Introduction

The SLC4 gene superfamily encodes three Na+-independent, Cl−/HCO3 − exchangers (SLC4A1-3), five Na+-dependent HCO3 − cotransporters or exchangers (SLC4A4, A5, A7, A8, and A10), a borate efflux transporter (SLC4A11), and SLC4A9, of still uncertain function. Mutations in human SLC4A1 cause hereditary spherocytic and stomatocytic anemias and familial distal renal tubular acidosis. A coding polymorphism in SLC4A3 is associated with susceptibility to seizure disorder. Mutations in SLC4A4 cause proximal renal tubular acidosis. Mutations in human SLC4A11 cause late-onset blindness due to several clinical variants of corneal endothelial dystrophy. Mouse knockouts of other SLC4 transporters produce additional disease phenotypes in mice [2, 3].

Although deficiency of AE2 has not yet been associated with Mendelian human disease, the Ae2 −/− mouse dies within 3–4 weeks after birth, and suffers osteopetrosis [51], hypochlorhydria, and intestinal hyposecretion [15, 16]. The hypomorphic Ae2a,b−/− mouse is grossly normal but exhibits male infertility [32], less severe bone disease [24], and serological signs reminiscent of human primary biliary cirrhosis [38].

SLC4A2/AE2 differs in its regulation from the more extensively studied SLC4 polypeptide SLC4A1/AE1 in several important ways. AE2-mediated Cl− transport is inhibited by intracellular and, independently, by extracellular protons in the physiological range, whereas Cl− transport by AE1 is inhibited only at non-physiologically acidic pH values [44, 56]. AE2 is activated by hypertonicity, but AE1 is insensitive to the same range of tonicity [23]. AE2 is activated by NH4 +, despite its intracellular acidification of Xenopus oocytes, but AE1 is NH4 +-insensitive [21]. The transmembrane domain of AE2 contributes importantly to these regulatory processes [10], but the relationship of residues controlling these regulatory processes to the still incompletely defined bidirectional anion translocation pathway remains unclear.

Structural information for the SLC4 gene family will thus be of substantial functional importance in understanding the varied transport mechanisms and substrate specificities exhibited by these widely expressed, phylogenetically ancient, SLC4 polypeptides. The crystal structure of part of the erythroid SLC4A1/AE1 N-terminal cytoplasmic domain has been reported [54], but the available structural information for the AE1 transmembrane domain is of inadequate resolution (7.5 A) for visualization of all transmembrane helices [52]. Cysteine scanning mutagenesis has provided important information on topographical accessibility and structure–function relationships for SLC4A1 [50, 59, 60, 63] and SLC4A4 [31, 58, 61, 62]. Interpretation of the mutagenesis data has been simplified for human SLC4A1 by the availability of variants in which all Cys residues (or, in the case of trout AE1, all transmembrane domain Cys residues) have been mutated to Ser [6, 30]. A preliminary report of Cys-less SLC4A4 has also appeared [64]. However, systematic Cys substitution and scanning mutagenesis studies of AE2 have not been reported. The substantial differences in regulation between AE1 and AE2 suggest structural differences expected to inform investigations of structure–function relationships across the ten-member SLC4 gene family.

We therefore generated a mouse AE2 (mAE2) expression construct in which all transmembrane domain cysteines have been substituted either with residues found in corresponding positions of AE1 and/or AE3, or with Ser or Ala.

Expression of this Cys-less mAE2 (mAE2Cys-less) in Xenopus oocytes revealed preservation of DIDS-sensitive anion exchange at the oocyte cell surface, with apparent AE2 affinity for extracellular Cl− indistinguishable from that of wildtype AE2. mAE2Cys-less also preserved wildtype activation by hypertonicity and by NH4 +, and maintained wildtype inhibition by acidic intracellular pH (pHi). However, the absence of transmembrane Cys residues enhanced the sensitivity of Cl− transport to inhibition by extracellular protons, such that the pHo at which inhibition was 50 % of maximum (pHo(50)) was >0.6–0.7 pH units higher than that of wildtype AE2. Missense substitution of all transmembrane domain Cys residues preserved both activation by extracellular protons of mAE2-mediated SO4 2− transport and the contrasting activation by alkaline pHo of mAE2 E1007Q-mediated SO4 2− transport. However, absence of transmembrane domain Cys residues abolished pH sensitivity of mAE2-mediated oxalate transport. The lack of transmembrane domain Cys residues also potentiated the reduction of Cl− transport by the mAE2 anion translocation pathway mutant E1007Q and by polypeptides carrying other substitutions at the same position.

Methods

Materials

Na36Cl and Na2 35SO4 were from Perkin-Elmer (Waltham, CA). 14C oxalate was from NEN-DuPont (gift of C. Scheid and T. Honeyman, University of Massachusetts Medical Center). Restriction enzymes were from New England Biolabs (Beverly, MA). EXPAND high-fidelity PCR System and T4 DNA ligase were from Roche Diagnostics (Indianapolis, IN). 4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) was obtained from Calbiochem (La Jolla, CA). Other chemicals (of reagent grade) were from Sigma (St. Louis, MO) or Fluka (Milwaukee, WI).

