2.A.31 The Anion Exchanger (AE) Family
Characterized protein members of the AE family are found in animals, plants and yeast. Uncharacterized AE homologues are present in bacteria (e.g., in Entercoccus faecium, 372 aas; gi 22992757; 29% identity in 90 residues). The animal AE proteins consist of homodimeric complexes of integral membrane proteins that vary in size from about 900 amino acyl residues to about 1250 residues. Their N-terminal hydrophilic domains may interact with cytoskeletal proteins and therefore play a cell structural role. The human AE1 binds carbonic anhydrase II (CAII) forming a 'transport metabolon' as CAII binding activates AE1 transport activity about 10 fold (Sterling et al., 2001). AE1 is also activated by interaction with glycophorin which also functions to target it to the plasma membrane (Young and Tanner, 2003). The membrane-embedded C-terminal domains may each span the membrane 13-16 times. According to the model of Zhu et al. (2003), it spans the membrane 16 times, 13 times as α-helix, and three times (TMSs 10, 11 and 14) possibly as β-strands. They preferentially catalyze anion exchange (antiport) reactions. Specific point mutations in human anion exchanger 1 (AE1) convert this electroneutral anion exchanger into a monovalent cation conductance. The same transport site within the AE1 spanning domain is involved in both anion exchange and cation transport (Barneaud-Rocca et al., 2011).
In humans, the AE family is composed of 10 paralogous members, among which are the proteins that perform Na+-independent Cl-HCO3- exchange (e.g., AEs 1-3), Na+-coupled anion exchange (e.g., NDCBE), and electroneutral (e.g., NBCn1) and electrogenic (e.g., NBCe1 and NBCe2) Na/HCO3- cotransport (Piermarini et al., 2007). These proteins are important for the regulation of intracellular pH (pHi) and play crucial roles in the epithelial absorption of HCO3- (e.g., in the renal proximal tubule) and secretion of HCO3- (e.g., in the pancreatic duct). All AE proteins are hypothesized to share a similar topology in the cell membrane. They have relatively long cytoplasmic N-terminal domains composed of a few hundred to several hundred residues, followed by 14 transmembrane (TM) domains, and end with relatively short cytoplasmic C-terminal domains composed of ~30 to ~90 residues (Vastermark et al. 2014). Although the C-terminal domain comprises a small percentage of the total protein, this domain in some cases (i) has PSD-95/Discs Large/ZO-1-binding motifs that may be important for protein-protein interactions (e.g., AE1, AE2, and NBCn1), (ii) is important for trafficking to the cell membrane (e.g., AE1 and NBCe1), and (iii) may provide sites for regulation of transporter function via protein kinase A phosphorylation (e.g., NBCe1).
Plasmalemmal Cl--HCO3- exchangers regulate intracellular pH and [Cl-] and cell volume. In polarized epithelial cells, they also contribute to transepithelial secretion and reabsorption of acid-base equivalents and of Cl-. Members of both the SLC4 and SLC26 mammalian gene families encode Na+-independent Cl--HCO3- exchangers. Human SLC4A1/AE1 mutations cause either the erythroid disorders spherocytic haemolytic anaemia or ovalocytosis, or distal renal tubular acidosis. SLC4A2/AE2 knockout mice die at weaning. Human SLC4A3/AE3 polymorphisms have been associated with seizure disorder. Although mammalian SLC4/AE polypeptides mediate only electroneutral Cl--anion exchange, trout erythroid AE1 also promotes osmolyte transport and increased anion conductance. Mouse AE1 is required for DIDS-sensitive erythroid Cl- conductance. A single missense mutation allows AE1 to mediate both electrogenic SO42--Cl- exchange or electroneutral, H+-independent SO42- -SO42- exchange (Alper 2006). In the Xenopus oocyte, the AE1 C-terminal cytoplasmic tail residues bind carbonic anhydrase II and are dispensable for Cl--Cl- exchange, but are required for Cl--HCO3- exchange. AE2 is acutely and independently inhibited by intracellular and extracellular H+, and this regulation requires integrity of the most highly conserved sequence of the AE2 N-terminal cytoplasmic domain.
