytes were injected with 50 nl
of 130 mM Na2SO4 without tracer, to maximize and
equalize intra-oocyte sulfate concentrations. For 35SO42
efflux experiments, the injected Na2SO4 included carrier-free tracer. The paired influx and efflux experiments
carried out with sulfate-loaded oocytes were conducted
on the same days (Table I).
Table I. |
In AE1 E699Q-expressing, Na2SO4-loaded oocytes (n = 7 frogs, 73 oocytes), AE1-mediated 36Cl influx was 0.43 ± 0.11 (SEM) pmol cell
1 s
1. In identically handled AE1
E699Q-expressing, sulfate-loaded oocytes (n = 40)
from the same seven frogs, AE1-mediated 35SO42
efflux was 0.37 ± 0.04 pmol cell
1 s
1. The ratio of the
mean sulfate efflux to the mean chloride influx was
0.86. The mean of the seven individual experimentally
determined ratios of sulfate efflux over chloride influx
was 1.13 ± 0.23 (Table I). These values were consistent
with a 1:1 stoichiometry of SO42
/Cl
exchange mediated by AE1 E699Q in Na2SO4-loaded oocytes.2
The SO42/SO42
exchange stoichiometry of AE1
E699Q was examined 5 or more days after cRNA injection in the absence of exogenous SO42
loading. AE1
E699Q-mediated SO42
efflux into 64 mM extracellular
SO42
was 0.091 ± 0.012 pmol cell
1 s
1 (n = 4 frogs,
20 oocytes). 35SO42
influx from 64 mM extracellular
SO42
into AE1 E699Q-expressing oocytes was 0.079 ± 0.012 pmol cell
1 s
1 (1 frog, 10 oocytes) These values
were statistically indistinguishable (P > 0.5), and yielded
a flux ratio of 1.15. Thus, under the nonequilibrium conditions used, AE1 E699Q also mediated SO42
/SO42
exchange with a stoichiometry consistent with 1:1 exchange.
Effect of AE1 E699Q on Membrane Potential
In WRK-BH4-modified, SO42-loaded red cells treated
with gramicidin, the acceleration of 86Rb+ efflux by addition of 5 mM extracellular Cl
suggested that the
modified AE1 mediated electrogenic anion exchange (Jennings, 1995
). Since the 1:1 stoichiometry of AE1
E699Q-mediated SO42
/Cl
exchange in oocytes resembled that of WRK-BH4-modified red cells, the effects of substrate anions on membrane potential were
directly examined in oocytes expressing either wt or
mutant AE1 polypeptides.
Upon pressure injection of ~50 nl 87 mM K sulfate,
E699Q-expressing oocytes underwent a depolarization
of 17 ± 3 mV that was maintained during 30 min of observation and was accompanied by a 66% decrease in
membrane resistance (Fig. 7). Estimated intracellular [SO42] concentration was 9-10 mM. Injection of equal
volumes of KCl or of K gluconate (87 mM) led to transient 3-mV hyperpolarizations accompanied by small
increases in membrane resistance. Wt AE1-expressing oocytes, in contrast, displayed minimal change in membrane potential in response to injection of either K sulfate or K gluconate. Similarly, water-injected oocytes
showed no voltage response to injection of K sulfate
(Fig. 7, Table II). The depolarization and decrease in
membrane resistance which characterized SO42
injection into AE1 E699Q-expressing oocytes suggested outward electrogenic flow of negative charge, and supported
the hypothesis of electrogenic SO42
/Cl
exchange.
Table II. |
To distinguish between AE1 E699Q-mediated electrodiffusive transport and electrogenic exchange, the
extracellular anion selectivity of membrane potential
changes was investigated in SO42-loaded oocytes (Fig.
8, Table III). Whereas resting potentials in Cl
medium
were
58 ± 4 mV and
55 ± 2 mV in water-injected (n = 11) and wt AE1-expressing oocytes (n = 20), respectively, oocytes expressing AE1 E699Q displayed a depolarized resting potential of
38 ± 2 mV (n = 60). Substitution of extracellular Cl
by isethionate produced
no change in membrane potential in wt AE1-expressing and water-injected oocytes, but caused AE1 E699Q-expressing oocytes to hyperpolarize by 28 ± 2 mV (n = 60). In contrast, replacement of extracellular isethionate by SO42
, produced little or no change in membrane potential (Table III). Addition of DNDS in the
continued presence of extracellular Cl
hyperpolarized
membrane potential in AE1 E699Q-expressing oocytes (Fig. 8 B) by 24 ± 2 mV (n = 25, 4 frogs). Taken together with the isotopic flux data, these results suggest
that AE1 E699Q mediated asymmetric, electrogenic 1:1
SO42
/Cl
exchange and electroneutral 1:1 SO42
/
SO42
exchange.
