Structure-function relationships of AE2 regulation by Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>-sensitive stimulators NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and hypertonicity

Marina N. Chernova, Andrew K. Stewart, Lianwei Jiang, David J. Friedman, Yune Z. Kunes, and Seth L. Alper

Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215


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We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH (pHi) and extracellular pH (pHo). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pHi: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. We find that both NH2-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the NH2-terminal cytoplasmic domain, an alanine scan of AE2 amino acids 336-347 identified residues whose individual mutation abolished or severely attenuated sensitivity to both or only one activating stimulus. Chelation of cytoplasmic Ca2+ (Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) diminished or abolished AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of Ca2+ signaling. Unlike AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain.

band 3; Xenopus oocyte; isotopic flux; site-directed mutagenesis; chloride-bicarbonate exchange; chloride-bicarbonate exchanger; intracellular calcium ion


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BICARBONATE IS TRANSPORTED across plasma membranes of higher vertebrate cells by the polytopic transmembrane proteins encoded by the SLC4 and SLC26 gene superfamilies. The SLC4 anion transporter gene superfamily includes at least three homologous genes encoding Na+-dependent Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers: AE1 (SLC4A1), AE2 (SLC4A2), and AE3 (SLC4A3). These polypeptides contribute to regulation of pH/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration ([HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]), volume, and [Cl-] in individual cells, in their surrounding extracellular space, and in compartments separated by epithelial cell monolayers (1, 2). AE transcripts are expressed in patterns that display specificity of tissue distribution and specificity of developmental and homeostatic regulation. Mutations in the AE1 gene, expressed in erythroid and renal intercalated cells, have been associated with inherited erythroid dyscrasias such as hereditary spherocytosis and with autosomal dominant and recessive forms of distal renal tubular acidosis (2). AE2 and AE3 are expressed more widely but have not been associated with human disease.

AE polypeptides are characterized by an NH2-terminal hydrophilic, cytoplasmic domain of ~400-700 amino acids (aa). This domain is believed, on the basis of studies of red blood cell AE1, to interact with several proteins of the cytoskeleton and with metabolic and signaling enzymes. This NH2-terminal cytoplasmic domain is followed by a polytopic transmembrane domain of ~500 aa that mediates anion exchange and (at least in AE1) binds the transmembrane carbonic anhydrase CAIV through interaction with an exofacial loop. Following the transmembrane domain is a short, hydrophilic COOH-terminal tail of 30 aa or longer that binds the cytosolic carbonic anhydrase CAII via an acidic motif (38) and terminates with a candidate type-2 PDZ interaction motif (14).

The nonerythroid anion exchanger AE2 (SLC4A2) and the erythroid/renal anion exchanger AE1 (SLC4A1) differ greatly in their patterns of acute regulation when expressed as heterologous polypeptides in Xenopus oocytes. Whereas acute decreases in intracellular (pHi) and extracellular (pHo) pH each inhibit AE2-mediated Cl-/Cl- and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (23, 31, 32, 43), AE1-mediated Cl- transport is much less sensitive to pHo and completely insensitive to pHi over the range tested (31, 32, 43). Similarly, whereas acute exposure to low concentrations of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (19) and to moderate hypertonicity (20) activate AE2, AE1 is unaffected by these stimuli.

Curiously, these two AE2 activators produce rapid but opposing changes in oocyte pHi. Hypertonicity alkalinizes oocytes through cJNK-dependent stimulation of a Cl--dependent Na+/H+ exchange activity (18). Thus activation of AE2 by hypertonicity is consonant with AE2 activation by alkaline pHi, and alkalinization accounts for at least part of AE2 activation by hypertonicity (20). In contrast, stimulation of AE2 by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> occurs in the presence of a pHi sufficiently acidic so as to inactivate AE2 completely if elicited in any other way (31, 32, 43). The mechanism by which NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulates AE2 in a way that overrides concomitant inhibition by acidic pHi remains unknown.

Although some of the AE2 structural elements required for AE2 to exhibit its wild-type sensitivity to inhibition by acidic pHo and to pHi have been defined (31, 32, 43), the structural elements required for stimulation by either NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or hypertonicity have not been reported. We therefore set out to define amino acid residues of AE2 whose presence is required for AE2 to display its wild-type sensitivity to the acute stimulators NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or hypertonicity. We hypothesized that these two AE2 stimulators with opposing effects on pHi would require the integrity of different regions of the AE2 polypeptide to produce stimulation.

Our studies of regulated 36Cl- transport by chimeric AE polypeptides showed that the presence of both the NH2-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 is required for functional activation either by hypertonicity or by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Systematic, progressive deletion mutagenesis studies indicated the importance for both modes of AE2 stimulation of a highly conserved region in the midportion of the AE2 NH2-terminal cytoplasmic domain. An alanine scan of AE2 aa 336-347 identified residues whose individual mutation abolished or severely attenuated both types or only one type of stimulation. Chelation of cytoplasmic Ca2+ (Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) diminished AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and abrogated stimulation by hypertonicity. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> elevated oocyte cytoplasmic [Ca2+] ([Ca2+]i) in the presence or nominal absence of bath Ca2+. Calmidazolium blocked both stimulated and basal AE2 activity. However, many other modifiers of cell signaling, including other calmodulin inhibitors, failed to inhibit AE2 or reduce its stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. In contrast to AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity, AE2 inhibition by calmidazolium did not require the presence of the AE2 NH2-terminal cytoplasmic domain.


