Structural Model of the Anion Exchanger 1 (SLC4A1) and Identification of Transmembrane Segments Forming the Transport Site

Abstract

Background: There is still no high resolution three-dimensional structure available for the membrane-spanning domain of anion exchanger 1 (AE1). Results: A three-dimensional model of AE1 membrane-spanning domain has been generated in silico and experimentally assessed. Conclusion: Transmembrane segments forming AE1 transport site have been identified. Significance: This is the first three-dimensional model of AE1 membrane-spanning domain based on a cation symporter.The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na ؉ and K ؉ conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracilproton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3-5 and TM8 that should function as an anion exchanger and a cation leak.The anion exchanger 1 (AE1, 2 SLC4A1, or band 3) is the main membrane protein in vertebrate red cells. It fulfills different tasks in these cells: a structural role by linking plasma membrane to cytoskeleton, a respiratory role by improving CO 2 transport capacity of red cells, and an antigenic function (Diego blood group and senescence), and it is also involved in cytokinesis and red cell volume regulation (1). At the molecular level, this protein is divided into two main structural domains: a cytoplasmic N-terminal domain (about 400 amino acids) and a membrane-spanning domain (about 450 amino acids) with a short C-terminal tail in the cytoplasm. These two entities seem to function independently: the large cytoplasmic domain is involved in interactions with enzymes, hemoglobin, and structural proteins, whereas the membrane-spanning domain is responsible for the transport activity of the protein (2, 3). In addition to red cells, AE1 is also expressed in kidney ␣-intercalated cells and cardiac myocytes (4). In physiological conditions, AE1 exchanges one chloride for one bicarbonate by an electroneutral transport mechanism with the driving force for ion movement provided by their electrochemical gradient. A few years ago, some specific point mutations were characterized and proposed to convert the electroneutral anion exchanger into a cation-conductive pathway (5, 6). These mutations are associated with human pathologies (hered...

Structure, Function, and Trafficking of SLC4 and SLC26 Anion Transporters

“…The N and C termini are in the cytosol and the N-glycosylation site (N642) faces the cell exterior. The model is supported with recent experimental findings of TM segments (TM3-5 and 8) involved in ion permeation (Barneaud-Rocca et al, 2013). TM segments 3 and 10 consist of a central b-strand joined to a short helical segment.…”

“…The membrane domain of AE1 has also been modeled on the E. coli UraA uracil permease (Barneaud-Rocca, Etchebest, & Guizouarn, 2013), which is a proton-uracil symporter. A search using the human AE1 sequence on the Phyre2 Protein Fold Recognition Server (Kelley & Sternberg, 2009) found only UraA (Reithmeier, unpublished).…”

“…The homology model for the membrane domain of AE1 based on the crystal structure of the E. coli UraA uracil permease(Barneaud-Rocca et al, 2013). (A) A threedimensional model for the membrane domain of the human AE1 monomer based on a Phyre2 homology search that identified UraA as the sole membrane protein in the PDB with a similar fold.…”

Structure of Bor1 supports an elevator transport mechanism for SLC4 anion exchangers

“…In lungs, the partial pressure of CO 2 is lower and the process is reversed, thus driving cellular respiration. Decades of biochemical characterization have provided a wealth of information about Band 3 topology and multimerization (11)(12)(13)(14)(15), as well as the identification of amino acid residues likely involved in substrate transport (16)(17)(18)(19)(20)(21)(22)(23). However, our understanding of transport by SLC4 anion exchangers remains limited by a paucity of structural data, for the SLC4 family in general and Bor1 in particular.…”

Senescent Sickle Erythrocytes and Endothelial Adhesion via Band 3 Peptides

“…First, consider the basic band 3 molecule (B3M), it is the third band seen when erythrocyte membrane proteins are examined by gel electrophoresis and it is a transmebrane protein important in the erythrocyte's structure that functions as a channel for exchanging bicarbonate ions for chloride ions [1]. The B3M's are normally dispersed on the erythrocytes surface but in senescent erythrocytes some of their Hemoglobin (Hgb) molecules have broken down and formed hemichromes that attach to the cytoplasmic portion of the erythrocyte's membrane causing the band 3 molecules in the vicinity to cluster above them on the erythrocyte's surface [2,3].…”