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CELLULAR METABOLISM
1Molecular and Vascular Medicine Unit and Renal Unit, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts; and 2Burdon Sanderson Cardiac Science Centre, University Laboratory of Physiology, University of Oxford, Oxford, United Kingdom
Submitted 12 May 2006 ; accepted in final form 17 September 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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pH regulation; histidine residues; Cl–/HCO3– exchange
500 amino acids (aa) with a short COOH-terminal cytoplasmic tail. The TMD is preceded by a less extensively conserved 400-aa (AE1) to 700-aa (AE2 and AE3) NH2-terminal cytoplasmic tail (1). The COOH-terminal TMD can mediate anion exchange in the absence of the NH2-terminal cytoplasmic domain (12, 21, 37), but the physiological regulation of AE2 transport activity by pH requires the NH2-terminal cytoplasmic domain. The anion exchanger polypeptides are expressed in tissue- and cell-specific patterns and differ in their acute response to a change of pH. Structural elements contributing to this pH sensitivity have been localized to the COOH TMD and NH2-terminal cytoplasmic domain (37, 46). AE1-mediated Cl–/HCO3– and Cl–/Cl– exchange in erythrocytes (10) and Xenopus oocytes exhibits a broad pH vs. activity profile (17, 46). In contrast, Na+-independent Cl–/HCO3– exchange in nonerythroid cells in culture is often characterized by strong pH sensitivity (24, 35) and is most likely mediated by AE2 and AE3 gene products and/or by polypeptides of the SLC26 gene family. A high sensitivity to intracellular pH (pHi) and/or extracellular pH (pHo) has been observed for recombinant AE2 and AE3 expressed in tissue culture cells (19, 23) and Xenopus oocytes (37, 46). AE2–/– mice are achlorhydric and fail to survive weaning (11). AE3–/– mice display enhanced susceptibility to pharmacologically induced seizures (14).
Structure-function studies have localized to the AE2 TMD a putative "pH sensor" that confers sensitivity of the anion transporter to changes in pHo and pHi (37, 46). The pHo at which AE2 activity is half-maximal [pHo(50)] is 6.9. Truncation of the NH2-terminal cytoplasmic domain acid shifts pHo(50) by 0.7 pH unit and abolishes pHi sensitivity (37). We have defined two noncontiguous regions of the NH2-terminal cytoplasmic domain, the highly conserved aa 336–360 and the less well conserved aa 391–410, in which mutations alter or abolish pH sensitivity (39). However, residues of the AE2 TMD whose mutation alters AE2 pH sensitivity have not been reported, and the molecular identity of the pH sensor(s) in the AE2 COOH-terminal TMD remains unknown.
With their imidazole side chain pKa of 6.0 as the free amino acid, AE2 histidine (His) residues are strong a priori candidate pH sensors within the TMD (29). In contrast to the established role of His residues in conferring pH sensitivity on various ion channels (5, 6), the potential role of TMD His residues in the regulation of acid/base transporters is less well understood. For example, TMD His residues are not essential for cation transport through the ubiquitous eukaryotic Na+/H+ exchanger NHE1, but their mutation alters the inhibitory potency of amiloride (42). In contrast, mutation of a single His residue in the prokaryotic Sod2 Na+/H+ exchanger of S. pombe impairs proton translocation (43). Juxtamembranous His residues of the NHE3 COOH-terminal cytoplasmic domain have also been implicated in sustaining basal transport activity and its regulation by pHi (4), but a role for His residues in the TMD has not been reported. Four of the five TMD His residues in mouse AE1 are essential for basal transport activity (13, 32), and, although all five are conserved among the eight His residues of the mouse AE2 TMD, the influence of those His residues on AE2 pH sensitivity has not been studied.
In the present work, we have investigated the role of TMD His residues in AE2-mediated Cl–/anion exchange and its regulation by pHo and pHi. Through functional assay of AE2 polypeptides into which we have introduced mutations of individual and multiple His residues, we have determined their contributions to basal anion flux and polypeptide accumulation and to the pH sensitivity of AE2-mediated anion transport.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of AE2 His mutants.
