A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity.

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.

Differential expression and regulation of AE2 anion exchanger subtypes in rabbit parietal and mucous cells.

1. The anion exchanger isoform 2 (AE2) gene encodes three subtypes (AE2a, b and c), which have different N-termini and tissue distributions. AE2 is expressed at high levels in the stomach, where it is thought to mediate basolateral base exit during acid production. The present study investigated if the three AE2 subtypes are differentially expressed and regulated in different cell types within the gastric mucosa. 2. The cloning strategy to obtain rabbit AE2a, b and c cDNAs combined genomic PCR and RT-PCR based on primers deduced from the rat sequences. Semiquantitative RT-PCR using homologous primers revealed much higher AE2 mRNA expression in rabbit parietal cells (PCs) than in mucous cells (MCs). The subtype expression pattern was AE2b >> AE2c > or = AE2a in PCs and AE2a >AE2b >> AE2c in MCs. Sequence analysis revealed the presence of a highly conserved protein kinase C (PKC) consensus sequence in the AE2a alternative N-terminus. 3. Maximal Cl(-)-HCO(3)(-) exchange rates, measured fluorometrically in BCECF-loaded cultured gastric cells, were much higher in PCs than MCs. PKC activation by phorbol ester stimulated maximal Cl(-)-HCO(3)(-) exchange rates in MCs but not in PCs, whereas forskolin had no effect in each cell type. 4. In summary, rabbit PCs and MCs, which originate from the same gastric stem cell population, display a completely different AE2 subtype expression pattern. Therefore, AE2 subtype expression is not organ specific but cell type specific. The different regulation of anion exchange in parietal and mucous cells suggests that AE2 subtypes may be differentially regulated.

VHL tumor suppressor regulates Cl-/HCO3- exchange and Na+/H+ exchange activities in renal carcinoma cells.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are thought to play a critical role in the pathogenesis of both sporadic and VHL disease-associated clear-cell renal carcinomas (RCC). Differential display-PCR identified the AE2 anion exchanger as a candidate VHL target gene. AE2 mRNA and polypeptide levels were approximately threefold higher in 786-O VHL cells than in 786-O Neo cells. In contrast, Cl(-)/HCO(3)(-) exchange activity in 786-O VHL cells was 50% lower than in 786-O Neo cells. Since resting intracellular pH (pH(i)) values were indistinguishable, we postulated that Na(+)/H(+) exchange activity (NHE) might be similarly reduced in 786-O VHL cells. NHE-mediated pH(i) recovery from acid load was less than 50% that in 786-O Neo cells, whereas hypertonicity-stimulated, amiloride-sensitive NHE was indistinguishable in the two cell lines. The NHE3 mRNA level was higher in 786-O VHL than 786-O Neo cells, but NHE1 mRNA levels did not differ. AE2 and NHE3 are the first transcripts reported to be upregulated by pVHL. Elucidation of mechanisms responsible for downregulation of both ion exchange activities will require further investigation.

AE anion exchangers in atrial tumor cells.

Intracellular pH homeostasis and intracellular Cl(-) concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular Cl(-) concentrations, Cl(-)/HCO(3)(-) exchange promotes intracellular acidification and Cl(-) loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal Na(+)-independent Cl(-)/HCO(3)(-) (but not Cl(-)/OH(-)) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell Cl(-)/HCO(3)(-) activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of Cl(-)/HCO(3)(-) exchange activity in an atrial cell type.

The cytoplasmic C-terminal fragment of polycystin-1 regulates a Ca2+-permeable cation channel.

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca(2+)-permeable cation current, which shifted oocyte reversal potential (E(rev)) by +33 mV. Whole cell currents were sensitive to inhibition by La(3+), Gd(3+), and Zn(2+), and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NP(o) of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NP(o) of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and (45)Ca(2+) flux experiments. Both Ca(2+) conductance and influx were inhibited by La(3+). Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca(2+)-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.

Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells.

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation.

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

Immunolocalization of AE2 anion exchanger in rat and mouse epididymis.