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Materials. Female Xenopus were purchased from NASCO (Madison, WI). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was from Molecular Probes. Bumetanide was the gift of P. Feit (Leo Pharmaceutical). Na36Cl was purchased from ICN (Irvine, CA). All other chemical reagents were of analytical grade and purchased from Sigma, Calbiochem, or Fluka. Restriction enzymes and T4 DNA ligase were purchased from New England BioLabs (Beverly, MA). Taq DNA polymerase and 2-deoxynucleotide 5'-triphosphates (dNTPs) were purchased from Promega (Madison, WI). Expand High Fidelity PCR System was obtained from Roche (Indianapolis, IN). The Sure Escherichia coli strain was from Stratagene (La Jolla, CA). Oligonucleotides were purchased from Bio-Synthesis (Lewisville, TX). Anti-calmodulin polyclonal and monoclonal antibodies were from Sigma.

Solutions. ND-96 medium consisted of (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, and 2.5 sodium pyruvate with 100 µg/ml gentamicin. All flux media contained 10 µM bumetanide and lacked pyruvate and gentamicin. Influx media contained 20-38 mM NaCl as Na36Cl (2.8-3.2 µCi/ml). When indicated, 10 or 20 mM NaCl was replaced with 10 or 20 mM NH4Cl. Hypertonic media for efflux assays were supplemented with Na isothionate to a calculated osmolarity of 362 mosM (verified with a µOsmometer 5004 microosmometer; Precision Systems, Natick, MA). All media were titrated to a final pH of 7.40 before use.

Mutagenesis, cRNA transcription, and polypeptide expression. Construction of the mutant AE2 cDNAs was as previously described (9, 31, 32, 43). The designation AE2Delta N# indicates a cDNA encoding an NH2-terminally truncated AE2 in which an introduced Met residue precedes native AE2 residue (#+1). Thus, in AE2Delta N248, the introduced initiator Met is followed by natural AE2 aa residue 249.

Capped cRNA transcripts were synthesized from linearized DNA templates with the MEGAscript kit (Ambion, Austin, TX), and resuspended in diethylpyrocarbonate-treated water. RNA integrity was confirmed and concentration was estimated by formaldehyde-agarose gel electrophoresis. Handling of Xenopus and preparation of oocytes were performed as previously described (9, 11, 18-20, 31, 32, 43) as approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center. cRNAs or water (50 nl) were injected into defolliculated oocytes and incubated for 2-5 days at 19°C in ND-96 to allow biosynthesis of heterologous protein.

36Cl- influx studies. Groups of 8-12 oocytes previously injected with cRNA or water were preincubated for 15 min in 150 µl of ND-96 or ND-96 in which 10 mM NaCl was replaced with 10 mM NH4Cl. Oocytes were then transferred into 150 µl of the same medium containing in addition 10 µM bumetanide and 2.8-3.2 µCi Na36Cl. Two 10-µl aliquots of influx medium were saved for later determinations of 36Cl- specific activity in each influx group. 36Cl- influx into oocytes was carried out for 15 min and then terminated by three washes in 25 ml of isotonic, Cl--free medium. Individual oocytes underwent scintillation counting of associated 36Cl-. AE-mediated 36Cl- influx was calculated by subtracting values for Cl- influx into water-injected oocytes subjected to identical flux conditions as part of each experiment. Tests of wild-type (wt) AE2 and/or wt AE1 were included for comparison in each experiment. cRNA quantities chosen for injection resulted in rates of AE-mediated 36Cl- influx that varied across a range of threefold among all tested constructs. AE-mediated 36Cl- influxes were compared in the absence and presence of NH4Cl by unpaired two-tailed t-test (Microsoft Excel).

36Cl- efflux studies. Efflux studies were carried out as previously described (28, 29). Individual oocytes injected with cRNA or water 2-5 days previously were injected in Cl--free ND-96 with 50 nl of 130 mM Na36Cl representing 8,000-12,000 counts per minute (cpm). After 5-10 min recovery from injection, the efflux assay was initiated by transfer of individual oocytes to 6-ml borosilicate glass tubes each containing 1 ml of efflux solution. At timed intervals of 2 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. After completion of the assay with a final efflux period marked by addition of 200 µM DIDS, each oocyte was lysed in 100 µl of 2% SDS. Samples were counted for 3-5 min. Values for 2 standard deviations were <6% of the mean.

Experimental data were plotted as ln(% cpm remaining) vs. time. 36Cl- efflux rate constants were measured from linear fits to data from the last three time points for each condition. Within each experiment, water-injected and AE2 cRNA-injected oocytes from the same frog were subjected to parallel measurements. Stimulation or inhibition of AE2 activity was calculated as percentage of control [(kpostmodulation/ kpremodulation) × 100, where k is rate constant]. All single values for 36Cl- efflux from AE2 cRNA-injected oocytes in Cl- medium exceeded 150 cpm. Efflux cpm values for water-injected oocytes or for AE2 cRNA-injected oocytes in the presence of DIDS differed less than threefold from machine background values. Rates of AE-mediated 36Cl- efflux from individual oocytes before and after addition of modulator were compared by paired, two-tailed t-test (Excel or JMP).

Measurements of [Ca2+]i in Xenopus oocytes. [Ca2+]i was measured by fluorescence excitation ratio in fura 2-AM-loaded oocytes or by fluorescence intensity measurement in dextran-coupled calcium green-injected oocytes, as previously described (35, 36).


