|
|
|
|
|
|
|
||
Article |
,|||| 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
 |
ABSTRACT |
|---|
 Top ABSTRACT introductionmethodsresultsdiscussionreferences |
|---|
Key Words: band 3 • chloride/bicarbonate exchange • Xenopus oocyte • BCECF • sulfate
|
introduction |
|---|
|
TopABSTRACTintroductionmethodsresultsdiscussionreferences |
|---|
; 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.
|
methods |
|---|
|
TopABSTRACTintroductionmethodsresultsdiscussionreferences |
|---|
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.
|
results |
|---|
|
TopABSTRACTintroductionmethodsresultsdiscussionreferences |
|---|
|
).
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.
|
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.
|
. 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.
|
). 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).
|
). 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.
|
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).
|
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.
|
|
|
|
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.
|
|
) 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. |
discussion |
|---|
|
TopABSTRACTintroductionmethodsresultsdiscussionreferences |
|---|
), 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 SO42–i/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.
1 Abbreviations used in this paper: AE1, anion exchanger 1; DNDS, 4,4'-dinitrostilbene-2,2'-disulfonic acid; WRK, Woodward's Reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate); wt, wild-type.
2 Exclusion of the "outlier" experiment 2 from the calculations of the flux ratios resulted only in small changes. Such an edited mean of the individual flux ratios, 1.30 ± 0.19, did not differ statistically from the value in Table I. Such an edited ratio of the individual experimental mean flux values, 1.24 ± 0.05, differed slightly from that in Table I, but both values were consistent with a 1:1 stoichiometry of SO42–i/Cl–o exchange. Jennings (1995) also observed a ratio of SO42– efflux to Cl– influx of 1.21 ± 0.1 (SEM) in WRK-BH4–treated human red cells in the presence of gramicidin (Table 1 of Jennings, 1995).
3 Though AE1 E699Q-mediated inward current at clamped resting potential was of sufficient magnitude to conclude that all anion exchange flux measured under open circuit conditions was electrogenic (Table IV), extracellular isethionate supported a rate of AE1 E699Q-mediated 35SO42– efflux of 0.2 relative to extracellular Cl– (Fig. 5) but supported no greater inward current than did extracellular sulfate. Several factors might account for this discrepancy without invoking an alternate transport mechanism. First, current values of 0.2 relative to those recorded in extracellular Cl– are near or below the threshold of detection against the variable background current of individual oocytes. Second, the two assays were performed under different experimental conditions. Sulfate efflux into extracellular isethionate (Fig. 5) was measured 10 min after injection of oocytes with 50 nl carrier-free Na235SO4 in 50 mM Na HEPES, pH 7.4, whereas membrane potential change in response to extracellular anion substitution (Fig. 8, Table III) was measured in oocytes maintained in isethionate medium for 1–6 h after injection with 50 nl of 130 mM Na2SO4, 50 mM Na HEPES, pH 7.4. Lastly, evaluation of the effect of extracellular isethionate on the membrane potential of AE1 E699Q-expressing oocytes requires comparison with the effect of an extracellular monovalent anion to which the oocyte is "absolutely" or maximally impermeable (at least as judged by 35SO42– efflux). Experiments to address this issue are underway.
|
ACKNOWLEDGMENTS |
|---|
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.