A low-bicarbonate concentration and an acidic pH in the luminal fluid of the epididymis and vas deferens are important for sperm maturation. These factors help maintain mature sperm in an immotile but viable state during storage in the cauda epididymidis and vas deferens. Two proton extrusion mechanisms, an Na(+)/H(+) exchanger and an H(+)ATPase, have been proposed to be involved in this luminal acidification process. The Na(+)/H(+) exchanger has not yet been localized in situ, but we have reported that H(+)ATPase is expressed on the apical membrane of apical (or narrow) and clear cells of the epididymis. These cells are enriched in carbonic anhydrase II, indicating the involvement of bicarbonate in the acidification process and suggesting that the epididymis is a site of bicarbonate reabsorption. Previous unsuccessful attempts to localize the Cl/HCO(3) anion exchanger AE1 in rat epididymis did not investigate other anion exchanger (AE) isoforms. In this report, we used a recently described SDS antigen unmasking treatment to localize the Cl/HCO(3) exchanger AE2 in rat and mouse epididymis. AE2 is highly expressed in the initial segment, intermediate zone, and caput epididymidis, where it is located on the basolateral membrane of epithelial cells. The cauda epididymidis and vas deferens also contain basolateral AE2, but in lower amounts. The identity of the AE2 protein was further confirmed by the observation that basolateral AE2 expression was unaltered in the epididymis of AE1-knockout mice. Basolateral AE2 may participate in bicarbonate reabsorption and luminal acidification, and/or may be involved in intracellular pH homeostasis of epithelial cells of the male reproductive tract.

Expression of AE2 anion exchanger in mouse intestine.

We have characterized expression of anion exchanger 2 (AE2) mRNA and protein in the mouse intestine. AE2 mRNA abundance was higher in colon than in more proximal segments. AE2a mRNA was more abundant than AE2b mRNA throughout the intestine, and AE2c mRNA was expressed at very low levels. This AE2 mRNA pattern contrasted with that in mouse stomach, in which AE2c > AE2b > AE2a. AE2 polypeptide abundance as detected by immunoblot qualitatively paralleled that of mRNA, whereas AE2 immunostaining exhibited a more continuous decrease in intensity from colon to duodenum. AE2 polypeptide was more abundant in colonic surface cells than in crypts, whereas ileal crypts and villi exhibited similar AE2 abundance. AE2 was also observed in mural and vascular smooth muscle. Localization of AE2 epitopes was restricted to the basolateral membranes of epithelial cells throughout the intestine with three exceptions. Under mild fixation conditions, anti-AE2 amino acids (aa) 109-122 detected nonpolarized immunostaining of ileal enterocytes and of Paneth cell granule membranes. An epitope detected by anti-AE2 aa 1224-1237 was also localized to subapical regions of Brunner's gland ducts of duodenum and upper jejunum. These localization studies will aid in the interpretation of anion exchanger function measured in epithelial sheets, isolated cells, and membrane vesicles.

Functional and molecular characterization of luminal and basolateral Cl-/HCO-3 exchangers of rat thick limbs.

Cl-/HCO-3 exchange was measured in luminal (LMV) and basolateral (BLMV) membrane vesicles purified from rat medullary thick ascending limb (MTAL). Cl-/HCO-3 exchange in BLMV and LMV was inhibited by DIDS, with respective IC50 values of 3.2 +/- 0.9 and 15.2 +/- 5.2 microM, whereas Cl- conductances were DIDS insensitive. At constant external pH, BLMV 36Cl-/HCO-3 and 36Cl-/Cl- exchanges exhibited a sigmoidal pattern of activation as internal pH (pHi) increased from 6.1 to 8.0, whereas LMV 36Cl-/Cl- exchange was unchanged between pHi 6.7 and 7.8. The 165-kDa AE2 polypeptide and approximately 115-kDa AE1-related polypeptide were present only in BLMV. In contrast, AE1-related polypeptides of approximately 90 and 95 kDa were present not only in BLMV but also (in variable abundance) in LMV. We conclude that rat MTAL BLMV and LMV express distinct anion exchange activities and distinct sets of AE polypeptides. AE2 (and perhaps AE1) in BLMV likely contribute to HCO-3 absorption. In contrast, LMV exchangers may contribute to NaCl absorption via parallel coupling with the luminal Na+/H+ antiporters and/or may provide negative feedback regulation of HCO-3 absorption.

Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney.