Solutions

MBS consisted of (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, and 10 HEPES (pH 7.40). ND-96 consisted of (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.40). In ND-96 at pH 5 and 6, Na HEPES was replaced by equimolar MES. In Cl−-free or partially Cl−-substituted solutions, NaCl was replaced mole for mole with Na cyclamate. Cl− salts of K+, Ca2+, and Mg2+ were substituted on an equimolar basis with the corresponding gluconate salts as needed. In some experiments 30 mM NaCl was replaced with 20 mM Na2SO4 as indicated. Oxalate-containing bath solutions were nominally Ca2+-free. Addition to flux media of the weak acid sodium butyrate (40 mM) was in equimolar substitution for Na cyclamate. Bath addition of NH4Cl (26 mM) was in equimolar substitution for NaCl.

cDNA mutagenesis

Mouse SLC4A2a/AE2a (mAE2) cDNA was subcloned into the Xenopus oocyte expression vector pXT7. All transmembrane domain Cys residues were subjected to missense mutagenesis by four primer polymerase chain reaction (PCR) as described [18, 47] to generate AE2Cys-less. AE2 C704 was changed to Val, C751 and C1042 were changed to Ala (as in the corresponding residues of mAE1), and C1211 was changed to Ala (as in AE3). AE2 C778 (highly conserved) and C851 (in the AE2-specific sequence of the third extracellular loop) were changed to Ser. Highly conserved AE2 C1169 and unconserved C1224 were also changed to Ala. AE2Cys-less(7/8) lacked all transmembrane domain Cys residues except C1211. Oligonucleotide primer sequences are presented in Table S1. PCR-amplified products were sequenced in entirety to ensure absence of unintended mutations. The sites of missense mutations in AE2Cys-less are summarized in Fig. 1. We define the “transmembrane domain” as extending from the start of TM1 to the C terminus and including, in addition to lipid bilayer-spanning or -embedded regions, all extracellular and intacellular loops and the cytoplasmic C-terminal tail.

Fig. 1
figure 1

Cysteine residues of the transmembrane domain of mouse AE2. a Schematic of mAE2 Cys residues. Numbered black circles indicate Cys residues. Boxed bold indicates Cys conservation among all SLC4 polypeptides (C778, C1169). C704 (*) is conserved in mAE3 but not mAE1. C1211 (**) is conserved in mAE1, but not mAE3. Other Cys residues are conserved among only mammalian AE2 polypeptides or (for 1224 only) not conserved. Consensus N-glycosylation sites are indicated in the third extracellular loop. b Comparison of mAE2 transmembrane domain Cys residues with the corresponding residues of hAE1. The mutagenic oligonucleotides and their encoded missense substitutions are presented in Table S1. The topographic model is derived from Zhu et al. (62)

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cRNA expression in Xenopus oocytes

Capped cRNA was transcribed from linearized cDNA templates with Megascript T7 RNA polymerase kit (Ambion, Austin, TX) and purified with an RNeasy mini-kit (Qiagen, Germantown, MD). cRNA concentration (A 260) was measured by Nanodrop spectrometer (ThermoFisher, Waltham, MA), and integrity was confirmed by formaldehyde agarose gel electrophoresis. Mature female Xenopus laevis frogs (Department of Systems Biology, Harvard Medical School) were subjected to partial ovariectomy under hypothermic tricaine anesthesia following protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center. Stage V–VI oocytes were prepared by overnight incubation of ovarian fragments in MBS with 1.5 mg/ml collagenase B (Alfa Aesar, Ward Hills, MA), followed by a 20 min rinse in Ca2+-free MBS, with subsequent manual selection and defolliculation as needed. Oocytes were injected on the same day with cRNA (0.5–50 ng as indicated), and maintained 72 h at 17.5 °C in MBS containing 10 μg/ml gentamicin until used for experiments. As uninjected and water-injected oocytes did not differ in anion transport at 72 h post-injection (data not shown), uninjected oocytes were used as controls for the current experiments.

Isotopic influx experiments

Unidirectional 36Cl− influx studies were carried out for periods of 30 min in 148 μl ND-96 and 2 μl of carrier-free 260 mM Na36Cl− (0.25 μCi), resulting in total bath [Cl−] of 103.6 mM. In experiments testing the effect of NH4 + on 36Cl− influx, ND-96 was substituted with ND-70 plus 26 mM NH4Cl. In experiments testing the effect of hypertonicity on 36Cl− influx, ND-96 (212 mOsm) was supplemented with mannitol to achieve calculated osmolarities of 280 or 400 mOsm, maintaining constant [Cl−]. In all 36Cl− influx experiments the carrier solution was supplemented with 10 μM bumetanide to impair Cl− influx by native oocyte NKCC1.

Unidirectional 35SO4 2− influx studies were carried out for periods of 30 min in a bath containing 148 μl of a carrier solution consisting of (in mM) 20 Na2SO4, 76 Na cyclamate, 2 K glutamate, 1.8 Ca glutamate, 1 Mg glutamate, and 2 μl of carrier-free 35SO4 2− (2 μCi, 47 nM) [18]. pH 5.0 solutions were buffered with 5 mM MES, and pH 8.0 solutions were buffered with 5 mM HEPES. Unidirectional 14C oxalate influx studies were carried out for 30 min periods in 148 μl of nominally Ca2+- and Mg2+-free influx medium containing (in mM) 96 mM Na cyclamate, 2 K glutamate, with 2 μl carrier-free 100 mM 14C oxalate (0.75 μCi), resulting in a final 14C oxalate concentration of 1.3 mM. pH 5.0 solutions were buffered with 5 mM MES, and pH 8.0 solutions were buffered with 5 mM HEPES.