Submitted: 1 May 1996
Accepted: 16 December 1996
|
references |
|---|
|
TopABSTRACTintroductionmethodsresultsdiscussionreferences |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  A. K. Stewart, D. H. Vandorpe, J. F. Heneghan, F. Chebib, K. Stolpe, A. Akhavein, E. J. Edelman, Y. Maksimova, P. G. Gallagher, and S. L. Alper The GPA-dependent, spherostomatocytosis mutant AE1 E758K induces GPA-independent, endogenous cation transport in amphibian oocytes Am J Physiol Cell Physiol, February 1, 2010; 298(2): C283 - C297. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Dai, A. K. Stewart, F. Chebib, A. Hsu, J. Rozenfeld, D. Huang, D. Kang, V. Lip, H. Fang, H. Shao, et al. Distinct and novel SLC26A4/Pendrin mutations in Chinese and U.S. patients with nonsyndromic hearing loss Physiol Genomics, August 7, 2009; 38(3): 281 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Alper Molecular physiology and genetics of Na+-independent SLC4 anion exchangers J. Exp. Biol., June 1, 2009; 212(11): 1672 - 1683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Chernova, A. K. Stewart, P. N. Barry, M. L. Jennings, and S. L. Alper Mouse Ae1 E699Q mediates SO42-i/aniono exchange with [SO42-]i-dependent reversal of wild-type pHo sensitivity Am J Physiol Cell Physiol, August 1, 2008; 295(2): C302 - C312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Stewart, C. E. Kurschat, R. D. Vaughan-Jones, B. E. Shmukler, and S. L. Alper Acute regulation of mouse AE2 anion exchanger requires isoform-specific amino acid residues from most of the transmembrane domain J. Physiol., October 1, 2007; 584(1): 59 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pushkin and I. Kurtz SLC4 base (HCO3-, CO32-) transporters: classification, function, structure, genetic diseases, and knockout models Am J Physiol Renal Physiol, March 1, 2006; 290(3): F580 - F599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L Alper Molecular physiology of SLC4 anion exchangers Exp Physiol, January 1, 2006; 91(1): 153 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. N. Chernova, D. H. Vandorpe, J. S. Clark, J. I. Williams, M. A. Zasloff, L. Jiang, and S. L. Alper Apparent receptor-mediated activation of Ca2+-dependent conductive Cl- transport by shark-derived polyaminosterols Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1644 - R1658. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. E. Shmukler, C. E. Kurschat, G. E. Ackermann, L. Jiang, Y. Zhou, B. Barut, A. K. Stuart-Tilley, J. Zhao, L. I. Zon, I. A. Drummond, et al. Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization Am J Physiol Renal Physiol, October 1, 2005; 289(4): F835 - F849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sherman, M. N. Chernova, J. S. Clark, L. Jiang, S. L. Alper, and K. Nehrke The abts and sulp families of anion transporters from Caenorhabditis elegans Am J Physiol Cell Physiol, August 1, 2005; 289(2): C341 - C351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. N. Chernova, L. Jiang, D. J. Friedman, R. B. Darman, H. Lohi, J. Kere, D. H. Vandorpe, and S. L. Alper Functional Comparison of Mouse slc26a6 Anion Exchanger with Human SLC26A6 Polypeptide Variants: DIFFERENCES IN ANION SELECTIVITY, REGULATION, AND ELECTROGENICITY J. Biol. Chem., March 4, 2005; 280(9): 8564 - 8580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. K. Dahl, L. Jiang, M. N. Chernova, A. K. Stuart-Tilley, B. E. Shmukler, and S. L. Alper Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer J. Biol. Chem., November 7, 2003; 278(45): 44949 - 44958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N Chernova, L. Jiang, B. E Shmukler, C. W Schweinfest, P. Blanco, S. D Freedman, A. K Stewart, and S. L Alper Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes J. Physiol., May 15, 2003; 549(1): 3 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Chernova, A. K. Stewart, L. Jiang, D. J. Friedman, Y. Z. Kunes, and S. L. Alper Structure-function relationships of AE2 regulation by Ca2+i-sensitive stimulators NH+4 and hypertonicity Am J Physiol Cell Physiol, May 1, 2003; 284(5): C1235 - C1246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.K. Stewart, M.N. Chernova, B.E. Shmukler, S. Wilhelm, and S.L. Alper Regulation of AE2-mediated Cl- Transport by Intracellular or by Extracellular pH Requires Highly Conserved Amino Acid Residues of the AE2 NH2-terminal