Table III. |
Stoichiometry of SO42 Efflux and Current Mediated
by AE1 E699Q
The hypothesis of 1:1 electrogenic SO42/Cl
exchange
predicts that AE1 E699Q should mediate outward flow
of negative charge from oocytes with properties identical to and of magnitude equal to AE1 E699Q-mediated
SO42
efflux from oocytes. Therefore, SO42
-loaded oocytes expressing wt and mutant AE1 cRNAs were subjected to two-electrode voltage clamp in order to characterize and to quantitate current flow mediated by the
AE1 E699Q polypeptide.
Fig. 9 A compares current-voltage relationships of
SO42-loaded oocytes expressing wt AE1 and AE1
E699Q. The ~1 µS conductance measured in oocytes
expressing wt mouse AE1 was similar to that of 0.7 µS
measured by Fievet et al. (1995)
. The oocytes expressing AE1 E699Q exhibited a small but significant linear
inward current not seen in oocytes expressing wt AE1
at membrane potentials more positive than
80 mV
(Fig. 9). The current-voltage relationship remained linear to test potentials as negative as
150 mV (not shown). The current in wt AE1-expressing oocytes displayed a reversal potential of
64 mV (Fig. 9 A; 20 oocytes from 6 frogs) similar to the value of
69 mV measured in water-injected oocytes (not shown; 8 oocytes
from 6 frogs). Oocytes expressing AE1 E699Q exhibited substantially increased conductance, with a depolarized reversal potential of
33 mV (Fig. 9 A; 54 oocytes from 8 frogs). Substitution of extracellular Cl
by
isethionate greatly reduced inward current in Na2SO4-loaded oocytes expressing AE1 E699Q, and restored
oocyte reversal potential to the more hyperpolarized
value of
74 mV (Fig. 9 B; 44 oocytes from 7 frogs).
Substitution of extracellular NaCl by isosmotic Na2SO4
reduced inward current and hyperpolarized oocyte reversal to the same relative extents (Fig. 9 B; 5 oocytes).
Exposure of Na2SO4-loaded oocytes in extracellular
chloride to DNDS (200 µM) similarly inhibited AE1
E699Q-associated inward current, and similarly hyperpolarized the reversal potential to
68 mV (Fig. 9 C; 24 oocytes from 4 frogs). Thus, the conductance of AE1
E699Q-expressing oocytes was restored to that typical
of wt AE1-expressing oocytes either by substitution of
extracellular chloride by isethionate or by sulfate, or by
addition of the anion exchange inhibitor, DNDS. Taken
together, these data are consistent with the presence of
AE1 E699Q-mediated electrogenic sulfate efflux at inside-negative oocyte resting potential.
To evaluate whether inward current carried by AE1
E699Q sufficed to account for AE1 E699Q-mediated
SO42 efflux, oocyte current at the resting potential in
extracellular Cl
medium was interpolated from the
measured I-V relationships. These values were compared to AE1 E699Q-mediated SO42
efflux into Cl
medium (Table IV). In 12 experiments examining 104 Na2SO4-loaded oocytes, AE1 E699Q-mediated SO42
efflux into extracellular Cl
was 0.48 + 0.15 pmol cell
1
s
1. SO42
efflux did not differ significantly (P > 0.5, two-tailed t test) from AE1 E699Q-mediated inward currents calculated by any of three criteria: 0.58 ± 0.15 pmol charge cell
1 s
1 (for current in extracellular Cl
minus that in extracellular isethionate); 0.37 ± 0.08 pmol charge cell
1 s
1 (for current in E699Q-expressing oocytes minus that in wt AE1-expressing oocytes);
and 0.43 ± 0.05 pmol charge cell
1 s
1 (for current in
absence of DNDS minus that in the presence of DNDS). Thus, the outflow of charge mediated by AE1
E699Q in sulfate-loaded oocytes at resting membrane
potential sufficed to account for SO42
efflux from similarly treated oocytes.
