title: A substrate access tunnel in the cytosolic domain is not an essential feature of the solute carrier 4 (SLC4) family of bicarbonate transporters.
abstract: Anion exchanger 1 (AE1; Band 3; SLC4A1) is the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters that includes chloride/bicarbonate AEs and Na(+)-bicarbonate co-transporters (NBCs). These membrane proteins consist of an amino-terminal cytosolic domain involved in protein interactions and a carboxyl-terminal membrane domain that carries out the transport function. Mutation of a conserved arginine residue (R298S) in the cytosolic domain of NBCe1 (SLC4A4) is linked to proximal renal tubular acidosis and results in impaired transport function, suggesting that the cytosolic domain plays a role in substrate permeation. Introduction of single and double mutations at the equivalent arginine (Arg(283)) and at an interacting glutamate (Glu(85)) in the cytosolic domain of human AE1 (cdAE1) had no effect on the cell surface expression or the transport activity of AE1 expressed in HEK-293 cells. In addition, the membrane domain of AE1 (mdAE1) efficiently mediated anion transport. A 2.1-Å resolution crystal structure of cdΔ54AE1 (residues 55-356 of cdAE1) lacking the amino-terminal and carboxyl-terminal disordered regions, produced at physiological pH, revealed an extensive hydrogen-bonded network involving Arg(283) and Glu(85). Mutations at these residues affected the pH-dependent conformational changes and stability of cdΔ54AE1. As these structural alterations did not impair functional expression of AE1, the cytosolic and membrane domains operate independently. A substrate access tunnel within the cytosolic domain is not present in AE1 and therefore is not an essential feature of the SLC4 family of bicarbonate transporters.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Shnitsar
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Xuyao
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Charles
authors.initials: C
authors.lastname: Calmettes
authors.affiliation: Department of Biochemistry and Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Arghya
authors.initials: A
authors.lastname: Basu
authors.affiliation: Department of Biochemistry and Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Trevor F
authors.initials: TF
authors.lastname: Moraes
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: r.reithmeier@utoronto.ca.
authors.forename: Reinhart A F
authors.initials: RAF
authors.lastname: Reithmeier
mesh_terms: D019943:Amino Acid Substitution; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000502:physiology; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; C454201:SLC4A1 protein, human; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters
keywords: Anion Exchanger 1 (AE1); Anion Transport; Band 3; Membrane Proteins; Protein Folding; SLC4; Site-directed Mutagenesis; Trafficking; Transporters; X-ray Crystallography