In this study, an epitope-unmasking technique was used to immunolocalize AE2 anion exchanger polypeptide to basolateral plasma membranes of tubular epithelial cells in mouse kidney. Kidney AE2 immunostaining in mouse kidney was less prominent than in rat, consistent with the relative levels of AE2 mRNA and polypeptide in these two species. Glomeruli showed faint but consistent AE2 immunostaining, whereas proximal tubules were generally unstained. Macula densa epithelial cells displayed bright AE2 immunostaining, and cortical thick limbs were stained at a lower intensity. AE2 immunostaining was weak or absent in type B intercalated cells and principal cells of the cortical collecting duct, but increased in intensity in principal cells of the inner stripe of the outer medulla. AE2 staining in medullary thick limbs was also of greater intensity than in cortical thick limbs. AE2 staining was strong and uniform in the epithelial cells of the inner medullary collecting duct, and in epithelial cells of the papillary surface, the ureter, and the urinary bladder. Extratubular and epithelial cells of the inner medulla also showed punctate intracellular AE2 staining in a Golgi-like distribution that, in contrast to cell surface staining, was sodium dodecyl sulfate-sensitive. Golgi localization of AE2 epitope was confirmed by immunoperoxidase electron microscopy. Reverse transcription-PCR analysis of mouse kidney RNA detected AE2a, AE2b, and an AE2c2 transcript, but an AE2c1 transcript was absent. Unlike in rat, the mouse AE2c2 mRNA splice variant encoded a polypeptide with a novel predicted N-terminal amino acid sequence.

Autosomal dominant distal renal tubular acidosis is associated in three families with heterozygosity for the R589H mutation in the AE1 (band 3) Cl-/HCO3- exchanger.

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed approximately 20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.

Functional consequences of mutations in the transmembrane domain and the carboxy-terminus of the murine AE1 anion exchanger.

We have characterized mouse AE1-mediated 36Cl- influx and surface AE1 polypeptide expression in Xenopus oocytes injected with cRNA encoding two classes of loss-of-function mutants. The first arose spontaneously. Chimeric mutants constructed with a functional AE1 cDNA localized the site of spontaneous mutation to the transmembrane domain, and DNA sequencing revealed two missense mutations encoding the double-mutant polypeptide V728F/M7301. Each mutation individually produced only partial loss of AE1 transport activity, and coexpression of the individual mutants did not restore full activity. The functional changes produced by the mutations correlated with reduced fractional accumulation of polypeptides at the oocyte surface. The V728F/M7301 polypeptide expressed in mammalian cells displayed complete endoH resistance and rapid degradation. We also examined the effect on AE1 function of engineered removal of its hydrophilic carboxy-terminus. Both delta(c)890 and the internal deletion delta(c)890-917 were functionally inactive in Xenopus oocytes. Lack of transport activity correlated with lack of detectable polypeptide accumulation at the oocyte surface. Coexpression with wt AE1 of some, but not all, of these AE1 mutants partially suppressed wt AE1-mediated 36Cl- uptake. In contrast, coexpression with wt AE1 of soluble N-terminal AE1 fragments was not inhibitory.

Immunolocalization of AE2 anion exchanger in rat kidney.

The cellular and subcellular localizations of the AE2 anion exchanger in rat kidney have remained elusive despite detection of moderately abundant AE2 mRNA and AE2 polypeptide in all kidney regions. In this report a simple epitope unmasking technique has allowed the immunolocalization of AE2 antigenic sites in basolateral membranes of several rat kidney tubular epithelial cells. AE2 immunostaining was faint or absent in the glomerulus and proximal tubule, present in descending and ascending thin limbs, and stronger in the medullary thick ascending limb (MTAL). A lower staining intensity was found in cortical thick ascending limbs and even less in the distal convoluted tubule. In contrast, there was an enhanced staining in the macula densa. In principal cells (PC) of the connecting segment, AE2 was undetectable but gradually increased in intensity along the collecting duct, with strongest staining in inner medullary collecting duct (IMCD) PC. A sodium dodecyl sulfate-sensitive AE2-related Golgi epitope was also detected in some interstitial and endothelial cells of the inner medulla and in epithelial cells of IMCD and MTAL. Colchicine treatment of the intact animal altered the distribution of this Golgi-associated epitope but left plasmalemmal AE2 undisturbed. Reverse transcription-polymerase chain reaction detected AE2a, AE2b, and AE2c2 but not AE2cl transcripts in rat kidney mRNA. The results suggest a widespread occurrence of the AE2 protein in several renal epithelial cell types.