Table IV. |
Estimate of AE1 E699Q-mediated H+ Transport during
SO42/Cl
Exchange
WRK-BH4-treated red cells mediate SO42/Cl
exchange without evidence for the H+/SO42
cotransport
(Jennings and Al-Rhaiyel, 1988
) that typifies wt AE1
(Milanick and Gunn, 1982
, 1984
). The electrogenicity
of 1:1 SO42
/Cl
exchange mediated by AE1 E699Q
also suggested the absence of H+ cotransport with
SO42
. Therefore, H+ efflux from SO42
-loaded oocytes
was estimated from the time-dependent change in BCECF fluorescence ratio of minimally buffered extracellular medium (Jaisser et al., 1993
). Water-injected
oocytes did not detectably acidify the extracellular medium (n = 3). dpHo/dt in the droplets surrounding
AE1 E699Q-expressing oocytes previously injected with Na2SO4 was
0.011 ± 0.003 min
1 (n = 5), corresponding to outward JH+ of 0.049 pmol cell
1 s
1. With
the simplifying assumption of no proton backleak, H+
efflux associated with expression of AE1 E699Q was
~10% of the level of AE1 E699Q-mediated SO42
efflux. However, JH+ had the same value in AE1 E699Q-expressing oocytes not previously injected with Na2SO4
(n = 6), suggesting that the proton efflux was not coupled to SO42
transport. Thus, SO42
transport by AE1
E699Q was largely unaccompanied by protons.
The experiments presented above demonstrate a complex phenotype of anion exchange in Xenopus oocytes
expressing mouse AE1 polypeptide with the E699Q
missense mutation. The AE1 E699Q polypeptide was
processed and delivered to the surface of the oocyte
(Fig. 3). There it exhibited both a loss-of-function phenotype with respect to exchange of intracellular Cl for
extracellular anion (Fig. 1) and a gain-of-function phenotype with respect to exchange of intracellular SO42
for extracellular anion (Figs. 2, 5, and 6). Thus, SO42
/
Cl
exchange was asymmetric: whereas exchange of intracellular SO42
for extracellular Cl
was increased,
exchange of extracellular SO42
for intracellular Cl
was undetectable. Though the sensitivity of anion exchange to inhibition by DNDS was unchanged (Fig. 4)
and the rank-order of exchange rates with various extracellular anions was little changed from the corresponding wt values (Fig. 5), the mechanism of SO42
/
Cl
exchange differed dramatically from that of wt
AE1. The Xenopus oocyte expression system allowed unambiguous discrimination between two possible mechanisms of SO42
/Cl
exchange in the absence of proton cotransport (Jennings, 1995
), electroneutral exchange of 2 Cl
for 1 SO42
and electrogenic exchange
of 1 Cl
for 1 SO42
.
Electrogenicity of Anion Exchange by AE1 E699Q
Murine AE1 E699Q-mediated exchange of intracellular
SO42 for extracellular Cl
with a stoichiometry of 1:1
in Xenopus oocytes (Table I). This exchange was unaccompanied by stoichiometric proton transport and was
electrogenic (Tables II and III, Figs. 7 and 8). In SO42
-loaded Xenopus oocytes at inside-negative membrane
potentials, AE1 E699Q mediated an inward current
that required the presence of intracellular SO42
and
extracellular Cl
and was inhibited by DNDS (Fig. 9).
AE1 E699Q-mediated inward current measured at the
resting membrane potential did not differ detectably in
magnitude from E699Q-mediated efflux of 35SO42
, and
so could account entirely for SO42
efflux (Table IV).
The mutation of glutamate to glutamine at position
699 converted the obligate electroneutrality of wt AE1-mediated anion exchange to a more flexible mechanism, in which exchange of intracellular SO42 for extracellular Cl
by AE1 E699Q was electrogenic, but AE1
E699Q-mediated SO42
/SO42
exchange was electroneutral (Fig. 9 B). The change in charge of this single
amino acid altered the capacity of AE1 to effect transmembrane transport of net charge, without alteration
of the wt stoichiometry of 1:1 anion exchange. Electrogenic anion transport by wt mouse AE1 was not observed in Xenopus oocytes (Fig. 9 A, Tables II and III),
in agreement with earlier observations of Grygorczyk et
al. (1987)
and Fievet et al. (1995)
. In human red cells
in which membrane potential was clamped with gramicidin and imposed K+ gradients, Jennings et al. (1990)
also found that neither Cl
/Cl
exchange nor Cl
/
HCO3
exchange displayed detectable potential dependence.