Murine AE2a encoded in plasmid p
X (46) was used as template for PCR. AE2a His substitution mutants were constructed by a four-primer PCR method as described elsewhere (15, 46). Integrity of PCR products and ligation junctions was confirmed by DNA sequencing of both strands. The AE2 mutant H846A/H849A constructed by the same method was cut with StuI and BsrGI and then ligated into the appropriate AE2 His mutant backbone (H1144Y, H1145A, or H1160A) to generate three-His mutants. The five- or six-His mutants were constructed from the appropriate three-His mutant templates by four-primer PCR or ligation of StuI/BsrGI insert fragments containing the desired His mutations into the similarly restricted three-His mutant backbone. The wild-type AE2a-green fluorescent protein (GFP) fusion protein with a COOH-terminal GFP was constructed by four-primer PCR. COOH-terminal GFP fusions of AE2 mutants were then generated by ligation of complementary restriction fragments. Oligonucleotide primers were obtained from Biosynthesis (Woodlands, TX); primer sequences are available on request.
cRNA expression in Xenopus oocytes. Mature female Xenopus (Xenopus One, Madison, WI) were maintained and subjected to partial ovariectomy as described (46). Stage V–VI oocytes were manually defolliculated after incubation of ovarian fragments with 2 mg/ml collagenase A (Boehringer Mannheim, Indianapolis, IN) for 60 min in ND-96 solution containing 50 ng/ml gentamicin and 2.5 mM sodium pyruvate. Oocytes were injected on the same day with cRNA or water in a volume of 50 nl. Capped cRNA was transcribed from linearized cDNA templates with the T7 MEGAscript kit (Ambion, Austin, TX) and resuspended in diethylpyrocarbonate (DEPC)-treated water. RNA integrity was confirmed by agarose gel electrophoresis in formaldehyde, and RNA concentration was estimated by absorbance at 260 nm. cRNA-injected oocytes were then maintained for 2–6 days at 19°C until they were used for functional assay (see below).
36Cl– efflux assay. Individual oocytes in Cl–-free ND-96 solution [in mM: 96 sodium isethionate, 2 potassium gluconate, 1.8 calcium gluconate, 1 magnesium gluconate, and 5 HEPES (pH 7.4)] were injected with 50 nl of 260 mM Na36Cl (10,000–12,000 cpm). After 5–10 min of recovery in the same medium, the efflux assay was initiated by transfer of individual oocytes to 1 ml of ND-96 efflux solution containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES in 6-ml borosilicate glass tubes. At 3-min intervals, 0.95 ml of this efflux solution was removed for scintillation counting and replaced with an equal volume of fresh efflux solution. After completion of the assay with a final efflux period in the presence of the anion transport inhibitor DIDS (200 µM), each oocyte was lysed in 100 µl of 2% SDS. 36Cl– was quantitated as counts per minute (cpm) over 3–5 min, such that 2 SDs <5% of the sample mean.
Experimental data were plotted as ln (%cpm remaining in the oocyte) vs. time. 36Cl– efflux rate constants were measured from linear fits to data from the last three time points sampled for each experimental condition. All single time-point values for 36Cl– efflux from AE2 cRNA-injected oocytes into Cl–-containing medium exceeded 150 cpm. Efflux values for water-injected oocytes (30–90 cpm) were indistinguishable from those for AE2 cRNA-injected oocytes in the presence of 200 µM DIDS and exceeded machine background (10–25 cpm). Within each experiment, water- and AE2 cRNA-injected oocytes from the same frog were subjected to parallel measurements. On each experimental day, bath Cl–-dependent 36Cl– efflux activity of the tested mutant AE2 polypeptides was compared at pHo 7.4 with wild-type AE2 activity ("basal anion exchange activity"). AE2 mutants exhibiting low or absent transport activity at pHo 7.4 are described in RESULTS as "low-expression" polypeptides. Each AE2 mutant was tested in oocytes from at least two, and usually three frogs (efflux data are summarized in Supplemental Table 1 in the online version of this article). The weak-acid salt sodium butyrate was added to flux media as an equimolar substitution for NaCl. Some oocytes were treated for the indicated times with 0.5 or 5.0 mM DEPC, and 10 or 50 mM hydroxylamine was added in several experiments after removal of DEPC.
pHo dependence of anion exchanger-mediated 36Cl– efflux.