Influx experiments were terminated with four washes in ice-cold isotonic Na cyclamate solution (nominally Mg2+- and Ca2+-free for oxalate influx studies). Washed oocytes were individually lysed in 150 μl 2 % sodium dodecyl sulfate (SDS). Triplicate 10-μl aliquots of the influx solution were used to calculate specific acitivity of radiolabeled substrate anions. Oocyte anion uptake was calculated from cpm values of washed oocytes and from bath specific activity.

Isotopic efflux experiments

For unidirectional 36Cl− efflux studies, individual oocytes were injected with 50 nl of 260 mM Na36Cl (6.25 nCi, 20,000–24,000 cpm). Following a 5- to 10-min recovery period in Cl−-free solution (cyclamate), the efflux assay was initiated by transfer of individual oocytes to 6-ml borosilicate glass tubes, each containing 1 ml efflux solution. At intervals of 1 or 3 min, 0.95 ml of this efflux solution was removed for scintillation counting and replaced with an equal volume of fresh efflux solution. Following completion of the assay with a final efflux period either in Cl−-free cyclamate solution or in the presence of the inhibitor DIDS (200 μM unless otherwise noted), each oocyte was lysed in 150 μl of 2 % SDS. Samples were counted for 3–5 min such that the magnitude of 2SD was <5 % of the sample mean.

To vary pHi at constant pHo, oocytes were pre-exposed to 40 mM Na butyrate (substituting for Na cyclamate) for 30 min prior to initiation of an efflux experiment, producing intracellular acidification of 0.5 pH units [44]. Upon removal of bath butyrate (with substitution by Na cyclamate) during the course of the efflux experiment, pHi rapidly alkalinized back towards initial pHi (t 1/2 = 6 min), while pHo remained constant. 40 mM butyrate is neither an inhibitor nor a substrate of AE2 [43].

Efflux data was plotted as the natural logarithm (ln) of the quantity (% cpm remaining in the oocyte) vs. time. Efflux rate constants for 36Cl− were measured from linear fits to data from the last three time points sampled within each experimental period. For each experiment, uninjected oocytes from the same frog were subjected to parallel measurements with cRNA-injected oocytes. Oocytes, with <15 % of injected 36Cl− remaining at the end of the assay were excluded from analysis [35]. Extracellular [Cl−] ([Cl−]o)–response curves were fit by the following Michaelis–Menten-type equation with baseline offset (Sigmaplot 11.0):

$$ {{K}_{{{\rm{eff}}}}} = \frac{{{{y}_{0}} + {{V}_{{\max }}} \times [{\rm{anion}}]}}{{({{K}_{{1/2}}} + [{\rm{anion}}])}} $$
(1)

where k eff is the 36Cl− efflux rate constant, y 0 is the efflux rate constant in the absence of extracellular substrate anion (in the presence of nominally impermeant anions cyclamate and gluconate), V max represents the maximal value of k eff, and K 1/2 the [Cl−]o at which k eff is half-maximal.

The pHo sensitivity of AE2-mediated 36Cl− efflux at nearly constant pHi was measured and reproduced our previously reported data [44]. Individual oocytes were exposed sequentially to ND-96 at pHo 5.0, 6.0, 7.0, 8.0, and 8.5, followed by addition of DIDS (200 μM) at pHo 8.5. Rate constants measured at each pHo value for each oocyte were fit with the following first order logistic sigmoid equation,

$$ V = \left( {{{V}_{{\max }}} \times {{{10}}^{{ - K}}}} \right)/\left( {{{{10}}^{{ - K}}} + {{{10}}^{{ - X}}}} \right) + d $$
(2)

where V, V max are as defined above, exponent X is the pHo at which the efflux rate constant was measured, exponent K is pHo(50) (pHo at which V is half-maximal), and d is the efflux rate constant in the absence of substrate anion, with a value ≥0. Equation 2 allowed calculation (Microsoft Excel) of V max and pHo(50) values for each individual oocyte. Rate constants measured at each pHo value were normalized to that oocyte's V max (set at 100 %). Mean normalized data for mAE2 and for mAE2Cys-less were fit (Sigma Plot version 11.0) to Eq. 2.

All experimental conditions were tested in oocytes from at least two frogs.

Confocal immunofluorescence microscopy

Oocytes were injected with cRNA encoding wildtype (10 ng) or mutant (50 ng) mAE2 bearing the HA epitope (YPYDVPDYA) inserted between Glu-858 and Ala-859 in the third extracellular loop. Uninjected oocytes and oocytes injected with cRNA 3 days prior were fixed at 4 °C for 30 min in phosphate-buffered saline (PBS) containing 1.5 % paraformaldehyde (PFA), then washed three times in PBS supplemented with 0.002 % sodium azide, exposed to 1 % SDS in 1× PBS for 5 min, and then blocked in PBS with 1 % bovine serum albumin (BSA) and 0.05 % saponin for 1 h at 4 °C. Oocytes were then incubated 2 h at 4 °C with rabbit monoclonal anti-HA peptide (dilution 1:1,600; Cell Signaling, Danvers, MA), followed by three washes with 1 % PBS–BSA. Oocytes were then incubated for 2 h with Cy3-conjugated secondary donkey anti-rabbit Ig (dilution 1:1,600; Jackson Immunochemicals, West Grove, PA) and again thoroughly washed in PBS–BSA. Oocytes were aligned in uniform orientation along a plexiglass groove and sequentially imaged through the 10× objective of a Zeiss LSM510 laser scanning confocal microscope, using the 543-nm laser line at 512 × 512 resolution, at constant filter, gain, and pinhole settings.

