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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Molecular and Vascular Medicine Unit and the Renal Division, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts; 2Department of Psychology, Wellesley College, Wellesley, Massachusetts; and 3Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Submitted 20 February 2008 ; accepted in final form 7 May 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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band 3; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; Xenopus oocyte; Woodward's reagent K
In addition to 1:1 monovalent anion exchange, AE1 can mediate cotransport of H+/SO42– in exchange for Cl– or as a self-exchange reaction (27, 28). The binding site for the cotransported proton during H+/SO42– cotransport is believed to be E681 of human AE1 (19). Treatment of intact human erythrocytes with Woodward's reagent K (WRK) followed by borohydride (BH4) reduction converts AE1 E681 to the corresponding alcohol (hAE1 E681OH). This modification produces multiple alterations in anion transport. These changes include severely reduced rates of electroneutral Cl–/Cl– exchange rates and dramatically increased H+-independent rates of electrogenic SO42–/Cl– exchange and electroneutral SO42–/SO42– exchange (15, 17). Similar changes were observed in the function of recombinant mouse AE1 (mAe1) mutated at the corresponding E699 to the amide Gln. In particular, the high rate of Cl–/Cl– exchange characteristic of wild-type Ae1 became undetectable. In addition, the low wild-type rates of H+/SO42– cotransport were converted to greatly accelerated rates of H+-independent, electroneutral SO42–/SO42– exchange and electrogenic SO42–/Cl– exchange (7).
In the course of investigating the altered properties of mAe1 E699Q-mediated SO42– transport, we observed a novel pH dependence of SO42– transport. We show in the present study that, in contrast to activation of wild-type AE1-mediated SO42– transport by H+ consistent with H+/SO42– cotransport, SO42– transport by the mAE1 mutant E699Q was inhibited by acidic extracellular pH (pHo) and activated by alkaline pHo. This pattern of regulation, diametrically opposed to that of wild-type AE1, was shared by SO42–i/Cl–o and SO42–/SO42– exchanges mediated by mAE1 E699Q, and by SO42–i/Cl–o exchange mediated by hAE1 E681OH. The extracellular pHo(50) for 35SO42– efflux (the pHo at which efflux was half-maximal) was not significantly alkaline-shifted in the presence of acidic intracellular pH (pHi). Moreover, changing pHi at constant pHo had no effect on either SO42–i/Cl–o or SO42–/SO42– exchange. Elevation of intracellular [SO42–] ([SO42–]i) greatly attenuated inhibition of 35SO42– efflux by acidic pHo. Elevated [SO42–]i also increased K1/2 for extracellular SO42– ([SO42–]o) in SO42–/SO42– exchange and greatly increased K1/2 for extracellular Cl– ([Cl–]o) in SO42–i/Cl–o exchange. mAE1 E699Q mediated exchange of SO42–i with a wide range of extracellular oxyanions at rates comparable to those measured for SO42–i/Cl–o exchange and for SO42–/SO42– exchange.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Solutions for Xenopus laevis oocytes. ND-96 medium consisted of (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and 2.5 sodium pyruvate, pH 7.40. Flux media lacked sodium pyruvate. pH values of 7.0, 8.0, and 8.5 in flux media were achieved with 5 mM HEPES. 4-Morpholino-ethanesulfonic acid (MES; 5 mM) was used for flux media of pH values 5.0 and 6.0. In Cl–-free solutions, NaCl was replaced isosmotically with 96 mM sodium isethionate, and equimolar K, Ca, and Mg gluconate substituted for the corresponding Cl– salts. Addition to flux media of the weak acid salt sodium butyrate or of NH4Cl was in equimolar substitution for NaCl.
Expression of cRNA in Xenopus oocytes. Transcription template was generated by linearizing plasmid cDNA with HindIII. cRNA transcription with T7 RNA polymerase was performed with Ambion's Megascript Kit (Austin, TX). Female Xenopus laevis were purchased from NASCO (Madison, WI). Manually defolliculated oocytes were prepared as previously described (13), in compliance with a protocol approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee, were microinjected with 10–20 ng cRNA encoding mAe1 E699Q, and were then incubated in ND-96 supplemented with gentamicin at 19°C for 2–7 days before use in efflux experiments (7).