Individual oocytes initially maintained in Cl–-free solution at pHo 7.4 were exposed sequentially to ND-96 buffered to pH 5.0 or 6.0 with 5 mM MES and then to ND-96 buffered to pH 7.0, 8.0, or 8.5 with 5 mM HEPES before addition of 200 µM DIDS to terminate the assay and confirm pharmacospecificiy of the 36Cl– efflux (32, 37). Rate constants measured at each pHo in individual oocytes expressing wild-type AE2 or the AE2 mutant were fit (Sigma Plot) to the following first-order logistic sigmoid equation
 | (1) |
is the AE2-mediated Cl– efflux rate constant, Vmax is the maximum AE2-mediated Cl– efflux rate constant, x is pHo at which the rate constant was measured, K is pHo(50) (i.e., pHo at which is half-maximal), and d is the y-axis point of intersection. Any value of d > 0 leads to an underestimate of the degree of acid shift of pHo(50) for the particular AE2 mutant compared with the wild-type AE2 pHo(50). Rate constants for each mutant were normalized to the fit parameter Vmax calculated for each individual oocyte (100%), and the normalized data were fit to Eq. 1. The pHo dependence of wild-type AE2-mediated 36Cl– efflux did not differ when the experiment was performed with the order of pHo change reversed or randomized (38). pHo dependence was also unchanged when pHi was clamped during pHo variation by simultaneous alteration of butyrate concentration in the bath (38) [see Supplemental Table 1 for pHo(50) data for wild-type and mutant AE2 polypeptides].
pHi dependence of AE-mediated 36Cl– efflux.
pHi was varied at constant pHo by preincubation of 36Cl–-injected oocytes for 30 min before the start of the experiment in pH 7.4 Cl–-free solution containing 40 mM sodium butyrate. Under these conditions, pHi falls by 0.5 pH unit. 36Cl– efflux was initiated by transfer of oocytes to Cl–-containing solution in the continued presence of butyrate. The oocytes were then transferred to efflux medium containing Cl– but lacking weak acid. In these conditions, pHi increases 0.50 ± 0.03 unit after butyrate removal, with a time constant of 6 min, and butyrate is neither an inhibitor of nor a substrate for AE2 (37). 36Cl– efflux rate constants were normalized as the ratio of the efflux activity in the presence of 40 mM butyrate to that in the absence of butyrate at constant pHo 7.4 (see Supplemental Table 1 for summary of normalized 36Cl– efflux rate constants for wild-type and mutant AE2 polypeptides).
Immunoblot analysis of mouse AE2 polypeptide in Xenopus oocytes. Ten oocytes previously injected with a single cRNA were suspended at 4°C in oocyte lysis buffer (10 µl/oocyte) containing 1% Triton X-100, Complete protease inhibitor (Roche Diagnostics, Indianapolis, IN), 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA. After the sample was shaken vigorously for 30 min at 4°C, the extract was centrifuged for 10 min at 4°C in a microcentrifuge. Clarified total oocyte lysate was fractionated by SDS-PAGE (8% gels), transferred to nitrocellulose, developed with affinity-purified rabbit polyclonal antibody to mouse AE2 COOH-terminal aa 1224–1237 (46), and visualized on Kodak XB-1 film by chemiluminescence (Perkin Elmer Life Sciences, Boston, MA).
