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.

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 (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 C-terminal 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).

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.

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.

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 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)