AE1 in human red blood cells has been shown to transport a variety of inorganic and organic anions. Divalent anions may be symported with H+. Additionally, it catalyzes flipping of several anionic amphipathic molecules such as sodium dodecyl sulfate (SDS) and phosphatidic acid from one monolayer of the phospholipid bilayer to the other monolayer. The rate of flipping is sufficiently rapid to suggest that this AE1-catalyzed process is physiologically important in red blood cells and possibly in other animal tissues as well. Anionic phospholipids and fatty acids are likely to be natural substrates. However, it should be noted that the mere presence of TMSs enhances the rates of lipid flip-flop (Kol et al., 2001; Sapay et al., 2010). Four point mutations (L687P, D705Y, S731P and H734R) are associated with hemolytic anemia, and they confer channel-like cation transport to the human AE1. These point mutations convert the electroneutral anion exchanger to a cation-specific channel: the exchangers are no longer able to exchange Cl- and HCO3-, whereas they transport Na+ and K+ through a conductive mechanism (Guizouarn et al. 2007).
The heterozygous missense mutation E758K occurs in the human AE1/SLC4A1/band 3 gene in two unrelated patients with well-compensated hereditary spherostomatocytic anemia (HSt). Oocyte surface expression of AE1 E758K, in contrast to that of wild-type AE1, required coexpressed glycophorin A (GPA). The mutant polypeptide exhibited strong GPA dependence of DIDS-sensitive Cl- influx, trans-anion-dependent Cl- efflux, and Cl-/HCO3- exchange activities at near wild-type levels. Expression was also associated with GPA-dependent increases of DIDS-sensitive SO42- and oxalate uptake with altered pH dependence. Bumetanide- and ouabain-insensitive Rb+ influx was largely GPA-independent in Xenopus oocytes. Most of the increased cation transport probably reflected activation of endogenous oocyte cation permeability pathways, rather than cation translocation through the mutant polypeptide.
Renal Na+:HCO3- cotransporters have been found to be members of the AE family. They catalyze the reabsorption of HCO3- in the renal proximal tubule in an electogenic process that is inhibited by typical stilbene inhibitors of AE such as DIDS and SITS. They are also found in many other body tissues. At least two genes encode these symporters in any one mammal. A 10 TMS model has been presented (Romero and Boron, 1999), but this model conflicts with the 14 TMS model proposed for AE1. The transmembrane topology of the human pancreatic electrogenic Na+:HO3- transporter, NBC1, has been studied (Tatishchev et al., 2003). A TMS topology with N- and C-termini in the cytoplasm has been suggested. An extracellular loop determines the stoichiometry of Na+-HCO3- cotransporters (Chen et al., 2011).
In addition to the Na+-independent anion exchangers (AE1-3) and the Na+:HCO3- cotransporters (NBCs) (which may be either electroneutral or electrogenic), a Na+-driven HCO3-/Cl- exchanger (NCBE) has been sequenced and characterized (Wang et al., 2000). It transports Na+ + HCO3- preferentially in the inward direction and H+ + Cl- in the outward direction. This NCBE is widespread in mammalian tissues where it plays an important role in cytoplasmic alkalinization. For example, in pancreatic β-cells, it mediates a glucose-dependent rise in pH related to insulin secrection.
In humans, the AE family is composed of 10 members, among which are the proteins that perform Na+-independent Cl-HCO3- exchange (e.g., AEs 1-3), Na+-coupled anion exchange (e.g., NDCBE), and electroneutral (e.g., NBCn1) and electrogenic (e.g., NBCe1 and NBCe2) Na/HCO3 cotransport (Piermarini et al., 2007). These proteins are important for the regulation of intracellular pH (pHi) and play crucial roles in the epithelial absorption of HCO3- (e.g., in the renal proximal tubule) and secretion of HCO3- (e.g., in the pancreatic duct).
All AE proteins are hypothesized to share a similar topology in the cell membrane. They have relatively long cytoplasmic N-terminal domains composed of a few hundred to several hundred residues, followed by 10-14 transmembrane (TM) domains, and end with relatively short cytoplasmic C-terminal domains composed of ~30 to ~90 residues. Although the Ct domain comprises a small percentage of the size of the protein, this domain in some cases (i) has PSD-95/Discs Large/ZO-1-binding motifs that may be important for protein-protein interactions (e.g., AE1, AE2, and NBCn1), (ii) is important for trafficking to the cell membrane (e.g., AE1 and NBCe1), and (iii) may provide sites for regulation of transporter function via protein kinase A phosphorylation (e.g., NBCe1).