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AE2 stimulation by NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and by hypertonicity requires presence of both NH2-terminal cytoplasmic and COOH-terminal transmembrane domains. We showed previously that 36Cl- influx mediated by AE2, but not by AE1, can be stimulated by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity (19, 20). Figure 1 confirms these findings and extends them to 36Cl- efflux measurements. Neither the chimera AE1cyto/AE2memb nor the chimera AE2cyto/AE1memb retains sensitivity to either activator. Thus the presence of both the NH2-terminal cytoplasmic and the COOH-terminal transmembrane domains of AE2 is required for NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or for hypertonicity to stimulate AE2 anion transport activity.


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Fig. 1.   Stimulation of AE2-mediated Cl- transport either by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or by hypertonicity requires both cytoplasmic and transmembrane domains of AE2. A: schematic of wild-type AE2 (gray bars), AE1 (open bars), and chimeric constructs. Residue numbers are black for AE2 and gray for AE1. wt, Wild type; cyto, cytoplasmic; memb, transmembrane. B: %stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> of Cl- influx. Means ± SE for n* influx experiments, each representing 8-10 oocytes, are shown. Also shown is Cl- efflux from n individual oocytes expressing the same cRNA constructs. Unstimulated (basal) AE-mediated Cl- influx rates for these oocytes ranged between 6 and 17 nmol/h. For n*/n in parentheses, n* indicates the no. of influx experiments and n indicates the no. of efflux experiments. C: %stimulation by hypertonicity of Cl- efflux from oocytes expressing the indicated cRNA constructs. Means ± SE are shown for n oocytes. P values compare to the basal condition. Mean rate constants for basal AE-mediated Cl- efflux were 0.014 (AE1), 0.017 (AE2), 0.028 (AE1cyto/AE2memb), and 0.010 (AE2cyto/AE1memb).

AE2 NH2-terminal cytoplasmic domain residues required for stimulation by NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and by hypertonicity reside beyond aa 311. Experiments were performed to define with gradually increasing precision the residues within the AE2 NH2-terminal cytoplasmic domain required for sensitivity to each of the two activators. The first stage of evaluation assessed AE2 constructs in which increasing portions of the cytoplasmic NH2-terminal domain were deleted (Fig. 2A). As shown in Fig 2, B and C, sensitivity to both stimuli was retained in the mutant AE2 polypeptides Delta N99, Delta N248, and Delta N311. However, stimulation was absent in the AE2 mutant polypeptides Delta N410, Delta N510, and Delta N659. Thus the presence of AE2 residues between aa 311 and aa 410 is required for wild-type AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or hypertonicity. AE2 Delta N659 actually exhibited moderate inhibition by the normally stimulatory (and acidifying) NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (Fig. 2B), but not by the alkalinizing stimulator, hypertonicity (Fig. 2C).


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Fig. 2.   Deletion scan of the AE2 NH2-terminal cytoplasmic domain region localizes a region critical for stimulation of Cl- transport by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. A: schematic of AE2 NH2-terminal cytoplasmic domain deletion constructs. B: %stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> of Cl- influx into oocytes expressing the indicated cRNAs. Means ± SE are shown for n experiments, each representing 8-10 oocytes. Unstimulated (basal) AE-mediated Cl- influx rates for these oocytes ranged between 6 and 20 nmol/h. [%Stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> of AE2 Delta N659 portrays Cl- efflux and can be directly compared with NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-stimulated Cl- efflux by wt AE2 of 344 ± 101% (n = 4).] C: %stimulation by hypertonicity of Cl- efflux from oocytes expressing the indicated cRNAs. Means ± SE are shown for n oocytes. P values compare to the unstimulated condition. Mean rate constants for basal AE-mediated Cl- efflux were between 0.011 and 0.020.

AE2 NH2-terminal cytoplasmic domain residues between aa 311 and 347 are required for stimulation by NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and by hypertonicity. Sequential NH2-terminal deletions between AE2 aa 311 and 410 were generated and studied to define more precisely the residues needed for wild-type patterns of AE2 stimulation (Fig. 3A). However, truncation of as few as 17 additional residues beyond aa 311 abrogated stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (Fig. 3B) or by hypertonicity (Fig. 3C). Internal deletions within the region between AE2 aa 311 and 347 were constructed and expressed to test whether deletion of these residues sufficed to abrogate stimulation. Stimulation by both NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (Fig. 3B) and hypertonicity (Fig. 3C) was abolished by internal deletion of aa 312-347 or by deletion of either half of that region in the mutants AE2Delta (312-328) and AE2Delta (329-347).


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Fig. 3.   Deletion scan of the AE2 NH2-terminal cytoplasmic domain region between residues 311 and 391 further refines localization of a region critical for stimulation of Cl- transport by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. A: schematic of AE2 NH2-terminal cytoplasmic domain deletions and internal deletions. B: %stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> of Cl- efflux from oocytes expressing the indicated cRNAs. Means ± SE for n oocytes are shown; P values compare to the unstimulated condition. Mean rate constants for unstimulated Cl- efflux ranged between 0.0067 and 0.0441. C: %stimulation by hypertonicity of Cl- efflux from oocytes expressing the indicated cRNAs. Means ± SE for n oocytes are shown; P values compare to the unstimulated condition. Mean rate constants for unstimulated Cl- efflux ranged between 0.007 and 0.020.