35SO42– efflux experiments in Xenopus oocytes.
Oocytes were injected with 50 nl of a solution containing Na235SO4 (0.25–0.5 µCi, 3–6 µM) in 130 mM of either HEPES, pH 7.4, MES, pH 5.0, or occasionally, Bis-Tris, pH 5.0. When indicated, the injectate additionally contained 130 mM cold Na2SO4, yielding a final estimated [SO42–]i of 
14 mM [including the endogenous [SO42–] of 1 mM (7)]. Following a 10-min recovery period in SO42–-free, Cl–-free medium containing Na isethionate, 35SO42– efflux was initiated by transfer of individual oocytes into 1 ml efflux medium containing 96 mM NaCl for assay of SO4i/Cl–o exchange, or containing 20 or 64 mM Na2SO4 for assay of SO42–i/SO42–o exchange. Experiments in which extracellular sulfate or chloride concentrations were varied used isethionate as substituting anion to maintain nominal isosmolarity. At regular intervals of 3 min, 950 µl of this medium was removed for scintillation counting and replaced with fresh medium. Experiments ended with a final efflux period in the presence of the AE1 inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 100 or 200 µM), before solubilization of the washed oocyte in 100 µl of 1% sodium dodecyl sulfate (SDS). All 35SO42– efflux experiments were conducted at room temperature.
Efflux cpm values for water-injected or cRNA-injected oocytes in the presence of DIDS were less than threefold above machine background values (20 cpm). Within each experiment, water-injected and cRNA-injected oocytes from the same frog were subjected to parallel measurements. With the exception of the SO42–/SO42– exchange experiment presented in Fig. 8B, each experiment was performed with oocytes obtained from at least two frogs.
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1 mM or 14 mM (7).
To describe the dependence of SO42–/anion exchange on pHo, efflux rate constants measured at each pHo value were fit (Ultrafit 3.0, Biosoft, Ferguson, MO; or Sigmaplot 8.0; Systat, San Jose, CA) by the following first-order logistic sigmoid equation:
 | (1) |
To describe the Cl–o dependence of SO42–i/Cl–o exchange and the SO42–o-dependence of SO42–/SO42– exchange, 35SO42– efflux rate constants plotted as functions of [anion]o were fit by the Michaelis-Menten equation modified to include an added constant, y0:
 | (2) |
1 mM were 0.99 for seven individual oocytes and 0.96 and 0.92 for two additional oocytes (Fig. 7B).
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Treatment of human erythrocytes with Woodward's reagent K.
Red blood cells from healthy adults were obtained by a venipuncture protocol approved by the University of Arkansas for Medical Sciences Institutional Review Board, washed, and modified with Woodward's reagent K (WRK) and NaBH4 as described previously (15). Cells were washed three times and suspended at 5% hematocrit in 150 mM KCl and 10 mM MOPS, pH 7.0. After the suspension was chilled on ice for at least 20 min, solid WRK (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 2 mM. The suspension was mixed gently and incubated 10 min further on ice. NaBH4 (Sigma-Aldrich) was added from a freshly prepared 1 M stock (in 0.1 N NaOH) to a final concentration of 2 mM. After 5 min on ice, an additional 2 mM NaBH4 was added, and the suspension was incubated another 5 min. Between the NaBH4 additions, 10 mM MOPS acid was added to the suspension to keep the pH from rising above 7. This procedure converts 80% of the copies of wild-type hAE1 to hAE1 E681OH (15).
35SO42– efflux from erythrocytes.