Wt AE1 in red cells cotransports H+ with SO42 in
electroneutral exchange for Cl
. This cotransport is reflected in stimulation of AE1-mediated SO42
transport
at acid pH (Milanick and Gunn, 1982
, 1984
). WRK-BH4 modification of red cells (Jennings, 1995
) led to AE1-mediated SO42
transport that was active at neutral pH,
not further stimulated at acid pH, and was unaccompanied by H+ cotransport. In the present study, the electrogenicity of 1:1 SO42
/Cl
exchange by AE1 E699Q
suggested the absence of H+ cotransport; indeed, AE1
E699Q-mediated SO42
efflux in Xenopus oocytes was
unaccompanied by stoichiometric H+ efflux (RESULTS).
Moreover, neither SO42
/Cl
exchange nor SO42
/
SO42
exchange were accelerated by intracellular or by
extracellular protons (Chernova and Alper, manuscript
in preparation).
Asymmetry of Sulfate Transport by AE1 E699Q
SO42/Cl
exchange in WRK-BH4-modified human red
cells was vectorially asymmetric: exchange of intracellular SO42
for extracellular Cl
was 10-fold faster than
was exchange of intracellular Cl
for extracellular
SO42
, though both rates were increased (Jennings,
1995
). Xenopus oocytes expressing AE1 E699Q displayed a qualitatively similar asymmetry. Whereas exchange of intracellular SO42
for extracellular SO42
or
Cl
was easily detected (Figs. 2, 5, and 6), exchange of
intracellular Cl
for extracellular SO42
or Cl
was undetectable not only at 48 h (Fig. 1 B) but also 15 d after cRNA injection (not shown).
These differences in the relative rates of oppositely
directed anion hetero-exchange in WRK-BH4-modified
red cells and in AE1 E699Q-expressing oocytes compared to wt AE1 function may arise from differences in
rate constants for both inward and outward translocation. The contribution of the outward translocation
step can be more easily estimated, since both wt and
mutant proteins transport extracellular Cl at operationally maximal rates when measured in the presence of appropriate intracellular anions. In extracellular
Cl
, rate constants for SO42
efflux reflect the contribution of the E699 negative charge to a local structure in
wt AE1 that maintains a low energy barrier for outward
translocation of the Cli
-carrier complex and a much
higher energy barrier for outward translocation of the
(H+/SO42
)i-carrier complex. Neutralization of the
E699 charge by chemical modification or by mutation
likely elevates the energy barrier for outward translocation of the Cli
-carrier complex. In contrast, this neutralization appears to lower the energy barrier for outward translocation of the SO42
i-carrier complex, despite the movement of a unit charge across the
membrane electric field associated with the mutant
transport cycle. The absence of evident H+/SO42
cotransport by AE1 E699Q is either due to a greatly elevated energy barrier for outward translocation of the
(H+/SO42
)i -carrier complex, or simply to loss of the
H+ binding site, with failure to form the (H+/SO42
)i - carrier complex.
Despite the minimal changes in ID50 for DNDS (Fig.
4) and in the rank order for most extracellular anions
(Fig. 5) produced by the E699Q mutation, it is possible
that the inward translocation step also contributes to
accelerated SO42i/Cl
o exchange by AE1 E699Q. A
role for acceleration of the inward translocation step is
more evident in SO42
i/ SO42
o exchange, which occurs at close to maximal (SO42
i/Cl
o) rates in AE1
E699Q-expressing oocytes. This contrasts with undetectable rates of SO42
efflux in oocytes expressing wt
AE1, whether in exchange for intracellular Cl
or SO42
.
Inward current associated with AE1 E699Q-mediated
exchange of intracellular SO42 for extracellular Cl
could arise from outward movement of substrate-associated negative charge during the SO42
efflux step or
from inward movement of protein-associated positive charge during the Cl
influx step. The oocyte experiments did not allow discrimination between these possible mechanisms of charge movement. The experiments of Jennings (1995)
with WRK-BH4-modified red
cells assessed the efflux of SO42
at varying membrane
potentials and varying extracellular Cl
concentrations.