Integrity of two noncontiguous regions within AE2 aa 312-347 is required for wild-type stimulation by NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and by hypertonicity. Because NH2-terminal or internal deletions might disrupt protein structure in unanticipated ways, we carried out a systematic hexa-alanine substitution scan within the region of aa 312-347 (Fig. 4A) implicated as important by the above deletion analysis, as well as in previous studies of AE2 regulation by pHi and pHo (31, 32). Figure 4, B and C, reveals similar patterns of alteration in the acute responses of these A6-substituted mutant AE2 polypeptides to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and to hypertonicity. Alanine substitution of residues 312-317, 330-335, and 336-341 retained significant stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, although of magnitude lower than wtAE2. In contrast, alanine substitution of AE2 residues 318-323 or 342-347 completely abolished stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Alanine substitution of AE2 residues 324-329 also led to a minimal stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> that failed to reach statistical significance (Fig. 4B).


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Fig. 4.   Hexa-alanine scan of the AE2 region between residues 312 and 348 refines still further localization of regions critical for stimulation of Cl- transport by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. A: schematic of the hexa-alanine substitutions in AE2. B: %stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> of Cl- efflux from oocytes expressing the indicated cRNAs. Means ± SE for n oocytes are shown; P values compare to the unstimulated condition. Mean rate constants for unstimulated AE2-mediated Cl- efflux ranged from 0.014 to 0.046, except for k = 0.0045 for AE2 (A)6318-323. C: %stimulation by hypertonicity of Cl- efflux from oocytes expressing the indicated cRNAs. Means ± SE for n oocytes are shown; P values compare to the unstimulated condition. Mean rate constants for unstimulated Cl- efflux ranged from 0.008 to 0.044, except k = 0.0044 for AE2 (A)6318-323.

Hypertonic stimulation of AE2 was of wild-type magnitude when residues 330-335 or 336-341 were substituted with alanine and only partially reduced when residues 312-317 were substituted with alanine. In contrast, AE2 was insensitive to hypertonic stimulation when alanines were substituted for AE2 residues 318-323, 324-329, or 342-347. Thus the integrity of these three short regions is of particular import to the ability of AE2 to undergo acute stimulation on exposure to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or to hypertonicity.

Individual mutation of AE2 residues within aa 336-347 can abrogate wild-type stimulation by NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and by hypertonicity. We chose to study the contributions to AE2 regulation of individual amino acid residues within the critical region of aa 342-347. We also examined individually the residues 336-341 in view of their shared importance in AE2 regulation by changing pHi and pHo (28, 29). Figure 5B shows that the AE2 mutant W336A retains considerable sensitivity to stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> whereas stimulation by hypertonicity is abolished (Fig. 5C). AE2 R337A remains sensitive to stimulation by both NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and hypertonicity, whereas AE2 E338A is barely responsive to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> but fully responsive to hypertonicity. Mutation of the candidate phosphorylation site T339 to either valine or to glutamate changed stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> minimally and stimulation by hypertonicity not at all (Fig. 5, B and C, right). Hypertonic stimulation of AE2 A340G was intact, but stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was greatly reduced, although not abolished. All other single alanine substitution mutations in the region of AE2 aa 336-347 abolished or nearly abolished stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity, with the sole exception of AE2 K344A, which retained minimal but significant stimulation by both regulators. The AE2 double mutant E346A/E347A was unresponsive to stimulation either by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (102 ± 5%, n = 8) or by hypertonicity (125 ± 12%, n = 12; not shown). Thus the single substitution mutation with the greatest discordance of response to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and hypertonicity was W336. AE2 A340G and E338A also differed in their responses to the two stimuli, although to a lesser degree.


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Fig. 5.   Alanine scan of the AE2 regulatory region between residues 336 and 347 assesses roles of individual amino acids in the stimulation of Cl- transport by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. A: schematic of the AE2 region in which the indicated residues were individually mutated to alanine. B: %stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> of Cl- efflux from oocytes expressing the indicated cRNAs. Left: alanine scan. Right: threonine mutants. Mean rate constants for basal AE-mediated Cl- efflux for each mutant ranged from 0.008 to 0.032. C: %stimulation by hypertonicity of Cl- efflux from oocytes expressing the indicated cRNAs. Left: alanine scan. Right: threonine mutants. Means of n oocytes are shown. Mean rate constants for basal AE-mediated Cl- efflux for each mutant ranged from 0.003 to 0.016. *P < 0.05 compared with initial basal rate in same oocytes; **P < 0.005.

Individual mutation of AE2 residues within aa 336-347 has distinct consequences for regulation by NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP>, hypertonicity, pHi, and pHo. The WRETARWIKFEE sequence of AE2 aa 336-347 is among the most highly conserved among the NH2-terminal cytoplasmic domains of the SLC4 gene family (27, 31). Its importance to regulation of AE2 by pHi and pHo has been demonstrated previously (32). Figure 6 summarizes the similarities and differences among the altered responses of these AE2 missense mutants to four acute regulatory stimuli of AE2. The W336A mutation disrupted or modified AE2 responses to pHi, pHo, and hypertonicity but retained substantial sensitivity to stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. The AE2 missense mutations E338A, I343A, and F345A exhibited wild-type responses only to changing pHo, but responses to changing pHi, NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, or hypertonicity were altered or abolished. In contrast, AE2 E346A maintained wild-type response to changing pHi but exhibited shifted sensitivity to pHo and loss of stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. The AE2 mutants R337A, T339V, and T339E maintained wild-type response patterns to all four acute regulatory stimuli. In contrast, the AE2 mutants R341A, W342A, E347A, and (not shown) the double mutant E346A/E347A exhibited mutant phenotypes in response to all four acute regulatory stimuli.


