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). Local attraction of substrates and co-substrates enhances weak acid and base transmembrane transport, and principles in the attraction and facilitation of weak acid and base transport substrates, including monocarboxylates, ammonium, bicarbonate, and arsenite, plus protons as a co-substrate have been elucidated (Epalle and Beitz 2022).

In humans, the AE family (also called the SLC4 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 (pH_i) 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 TMSs, 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).

As noted above, the AE or solute carrier 4 (SLC4) family includes 10 members (SLC4A1-5, SLC4A7-11), which are expressed in multiple tissues in the human body. SLC4 family members differ in their substrate dependence, charge transport stoichiometry, and tissue expression. Their common function is responsible for the transmembrane exchange of multiple ions, which is involved in many important physiological processes, such as erythrocyte CO₂ transport and the regulation of cell volume and intracellular pH (Dong et al. 2023), and they play roles in human genetic diseases, with functional disorders causing diseases. Progress about the structures, functions and disease correlations of SLC4 members have been reviewed (Dong et al. 2023).

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 SO₄^2--Cl^- exchange or electroneutral, H⁺-independent SO₄^2- -SO₄^2- 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). Carbonate, rather than bicarbonate, is the species absorbed across the proximal tubule basolateral membrane, whereas in the collecting duct, bicarbonate is transported (Kurtz and Schwartz 2023).

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 SO₄^2- 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 (pH_i) 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)