Confocal fluorescence microscopy. At least 10 oocytes expressing each GFP fusion construct to be tested were fixed overnight in PBS containing 3% paraformaldehyde, washed three times in PBS, incubated overnight in 30% sucrose in PBS, and then mounted in OCT compound and frozen in liquid N2. Cryosections (20 µm) were imaged with a laser scanning confocal microscope (model MRC1024, Bio-Rad). Representative sections imaged at uniform laser intensity and filter settings were compiled in Microsoft PowerPoint.
Statistical analysis. Values are means ± SE. Values for individual mutants and multiple groups of mutants were compared with that of wild-type AE2 by Dunnett's two-way t-test. The level of significance was taken as P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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85% of the initial rate on return to pHo 7.4. In the presence of 5 mM DEPC at pH 6.0, AE2-mediated 36Cl– efflux was inhibited by 98% and recovered to only 20% of the initial rate after pHo was restored to 7.4 (Fig. 1B).
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20% of residual activity (Fig. 1B). The greater inhibition at pHo 6.0 likely reflects combined inhibitory effects of acidic pHo and DEPC. Recovery of AE2-mediated Cl– transport on DEPC washout was independent of pHo during DEPC exposure but was proportional to DEPC concentration. Recovery of AE2 function was 75–80% after exposure to 0.5 mM DEPC but only 10–20% after exposure to 5 mM DEPC. We next tested whether hydroxylamine, a reagent that removes N-carbethoxy groups from DEPC-modified His (29), could reverse the effects of DEPC modification of AE2. However, the persistent inhibition after removal of 5 mM DEPC was not reversed by subsequent 12-min exposure of oocytes to 10 or 50 mM hydroxylamine (n = 12 and 4, respectively; data not shown). DEPC acid-shifts pHo sensitivity of AE2-mediated anion transport. Oocytes expressing wild-type AE2 were pretreated with 5 mM DEPC for 10 min at pHo 6.0 before assay of pHo-dependent 36Cl– efflux (see MATERIALS AND METHODS). The normalized efflux data revealed an apparently acid-shifted pHo sensitivity of anion transport (Fig. 1C). In contrast to the control wild-type AE2 pHo(50) of 6.91 ± 0.05, DEPC-treated AE2 exhibited a pHo(50) of 5.92 ± 0.22 (P < 0.005; Fig. 1D). As these data suggest a possible role for TMD His residues in setting the pHo sensitivity of AE2, we studied AE2 mutants with individual amino acid substitutions in each of the eight His residues of the TMD, as well as in eight multiple-His mutants of AE2.
TMD His residues are important for basal levels of AE2-mediated Cl– transport.
The schematic diagram in Fig. 2A shows the locations of the eight His residues within the AE2 TMD, mapped according to the topographical models of AE1 proposed by Zhu et al. (47) and Fujinaga et al. (9). Figure 2B shows that AE2 polypeptide levels in oocytes expressing wild-type or mutant cRNAs did not consistently correlate with basal transport activity at pHo 7.4 (Fig. 2C). In particular, AE2 mutants H1029A and H1060A showed similar low levels of 36Cl– efflux, despite very different levels of protein accumulation. However, the six other single-His mutants displayed sufficient 36Cl– efflux activity to allow analysis of their sensitivities to pHi and pHo. The low activity of AE2 mutant H1060A contrasted with the near-wild-type activity of mutant H1060E. The low activity of AE2 H1144A contrasted with the 50% wild-type activity of mutant H1144Y, substituting of the Tyr residue present in the corresponding position of the relatively pH-insensitive AE1 (Fig. 2C).