Polypeptide abundance at or near each oocyte surface was estimated by quantitation of specific fluorescence intensity (FI) at the periphery of one quadrant of an equatorial focal plane (Image J v. 1.38, National Institutes of Health). The mean background FI of uninjected oocytes was subtracted from each single oocyte FI. Normalized means and standard errors were calculated for each group. Images of median intensity were selected from each group for presentation.

Statistics

Data are reported as mean ± SEM. Flux data were compared by Student's paired t-test, or as appropriate by ANOVA with Dunnett’s pairwise multiple comparison or Tukey’s post-hoc analysis (SigmaPlot 11.0). A p value <0.05 was interpreted as significant.

Results

Missense substitution of all transmembrane domain cysteine residues of mAE2 preserves DIDS-sensitive Cl−/Cl− exchange activity

The mAE2 transmembrane domain (defined to encompass the short cytoplasmic C-terminal tail of the protein) includes eight Cys residues (Fig. 1a), only three of which are conserved in hAE1 (Fig. 1b). To prepare a construct useful for future Cys-insertion structure–function studies, all eight Cys residues were mutated as described in the Methods section and Table S1. Xenopus oocytes expressing mAE2Cys-less retained the ability to mediate Cl− uptake sensitive to inhibition by DIDS (Fig. 2a), although at rates lower than exhibited by comparable oocytes injected with wildtype mAE2 cRNA. The Cl− uptake by oocytes expressing mAE2Cys-less represented Cl−/Cl− exchange, as evidenced by trans-anion-dependent 36Cl− efflux that was DIDS-sensitive (Fig. 2b,c). mAE2Cys-less(7/8) also retained these activities (data not shown).

Fig. 2
figure 2

Transmembrane domain cysteines are not essential for mAE2-mediated, DIDS-sensitive Cl−/Cl− exchange. a 36Cl− influx in the absence (−) or presence (+) of 500 μM DIDS was measured in uninjected oocytes (Uninj.) or in oocytes previously injected with 10 ng cRNA encoding wildtype mAE2 or mAE2Cys-less. Values are means ± SEM for (n) oocytes. *p < 0.05 vs. absence of DIDS. Similar results were observed in another experiment 34 additional oocytes (not shown). b Representative 36Cl− efflux traces of individual oocytes into sequential baths of Na cyclamate (Nacyc), NaCl (ND96) and ND96 + 500 μM DIDS (DIDS). c 36Cl− efflux rate constants of (n) uninjected oocytes or oocytes previously injected with 10 ng of cRNA encoding mAE2 or mAE2Cys-less, and subjected to the sequential bath changes of panel b. *p < 0.05 vs. mAE2; ^p < 0.05 vs. absence of DIDS; †p < 0.05 vs. presence of Cl−. d Cl− transport by mAE2Cys-less does not require the AE2 N-terminal cytoplasmic domain. 36Cl− influx into (n) uninjected oocytes or oocytes previously injected with 10 ng of cRNA encoding mAE2, mAE2Cys-less, mAE2 ΔN659, or mAE2Cys-less ΔN659. *p < 0.05 vs. corresponding Cys-replete polypeptides

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Anion exchange by mAE2Cys-less did not require the presence of the AE2 N-terminal cytoplasmic domain, as illustrated by 36Cl− uptake into oocytes expressing mAE2Cys-less ΔN659 (Fig. 2d) and found previously for mAE2 [43]. Absence of the N-terminal cytoplasmic domain diminished the difference in influx between polypeptides containing and lacking transmembrane domain Cys residues. mAE2Cys-less was expressed at or near the oocyte surface with near-normal abundance (Fig. 3), suggesting the possibility that the observed reduced transport activity might in part represent decreased turnover number for Cl−/Cl− exchange, rather than decreased surface abundance alone.

Fig. 3
figure 3

Absence of transmembrane domain cysteines does not prevent mAE2 expression at or near the oocyte surface. Representative median intensity confocal immunofluorescence images of uninjected oocytes or oocytes previously injected with cRNA encoding mAE2 (20 ng) or mAE2Cys-less (50 ng). The lower right panel shows mean normalized fluorescence intensities for (n) oocytes (p < 0.05 of each construct vs. uninjected oocytes (uninj.)

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Missense substitution of mAE2 transmembrane domain Cys residues maintains the K 1/2 for extracellular Cl− of Cl−/Cl− exchange

The mAE2-mediated unidirectional influx of 36Cl− exhibited a K 1/2 value for extracellular [Cl−] of 5.6 mM [22]. To measure K 1/2 based on efflux, we assessed the extracellular [Cl−] dependence of the 36Cl− efflux rate constant in individual mAE2-expressing oocytes, and found it to be 5.5 ± 0.9 mm (n = 15; Fig. 4a). This value was indistinguishable whether derived from a fit to data from all oocytes or as the mean of fits to single oocyte efflux data. mAE2Cys-less-mediated 36Cl−/Cl− exchange exhibited a K 1/2 for extracellular [Cl−] of 3.7 + 0.9 mM (n = 9; p = 0.53 vs. mAE2; Fig. 4b). Thus, the absence of transmembrane domain Cys residues does not change the apparent extracellular Cl− affinity of mAE2.