Modulation of c-fos and egr-1 expression in the isolated perfused kidney by agents that alter tubular work.

The isolated perfused rat kidney provides a model of selective hypoxia to the medullary thick ascending limb. To investigate the relationship between immediate early gene expression and the extent of hypoxic damage, we determined expression of the immediate early genes (IEG) c-fos and egr-1 in isolated perfused kidneys during standard perfusion and after various measures shown previously to be protective. mRNA levels of c-fos and egr-1 were markedly increased in kidneys after 90 minutes of standard perfusion with Krebs-Henseleit buffer containing albumin. Gene expression was most prominent in the outer medulla followed by papilla and cortex, a pattern reflected by the immunohistochemical demonstration of a prominent accumulation of both egr-1 and c-fos polypetides mainly in the medullary thick ascending limb (mTAL). Protective measures known to minimize morphological damage to the mTAL, including hyperoncotic perfusion, perfusion with glycine, or perfusion with a mixture of amino acids, decreased mRNA levels of c-fos and egr-1 in the outer medulla (by 50% and 35%, respectively) and the papilla (by 60 and 30%, respectively). Renal cortex showed only minor changes. In contrast, prevention of tubular transport by perfusion with 1 mM ouabain increased mRNA levels of c-fos and egr-1 in the outer medulla by 100% and 60%, respectively. Ouabain also dramatically increased mRNA levels of both IEGs in two lines of cultured renal epithelial cells. Changes in the level and distribution of the protein products of these IEGs were not detectable in perfused kidneys by immunohistochemistry. Hypoxic injury of the kidney stimulates IEG expression even in the absence of reperfusion. Protection against hypoxic injury in the mTAL correlates with suppression of IEG mRNA levels when protection is provided by amino acids or hyperoncotic perfusion, but not when provided by inhibition of Na,K-ATPase, which stimulates IEG expression. We conclude that diminished IEG expression is not a necessary concomitant of protection against hypoxic injury.

AE anion exchanger mRNA and protein expression in vascular smooth muscle cells, aorta, and renal microvessels.

Intracellular pH (pHi) is an important regulator of vascular smooth muscle cell (VSMC) tone, contractility, and intracellular Ca2+ concentration. Among the multiple transport processes that regulate VSMC pHi, Na(+)-independent Cl-/HCO3- exchange is the major process that acidifies VSMCs in response to an alkaline load. Here, we characterize, in native and cultured VSMCs, the expression of the AE family of band 3-related anion exchangers, the best studied of these Cl-/HCO3- exchangers. A 4.2-kb AE2 mRNA was present in aorta and in all cultured VSMCs tested. Cultured VSMCs and aorta both expressed a approximately 165-kDa AE2 polypeptide, but a approximately 115-kDa polypeptide was the major AE2-related protein in aorta. AE3 mRNA levels in VSMCs and in arterial tissue were significantly lower than those for AE2, but AE3 or related polypeptides were readily detected by immunoblot and immunolocalization experiments. The approximately 125-kDa AE3 polypeptide was present in an immortalized aortic VSMC line, but the predominant AE3 epitope in aorta and most cultured cells was associated with a polypeptide of M(r) approximately 80 kDa. These data demonstrate the expression in native arteries and in VSMCs of products of the AE2 and AE3 genes, which may contribute to Na(+)-independent Cl-/HCO3- exchange activity in these tissues and cells.

HCO3(-)-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid.

Although the AE1 chloride/bicarbonate exchanger of the red blood cell is among the most thoroughly investigated of membrane transport proteins, less is known about the related AE2 polypeptide of parietal cells. We have studied enzymatic deglycosylation of native AE2 polypeptide in gastric mucosal membranes from pig and rabbit. Deglycosylation of AE2 was maximal at low ionic strength. Deglycosylation of AE2 in membranes was preferentially inhibited by bicarbonate compared with other anions. This inhibition was maximal at alkaline pH and was not evident after detergent solubilization of AE2. Deglycosylation of AE2 increased its susceptibility to proteolytic degradation, but the presence of bicarbonate protected against this degradation. Bicarbonate failed to inhibit deglycosylation of the membrane glycoproteins AE1 and gastric H(+)-K(+)-adenosinetriphosphatase beta-subunit or deglycosylation of the soluble glycoproteins fetuin and ribonuclease B. These data suggest that bicarbonate induces a conformational change in AE2 that can protect the polypeptide from deglycosylation and proteolysis. Pig AE2 was purified in sodium dodecyl sulfate, and its monosaccharide composition was determined after blotting onto polyvinylidene fluoride membrane. AE2 was found to be devoid of sialic acid, with a composition suggestive of the presence of lactosamine-type chains.

Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).

A simple method for antigen retrieval in tissue sections and cell cultures is described. Because many antibodies recognize denatured proteins on western blots, but are poorly reactive by immunocytochemistry, the effect of applying sodium dodecyl sulfate (SDS) to cryostat sections of tissues and to cell cultures prior to immunostaining was examined. In many cases, a 5-min pretreatment with 1% SDS produced a dramatic increase in staining intensity by indirect immunofluorescence. Among the antibodies tested that showed a positive effect of SDS were an anti-Na/K-ATPase monoclonal antibody, an anti-AE1/2 anion exchanger polyclonal antipeptide antibody, a monoclonal anti-caveolin antibody, and an anti-rab4 monoclonal antibody. In other cases, including antibodies against gp330, aquaporin 1, and aquaporin 2, no effect of SDS was detected. The results show that SDS treatment can be used as a simple method of antigen retrieval in cryostat sections and on cultured cells. In some cases, antigens were not detectable without pretreatment with SDS.

The cytoplasmic and transmembrane domains of AE2 both contribute to regulation of anion exchange by pH.

We have compared regulation by pH of AE1 (band 3)- and AE2-mediated 36Cl- uptake into Xenopus oocytes. 36Cl- influx was assayed at varying extracellular pH (pHo) values between 9.0 and 5.0 under conditions in which corresponding intracellular pH (pHi) values were at or near steady-state. Wild type (WT) AE1 displayed a broad convex pH versus activity curve, with peak activity at pHo 7.0 and 63% of maximal activity at pHo 5.0. In contrast, WT AE2 displayed a steep pH versus activity curve, with peak activity at pHo9.0 and full suppression at pHo 5.0. The structural basis of these differing pH sensitivities was examined by expression of cRNAs encoding chimeric and truncated proteins. Mutant polypeptides were expressed in oocytes and detected at the cell surface. The AE2cyto/AE1memb polypeptide displayed a broad pH versus activity curve similar to that of WT AE1. In contrast, the AE1cyto/AE2memb polypeptide displayed a steep pH versus activity curve, which was shifted toward acid pH values from that of WT AE2 by 0.69 +/- 0.04 pHo units. Moreover, whereas the pH versus activity curves of AE2 Delta99 and WT AE2 were indistinguishable, AE2 Delta510 exhibited a pH versus activity curve acid-shifted from that of WT AE2 by 0.66 +/- 0.13 pHo units (indistinguishable from that of AE1cyto/AE2memb). The data suggest that a pH sensor resides within the transmembrane region of AE2. The affinity for protons of this pH sensor is influenced by a modifier site located between residues 99 and 510 of the N-terminal cytoplasmic domain of AE2. Acidification of oocytes with acetate suggested that pHi accounted for some but not all of the measured pH dependence of AE2.

Resistance to osmotic lysis in BXD-31 mouse erythrocytes: association with upregulated K-Cl cotransport.

The decreased osmotic fragility and reduced K+ content of BXD-31 mouse erythrocytes arise from variation at a single genetic locus. We compared ion transport in erythrocytes from BXD-31 mice and the parental strain, DBA/2J. The strains had similar rates for Na-K pump, Na/H exchange, Na-K-2Cl cotransport, Ca2+ activated K+ channel, or AE1-mediated SO4 transport. In contrast, K-Cl cotransport was twice as active in BXD-31 as in DBA/2J cells. Cl- dependent K+ efflux from BXD-31 cells displayed steep activation by acid pH (with maximal transport occurring at pH 6.75), whereas DBA/2J erythrocytes displayed a far less dramatic response to pH. Both strains displayed regulatory volume decrease in response to cell swelling. However, a 62% greater loss of cell K+ via K-Cl cotransport was observed in the BXD-31 strain. Furthermore the decreased osmotic fragility of BXD-31 red blood cells was normalized by treatment with nystatin to achieve normal cell K+ and water content. Thus upregulated K-Cl cotransport induces cell dehydration and K+ deficit in BXD-31 erythrocytes and causes their characteristic resistance to osmotic lysis.