Cytoplasmic Domain J. Gen. Physiol., October 29, 2002; 120(5): 707 - 722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Xie, R. Welch, A. Mercado, M. F. Romero, and D. B. Mount Molecular characterization of the murine Slc26a6 anion exchanger: functional comparison with Slc26a1 Am J Physiol Renal Physiol, October 1, 2002; 283(4): F826 - F838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Goss, L. Jiang, D. H. Vandorpe, D. Kieller, M. N. Chernova, M. Robertson, and S. L. Alper Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1978 - C1990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Casula, B. E. Shmukler, S. Wilhelm, A. K. Stuart-Tilley, W. Su, M. N. Chernova, C. Brugnara, and S. L. Alper A Dominant Negative Mutant of the KCC1 K-Cl Cotransporter. BOTH N- AND C-TERMINAL CYTOPLASMIC DOMAINS ARE REQUIRED FOR K-Cl COTRANSPORT ACTIVITY J. Biol. Chem., November 2, 2001; 276(45): 41870 - 41878. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zhao and R. A. F. Reithmeier Expression and characterization of the anion transporter homologue YNL275w in Saccharomyces cerevisiae Am J Physiol Cell Physiol, July 1, 2001; 281(1): C33 - C45. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Beckmann, J. S. Smythe, D. J. Anstee, and M. J. A. Tanner Coexpression of band 3 mutants and Rh polypeptides: differential effects of band 3 on the expression of the Rh complex containing D polypeptide and the Rh complex containing CcEe polypeptide Blood, April 15, 2001; 97(8): 2496 - 2505. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Stakisaitis, M. S. Lapointe, and D. Batlle Mechanisms of chloride transport in thymic lymphocytes Am J Physiol Renal Physiol, February 1, 2001; 280(2): F314 - F324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Knauf, N. M. Raha, and L. J. Spinelli The Noncompetitive Inhibitor Ww781 Senses Changes in Erythrocyte Anion Exchanger (Ae1) Transport Site Conformation and Substrate Binding J. Gen. Physiol., February 1, 2000; 115(2): 159 - 174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Su, B. E. Shmukler, M. N. Chernova, A. K. Stuart-Tilley, L. de Franceschi, C. Brugnara, and S. L. Alper Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation Am J Physiol Cell Physiol, November 1, 1999; 277(5): C899 - C912. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bahar, C. T. Gunter, C. Wu, S. D. Kennedy, and P. A. Knauf Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3 Am J Physiol Cell Physiol, October 1, 1999; 277(4): C791 - C799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-B. Tang, M. Kovacs, D. Sterling, and J. R. Casey Identification of Residues Lining the Translocation Pore of Human AE1, Plasma Membrane Anion Exchange Protein J. Biol. Chem., February 5, 1999; 274(6): 3557 - 3564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. T. Timmer and R. B. Gunn Inducible expression of erythrocyte band 3 protein Am J Physiol Cell Physiol, January 1, 1999; 276(1): C66 - C75. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-B. Tang, J. Fujinaga, R. Kopito, and J. R. Casey Topology of the Region Surrounding Glu681 of Human AE1 Protein, the Erythrocyte Anion Exchanger J. Biol. Chem., August 28, 1998; 273(35): 22545 - 22553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Vandorpe, B. E. Shmukler, L. Jiang, B. Lim, J. Maylie, J. P. Adelman, L. de Franceschi, M. D. Cappellini, C. Brugnara, and S. L. Alper cDNA Cloning and Functional Characterization of the Mouse Ca2+-gated K+ Channel, mIK1. ROLES IN REGULATORY VOLUME DECREASE AND ERYTHROID DIFFERENTIATION J. Biol. Chem., August 21, 1998; 273(34): 21542 - 21553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Jarolim, C. Shayakul, D. Prabakaran, L. Jiang, A. Stuart-Tilley, H. L. Rubin, S. Simova, J. Zavadil, J. T. Herrin, J. Brouillette, et al. Autosomal Dominant Distal Renal Tubular Acidosis Is Associated in Three Families with Heterozygosity for the R589H Mutation in the AE1 (Band 3) Cl-/HCO3- Exchanger J. Biol. Chem., March 13, 1998; 273(11): 6380 - 6388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Vandorpe, M. N. Chernova, L. Jiang, L. K. Sellin, S. Wilhelm, A. K. Stuart-Tilley, G. Walz, and S. L. Alper The Cytoplasmic C-terminal Fragment of Polycystin-1 Regulates a Ca2+-permeable Cation Channel J. Biol. Chem., February 2, 2001; 276(6): 4093 - 4101. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