Fig. 4
figure 4

K1/2(Cl− o) of mAE2-mediated 36Cl− efflux is minimally affected by absence of transmembrane domain Cys residues. a Extracellular [Cl−]-dependence of normalized rate constants of mAE2-mediated 36Cl− efflux (n = 15, oocytes previously injected with 2 ng cRNA). b Extracellular [Cl−]-dependence of normalized rate constants of mAE2Cys-less-mediated 36Cl− efflux (n = 11, oocytes previously injected with 10 ng cRNA). Na cyclamate was substituted mole for mole by NaCl to achieve bath [Cl−] of 0.3, 1, 3, 10 and 60 mM. Mean 36Cl− efflux rate constants of uninjected oocytes (n = 5) were subtracted from efflux rate constants of each groups, and data was fit as described in Methods. The K 1/2 values did not differ between groups (p = 0.53)

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Missense substitution of mAE2 transmembrane domain Cys residues preserves pHi sensitivity but alkaline-shifts pHo sensitivity of Cl−/Cl− exchange activity

DIDS-sensitive Cl−/Cl− exchange by mAE2-expressing Xenopus oocytes is inhibited by intracellular acidification to pHi ~6.7 by replacement of pre-equilibrated 40 mM butyrate with impermeant anion [44]. This property of anion exchange inhibition by acidic intracellular pH is maintained in the absence of transmembrane domain Cys residues (Fig. 5a,b). Extracellular acidification independently inhibits mAE2 (Fig. 5c) in the presence of minimal [43] or no intracellular acidification [44]. Comparison of this inhibition in oocytes expressing mAE2 or mAE2Cys-less revealed an alkaline-shifted pHo(50) value for inhibition by extracellular protons in the absence of transmembrane domain Cys residues, from pH 6.85 ± 0.06 (n = 14) for mAE2 to a minimum value of pH 7.56 ± 0.13 for mAE2Cys-less (Fig. 5d; calculated from individual oocyte pHo vs. activity curves). The mean values calculated from individual oocye pHo(50) values differed to a similar degree, 6.94 ± 0.04 for mAE2 and at least 7.47 ± 0.08 for mAE2Cys-less (p < 0.001). The true pHo(50) value for mAE2Cys-less may be higher than these estimates, since the normalized pH vs. activity curves did not reach definitive plateau values.

Fig. 5
figure 5

Absence of transmembrane domain cysteines does not grossly alter mAE2 pHi sensitivity, but shifts mAE2 pHo sensitivity curve to more alkaline pH values. a 36Cl− efflux traces from representative individual oocytes previously uninjected (open squares) or injected with cRNA encoding mAE2 (1 ng; open circles) or mAE2Cys-less (10 ng; filled squares), and subjected to sequential exposure to baths containing ND-56 plus 40 mM Na butyrate, then ND-56 plus 40 mM Na cyclamate (0 butyrate), then ND-56 plus 40 mM Na cyclamate with added 200 μM DIDS (DIDS). b Mean 36Cl− efflux rate constants of (n) oocytes subjected to the experimental conditions in panel a. *,^ p < 0.05 vs. cyclamate (0 butyrate). Values are means ± SEM. c 36Cl− efflux traces from representative individual oocytes previously uninjected (open squares) or injected with cRNA encoding mAE2 (1 ng; open circles) or mAE2Cys-less (10 ng; filled squares), and subjected to sequential exposure to baths containing ND-96 at the indicated pH values, followed by exposure to 200 μM DIDS (DIDS). d Mean normalized 36Cl− efflux rate constants (±SEM) of oocytes previously injected with cRNA encoding mAE2 WT (0.5–2 ng, n = 14) or mAE2Cys-less (5–20 ng, n = 23) as a function of bath pH values, measured as in panel c. All values reflect subtraction of the mean 36Cl− efflux rate constant of uninjected oocytes measured during the same experiments (n = 9). Mean pHo(50) values were 6.94 for mAE2 and a minimum value of 7.47 for mAE2Cys-less (p < 0.001). (Note that the mAE2Cys-less pHo vs. activity curve did not reach a definitive plateau)

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Missense substitution of mAE2 transmembrane domain Cys residues preserves stimulation of AE2-mediated Cl−/Cl− exchange activity by hypertonicity and by NH4 +

mAE2-mediated Cl−/Cl− exchange activity in Xenopus oocytes is activated by bath hypertonicity [23], which slightly alkalinizes oocyte pHi through activation of endogenous NHE1-like activity [17]. Exposure of oocytes expressing mAE2Cys-less exhibited similar activation by an increase in nominal bath tonicity from an isotonic value of 212–280 mOsm, and with further increased activity in the presence of 400 mOsm bath (Fig. 6a).

Fig. 6
figure 6

Absence of transmembrane domain cysteines does not grossly alter mAE2 activation by hypertonicity or by NH4 +. a 36Cl− influx into (n) uninjected oocytes or oocytes previously injected with 10 ng of cRNA encoding mAE2Cys-less in bath solutions of indicated osmolarity. ND-96 osmolarity is 212 mOsm. *p < 0.05 vs. 212 mOsm; **p < 0.05 vs. 212 or 280 mOsm. b 36Cl− influx into (n) uninjected oocytes or oocytes previously injected with 10 ng of cRNA encoding mAE2Cys-less in baths of ND96 (−) or ND-70 plus 26 mM NH4Cl (+). *p < 0.001 vs. ND-96. Values are means ± SEM

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mAE2-mediated Cl−/Cl− exchange activity also exhibits unusual activation by addition of bath NH4Cl, despite the atypical intracellular acidification produced in Xenopus oocytes by extracellular NH4 + addition [21]. As shown in Fig. 6b, mAE2Cys-less preserves this ability to respond to the acidifying stimulus of extracellular NH4 + addition with activation of Cl−/Cl− exchange.