After treatment with WRK and NaBH4, cells were washed three times in at least 20 volumes of 80 mM K2SO4 and 10 mM HEPES, pH 7.45, with a 10-min incubation at 37°C to allow SO42– to replace cellular Cl–. Cells were then loaded with 35SO42– by incubating 1 h, 37°C in the same medium containing 1 µCi Na35SO4/ml. Red blood cell [SO42–]i after this protocol of sulfate loading and labeling was 
40 mM. To measure 35SO42–/Cl– exchange, loaded and labeled cells were washed twice at 0°C in K2SO4/HEPES medium containing no radioactivity and resuspended in media consisting of 120 mM KCl, or 60 mM KCl, 120 mM sucrose, in each case buffered with one of the following: 20 mM Na-HEPES (pH 7.5), 20 mM Na-MES, pH 6.4, 10 mM Na-MES/10 mM Na-glutamate, pH 5.7, or 20 mM Na-glutamate, pH 4.4. The suspension was incubated at 20°C, and aliquots were centrifuged at various times (4 time points per efflux) for determination of extracellular radioactivity. The rate constants for efflux were determined as previously described (15). Data plotting efflux rate constant vs. pHo data were fit using equation 1, with d = 0.
Statistical analysis. Data are reported as means ± SE. Statistical significance of pairwise differences was assessed by Student's paired and unpaired t-tests. pHo(50) values were compared by Dunnett's two-way t-test. The level of significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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0.45 units does not significantly alter pHo dependence of mAe1 E699Q-mediated 35SO42–i/Cl–o exchange.
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1 mM), intracellular acidification by 0.5 pH units at constant pHo 7.4 did not alter rates of mAe1 E699Q-mediated SO42–/SO42– exchange or of SO42–i/Cl–o exchange, whether acidification was imposed by exposure to 40 mM sodium butyrate or to 20 mM NH4Cl (12, 40). Exposure to 20 mM KCl to assess the effect of moderate membrane depolarization without acidification also had no effect (n = 4, not shown).
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hAE1 E681OH-mediated SO42–/Cl– exchange is also inhibited by acidic pHo.
To test the possibility that the reversed pH dependence of mAe1 E699Q-mediated SO42– transport might be a cell type-specific property of the Xenopus oocyte expression system, we tested pHo dependence of hAE1 E681OH-mediated 35SO42– efflux from intact erythrocytes. As shown in Fig. 5, acidic pHo inhibited 35SO42– efflux from WRK-BH4-treated human red blood cells, with pHo(50) value 5.6 tested at [Cl–]o of 60 and 120 mM. In these conditions, red blood cell pHi is expected to be close to neutral at neutral pHo. Thus hAE1 E681OH-mediated SO42–i/Cl–o exchange is also inhibited by extracellular H+, a pattern opposite to that of wild-type hAE1 in erythrocytes (27).
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10-fold higher K1/2 for Cl–o of 40 ± 10 mM (n = 5). This increase of extracellular K1/2 in response to elevation of intracellular substrate concentration is consistent with a ping-pong mechanism of SO42–i/Cl–o exchange (11).
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1 mM, n = 4) lower than the rates at 20 mM [SO42–]o. Such apparent autoinhibition was not evident for SO42–i/Cl–o exchange for [Cl–]o as high as 103 mM.
Oocytes with nominal [SO42–]i of 14 mM (Fig. 7, C and D) exhibited a similarly hyperbolic Cl–o dependence of SO42–/SO42– exchange, but with a 2.7-fold higher K1/2 for SO42–o of 3.4 ± 0.68 mM (n = 8). Thus elevation of [SO42–]i increases the K1/2 of mAe1 E699Q for extracellular SO42– (P < 0.02), but to a lesser degree than for extracellular Cl–. But the increases in both values are qualitatively consistent with a ping-pong mechanism for both electrogenic and electroneutral anion exchange by mAE1 E699Q. Measurements of SO42–/SO42– exchange from similar oocytes with nominal [SO42–]i of 14 mM during the transition from 20 to 64 mM [SO42–]o showed apparent autoinhibition of 39 ± 7% (n = 12). Thus, changes in [SO42–]i did not detectably alter the apparent affinity of the autoinhibitory site for SO42–o.
Elevated [SO42–]i severely attenuates inhibition by pHo of both SO42–i/Cl–o exchange and SO42–/SO42– exchange mediated by mAe1 E699Q.