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Fig. 6.   Effects of mutation of individual residues within the conserved regulatory region of AE2 [amino acids (aa) 336-347] on acute regulation by 4 stimuli. Most residues were mutated to Ala. A340 was mutated to Gly. Thr339 was mutated to Glu and to Val, with indistinguishable results. +, Wild-type (qualitatively unmodified) phenotype; -, regulatory effect abolished or [in the case of extracellular pH (pHo)] shifted. pHi, intracellular pH.

Chelation of Ca<UP><SUB>i</SUB><SUP>2<UP>+</UP></SUP></UP> in oocytes attenuates AE2 stimulation by NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and by hypertonicity. The mechanism by which NH<UP><SUB>4</SUB><SUP>+</SUP></UP> activates AE2 despite acidic pHi remains obscure. Previous studies suggested several factors that were not sufficient to account for the stimulation. Thus stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was not mimicked by equally depolarizing concentrations of KCl, by concentrations of chloroquine sufficient to alkalinize the pH of intracellular organelles, or by incubation with or injection of polyamines (19). The same degree of stimulation is observed whether NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is added as NH4Cl, as the NH<UP><SUB>4</SUB><SUP>+</SUP></UP> salt of the permeant weak acid acetate, or as the NH<UP><SUB>4</SUB><SUP>+</SUP></UP> salt of the impermeant (31) weak acid formate (n = 4 for each; not shown).

Our previous finding (18) that chelation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> inhibits hypertonic activation of the endogenous Cl--dependent Na+/H+ exchange activity of the oocyte prompted examination of the role of [Ca2+]i in the acute stimulation of AE2. Prior injection into oocytes of BAPTA (5 mM) or EGTA (5 mM) attenuated subsequent AE2 stimulation by exposure to 20 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (Fig. 7A). Thirty-minute exposure to 20 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP> modestly elevated [Ca2+]i by 28 ± 2 nM, as measured in water-injected oocytes loaded with fura 2-AM (n = 3; mean ± SE) or by 33 ± 2 nM in oocytes injected with 70-kDa dextran-coupled calcium green (n = 4), and elevated [Ca2+]i by 32 ± 1 nM in AE2-expressing oocytes (n = 6; fura 2-AM). Injected EGTA inhibited NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced elevation of [Ca2+]i by 85% (P < 0.001, n = 8; fura 2-AM), whereas extracellular EGTA alone reduced neither NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-stimulated 36Cl- efflux nor elevation of [Ca2+]i.


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Fig. 7.   Role of cytoplasmic Ca2+ (Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) in AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. A: chelation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> attenuates stimulation of AE2 by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. *P < 0.005. Mean rate constants for basal Cl- efflux from individual oocytes ranged from 0.017 to 0.029. B. chelation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> abolishes or attenuates stimulation of AE2 by hypertonicity. *P = 0.01; **P < 0.0001. Mean rate constants for basal Cl- efflux from individual oocytes ranged from 0.007 to 0.017. BAPTA and EGTA solutions titrated to pH 7.40 were injected to final concentrations of 5 mM. Means ± SE for n oocytes are shown.

Prior injection into oocytes of BAPTA (5 mM) or EGTA (5 mM) also attenuated or abrogated subsequent AE2 stimulation by exposure to hypertonic medium (Fig. 7B). Thirty-minute exposure to hypertonicity (282 mosM) increased oocyte [Ca2+]i by 80 ± 18 nM (n = 6; mean ± SE) in water-injected oocytes and by 70 ± 5 nM (n = 6) in AE2-expressing oocytes (fura 2 measurements). However, extracellular EGTA inhibited this hypertonic elevation of [Ca2+]i by 79% (n = 6), in contrast to its lack of effect on NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced elevation of [Ca2+]i.

Calmidazolium inhibits both basal and stimulated AE2 activity by acting on COOH-terminal transmembrane domain of AE2. The ability of injected Ca2+ chelators to inhibit NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulation of AE2-mediated Cl- transport led us to test the calmodulin dependence of this stimulation. The calmodulin inhibitor calmidazolium also inhibited AE2-mediated 36Cl- uptake (ID50 ~0.5 µM; not shown). Interestingly, calmidazolium at slightly higher concentrations also inhibited basal AE2 function in the absence of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. As shown in Fig. 8, 10 µM calmidazolium inhibited basal activity of wtAE2 to 30% of the uninhibited value. Higher concentrations of calmidazolium did not further inhibit AE2. In contrast, wtAE1-mediated 36Cl- efflux in the presence of 10 µM calmidazolium retained 79 ± 1% of its uninhibited value (n = 7; not shown). Although 5 mM EGTA added to a nominally Ca2+-free bath only minimally reduced AE2 inhibition by 10 µM calmidazolium (39 ± 11% of control; n = 3), that inhibition was greatly attenuated (79 ± 19% of control; n = 3) in oocytes in the same Ca2+-free bath also injected with EGTA (5 mM final).


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Fig. 8.   Analysis of chimeras and of NH2-terminal cytoplasmic domain sequential deletion constructs defines the AE2 transmembrane domain as critical for inhibition by calmidazolium. Means ± SE for n oocytes are shown; P values compare each group with the uninhibited condition. Mean rate constants for uninhibited Cl- efflux ranged from 0.064 to 0.009.