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The active His substitution mutants of AE2 allowed a test of the hypothesis that inhibition of AE2 by DEPC might be mediated entirely, or in large part, by modification of a single His residue of the AE2 TMD. As shown in Fig. 3A, anion transport by wild-type AE2 and AE2 H1160A was nearly completely inhibited by exposure to 5 mM DEPC at pHo 6.0, with only 20% recovery after DEPC removal at pHo 7.4. (Initial DEPC exposure at pHo 7.4 produced similar degrees of inhibition and recovery, as shown for AE2 H1160A in Supplemental Fig. 1C). In contrast, the AE2 mutant H1144Y was the only tested polypeptide that showed substantially increased post-DEPC recovery at pHo 7.4 (Fig. 3B). This enhanced recovery was also evident after DEPC exposure at pHo 7.4 and in the setting of a triple-His mutant (Fig. 3C; see Supplemental Fig. 1B). These data thus suggest that the nonconserved H1144 of AE2 is an important participant in irreversible AE2 inhibition by DEPC, although modification of this single-His residue does not mediate the full effect of DEPC.
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45% but was without effect on wild-type AE1. This selective AE2 inhibition also appeared to be mediated in part by H1144, since the AE2 mutant H1144Y was inhibited to a lesser degree by 1 mM Zn2+ (as was the triple mutant H846A/H849A/H1144Y; see Supplemental Fig. 3). These data reinforce the importance of H1144 in regulating AE2 activity. His residues of the AE2 TMD are involved in regulation of AE2 by pH. We showed previously that His residues of the AE2 NH2-terminal cytoplasmic domain are important for pH sensitivity (39). Figure 4A profiles normalized 36Cl– efflux activity as a function of pHo for wild-type AE2 and the H1144Y mutant. The alkaline-shifted pHo(50) for H1144Y (7.15 ± 0.08, n = 15) was significantly different from that of wild-type AE2 (6.82 ± 0.04, n = 62, P < 0.005). Figure 4B summarizes pHo(50) values measured for several AE2 His substitution mutants. In addition to AE2 H1144Y, the mutant H1145A also exhibited a modestly alkaline-shifted pHo(50) (P < 0.005). The apparently alkaline-shifted pHo(50) values of AE2 mutants H846A and H849A did not reach statistical significance (Fig. 4B).
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We previously demonstrated that double mutations of selected residues in the AE2 NH2-terminal cytoplasmic domain can have effects on the pH sensitivity of AE2 that are different from the effects of their component single-mutant counterparts (39). We therefore tested the effect of mutating multiple-His residues in the TMD, as schematized in Fig. 5A. Figure 5B shows that mutant polypeptides containing three or fewer His substitutions retained 36Cl– efflux activity at pHo 7.4 sufficient for analysis of pH sensitivity (mutants His2, His3a, His3b, and His3c). In contrast, mutants containing five or more His substitutions exhibited very low anion transport activity, which precluded assessment of pH sensitivity (mutants His5a, His5b, His6a, and His6b). Among the multiple-His mutants that exhibited sufficient basal activity, the double mutant H846A/H849A (His2; Fig. 5C) displayed a near-wild-type pHo dependence (P = 0.07), whereas the triple mutants H846A/H849A/H1145A (His3b) and H846A/H849A/H1160A (His3c) exhibited alkaline-shifted pHo(50) values of 7.29 ± 0.09 and 7.30 ± 0.09, respectively (each n = 22, P < 0.02 compared with wild-type AE2). Figure 5D summarizes the pHo(50) values derived from experiments similar to those shown in Fig. 5C. AE2 residues H846 and H849 are likely to have contributed to the alkaline-shifted pHo(50) of mutant His3c, inasmuch as the pHo(50) of mutant H1160A did not differ from the wild-type value (Fig. 4B). Multiple-His mutations also affected sensitivity to pHi. Thus inhibition by low pHi was enhanced in AE2 triple mutants His3b and His3c (P < 0.02), although the mutants His3a and His2 retained wild-type sensitivity (see Supplemental Fig. 2C).
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80–85% by 200 µM DIDS in the presence or absence of extracellular Cl–. In contrast, AE2 H1060E-mediated 36Cl– efflux was inhibited by only 20% (n = 15, P < 0.05) in the presence of extracellular Cl– (although, in the absence of Cl–, inhibition resembled the wild-type phenotype). This finding is consistent with an influence of AE2 H1060 on the DIDS isothiocyanate covalent reaction site K838, reflected in an apparent decrease in inhibitory potency of DIDS.