Kinetic analysis with assumption of a ping-pong transport mechanism suggested that the principal charge-carrying limb of the anion transport cycle of WRK-BH4-modified AE1 was the influx of Cl
rather than
the efflux of divalent SO42
. The resulting model of anion translocation by wt AE1 proposed outward transfer
through the transbilayer electric field of a neutral complex of two protein-associated positive charges with the
two negative charges of transported SO42
. In contrast,
during the inward flux of monovalent Cl
, one of the
protein-associated positive charges (which in wt AE1
may be paired with and neutralized by the negative
charge of E699) remains unpaired. Muller-Berger et al.
(1995b)
have proposed H752 as a candidate residue to
ion pair with E699, based on the indirect evidence of
similar changes in the apparent pK for AE1-mediated Cl
efflux produced by the independent mouse AE1
mutations H752S and E699D. Discovery of intragenic
second site revertants within AE1 E699 mutants will test
more stringently this and other possible charge pairs.
Consequences of the E699Q Mutation to Anion Selectivity and Stilbene Sensitivity
Despite human E681's putative location at the COOH
terminus of a stretch of nearly 25 hydrophobic amino
acids extending from the exofacial terminus of AE1's
putative transmembrane span 8, its WRK-reactivity in
red cells suggested its accessibility to the extracellular
space (Jennings and Smith, 1992). However, in contrast to the dramatic changes in outward anion translocation rates produced by the E699Q mutation in mouse
AE1, changes in inward translocation rates in the presence of elevated intracellular sulfate concentration
were minimal for tested anions other than sulfate. The
rates of SO42
efflux in exchange for extracellular gluconate and isethionate were higher than expected
from red cell studies, but similar to those measured in
oocytes expressing wt AE1 and AE2. It remains unclear
why isethionate and gluconate are not impermeant anions with respect to anion exchange in Xenopus oocytes.3 The phenomenon could reflect altered conformation of the anion translocation pathway of either wt
or mutant AE1 expressed in oocytes compared to red
cells, or activation of endogenous anion transport pathways of the oocyte by expression of heterologous AE
polypeptides. The presence of substantial endogenous
oocyte permeabilities to extracellular gluconate and
isethionate has been suggested by Costa et al. (1989)
.
The approximately equipotent inhibition by DNDS
of AE1 E699Q-mediated SO42/Cl
exchange and of wt
AE1-mediated Cl
/Cl
exchange suggests preservation
in the mutant of those exofacial structures of AE1
which interact with stilbene inhibitors.
Influence of the Host Cell
The origins of the differences between the activities of
AE1 E699Q in Xenopus oocytes and of WRK-BH4-modified red cell AE1 likely reside in the chemical and steric
differences at position 699 between the amide group of
glutamine and the hydroxyl group of hydroxynorvaline, in addition to differences in host cell environments. A role for charge-independent steric factors in
the interaction between E699 and transported anions
was suggested by the loss of SO42/SO42
exchange in
microsomes from 293 cells expressing AE1 E699D, in contrast to the increased activity observed in microsomes from cells expressing either E699Q or E699K
(Sekler et al., 1995
). However, expression of these mutant AE1 polypeptides in Xenopus oocytes had different
consequences. AE1 E699K expressed in Xenopus oocytes did not confer measurable SO42
/SO42
exchange either 2 d (Fig. 2) or 2 wk after cRNA injection
(data not shown). In addition, AE1 E699D expressed in
Xenopus oocytes retained ~40% of wt levels of 36Cl
influx activity (Muller-Berger et al., 1995a
). Thus, the
consequences of selected mutations in AE1 E699 differed in different host cell expression systems. Since
the WRK-BH4 protocols optimized for the human red
cell (Jennings, 1995
) did not inhibit wt AE1-mediated Cl
/Cl
exchange in Xenopus oocytes (Chernova and
Alper, unpublished observations), direct comparison
of WRK-BH4-modified AE1 in red cells and in oocytes
was not possible.
Relationship to Chloride Conductance Attributed to Wt AE1
The DIDS-inhibitable portion of human red cell Cl
conductance has been attributed to conductive anion
"tunneling" through the AE1 protein (Frohlich, 1988
).
Jennings (1995)
noted that WRK-BH4-modification of
human red cells, in addition to its effects on anion exchange, increased Cl
conductance 8-10-fold. In addition to the absence of trans-anion dependence, a property distinguishing red cell Cl
conductance from anion exchange is the insensitivity of Cl
conductance to
concentrations of phloretin that inhibit anion exchange (Frohlich, 1988
). However, 200 µM phloretin
inhibits both SO42
/Cl
exchange (n = 6) and inward
current (n = 4, not shown) mediated by AE1 E699Q.