Animal cells in tissue culture expressing the gene-encoding the ABC-type chloride channel protein CFTR (TC #3.A.1.202.1) in the plasma membrane have been reported to exhibit cyclic AMP-dependent stimulation of AE activity. Regulation was independent of the Cl- conductance function of CFTR, and mutations in the nucleotide-binding domain #2 of CFTR altered regulation independently of their effects on chloride channel activity. These observations may explain impaired HCO3- secretion in cystic fibrosis patients.
Plants and yeast have anion transporters that in both the pericycle cells of plants and the plasma membrane of yeast cells export borate or boric acid (pKa = 9.2) (referred to below as 'boron') (Takano et al., 2002). In A. thaliana, boron is exported from pericycle cells into the root stelar apoplasm against a concentration gradient for uptake into the shoots. In S. cerevisiae, export is also against a concentration gradient. The yeast transporter recognizes HCO3-, I-, Br-, NO3- and Cl- which may be substrates. Tolerance to boron toxicity in cereals is known to be associated with reduced tissue accumulation of boron. Expression of genes from roots of boron-tolerant wheat and barley with high similarity to efflux transporters from Arabidopsis and rice lowered boron concentrations due to an efflux mechanism (Reid, 2007). The mechanism of energy coupling is not known, nor is it known if borate or boric acid is the substrate. Several possibilities (uniport, anion:anion exchange and anion:cation exchange) can account for the data (Takano et al., 2002).
The SLC4 family consists of 10 genes (SLC4A1-5; SLC4A7-11). All have similar hydropathy plots-consistent with 10-14 transmembrane segments. Nine encode proteins that transport HCO3-. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3- exchangers (AE1-3) and five Na+-coupled HCO3- transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na+-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na+-coupled HCO3- transporters and all three AEs are electroneutral (Romero et al. 2013). Two others (AE4, SLC4A9 and BTR1, SLC4A11) are not characterized. Most, though not all, are inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 proteins play roles in acid-base homeostasis, transport of H+ or HCO3- by epithelia, as well as the regulation of cell volume and intracellular pH (Romero et al. 2013).
The crystal structure of AE1(CTD) at 3.5 angstroms has been determined (Arakawa et al. 2015). The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed identification of the likely anion-binding position in the AE1(CTD), and to propose a possible transport mechanism that could explain why selected mutations lead to disease. The 3-d structure confirmed that the AE family is a member of the APC superfamily (Vastermark et al. 2014), and this conclusion has been further confirmed (Chang and Geertsma 2017).
Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Ficici et al. 2017 hypothesized that the unknown, inward-facing conformation results from inversion of this asymmetry, and the proposed a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, they further predicted that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which are reviewed (Ficici et al. 2017).
The physiologically relevant transport reaction catalyzed by anion exchangers of the AE family is:
Cl- (in) + HCO3- (out) ⇌ Cl- (out) + HCO3- (in).
That for the Na+:HCO3- cotransporters is:
Na+ (out) + nHCO3- (out) → Na+ (in) + nHCO3- (in).
That for the Na+/HCO3-:H+/Cl- exchanger is:
Na+ (out) + HCO3- (out) + H+ (in) + Cl- (in) ⇌ Na+ (in) + HCO3- (in) + H+ (out) + Cl- (out).
That for the boron efflux protein of plants and yeast is:
boron (in) → boron (out)
Anion exchanger (HCO3-:Cl- antiporter; also called Band 3, transports a variety of inorganic and organic anions. Anionic phospholipids are ''flipped'' from one monolayer to the other in erythrocytes and the nephron). Mutations cause Southeast Asian ovalocytosis (SAO) hereditary spherocytosis and distal renal tubular acidosis (dRTA) with impaired acid secretion in humans (Chu et al., 2010; Kittanakom et al., 2008; Toye et al., 2008). Activated by glycophorin A (Stewart et al., 2011). Kanadaptin (SLC4a1; Q9BWU0) has been reported to interact with kidney AE1 (Chen et al. 1998), but Kittanakom et al. 2005 could not detect this interaction. Hübner et al. 2002, 2003 reported nuclear and mitochondrial targetting of kanadaptin. Some point mutations allow the normally electroneutral anion exchanger to catalyze Na+ and K+ conductance or induce a cation leak in the still functional anion exchanger. A structural model of the AE1 membrane spanning domain, based on the structure of Uracil-proton symporter, suggests that there is a unique transport site comprising TMSs 3-5 and 8 that may function in anion exchange and cation leak (Barneaud-Rocca et al. 2013). The spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions with ankyrin, which binds to both spectrin and a beta-hairpin loop in the cytoplasmic domain of band 3 (Stefanovic et al. 2007). A detailed transport mechanism has been proposed: It involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane (Ficici et al. 2017). The structure-function relationships of band 3 have been reviewed (Abbas et al. 2018).