The AE2 structural requirements for inhibition by calmidazolium were compared with those of stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. Figure 8 shows that although the AE1cyto/AE2memb chimera was inhibited by calmidazolium, the converse chimera AE2cyto/AE1memb was insensitive to inhibition by the drug. These data suggested both the importance of the AE2 membrane domain for calmidazolium action and the lack of importance of the NH2-terminal cytoplasmic domain of AE2. Analysis of mutants progressively truncated across the NH2-terminal cytoplasmic domain supported this hypothesis. Removal of the NH2-terminal 99 aa was without any effect on calmidazolium inhibition. Even with removal of the NH2-terminal 311, 410, 510, or 659 aa (nearly all of the NH2-terminal cytoplasmic domain), most of the inhibitory effect of calmidazolium was maintained. Thus the AE2 transmembrane domain was both necessary and sufficient to exhibit sensitivity to inhibition by calmidazolium. This differed from the requirement of both cytoplasmic and membrane domains of AE2 for stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or hypertonicity.

Because calmodulin sometimes acts via CaM-kinases or calcineurin phosphatases, the AE2 mutants T339V and T339E were also tested for calmidazolium responsiveness. As shown in Fig. 8, AE2 T339V exhibited wild-type sensitivity to calmidazolium. AE2 T339E showed calmidazolium sensitivity equivalent to that shown by the NH2-terminal truncation mutants Delta N311, -410, -510, and -659.

Effect of agents that modulate [Ca<UP><SUB>2</SUB><SUP><UP>+</UP></SUP></UP>]i signaling on acute stimulation of AE2. Although calmidazolium potently inhibited both basal and NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-stimulated AE2-mediated Cl- transport, the calmodulin inhibitor W13 was inactive as an inhibitor of basal or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-stimulated AE2 activity when exposed in the bath solution (92 µM; n = 3) or injected to an estimated final intracellular concentration of 200 µM (n = 9). However, injection of CaMKII inhibitor peptide (40) to final intracellular concentrations of 1 (n = 5) or 50 (n = 5) µM in each case modestly inhibited AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> by 30% (P < 0.05 for the pooled data; n = 10). Nonetheless, stable interaction of calmodulin with AE2 was not detected by several approaches (not shown). Calmodulin-agarose did not bind AE2 in 1% Triton X-100 lysates of transiently transfected and muristerone-induced 293-EcR cells or in lysates of light membranes from pig gastric mucosa (45), under conditions in which the matrix binds KCNN1 (SK1) K+ channel polypeptide (30). Moreover, anti-calmodulin antibody (Sigma) failed to coimmunoprecipitate AE2 from these same lysates.

Several modifiers of organellar Ca2+ stores were without significant effect on basal or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-stimulated AE2-mediated 36Cl- efflux (not shown). Among antagonists of inositol 1,3,4-trisphosphate receptor-mediated Ca2+ release tested were heparin sulfate [injected to final 5 mg/ml, indistinguishable in the presence (n = 22) or absence (n = 21) of bath Ca2+ from the inactive analog heparan sulfate]; pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (injected to final 50 µM, n = 5); 2 aminophenylborate [bath 250 µM (n = 5) or injected to final 100 µM (n = 4)]; xestospongin C (injected to final 10 µM; n = 5); and MDL-12,330A (injected to 100 µM; n = 5). Agents that elevate oocyte [Ca2+]i without altering basal or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-stimulated AE2-mediated 36Cl- efflux included thapsigargin (bath 10 µM; n = 5) and A-23187 (bath 30 µM; n = 5). These data suggest that, although elevation of [Ca2+]i is required for maximal NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulation of AE2, it is not sufficient itself to mediate AE2 activation.

The c-JNK inhibitor SP-600125 blocks hypertonic activation of the endogenous oocyte Cl--dependent Na+/H+ exchanger (18). In contrast, SP-600125 (final injected concentration 25 µM) did not inhibit basal function (n = 13) or hypertonic stimulation (n = 14) of AE2 whether injected acutely or 2 h before the efflux assay. At the nonspecific (final injected) concentration of 50 µM SP-600125, hypertonic stimulation was reduced by 50% (n = 3), whereas NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulation of AE2 was unaffected (n = 11). Neither the p42/44 MAPK inhibitor PD-98059 (10 µM injected; n = 5) nor the p38 MAPK inhibitor SB-220025 (2 µM injected; n = 4) reduced hypertonic stimulation of AE2. The general protein kinase inhibitor staurosporine (1 µM in bath; n = 6) modestly inhibited NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-stimulated AE2-mediated 36Cl- efflux by 24 ± 6% (P < 0.01).

Additional modifiers of intracellular signaling that were without effect on basal or NH4+-stimulated AE2-mediated 36Cl- efflux (data not shown) included the phospholipase C inhibitor U-73122 (injected to final 12 µM; n = 5) and calcineurin (protein phosphatase 2B, injected 0.025 U, nominal 7.5 µM; n = 5). Among additional bath-applied drugs without effect on basal AE2-mediated 36Cl- influx were the phosphatidylinositol-3-kinase inhibitor wortmannin (10 µM; n = 6), the calcineurin inhibitor cyclosporin A (1 µM; n = 6), the protein serine phosphatase inhibitor okadaic acid (100 nM; n = 7), the protein kinase C inhibitor chelerythrine (10 µM; n = 8), the tyrosine kinase inhibitors genistein (10 µM; n = 8), calphostin (1 µM; n = 8), tyrphostin AG126 (n = 8), and tyrphostin S1 (n = 11), and the lipid mediators sphingosine (100 µM, n = 4 in bath, n = 4 injected) and its antagonist, dihydrosphingosine (100 µM; n = 4 in bath, n = 4 injected).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have defined structural elements of AE2 required for wild-type stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity. Each stimulation requires both NH2-terminal cytoplasmic and transmembrane domains of AE2. The regions of the NH2-terminal cytoplasmic domain required for each stimulation are the same. However, within the highly conserved AE2 aa 336-347, stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity each exhibit slightly different structural requirements. Chelation of intracellular Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> inhibits both stimuli. Calmidazolium inhibits AE2 profoundly but inhibits AE1 only minimally. AE2 inhibition by calmidazolium does not require the presence of the AE2 NH2-terminal cytoplasmic domain but does require the AE2 transmembrane domain.