Missense substitution of mAE2 transmembrane domain Cys residues preserves reversed pH sensitivity of SO4 2− transport by the E1007Q mutant of AE2

Human erythrocytes exhibit AE1-mediated H+/SO4 2− cotransport in exchange for Cl− [33]. Human AE1 E681 [25] and the corresponding mouse AE1 E699 [8] are critical residues for this and other anion exchange modes. The mAE1 E699Q mutation also reverses the normal stimulation of SO4 2− transport by extracellular protons, such that SO4 2− transport is stimulated by alkaline pHo rather than by acidic pHo [9].

The pH dependence of SO4 2− transport by plasmalemmal AE2 in Xenopus oocytes has not been reported. Figure 7a shows that acidic pHo indeed activates mAE2-mediated SO4 2− uptake, as was previously shown for SO4 2− transport by AE2 in HEK-293 cells [40] and with AE1-mediated transport of SO4 2− in oocytes [9]. mAE2Cys-less exhibits ~4-fold lower rates of SO4 2− transport but preserves some stimulation by acidic pHo. Introduction into mAE2 of the missense mutation E1007Q, corresponding to mAE1 E699Q, reverses pHo dependence of AE2-mediated SO4 2− transport such that alkaline pHo stimulates it. The absence of transmembrane domain Cys residues similarly decreases SO4 2− transport rates by mAE2 E1007Q, but preserves the “reversed” stimulation by alkaline pHo (Fig. 7a).

Fig. 7
figure 7

Absence of mAE2 transmembrane domain cysteines does not prevent acidic pHo activation of SO4 2− transport by mAE2 E1007Q, but abolishes H+-stimulated oxalate transport by mAE2. a 35SO4 2− influx into (n) uninjected oocytes or oocytes previously injected with 50 ng cRNA encoding mAE2 or mAE2Cys-less without or with the missense substitution mutation E1007Q, measured in baths of pH 5.0 (black bars) or 8.0 (white bars). *p < 0.001 vs. pH 5; = **p < 0.005 vs. pH 5; §p < 0.001 vs. pH 5; †p < 0.01 vs. pH 5. b 14C oxalate influx into (n) uninjected oocytes or oocytes previously injected with 50 ng cRNA encoding mAE2 or mAE2Cys-less. *p < 0.001 vs. pH 5. Values are means ± SEM

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AE1-mediated oxalate transport in erythrocytes is also activated by extracellular protons, consistent with a mechanism of H+/oxalate vs. Cl− exchange [26]. Figure 7b shows that mAE2-mediated oxalate uptake is also strongly stimulated by acidic pHo. However, although oxalate uptake by mAE2Cys-less-expressing oocytes was proportionately reduced to the same degree as was SO4 2− uptake by mAE2 (Fig. 7a), activation of oxalate uptake by acidic pHo was not evident in oocytes expressing mAE2Cys-less.

Missense substitutions at mAE2 E1007 produce less severe impairment of Cl− transport than do corresponding substitutions at mAE1 E699, but loss-of-function is exacerbated by absence of AE2 transmembrane domain Cys residues.

Xenopus oocytes expressing mAE1 E699Q or any of several other missense substitutions at the same position exhibited complete loss of Cl− transport, strengthening the contention that E699 constitutes part of the AE1 anion translocation pathway [8]. Figure 8 shows that the corresponding mAE2 missense mutation E1007Q produced less than complete loss of Cl− transport function that can be partially overcome by increasing the mass of injected cRNA. We therefore tested the effect of other substitutions at residue 1007. Substitution of Cys or His also produced less than complete loss of Cl− transport, again partially overcome by increasing the mass of injected cRNA. mAE2 E1007K exhibited the greatest loss of Cl− transport function. Absence of AE2 transmembrane domain Cys residues exacerbated the reduced Cl− influx phenotype of each E1007 substitution tested (Fig. 8).

Fig. 8
figure 8

Substitution mutations at mAE2 E1007 produce partial loss of Cl− transport, exacerbated by absence of transmembrane domain Cys residues. Role in 36Cl− influx of amino acid substitutions at mAE2a position E1007 in (n) oocytes previously injected with 10 ng (black bars) or 50 ng (white bars) of mAE2 or mAE2Cys-less cRNAs, or their indicated E1007 missense substitution mutants. 36Cl− influx into uninjected oocytes is indicated by the hatched bar at the far right. Values are means ± SEM. *p < 0.05 vs. mAE2 10 ng; **p < 0.001 vs. mAE2 10 ng; ^p < 0.05 vs. mAE2Cys-less 10 ng

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Discussion

In this work we have shown that DIDS-sensitive Cl−/Cl− exchange mediated by mAE2Cys-less retained nearly all native regulatory properties of wildtype mAE2, but with three notable differences. First, in the absence of transmembrane domain Cys residues the AE2-mediated Cl−/Cl− exchange rate was reduced ~50 %, in the context of near-normal polypeptide surface abundance. Second, although both wildtype AE2 and AE2Cys-less were inhibited by acidic intracellular pH, the pHo(50) value of mAE2Cys-less for inhibition by extracellular protons was alkaline-shifted >0.6–0.7 pH units. Third, activation of mAE2-mediated oxalate transport by acidic pHo was abolished in mAE2Cys-less.

Effects of Cysteine substitution on membrane transport proteins

The prototype for cysteine scanning mutagenesis studies as “an approach to membrane transport protein structure without crystals” has been the LacY H+/lactate cotransporter of Escherichia. coli [14, 41]. After demonstration that Cys residues are not essential to LacY transport function, Cys substitution was applied to nearly every residue of Cys-less LacY. Investigation of these mutants through a range of biochemical and biophysical approaches has yielded an extensive set of results consistent with insights available from crystal structures [1, 7].