SLC4A1 of Homo sapiens
Anion exchanger-2 (AE2; 1241 aas); catalyzes solium-independent Cl-:HCO3- exchange; forms a metabolon with the carbonic anhydrase, Car2 (AAH11949) (Gonzalez-Begne et al., 2007) and with another carbonic anhydrase, CAIX (Q16790) which also forms complexes with AE1 and AE3, activating all of these transporters about 30% (Morgan et al. 2007). Cys residues play a role in pH sensitivity, but are not essential for activity (Reimold et al. 2013).
SLC4A2 of Homo sapiens
Anion exchange protein 3 (AE 3; AE3) (Anion exchanger 3) (CAE3/BAE3) (Cardiac/brain band 3-like protein) (Neuronal band 3-like protein) (Solute carrier family 4 member 3). The motif LDADD is a binding site for the N-terminal basic region of carbonic ahnydride II (not I), facilitating bicarbonate transport (Moraes and Reithmeier 2012). An isoform of this Na+-independent Cl-/HCO3- exchanger is involved in myocardial pHi recovery from intracellular alkalization (Chiappe de Cingolani et al. 2006).
SLC4A3 of Homo sapiens
Anion exchanger 1 (AE1, Band 3 anion exchanger, Slc4a1) of 918 aas and 13 TMSs in a 1 + 12 TMS arrangement that looks like a 1 + 6 + 6 arrangement. The protein functions both as a transporter that mediates electroneutral inorganic anion exchange (e.g., Cl- against HCO3-) across the cell membrane and as a structural protein. It is a major integral membrane glycoprotein of the erythrocyte membrane required for normal flexibility and stability of the erythrocyte membrane and for normal erythrocyte shape via the interactions of its N-terminal cytoplasmic domain with cytoskeletal proteins, glycolytic enzymes, and hemoglobin. It mediates chloride-bicarbonate exchange in the kidney, and is required for normal acidification of the urine. A mutant containing the sole C462 can drive a marginal Cl- current, but the minimal configuration necessary to get optimal functional expression includes residues C462, C583 and C588 (Martial et al. 2007). Trout AE1 can function both as a antiporter or a channel, and mutations affecting one or the other function or both have been isolated (Martial et al. 2006).
AE1 of Oncorhynchus mykiss (Rainbow trout) (Salmo gairdneri)
The Na+-driven Cl--HCO3- exchanger, ABTS-1 (extrudes Cl- from the cell) (Bellemer et al., 2011).
ABTS-1 of Caenorhabditis elegans (B3WFV9)
Electrogenic sodium bicarbonate cotransporter 1, NBCe1 (Sodium bicarbonate cotransporter, NBC) (Na+/HCO3- cotransporter) (Solute carrier family 4 member 4) (kNBC1) of 1079 aas (Boron et al. 2009). Mutations cause proximal renal tubular acidosis and ocular pathology (Demirci et al. 2006). NBCe1, together with carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes (Theparambil et al. 2017).
SLC4A4 of Homo sapiens
Sodium bicarbonate cotransporter, NBC, of 1194 aas and 12 TMSs. It can transport HCO3- (or a related species, such as CO32-). It functions in adaptation to pH stress, both acid- and base-stress (Cai et al. 2017).
NBC of Litopenaeus vannamei (Pacific white shrimp)
Probable electroneutral Na+-driven bicarbonate transporter, NBC, of 1214 aas and 13 putative TMSs. It possibly functions by Cl- exchange as does the human ortholog (TC# 2.A.31.2.4). It is present in the flagellar plasma membrane (Gunaratne et al. 2006).
