Abscisic acid influx into human nucleated cells occurs through the anion exchanger AE2

Abscisic acid (ABA) is a hormone conserved from cyanobacteria to higher plants, where it regulates responses to environmental stimuli. ABA also plays a role in mammalian physiology, pointedly in inflammatory responses and in glycemic control. As the animal ABA receptor is on the intracellular side of the plasma membrane, a transporter is required for the hormone’s action. Here we demonstrate that ABA transport in human nucleated cells occurs via the anion exchanger AE2. Together with the recent demonstration that ABA influx into human erythrocytes occurs via Band 3, this result identifies the AE family members as the mammalian ABA transporters.     

How pH regulates a pH regulator: a regulatory hot spot in the N-terminal cytoplasmic domain of the AE2 anion exchanger

Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na⁺-independent chloride-bicarbonate exchanger genes and multiple Na⁺-bicarbonate cotransporter and Na⁺-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na⁺-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.     

Identification of the full-length AE2 (AE2a) isoform as the Golgi-associated anion exchanger in fibroblasts.

Na(+)-independent Cl(-)/HCO(3)(-) exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid-base balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that show prominent Golgi staining with antibodies to AE2. Third, antisense oligonucleotides directed against the translational initiation site of the AE2a mRNA markedly inhibited the expression of the endogenous AE2 protein in the Golgi. Finally, transient expression of the GFP-tagged full-length AE2a protein resulted in predominant accumulation of the fusion protein in the Golgi membranes in COS-7 and CHO-K1 cells. Golgi localization of the AE2a probably involves its oligomerization and/or association with the recently identified Golgi membrane skeleton, because a substantial portion of both the endogenous AE2a and the GFP-tagged fusion protein resisted detergent extraction in cold. (J Histochem Cytochem 49:259-269, 2001)     

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.     

Novel topology in C-terminal region of the human plasma membrane anion exchanger,AE1

Human AE1 performs electroneutral exchange of Cl(-) for HCO(3)(-) across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between Phe(806) and Cys(885), were expressed by transient transfection of HEK293 cells. Topology of the region was determined by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent biotin maleimide, with or without prior labeling with the membrane-impermeant reagents, bromotrimethylammoniumbimane bromide (qBBr) and lucifer yellow iodoacetamide (LYIA). Phe(806)-Leu(835), Ser(852)-Ala(855), and Ile(872)-Cys(885) were labeled by biotin maleimide, suggesting their location in an aqueous environment. In contrast, Phe(836)-Lys(851) and Ser(856)-Arg(871) were not labeled by biotin maleimide and therefore localize to the plane of the bilayer, as transmembrane segments (TM). Labeling by qBBr revealed that Pro(815)-Lys(829) and Ser(852)-Ala(855) are accessible to the extracellular medium. Pro(815)-Lys(829) mutants were also labeled with LYIA. Mutants Ile(872)-Cys(885) were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.     

Alkaline-shifted pHo sensitivity of AE2c1-mediated anion exchange reveals novel regulatory determinants in the AE2 N-terminal cytoplasmic domain

The mouse anion exchanger AE2/SLC4A2 Cl(-)/HCO(-)(3) exchanger is essential to post-weaning life. AE2 polypeptides regulate pH(i), chloride concentration, cell volume, and transepithelial ion transport in many tissues. Although the AE2a isoform has been extensively studied, the function and regulation of the other AE2 N-terminal variant mRNAs of mouse (AE2b1, AE2b2, AE2c1, and AE2c2) have not been examined. We now present an extended analysis of AE2 variant mRNA tissue distribution and function. We show in Xenopus oocytes that all AE2 variant polypeptides except AE2c2 mediated Cl(-) transport are subject to inhibition by acidic pH(i) and to activation by hypertonicity and NH(+)(4). However, AE2c1 differs from AE2a, AE2b1, and AE2b2 in its alkaline-shifted pH(o)((50)) (7.70 +/- 0.11 versus 6.80 +/- 0.05), suggesting the presence of a novel AE2a pH-sensitive regulatory site between amino acids 99 and 198. Initial N-terminal deletion mutagenesis restricted this site to the region between amino acids 120 and 150. Further analysis identified AE2a residues 127-129, 130-134, and 145-149 as jointly responsible for the difference in pH(o)((50)) between AE2c1 and the longer AE2a, AE2b1, and AE2b2 polypeptides. Thus, AE2c1 exhibits a unique pH(o) sensitivity among the murine AE2 variant polypeptides, in addition to a unique tissue distribution. Physiological coexpression of AE2c1 with other AE2 variant polypeptides in the same cell should extend the range over which changing pH(o) can regulate AE2 transport activity.     