Together with the demonstrated trans-anion dependence, this result suggests that anion tunneling contributed minimally to AE1 E699Q-mediated electrogenic
anion transport in oocytes.
Expression in oocytes of wt trout AE1 cRNA was reported to confer on oocytes a Cl conductance (Fievet
et al., 1995
). This conductance differed in at least three
ways from mouse AE1 E699Q-mediated electrogenic exchange of intracellular SO42
for extracellular Cl
.
First, trout AE1-mediated Cl
conductance took place
in the presence of the conserved glutamate at the position corresponding to mouse E699. Second, Cl
conductance and Cl
exchange by trout AE1 displayed very
different stilbene sensitivities, whereas electrogenic
and electroneutral variants of anion exchange by
mouse AE1 E699Q exhibited similar stilbene sensitivities. Third, trout AE1 expressed in Xenopus oocytes exhibited maximal anion exchange activity comparable in
magnitude to that of mouse AE1 but exhibited a Cl
conductance ~50-fold larger than the AE1 E699Q-mediated
currents in the present study. Interestingly, however,
trout AE1 expression at levels lower than 15% of maximal anion exchange rates was unassociated with Cl
current, whereas large currents accompanied higher
levels of AE1 expression (Fievet et al., 1995
). Though
the mechanistic relationship between trout AE1-associated Cl
conductance and AE1 E699Q-mediated electrogenic anion exchange is unclear, each may require
for expression of electrogenic transport some minimal
level of electroneutral exchange activity.
Trout AE1 expression in oocytes was also associated
with increased taurine transport (Fievet et al., 1995).
Similarly, skate erythrocyte AE1 has been proposed to
mediate taurine transport (Musch et al., 1994
). However, expression in oocytes of neither wt mouse AE1
nor of AE1 E699Q was associated with increased 3H-taurine efflux, whether measured in isotonic or in hypotonic chloride medium (Chernova and Alper, unpublished results).
Relationship to Other Sulfate Transporters
AE1 is thought to be the principal sulfate transporter of
red cells. In addition to decreased red cell SO42 transport noted in the setting of heterozygous AE1 loss-of-function mutations in hereditary spherocytosis (Jarolim
et al., 1994
; Tanner, 1993
), increased red cell SO42
transport has been associated with the human AE1 mutation P868L in hereditary acanthocytosis (Bruce et al.,
1993
). These data, along with the broad spectrum of
anions transported by AE1 in erythrocytes, has encouraged speculation that nonerythroid AE proteins serve
as physiologically important transporters of anions other than Cl
and HCO3
, including SO42
. Other
Na+-independent SO42
transporters and related proteins from mammalian tissues cloned by functional expression (Bissig et al., 1994
), by differential expression
(Silberg et al., 1995
), or by positional cloning (Hastbacka et al., 1994
) comprise a distinct gene family unrelated in sequence to the AE anion exchanger gene family, despite evidence for SO42
/HCO3
exchange mediated by the transporter Sat1 (Bissig et al., 1994
). At
least one member of this gene family has been implicated in Cl
/HCO3
exchange by genetic linkage (Hoglund et al., 1996
). In contrast, a role for any endogenous AE polypeptide in physiological SO42
transport
by nonerythroid cells remains to be demonstrated.
Original version received 1 May 1996 and accepted version received 16 December 1996.
Address correspondence to Dr. S.L. Alper, Molecular Medicine Unit, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Fax: 617-667-2913; E-mail: salper{at}bidmc.harvard.edu
Portions of this work were presented at the Forty-ninth annual meeting of the Society of General Physiologists (1995. J. Gen. Physiol. 106: 31a) and at the Twenty-eighth annual meeting of American Society of Nephrology (1995. J. Am. Soc. Nephrol. 6:303a).We are grateful to J. Zeind for sulfate determinations, G. Jacquet for technical assistance, and to Dr. M. Gola and A. Stuart-Tilley for discussion.
This work was supported by National Institutes of Health grants DK43495 (S.L. Alper), DK34854 (Harvard Digestive Diseases Center to S.L. Alper), RR01032 (Beth Israel Hospital General Clinical Research Center Core Laboratory), NS30591, and DK45628 (K. Strange), DK77726 (The Children's Hospital Renal Training Grant to M. Hand), and the CNRS (M. Crest). S.L. Alper and K. Strange are Established Investigators of the American Heart Association.