With a similar approach to the study of hAE1 in mind, Casey et al. substituted all five Cys residues of hAE1 (three in the transmembrane domain) with serine residues [6]. At that time, the role of cysteine residues in AE1 function remained unclear. Previous studies in human red cells had shown hAE1 to be stably palmitoylated on C843 [36], but mutation of the corresponding C841 in mAE1 to Ser or Met did not impair stilbene-sensitive anion exchange in Xenopus oocytes [27]. Moreover, C843 was not palmitoylated in heterologous hAE1 overexpressed in HEK293 or COS7 cells. Indeed, in these cell types, overexpressed hAE1 C843A underwent apparently normal N-glycosylation and trafficked normally to the cell surface [11].

More recently, combined distal renal tubular acidosis and hereditary spherocytosis was shown to be associated with the hAE1 missense substitution C479W, in compound heterozygosity with recessive mutation G701D. The mutant C479W polypeptide was retained in the endoplasmic reticulum of polarized MDCK cells, and contributed to reduced AE1 abundance in red cells [12]. Mutations to Cys of hAE1 residues R490, R518, and R808 also cause hereditary spherocytosis. In addition, the hAE1 mutation R730C causes stomatocytosis [45], and hAE1 mutation R589C causes distal renal tubular acidosis [42]. hAE1 Cys residues 201 and 317 of the cytoplasmic domain and Cys 843 of the transmembrane domain have (more speculatively) also been proposed to provide red cell efflux shuttle pathways for selenious acid and for nitric oxide [20], pathways of undefined relationship to the anion translocation pathway utilized by the anion exchange mechanism.

Cys-less AE1 retained about half of wildtype Cl−/HCO3 − exchange activity when expressed in HEK-293 cells, but normalization of activity to surface abundance suggested that exchange turnover number was unchanged in the absence of AE1 Cys residues [6, 49]. In contrast, Cl−/HCO3 − exchange activity in Xenopus oocytes expressing Cys-less hAE1 was later reported to be undetectable due to surface expression below the detection threshold [5]. More dramatically still, missense substitution of all seven Cys residues of the trout AE1 transmembrane domain led to absence of detectable tAE1 polypeptide in cRNA-injected oocytes, with consequent loss of tAE1-mediated Cl− conductance [30]. Mutation of the single C462 of tAE1 sufficed to lead to intracellular retention of polypeptide, similar to the phenotype of the corresponding dRTA mutation C479W of hAE1. Moreover, two single-Cys mutants of tAE1 (in positions at which mammalian AE1 lacks Cys residues) exhibited proportionately greater loss of the tAE1-associated Cl− conductance than of Cl−/HCO3 − exchange [30].

SLC4A4/NBCe1 was shown to be an ecto-disulfide-linked dimer [28], very much unlike the noncovalent SLC4 family homodimers, AE1 and AE2. Cys scan mutations of SLC4A4/NBC31 have not been performed in oocytes, but that performed in HEK-293 cells has predicted a structure for the C-terminal half of the NBCe1 transmembrane domain quite different from that of AE1 [61]. The functional capacity of Cys-less SLC4A4/NBCe1 has not been reported, although the Cys-less polypeptide has been expressed. SLC4A4/NBCe1 was functional in the absence of five of its 15 Cys residues (five of its 12 transmembrane domain Cys residues as defined as in this paper on AE2), but rates were not directly compared to those of wildtype NBCe1 [62]. The above results for Cys substitution and Cys scan experiments in NBCe1 show that results obtained with one SLC4 protein cannot necessarily be extrapolated with confidence to another SLC4 protein, even when closely related.

Indeed, the functional importance of transmembrane domain Cys residues can vary according to gene product, species of origin, and expression system. For example, whereas engineered removal of all Cys residues from nucleobase transporter hENT1 led to complete loss-of-function [53], Cys-less forms of glucose transporter GLUT1 [34], organic cation transporter MATE1 [55], glutamate transporter EAAT1 [39], and the H+-coupled folate transporter [57] retained all or nearly all functional activity. More subtle phenotypes of Cys-less proteins as compared to their corresponding wildtype polypeptides included decreased plasmalemmal trafficking and single channel conductance in Cys-less CFTR [19], loss of 5-fluoro-maleimide inhibition of otherwise intact channel activity of Cys-less mitochondrial porin VDAC [4], and increased susceptibility to loss-of-function by otherwise inactive second-site mutations in the H+-coupled folate transporter [57].

Absence of transmembrane Cys residues preserves many normal functions of mAE2

The absence of transmembrane domain Cys residues decreases rates of AE2-mediated Cl−/Cl− exchange in Xenopus oocytes from 40 % to 75 % (Fig. 2), in the context of near-normal polypeptide surface abundance (Fig. 3). The removal from mAE2Cys-less of most of its N-terminal cytoplasmic domain does not further decrease transport activity, and may even stimulate it (Fig. 3). The apparent affinity of AE2 for extracellular Cl− is not significantly altered in the absence or transmembrane Cys residues (Fig. 4). The ability of AE2 to be activated by hypertonicity or by isotonic NH4Cl is also preserved in the absence of transmembrane domain Cys residues (Fig. 6). In addition, the inhibition of AE2 by intracellular protons and removal of inhibition during recovery from intracellular acidosis (by exposure to and withdrawal from bath butyrate) is preserved apparently intact in the absence of transmembrane domain Cys residues (Fig. 5a,b). The integrity of these regulatory properties in mAE2Cys-less suggests that transmembrane domain Cys substitution does not result in large-scale conformational change.