Role of NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> in physiological function of AE2. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is present in several tissues that express AE2, although often absent from in vitro studies of cellular ion transport. AE2 in the basolateral membranes of surface enterocytes (3) is exposed in the interstitium of the portal circulation to 1-3 mM (total [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]), reflecting much higher concentrations in the gut lumen. These concentrations approximate the ED50 for stimulation of AE2 by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (19). AE2 in the basolateral membranes of parietal cells is likely exposed to similar [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>], because it resides on the other side of the tight junction from gastric luminal [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>] of up to 20 mM when the lumen is colonized with urease-positive Helicobacter pylori (19).

NH<UP><SUB>4</SUB><SUP>+</SUP></UP> can reach high concentrations in the renal medullary interstitium, which provides NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> for secretion and "trapping" in the acidic collecting duct lumen. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by isolated perfused rabbit cortical collecting duct, due in part to activation of basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (41). Luminal NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in control or acidotic rat cortical tubule segments sampled by micropuncture lies near or below the ED50 for AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (23a). Basolateral NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulates Cl- secretion in isolated, perfused rat inner medullary collecting duct (39), consistent with stimulation of basolateral AE2 (4). AE2 is also abundant in the medullary thick ascending limb of rat (4, 16), where it can impact on uptake of residual tubular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Renal medullary [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>] likely remains at levels that are maximal with respect to AE2 stimulation, rendering AE2 insensitive to inhibition by acidic pHi (23a). Both in gut and in kidney, AE2 may rely on NH<UP><SUB>4</SUB><SUP>+</SUP></UP> to maintain function during fluctuations in pHo and/or pHi. The apical membranes of these gastrointestinal and renal epithelia potentially exposed to very high luminal [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>] tend to share with the Xenopus oocyte plasma membrane its high ratio of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> to NH3 permeabilities (7, 8, 19, 25, 29). This similarity supports the relevance to mammalian epithelial physiology of AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in Xenopus oocytes. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> modulation of AE2 function may be relevant not only at the plasma membrane but also in the Golgi apparatus (4, 18a), where AE2 may play a role in the regulation of organellar pH or [Cl-].

Regulation of anion exchange by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is not restricted to SLC4 anion exchangers. The SLC26A3 (DRA) anion exchanger of the ileocolonic apical membrane is also stimulated acutely by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and inhibited by acidic pHi (Chernova et al., unpublished observations), whereas the SLC26A4 (pendrin) anion exchanger of the type B intercalated cell apical membrane appears insensitive both to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and acidic pHi (A. K. Stewart and S. L. Alper, unpublished observations). NH<UP><SUB>4</SUB><SUP>+</SUP></UP> regulates anion exchange chronically as well as acutely. Thus prolonged hyperammonemia increased intracellular [Cl-] in hippocampus by mechanisms sensitive to inhibition by stilbene disulfonates and by acetazolamide. This change coincided with upregulation of AE3 mRNA and protein. Both the changes in intracellular [Cl-] and in AE3 abundance were blocked by inhibition of protein kinase C and stimulated by phorbol ester (21). Interestingly, dietary NH<UP><SUB>4</SUB><SUP>+</SUP></UP> loading exacerbates polycystic kidney disease in the Han:SPRD rat and in the CD1:pcy/pcy mouse (34), although the mechanisms remain unknown.

Structural regions of AE2 required for responsiveness to NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and hypertonicity. Although NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and hypertonicity change oocyte pHi in opposing directions, the same regions of AE2 are required for AE2 to exhibit sensitivity to activation in their presence. Effectiveness of both stimuli requires the presence of the AE2 NH2-terminal cytoplasmic and COOH-terminal transmembrane domains. This fact is emphasized by the unequivocal inhibition of AE2 Delta N659 by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by a trend toward inhibition of the chimera AE1cyto/AE2memb. This inhibition may represent unmasking of AE2 inhibition by acidic pHi in the absence of residues crucial for AE2 activation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Moreover, a wide series of truncation and alanine substitution mutations within the NH2-terminal cytoplasmic domain preserve, reduce, or abolish AE2 responses to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and to hypertonicity in strict parallel.

Only AE2 aa 336-347 (32) reveals a single difference in structural dependence of these two stimuli: the AE2 mutant W336A lacks responsiveness to hypertonicity while maintaining sensitivity to activation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (Figs. 5 and 6). In addition, the alanine scan of this region uncovers one difference in the structural dependence of sensitivity to hypertonicity and to pHi, evident in the AE2 mutant E346A, which retains sensitivity to inhibition by acidic pHi but loses responsiveness to hypertonicity. This difference suggests that intracellular alkalinization may not suffice to explain activation of AE2 by hypertonicity, despite its requirement for oocyte Na+/H+ exchange activity (18).