In mAe1 E699Q-expressing oocytes containing 14 mM [SO42–]i and 13 mM HEPES, pH 7.4, stimulation of SO42–i/Cl–o exchange by the pHo transition from 5.0 to 7.4 (Fig. 8A) was reduced to 1.7 ± 0.3-fold (n = 17; Fig. 8B). In mAe1 E699Q-expressing oocytes containing 14 mM [SO42–]i and 13 mM MES, pH 5.0, stimulation of SO42–i/Cl–o exchange by the pHo 5.0–7.4 transition (Fig. 8C) was reduced further to 1.2 ± 0.2-fold (n = 9, Fig. 8D). In mAe1 E699Q-expressing oocytes containing 1 mM [SO42–]i without injected buffer, SO42–/SO42– exchange was stimulated eightfold by the pHo 5.0-to-8.0 transition, but only 1.2 ± 0.4-fold in oocytes containing 14 mM [SO42–]i (Fig. 9). Thus, elevated [SO42–]i attenuated the pHo sensitivity of both SO42–/SO42– exchange and SO42–i/Cl–o exchange. In separate experiments, elevation of oocyte [SO42–]i to 26 mM similarly abolished inhibition of both SO42–/SO42– and SO42–i/Cl–o exchange by acidic pHo (n = 5–7, not shown).
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65–70% the rate of SO42–i/Cl–o exchange. SO42–i/SeO
o was 65% the rate of SO42–i/Cl–o exchange, but SO42–i/SeO
o exchange was roughly comparable in rate to SO42–i/Cl–o exchange. As true for wild-type hAE1 in red blood cells (8), SO42–i/POo exchange was much slower than SO42–i/POo exchange. SO42– exchange rates for extracellular NO3– or NO2– were nearly equivalent to those for SO42–i/Cl–o exchange.
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o exchange was activated approximately twofold by extracellular alkalinization of oocytes injected with HEPES, pH 7.4, and nearly threefold in oocytes injected with MES, pH 5.0 (Fig. 10B). Thus, acute inhibition of mAe1 E699Q-mediated 35SO42– efflux by pHo is also a property of exchange with other oxyanions recognized by the extracellular substrate site of this mutant polypeptide. |
DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Thus, the E699Q mutation unmasks a novel pHo-regulatory site on mAE1 that can be transregulated by intracellular substrate SO42–. We also show for mAe1 E699Q that elevated [SO42–]i increases apparent K1/2 10-fold for Cl–o and 2.5-fold for SO42–o, consistent with sequential (ping-pong) mechanisms for both SO42–i/Cl–o exchange and for SO42–/SO42– exchange. In addition, we document in mAe1 E699Q enhanced SO42– affinity of an autoinhibitory site for SO42–.
Opposite pH sensitivities of SO42–/anion exchange by wild-type AE1 and mAE1 E699Q or hAE1 E681OH.
Cotransport of H+ and SO42–o in exchange for Cl–i by wild-type hAE1 in human red blood cells is activated by pH, with pKa values of 5.5 (27, 28). However, whereas SO42–/SO42– exchange in human (27, 36) and in mouse red blood cells (31) peaked at pHo values between 5.9 and 6.5, with progressive inhibition upon further extracellular acidification, Cl–i/SO42–o exchange continued to increase upon further acidification until peaking at pH 4.5–4.0, with transport rates 20-fold higher than peak rates of SO42–/SO42– exchange (26). In contrast, both SO42–i/Cl–o exchange and SO42–/SO42– exchange mediated by mAe1 E699Q are inhibited by extracellular H+, with peak transport rates observed at values pHo 8. Sulfate transport by hAE1 E681OH in WRK/BH4-treated human red blood cells exhibited similar inhibition by extracellular protons, with pHo(50) 5.6 for SO42–i/Cl– exchange (Fig. 5) similar to the pHo(50) value for mouse AE1 E699Q-mediated SO42–i/Cl–o exchange (Fig. 1D) as fit by Eq. 1 with parameter d = 0, but lower than the pHo(50) value of 6.2 predicted from the better curve fit (Fig. 1D). Thus, the reversed pH dependence of mAe1 E699Q is not a function of the expression system or of the E-to-Q mutation, but rather results from replacement of the catalytically important carboxylate of E699/E681 with polar amide or alcohol residues,