NBC of Strongylocentrotus purpuratus
Electrogenic Na+:HCO3- cotransporter, rkNBC (NBCI, NBCe1, SLC4A4). The human orthologue, NBCe1/SLC4A4;Q9Y6R1, is stimulated by carbonic anhydrase II and IX which together form a transport metabolon (Becker and Deitmer, 2007; Orlowski et al., 2012). The bicarbonate channel in the C-terminal two-thirds of the protein is regulated by the N-terminal hydrophilic domain (Chang et al., 2008) which may actually form part of the channel (Zhu et al. 2013). The topological location and structural importance of the NBCe1-A residues mutated in proximal renal tubular acidosis have been identified (Zhu et al., 2010). Defective membrane expression of the Na+/HCO3- cotransporter NBCe1 is associated with familial migraines (Suzuki et al., 2010). This transporter plays a role in pH regulation and bicarbonate transport in the kideny proximal tubule (Yamazaki et al., 2011; Zhu et al. 2013). The three lysyl residues in the KKMIK motif in TMS5 plays a role in DIDS inhibition (Lu and Boron 2007). Electrogenicity of NBCe1 probably depends on interactions between TM1-5 and TM6-13 (Choi et al. 2007).
rkNBC (NBCl) of Rattus norvegicus
Electrogenic Na+:HCO3- symporter/Cl- antiporter, NCBE (regulates intracellular pH) (Wang et al., 2000; Damkier et al., 2010). Expressed in specific brain cell types; glycosylation required for functional expression (Chen et al., 2008). Loss reduces brain ventricle volume, impairs visual function and protects against fatal epileptic seizures in mice (Hilgen et al. 2012). NCBE is involved in the control of neuronal pH and excitability; may contribute to the secretion of cerebrospinal fluid (Jacobs et al., 2008). The human orthologue (Q6U841) is an electroneutral Na+/HCO3- cotransporter (NBCn2 or NCBE) with Cl- self exchange activity (Parker et al., 2008). NCBE/NBCn2 is predominantly expressed in the central nervous system (CNS) with highest concentrations in the choroid plexus. Its primary function is to regulate intracellular neuronal pH and to maintain the pH homeostasis across the blood-cerebrospinal fluid barrier. NCBE is predicted to contain at least 10 transmembrane helices. The N- and C- termini are both cytoplasmic, with a large N-terminal domain (Nt-NCBE) and a relatively small C-terminal domain (Ct-NCBE). The Nt-NCBE is likely to be involved in bicarbonate recognition and transport and contains key areas of regulation involving pH sensing and protein-protein interactions. It has an intrinsic disordered structure (Bjerregaard-Andersen et al. 2013).
NCBE of Mus musculus
Electroneutral Na+-driven HCO3-/Cl- (+ H+) exchanger, NDCBE1; SLC4A8; NBCn1 (Boron et al. 2009).
SLC4A8 of Homo sapiens
Squid Na+-dependent Cl-/HCO3- symporter, NDCBE (1198 aas) (Piermarini et al., 2007b)
NDCBE of Loligo pealei (Q8I8G6)
Electrogenic HCO3-:Na+ symporter, NBCe2 (3:1 stoichiometry) (Millar and Brown, 2008)
SLC4A5 of Homo sapiens
The electrogenic Na+ bicarbonate cotransporter (NBCe1) (Sussman et al., 2009).
NBCe1 of Danio rerio (Q3ZMH2)
Boron efflux transporter for xylem loading (Takano et al., 2002; Miwa et al., 2010)
BOR1 of Arabidopsis thaliana
Borate/boric acid (boron) efflux transporter, Bor1. Involved in boron deficiency tolerance (Cañon et al. 2013).
Bor1 of Citrus macrophylla
Borate exporter of 666 aas and 13 TMSs with 5 cytooplasmic α-helices, Bot1. Also known as the barley root anion-permeable transporter. Confers tolerance to boron. It is believed to be trimeric. Transport is dependent on Na+ (Nagarajan et al. 2016).
Bot1 of Hordeum vulgare
Boron transporter, NcBC1 or SLC4a11. In the absence of borate, it functions as a Na+ and OH- (H+) channel; in the presence of borate, it functions as an electrogenic Na+-coupled borate cotransporter (Park et al., 2004). Three genetic corneal dystrophies (congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy) arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. It can mediate water flux at a rate comparable to aquaporin in a process that is independent of solute transport (Vilas et al. 2013). Reviewed by Patel and Parker 2015. A 3-d homology model rationalizes vaious pathology-causing mutations (Badior et al. 2016).
SLC4A11 of Homo sapiens