The X-ray structure of the AE1 region homologous to AE2 aa 336-347 (beta 2-loop-beta 3) predicts a similar beta -sheet-loop-beta -sheet structure in AE2 (32, 42). We postulate that the various acute regulatory stimuli of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, hypertonicity, pHi, and pHo alter the structure of this region in AE2 and/or the structure of one or more complementary interaction surfaces. These interaction surfaces may reside within AE2 or in one or more distinct polypeptides.

Mechanism of NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> stimulation of AE2. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is a first messenger for the slime mold Dictyostelium. In yeast, extracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> currently is thought to signal nitrogen balance only via transmembrane transport and metabolism (17). Although similar NH<UP><SUB>4</SUB><SUP>+</SUP></UP> signaling in mammalian cells has not yet been described, the Mep/Amt gene family of alkylammonium/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport proteins may provide new approaches to investigation of this possibility. Extracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> enters the oocyte via nonspecific cation channel(s) (7, 8, 25) that can be activated by reactive oxygen species (13). A possible link between NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry into the cell and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulation of AE2 is suggested by the interaction (direct or indirect) between AE1 and the RhAg polypeptide in the erythrocyte (6), likely mediated in part by protein 4.2 (24). Analogous interaction might exist in the type A intercalated cell basolateral membrane between AE1 and the putative NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transporter RhBG (37) and between AE2 and a related transporter.

Although chelation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> inhibits AE2 stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity, additional data show that elevation of [Ca2+]i does not suffice to explain stimulation of AE2. These include the lack of AE2 stimulation by thapsigargin or by A-23187, the slow, modest increase in [Ca2+]i observed during exposure to NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, the modest (although significant) inhibitory effects of CaMKII inhibitory peptide on NH<UP><SUB>4</SUB><SUP>+</SUP></UP> stimulation of AE2, and the lack of effect of many pharmacological modifiers of Ca2+ signaling. Although pharmacological inhibition of cJNK, p38, and p42/44 did not support a role for MAPKs in the action of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or hypertonicity on AE2, a modest inhibitory effect of staurosporine suggested possible additional involvement of a kinase. Direct evidence suggesting regulatory phosphorylation of recombinant bAE3 has been reported (5), and suggestive evidence favors phosphoregulation of native AE2 (28).

Mechanism of AE2 inhibition by calmidazolium. The requirement of [Ca2+]i elevation for stimulation of AE2 by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and by hypertonicity led to the test of calmidzolium as an AE2 modulator. Although at lower concentrations calmidazolium indeed inhibited stimulation by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, at modestly higher concentrations inhibition of basal activity was also evident. The AE2 structure-function relationship of the inhibition of basal activity suggested a mechanism of action distinct from that of the stimulators. Although both cytoplasmic and transmembrane domains of AE2 are required for wild-type patterns of stimulation, only the transmembrane domain appears to be required for inhibition by calmidazolium.

Despite the ability of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> chelation to attenuate inhibition of basal AE2 function by calmidazolium, several observations raise the question of an inhibitory mechanism unrelated to calmodulin. These include calmidazolium's moderate potency and lack of inhibition by other calmodulin inhibitors. Calmidazolium is an imidazole, and it has a host of pharmacological activities in addition to inhibition of calmodulin. These include inhibition of P-450 cytochromes (44), the P2X7 receptor (12), the maitotoxin receptor (15), and constitutive nitric oxide synthase (33). Although Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> was elevated in MDCK cells by 2-5 µM (22), 25 µM calmidazolium did not suffice to activate Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>-dependent Cl- current in oocytes (26). It also remains possible that calmidazolium directly inhibits AE2-mediated Cl- transport, with either an internal or external site of action.

In conclusion, at least three conditions differentially regulate AE2-mediated anion exchange via mechanisms influenced by [Ca2+]i. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and hypertonicity both stimulate AE2 despite their opposing effects on oocyte pHi. Each stimulus requires the presence of both the AE2 NH2-terminal cytoplasmic domain and the transmembrane domain. Within the AE2 NH2-terminal cytoplasmic domain, each stimulus requires the integrity of select (but not identical) amino acid residues in the critical region of aa 336-347. Each stimulus is inhibited by chelation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>. Investigation of the role played by Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> in AE2 regulation led to the discovery that calmidazolium inhibits AE2 function through a process requiring only the AE2 transmembrane domain. This inhibition is itself attenuated by chelation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>. Additional experiments will be required to elucidate the mechanisms of these AE2 regulators and to define their relationship to the physiological functions of AE2-mediated anion exchange.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health (NIH) Grants DK-43495 and DK-34854 (the Harvard Digestive Diseases Center) to S. L. Alper and NIH fellowships T32-HL-07516 and F32-DK-09136 to Y. Z. Kunes. A. K. Stewart was supported by an International Prize Traveling Fellowship of the Wellcome Trust.


    FOOTNOTES

Present addresses: A. K. Stewart, University Laboratory of Physiology, Parks Rd., Oxford OX1 3PT, UK; Y. Z. Kunes, Millenium Pharmaceuticals, 640 Memorial Dr., Cambridge, MA 02139

Address for reprint requests and other correspondence: S. L. Alper, Molecular Medicine and Renal Units, RW763 East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: salper{at}bidmc.harvard.edu).

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.

First published January 15, 2003;10.1152/ajpcell.00522.2002

Received 11 November 2002; accepted in final form 9 January 2003.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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