The H+ binding site for H+/SO42– cotransport in wild-type AE1 is likely E681 in the human protein (15) and E699 in the mouse protein (7). The identity of the protonation site mediating inhibition of SO42– transport by mAe1 E699Q and by hAE1 E681OH is unknown, but it may be the inhibitory protonation site for wild-type AE1 SO42–/SO42– exchange, apparently inactive or inaccessible to protonation in the presence of Cl–o, and unmasked by mutation of E699/E681. Existence of the site was predicted from reversal of carrier asymmetry in hAE1 E681OH (15) and may relate to the inhibitory titratable side chain carboxylate modifiable by carbodiimides (5, 6). Similarly uncertain is a possible relationship to any among the conserved amino acid residues of mAe2 whose titration contribute to its H+-sensitive inhibition of monovalent anion transport (40–43, 45, 46). Intramolecular cross-linking of hAE1 with the cross-linker bis(sulfosuccinimidyl)-suberate (BS3) applied to intact erythrocytes also reveals an inhibitory protonation site coextant with the wild-type activating protonation site. The inhibitory site may reflect BS3's acid-shifting the pHo dependence of monovalent anion exchange by 5 pH units such that Cl–/Br– exchange, very slow at pHo >8, is activated to maximal rates at pH 5–6 (18).
The ability of 14 mM SO42–i to attenuate or prevent inhibition by acidic pHo of mouse AE1 E699Q-mediated exchange of SO42–i for either Cl–o or SO42–o might represent a transmembrane conformational effect of either an internal sulfate modifier site or, alternatively, of saturation of the intracellular substrate binding site. In either case, the effect must reflect the increased outward asymmetry of the carrier induced by elevated intracellular substrate. The ability of elevated [SO42–]i to attenuate inhibition by acidic pHo of hAE1 E681OH-mediated SO42–i/Cl–o exchange may be less than for mouse AE1 E699Q in oocytes, since red blood cell [SO42–]i at the onset of the efflux period approximated 40 mM. This apparent difference between AE1 E681OH and mouse AE1 E699Q may reflect in part the more extensive acid pHo range tested for WRK-BH4 red blood cells (Fig. 5), likely accompanied by much larger pHi excursions in red blood cells than in Xenopus oocytes (13, 41). Alternatively, the difference may reflect distinct SO42–i affinities at the intracellular substrate site and/or the putative internal modifier site (21) in the mouse and human proteins.
Proton-mediated inhibition of SO42–/anion exchange among SLC4 polypeptides. The Glu residue corresponding to mAe1 E699 and hAE1 E681 is conserved in the Na+-independent anion exchangers SLC4A2/AE2 and SLC4A3/AE3, and in the borate transporter, SLC4A11/BTR1. Preliminary results indicate that mutation to Gln of the corresponding mAe2 residue E1007 similarly abolishes Cl–/Cl– and Cl–i/HCO3–o exchange activities, and greatly accelerates activities of SO42–i/Cl–o and SO42–/SO42– exchange. mAe2 E1007Q-mediated SO42–/SO42– exchange is also inhibited by acidic pHo, exhibiting eightfold activation upon pHo shift from 5.0 to 8.0 (n = 4; Stewart AK and Alper SL, unpublished observations). These results differ from an earlier report that 35SO42– uptake by proteoliposomes reconstituted from microsomes of HEK-293 cells transiently transfected with mouse AE2 E1007Q was no slower at pHo 5.5 than at pHo 7.5 (38).
A conservative Asp substitution is present in all other SLC4 polypeptides, but sequence polymorphisms at this codon of SLC4 genes have not yet been described in the Single Nucleotide Polymorphism database. Mutation of the corresponding D754 to Cys in rat NBCe1/Slc4a4 abolished Na/HCO3– cotransport current despite normal oocyte surface expression (25). Mutation of the same Asp residue in human NBCe1 to Glu, Asn, and Arg resulted in Na+-dependent base flux at 23%, 9%, and 0% of wild-type rates (1). Possible alteration in substrate selectivity was not evaluated in either study.
Evidence favoring a ping-pong transport mechanism for mAe1 E699Q.