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We tested this hypothesis for AE2. The mutant H1060E retained wild-type 36Cl– efflux activity at pHo 7.4 (Fig. 6B) and wild-type pHi sensitivity (Fig. 6C), in contrast to the loss-of-function mutant H1060A (Fig. 3). Figure 6B also shows that the AE2 mutant E1007H exhibited loss of function (as did also the AE2 mutants E1007K and E1007C, not shown). The AE2 double mutant E1007H/H1060E, rather than rescuing wild-type function, was inactive (Fig. 6B) and not further stimulated by changes in pHi (Fig. 6C). These changes were not explained by reduced accumulation of mutant polypeptides (Fig. 6D) or by low surface expression of the inactive mutant E1007H (Fig. 6E). The corresponding AE1 single and double mutants are well expressed in oocytes (31), and the AE2 single mutants E1007Q, E1007K, and E1007D are also abundantly expressed in HEK 293 cells (36) and oocytes (unpublished data). Thus, although reduced surface expression of the H1060E/E1007H double mutant remains a possibility, these results are consistent with absence in the AE2 TMD of an interhelical functional interaction corresponding to that proposed between H752 and E699 of mouse AE1 (31).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Taken together, the data suggest that multiple His residues of the AE2 TMD contribute to the modulation of AE2 activity by pH.
DEPC sensitivity of AE2-mediated anion exchange suggests involvement of His residues.
The His-modifying agent DEPC is commonly used to assess possible involvement of His residues in native or recombinant ion channel or transporter function and, in particular, their involvement in pH sensitivity. DEPC carboxyethylates the proton-titratable imidazole group of His in a pH-sensitive reaction (7, 29), and DEPC specificity for His modification has been claimed at pH 5.5–7.5 (3). We have shown that DEPC can reversibly inhibit AE2-mediated Cl– transport in Xenopus oocytes, in contrast to its irreversible inhibition of kAE1 (Fig. 1B; see Supplemental Fig. 1A). DEPC treatment also results in an acid shift of the pHo(50) value of AE2-mediated Cl– transport. These findings suggest that His residues are involved in the regulation of AE2 by pHo. DEPC (5 mM) is known to inhibit AE1-mediated Cl– binding and anion transport by 70% in resealed erythrocyte ghosts at pHo 6.0 and pHi 7.4 but has little or no effect when pHi and pHo are 6.0 (13, 18). Results with the former condition resemble data obtained for kAE1-mediated Cl– transport in Xenopus oocytes at pHi 7.25 and pHo 6.0 (see Supplemental Fig. 1A) (37, 46). Therefore, the DEPC inhibition of wild-type AE1 is consistent with previous studies.
DEPC also affects the activity of other solute transporters in a pH-dependent manner, purportedly by interfering with His residues. For example, DEPC inhibited NHE1 activity (42) and aquaporin 1-mediated water transport (33, 34) in Xenopus oocytes at pHo 6.0. In contrast, DEPC inhibition of the ClC-1 Cl– channel was pHo independent between pHo 5.5 and 8.5 (22).