AE2 sensitivity to pHo is altered by absence of transmembrane domain cysteines

The pHo(50) value of mAE2Cys-less is at least 0.6–0.7 pH units higher than that of mAE2 (Fig. 5c,d). The absence of the first 199 N-terminal amino acids encoded by the naturally occurring variant transcript AE2c1, abundantly expressed only in gastric mucosa, produces an alkaline shift of comparable magnitude in the pHo(50) of mAE2 [29]. The physiological rationale for expression of the Ae2c1 transcript may be to extend the extracellular (interstitial space) pH range across which gastric parietal cell AE2 remains fully active during the alkaline tide generated during maximal stimulation of lumenal H+ secretion. However, the mechanism by which both absence of the first 199 N-terminal amino acids of AE2a and absence of transmembrane domain Cys residues increase the apparent affinity of extracellular proton-associated inhibition of AE2-mediated anion exchange activity remains obscure, and the structure of neither AE2 region is known. The slow onset and progression of AE2-deficiency phenotypes in AE2 a,b−/− mice [24, 32, 37, 38] compared to the complete AE2 knockout [16] suggests compensatory ectopic expression of AE2c1 that prevents the perinatal death associated with complete AE2 deficiency [16]. The alkaline-shifted pHo sensitivity of AE2c1 in these processes has not been examined. (See Fig. 1a of ref. 28 for a comparison of the N-terminal amino acid sequences of the five variant transcripts of mAE2.)

Missense mutations on either intracellular or extracellular faces of AE2 can regulate anion transport sensitivity to both cis and trans protons, suggesting conformational responses in both domains in response to changes in pH that extend beyond the site of substitution [46, 48]. In particular, mAE2 pHo(50) was alkaline-shifted to 7.6 in the mAE2 mutant I1079C, located in the first re-entrant loop of the transmembrane domain [47]. In addition, mAE2 pHo(50) values were slightly alkaline-shifted to ~7.2 in mutants K889A, H1144A, H1145A, and double mutants H1144/H1145 and A1041/A1053 [46, 48]. The mechanistic relationship between the alkaline-shifted pHo(50) caused by these mutations and that caused by complete transmembrane domain Cys substitution remains unknown.

Absence of transmembrane domain Cys residues differentially alters regulation of SO4 2− and oxalate exchange by pHo

The effect of the absence of transmembrane Cys residues on the pH sensitivity of Cl− transport led us to examine the effects on pH-sensitive transport of divalent anions. We showed previously that the mAE1-mediated electroneutral exchange of Cl− for the H+/SO4 2− ion pair, activated by acidic pHo, is converted by the mAE1 E699Q mutation to electrogenic SO4 2−/Cl− exchange [8] activated by alkaline pHo [9]. Activation by acidic pHo of AE2-mediated SO4 2− uptake was preserved in the absence of transmembrane domain Cys residues, as was activation by alkaline pHo of AE2 E1007Q-mediated SO4 2− uptake (Fig. 7a). The absence of transmembrane domain cysteines exacerbated reduction in transport function by all E1007 missense substitutions tested for mAE2 (Fig. 8). In addition to reducing the rate of oxalate transport, the absence of transmembrane domain cysteines also resulted in loss of activation by acidic pHo of AE2-mediated oxalate uptake (Fig. 7b). The reasons for the differing responses of pH-sensitive transport of SO4 2− and oxalate are not clear, but may relate to ionic radius and/or to variable oxalate geometry in free and liganded states [13].

It is of interest to note that mAE2 mutation E1007Q exhibits a less severe oocyte Cl− transport phenotype (Fig. 8) than does the corresponding mAE1 E699Q mutant [8]. However, other missense substitutions at mAE2 E1007 exhibit total loss of Cl− transport, suggesting that this Glu residue is very likely situated along the anion translocation pathway in both AE1 and AE2. mAE1 E699 and mAE2 E1007 are believed to reside at or near the cytoplasmic end of transmembrane helix 8. The less severe mAE2 phenotype may reflect local differences in their steric and electrostatic environments, as depicted in Fig. S1. mAE2 E1007 is bracketed by M1006 and T1008, whereas hAE1 E681 is bracketed by L680 and S682. (This region of mAE1 is identical in amino acid sequence.) Integration of these sequence differences into a context of neighboring transmembrane helices and intracellular loops remains elusive, due to the inadequately resolved transmembrane domain structure of any SLC4 polypeptide.

Prospects

The availability of mAE2Cys-less will allow directed cysteine scanning mutagenesis throughout the AE2 transmembrane domain in the absence of endogenous Cys residues. Such single Cys insertions will be important to test the predictive utility of electron microscopic and crystal structures of increasing resolution. Most regulatory properties of AE2Cys-less will be amenable to study in this fashion, including the inhibitory regulation by intracellular protons. However, the inhibitory regulation of AE2 by extracellular protons is alkali-shifted by the absence of transmembrane domain Cys residues. Targeted reintroduction of individual or multiple native Cys residues may identify those of greatest importance in setting pHo(50) of mAe2. In the meantime, this difference in regulation will need to be taken into account when interpreting the results of single Cys substitutions in the AE2Cys-less background. An understanding of the similarly shifted pHo(50) values of AE2c1 and AE2Cys-less may emerge from atomic-level resolution structures of AE1 or another member of the SLC4 superfamily. Such understanding should serve to enhance models of cellular homeostasis that balance the sometimes conflicting demands on Cl− transport in the service of regulating intracellular and systemic pH and in volume regulation.