Many characteristics of anion exchange by wild-type human red blood cell AE1 and by hAE1 E681OH can be described by simple ping-pong kinetics with translocation as the rate-limiting step (15, 22). Preservation of ping-pong kinetics in settings of mAe1 E699Q's altered substrate selectivities and substrate-conditional electrogenic anion exchange had not been tested. The 10-fold increase in mAe1 E699Q K1/2 for Cl–o (Fig. 6) and the smaller increase in K1/2 for SO42–o produced by elevation of [SO42–]i from 1 mM to 14 mM (Fig. 7) are both compatible with a substantial degree of intracellular substrate-driven polypeptide reorientation from inward to outward facing conformations of substrate binding sites. The smaller increase in K1/2 for SO42–o than for Cl–o may reflect the predominance of outward facing sites in SO42– medium, the large difference in magnitude of charge transfer during the inward anion translocation step (15), or intrinsic changes in carrier conformation resulting from occupancy of the outward facing substrate site by SO42–o or Cl–o. This proposed reorientation from inward to outward conformation was not very much influenced by pHi (Fig. 9). Unlike hAE1 E681OH red blood cells, the [Cl–]o dependence of 35SO42– efflux from oocytes by mAe1 E699Q was not sigmoidal (15) but hyperbolic. This difference may reflect different intrinsic consequences of mutation of the critical Glu residue to the alcohol as opposed to the amide. Alternatively, the difference may relate to the larger fractional contribution of electrogenic SO42–i/Cl–o exchange by hAE1 E681OH to total red blood cell conductance than that of mAe1 E699Q to total oocyte conductance.
Substrate selectivity of mAe1 E699Q.
The oxyanion substrate selectivity of mAe1 E699Q shows substantial differences from that of wild-type hAE1. Thus, whereas wild-type hAE1 exchanges Cl–i for SO42–o 100-fold more slowly than SOo at neutral pHo (8), mAe1 E699Q exchanges SO42–i for SO42–o and for SOo at equivalent rates (Fig. 10). Whereas wild-type hAE1 exchanges Cl–i for SeOo 100-fold more slowly than for SeOo (8), mAe1 E699Q exchanges SO42–i for SeOo only 35% more slowly than SeOo. Wild-type hAE1 exchanges Cl–i for POo 100-fold more slowly than for POo, mAe1 exchanges SO42–i for POo only 10-fold more slowly than for POo. These differences suggest that the transition state in the E699Q mutant can accommodate certain oxyanion substrates larger in the z dimension (8) than tolerated by the wild-type protein, and that this flexibility can be modified by pHo. The smaller monovalent anions NO2– and NO3– are transported by wild-type and mutant proteins at similar rates (50–70% those of Cl–o), whereas the bulky monovalent anions sulfamate and methanesulfonate are transported at negligible rates by both proteins (Fig. 10).
Wild-type SLC4 polypeptides also transport CO2 equivalents as the divalent oxyanion, CO. Wild-type AE1 in the human red blood cell mediates slow exchange of Cl–i for the extracellular ion pairs lithium carbonate (Li:CO3)– and lithium sulfite (Li:SO3)–. The even slower exchange of Cl–i for the extracellular Li/oxalate ion pair is more rapid at pHo 7.5 than at pHo 6.0 (4), likely reflecting competition for H+/oxalate cotransport, with its opposite pHo dependence (16). Electrogenic Na+/HCO3– cotransporter activity measured in basolateral membrane vesicles from rabbit kidney proximal tubule was postulated to cotransport Na+, HCO3–, and CO
functioning with 3:1 charge stoichiometry as in the native proximal tubule. SO was found to stimulate HCO3–-dependent Na+ uptake, with characteristics suggesting occupancy of a proposed CO site distinct from the HCO3– site (39). However, recombinant rat kidney NBCe1 expressed in Xenopus oocytes, with its 2:1 charge stoichiometry attributed at first to Na+/2HCO3– cotransport (37) and later reinterpreted as Na/CO cotransport (10), revealed no evidence for either binding or transport of SO(9). Gln substitution of hNBCe1, the residue corresponding to hAE1 E681/mAe1 E699, has not been reported (1, 25), and the hNBCe1 mutant D754N was not tested for upregulation of SO transport (1).