In the present work, AE2 differs from kAE1 in the extent and reversibility of inhibition by DEPC. These differences might be attributed to any of the three His residues of the AE2 TMD present in addition to the five TMD His residues also conserved in AE1. Inhibition of AE1 by DEPC has previously been attributed to modification of two separate His residues. Muller-Berger et al. (31) concluded from studies on murine erythroid AE1 expressed in oocytes that H752 (equivalent to H1060 in AE2) was the main site of DEPC action. In contrast, Jin et al. (20) concluded, on the basis of mass spectroscopic analysis of native human AE1 tryptic fragments from resealed red cell ghosts, that H834 (equivalent to H1160 in AE2) was the principal site of irreversible inhibition by DEPC. In the present study, DEPC inhibition and post-DEPC recovery at pHo 7.4 were indistinguishable for wild-type AE2 and for H1060E, H1160A, and His3c (Fig. 3, B and C). Mutation of the nonconserved residues H1136 and H1145 also failed to modify AE2 inhibition by DEPC (Fig. 3B). These data suggest that the different patterns of DEPC inhibition of AE2 and AE1 may arise, at least in part, from DEPC modification of the nonconserved AE2 H1144. This conclusion is strengthened by the difference in AE2 transport rate produced by mutation of H1144 to Ala or to the corresponding AE1 residue Tyr (Fig. 2C), despite equivalent levels of polypeptide accumulation (not shown). The reduced extent and increased reversibility of DEPC inhibition of mutant AE2 H1144Y may reflect reduced adduct formation with H1144Y, a reduced association constant, and/or an increased dissociation constant. Since DEPC inhibition of AE2 H1144Y is incompletely reversible, H1144 is not likely the sole target of DEPC in wild-type AE2.
DEPC may also affect residues H1029 and H1060, inasmuch as Ala substitution of the former decreased polypeptide accumulation and surface expression, whereas Ala substitution of the latter inhibited transport function, despite maintained surface expression (Fig. 2). Thus the properties of AE2 inhibition by DEPC are consistent with involvement of His residues in protein stability or folding, in the basal transport process, and in its regulation by pH.
Mutation of individual His residues can modify AE2 pH sensitivity. Protonation of His residues can inhibit ion transport, as in the case of the Kv1.4 channel (6), or can stimulate it, as for lens aquaporin 0 water channels (40) and the influenza M2 proton channel (16). Muller-Berger et al. (32) suggested that four conserved His residues are individually required for wild-type anion exchange activity of mouse AE1. Although the conserved AE2 residue H1029 may be critical for AE2 activity (Fig. 2C), individual mutation of the other seven His residues of the AE2 TMD retained partial or full Cl– transport activity. However, combined mutation of five or more His residues in AE2 leads to loss of function (Fig. 5B). The region in AE1 corresponding to that encompassing AE2 residues H1144, H1145, and H1160 has been proposed, on the basis of scanning Cys accessibility studies, to adopt a reentrant loop structure (Fig. 2A) (47). In this AE1-derived model, AE2 H1136 resides at the extracellular side of the lipid bilayer and H1160 at the cytosolic side of the lipid bilayer. The adjacent AE2 residues H1144 and H1145 are situated such that they might be accessible to either side of the permeability barrier. Interestingly, mutation of mouse AE1 K832 and K835 (corresponding to AE2 M1140 and K1143) partially or completely inhibited anion transport (32). The results together show the importance of this distal TMD region and its His residues in anion transport and its regulation. These TMD His residues may combine their influence with that of AE2 His residues of the NH2-terminal cytoplasmic domain shown previously to contribute to pH regulation of AE2. The latter include an important contribution by H360 and more limited roles of H314 and H317 (39). In contrast, NH2-terminal cytoplasmic domain residues H78, H79, H81, H82, H423, and H449–H451 can be mutated individually or in combination (in the case of H78, H79, H81, and H82) without impact on AE2 function or regulation by pHi or pHo (unpublished data).
Whereas DEPC treatment induces an acid shift in the pHo(50) of wild-type-AE2, mutation of individual His residues within the AE2 TMD can induce an alkaline shift of pHo sensitivity. Thus the individual AE2 mutants H1144Y (in which Tyr is the corresponding AE1 residue) and H1145A each exhibited an increase in pHo(50) (Fig. 4B). In addition, AE2 H1145A exhibited enhanced inhibition in response to a fall of pHi (Fig. 4D). The altered regulatory properties of these mutants are consistent with accessibility of the proposed reentrant loop to extracellular and intracellular solute. Because the presence of Tyr in the AE1 position corresponding to AE2 H1144 does not suffice to confer AE2-like pHo sensitivity on AE1, but substitution of AE2 H1144 with Tyr does suffice for an alkaline-shifted pHo(50) of AE2, it is more likely that the absence of His, rather than the presence of Tyr, alters the AE2 pHo sensitivity phenotype.