Basolateral localization of anion exchanger 2 (AE2) and actin in acid-secreting (parietal) cells of the human stomach

Basolateral uptake of chloride by the HCl-secreting parietal cells of the gastric (oxyntic) glands is most likely mediated by a HCO^– ₃/Cl^– anion exchange mechanism. Circumstantial evidence indicates that in rodents the anion exchange proceeds through an anion exchanger 2(AE2)-like membrane protein. In the present study, we raised antibodies against a bacterial fusion protein expressing a -26-kDa portion of the human AE2 sequence. These antibodies were used to identify and localize AE2 in the human stomach. Here we report that the mucosa of the human stomach expresses an 160-kDa immunoreactive form of AE2 containing the AE2-specific exoplasmic domain (Z-loop) as identified by polymerase chain reaction. Immunostaining specific for AE2 was restricted to the basolateral membrane domain of parietal cells and was also detected in small epithelial cells localized in the glandular isthmus region. The latter cells most likely represent pre-parietal cells. Parietal cells were identified by simultaneous and sequential labelling with antibodies against the gastric H⁺, K⁺-ATPase and actin, respectively. Both actin and the H⁺, K⁺-ATPase were localized along the apical membrane of parietal cells and the membrane of their secretory intracellular canaliculi. In addition, actin was shown to be colocalized with AE2 along the basolateral cell surface. Discontinuous staining for AE2 coincided with infoldings of the basolateral plasma membrane labelled by the actin antibody. These observations indicate that AE2 might be placed at specialized (folded) microdomains of the basolateral cell surface by linkage to the actin-based cytoskeleton.Large parts of this publication belong to the MD thesis of B. Warrings. B. Warrings and T. Jöns should be considered alphabetically listed first authors who made equally strong contributions to this study     

钠-氢交换蛋白 mRNA在人胎儿两个发育阶段中的组织特异性表达

钠－氢交换蛋白（Na^ -H^ exchangers,NHE）至少包含6个不同的亚型,生长因子可激活其表达。目前,对在发育过程中NHE的表达了解甚少。本文利用RT－PCR观察了4种NHE亚型的mRNA在人胎儿的两个不同发育阶段（11周、16周）在不同组织中的表达,以研究它们的发育调控。结果显示,NHE1 mRNA在两种胎龄的多种组织中均有表达,和16周胚胎相比,11周的胚胎的NHE1 mRNA的表达较弱,并且表现出明显的组织差异。据此推测,NHE1的管家（house-keeping）功能可能至少在11周就开始形成,而最迟在16周已基本建立;NHE2和NHE3 mRNA在11周和16周的胚胎组织中的特异性表达呈现相反的变化趋势及组织分布上的重叠,后者与NHE2和NHE3在成人组织中的分布及功能的重叠的特点相吻合;NHE5 mRNA的表达在11周的胚胎组织中比较普遍,而在16周的胚胎组织中则局限在小脑组织中,本研究表明,在人胚胎发育11－16周期间,NHE的组织特异性表达表现出时间依赖性的调控,而在不迟于胚胎发育的第16周,具有“管家功能”的NHE1的基因表达已与成人相似。     

The AE2 anion exchanger is necessary for the structural integrity of the Golgi apparatus in mammalian cells

The structural integrity of the Golgi apparatus is known to be dependent on multiple factors, including the organizational status of microtubules, actin and the ankyrin/spectrin-based Golgi membrane skeleton, as well as vesicular trafficking and pH homeostasis. In this respect, our recently identified Golgi-associated anion exchanger, AE2, may also be of importance, since it potentially acts as a Golgi pH regulator and as a novel membrane anchor for the spectrin-based Golgi membrane skeleton. Here, we show that inhibition (>75%) of AE2 expression by antisense oligonucleotides in COS-7 cells results in the fragmentation of the juxtanuclear Golgi apparatus and in structural disorganization of the Golgi stacks, the cisternae becoming generally shorter, distorted, vesiculated and/or swollen. These structural changes occurred without apparent dissociation of the Golgi membrane skeletal protein Ankyrin(195), but were accompanied by the disappearance of the well-focused microtubule-organizing center (MTOC), suggesting the involvement of microtubule reorganization. Similar changes in Golgi structure and assembly of the MTOC were also observed upon transient overexpression of the EGFP-AE2 fusion protein. These data implicate a clear structural role for the AE2 protein in the Golgi and in its cytological positioning around the MTOC.