Extracellular modifier site of mAe1 E699Q. The extracellular modifier site of erythroid hAE1 is believed not to block access to the substrate site, but rather to slow the rate of the anion transporting conformational change. The modifier site likely has no physiological significance in the red blood cell, since Cl– and HCO3– at their physiological systemic plasma concentrations interact with it only weakly. Model-dependent estimates of modifier site K1/2 for Cl–o range from 270 to 375 mM (20). However, the modifier site on kidney AE1 of the type A intercalated cell might gain importance under antidiuretic conditions, in which renal medullary interstitial [Cl–] can be sustained at 300 mM in the human and at 600–1,200 mM or more in rodents. The modifier site on erythroid AE1 might thus exert a significant effect also on red blood cells retarded in their passage through the antidiuretic renal medulla. An internal inhibitory modifier site requiring high concentrations of Cl–i (21) may also become meaningful in these high ionic strength conditions.
The wild-type hAE1 extracellular modifier site exhibits highest affinity for I– [although K1/2 estimates range between 8 and 120 mM (18, 23)], with lesser affinity for Br–. The binding sites of the noncompetitive anion transport inhibitors NAP-taurine and NIP-taurine overlap the extracellular modifier site (23), and BS3 treatment attenuates or abolishes binding of I– or NAP-taurine to the modifier site (18). SO42– interaction with the modifier site has been little studied. However, no self-inhibition by SO42– of human red blood cell anion exchange has been noted at concentrations up to 100 mM. Thus, the 40% inhibition of mAe1 E699Q-mediated SO42–/SO42– exchange upon increasing [SO42–]o from 20 to 64 mM represents a change in both the anion selectivity as well as apparent affinity of the external modifier site, if not the emergence of a novel modifier site. The existence of a similar modifier site in mAe1 E681OH, preferring occupancy with SO42– over Cl–, was proposed to explain the transacceleration of SO42–i efflux by Cl–o to a degree higher than explicable by simple ping-pong kinetics (15). The interaction of SO42– with a similar modifier site in hAE1 E681OH may mediate the cis-stimulation of SO42–o uptake into WRK-BH4-modified human red blood cells by 10–20 mM Cl–o, possibly via displacement of SO42–o from the modifier site by noninhibitory Cl–o, but more likely via Cl–o-accelerated post-outward translocation release of SO42– from hAE1 E681OH (14).
The relationship of the inhibitory substrate modifier site of wild-type hAE1 to the inhibitory protonation site of mAe1 E699Q is not clear. Similarly unknown is the modifier site's relationship with either the second Cl– binding site of hAE1 E681OH (14, 34), the second stilbene binding site of hAE1 E681OH (33), or the distinct binding sites for HCO3– and CO postulated in some models of NBCe1 activity (10, 29, 39). Nonetheless, the presumed structural similarities among homologous SLC4 polypeptides, most prominent in their transmembrane domains, encourages consideration and testing of these possibilities.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* M. N. Chernova and A. K. Stewart contributed equally to this work. 
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, Cl– bath) or with mAe1 E699Q cRNA (bottom two traces). Oocytes preequilibrated with 40 mM butyrate in Cl–-free conditions were exposed to baths containing either Cl– (ND-96, 
) or SO42– (20 mM, 
), and subjected to subsequent butyrate removal and 200 µM DIDS exposure. B: mean efflux rate constants for SO42–/Cl– exchange and SO42–/SO42– exchange in the presence (black bars) and absence (gray bars) of butyrate, measured as depicted in A. C: 35SO42– efflux time course of a representative individual mAe1 E699Q-expressing oocyte exposed to the absence and subsequent presence of NH4Cl, followed by 200 µM DIDS. D: mean efflux rate constants for SO42–/Cl– exchange in the absence (black bars) and presence (gray bars) of NH4Cl, measured as depicted in C.
). Indicated pHo values were as measured in the efflux supernatants immediately after the final efflux time point. The curves represent fits with 
, top trace) or with mAe1 E699Q cRNA [estimated [SO42–]i = 1 mM (