Although the AE2 triple-mutant His3c (Fig. 5A) showed an alkaline-shifted pHo(50), the "component" mutants H1160A and His2 exhibited wild-type pHo sensitivity. Thus individual mutation of H1144 and H1145 can alter pHo sensitivity of AE2-mediated Cl– transport, whereas mutation of H1160 [corresponding to human AE1 DEPC target H834 (20)] alters pHo sensitivity only in cooperation with Ala substitutions at H846 and H849. A similar result was shown for the enhanced pHi sensitivity of the multi-His mutants His3c and His5a, in which some of the component single-His mutants had previously shown wild-type pHi sensitivity. Multiple-His residues were also shown to comprise a Zn2+-binding site on the dopamine D2 receptor, whereas single-His mutations had no effect (26).
The above results suggest the involvement of multiple AE2 His residues in control of pHo and pHi sensitivity of anion transport, although not in a way predicted by the acid-shifted pHo(50) produced by DEPC treatment of AE2. The inability of hydroxylamine to reverse AE2 inhibition by DEPC suggests that the DEPC modification(s) responsible for inhibition was His imidazole ring cleavage following its bis-carbethoxylation (25). The contrast between the DEPC-induced acid shift in AE2 pHo(50) and the His substitution-induced alkaline shift in pHo(50) of AE2 may represent the different consequences to pH sensitivity of Ala substitution and of imidazole ring cleavage. It is also possible that DEPC treatment or mutation of individual or multiple His residues could alter the oligomeric structure or the interprotomeric interface of AE2 (45, 48), with possible consequences to regulatory properties and/or interaction with regulatory proteins. Alternatively, the acid-shifted pH-sensitivity of DEPC-treated wild-type AE2 may reflect a contribution of DEPC modification of non-His residues such as Cys, Tyr, Trp, or Lys (27–29). Examples of pH sensitivity control by residues other than His include Lys and Leu in renal outer medullary K+ channels (8), glutamate in TRPV5 (44), and a combination of five non-His-charged residues in TASK2 (30).
Muller-Berger et al. (31) postulated that mouse AE1 sustains anion exchange at pH values inhibitory to AE2 function via hydrogen bonding between the protonated conserved AE1 residue H752 and (in a separate helix) the conserved E699, at or near the permeability barrier. However, the role of mouse AE1 E699 in anion translocation suggests that such postulated hydrogen bonding itself might be regulated during the transport cycle. Our test of this proposal in AE2 is consistent with the absence of functional interaction between the two corresponding residues, or with altered folding and/or stability of the double mutant AE2 polypeptide (Fig. 6). Muller-Berger et al. also noted rescue of the mouse AE1 loss-of-function mutant H852Q with the second site reversion mutation K558N. However, a corresponding interaction in AE2 could not be tested in the setting of near-normal function of the AE2 mutant H1160A (Fig. 2C).
In conclusion, we have found that some His residues of the AE2 COOH-terminal TMD are necessary for protein stability and maintenance of basal AE2 activity. We have also found that certain TMD His residues are important for the regulation of AE2 activity by pH. No single His residue, however, seems to function as a unique TMD pH sensor. Rather, the presence of multiple His residues within the TMD defines a range of pHi and pHo values within which physiological regulation of anion transport may occur. Proton titration of the residues may, for example, be necessary for coordinating appropriate interaction between the TMD and pH-sensitive regions of the cytoplasmic NH2-terminal portion of the AE2 protein (37–39). The opposing shifts in pHo sensitivity of AE2 produced by DEPC treatment and by single- or multiple-His substitution mutations remain unexplained, but the possibility that DEPC exerts additional effects on non-His residues cannot be excluded. Thus, in contrast to some ion channels in which protonation/deprotonation of one or a few His residues suffices to mediate pH sensitivity, the present study highlights a more complex molecular anatomy governing the pH sensitivity of an acid/base transporter.
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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