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.
Highlights
A mutation (R298S) in NBCe1 induces a transport defect and causes proximal renal tubular acidosis
Mutation of the single N-glycosylation site in Anion exchanger 1 (AE1) (N642D) produced a single band that ran slightly faster than the lower high mannose form, confirming the presence of N-linked oligosaccharide on AE1 expressed in HEK cells (Fig. 2, lane 9)
AE1 expressed in HEK-293 cells contains mainly high mannose oligosaccharide, about one-third of this form as well as the complex form is present at the cell surface with the remaining two-thirds localized in the endoplasmic reticulum (ER) [11]
Summary
A mutation (R298S) in NBCe1 induces a transport defect and causes proximal renal tubular acidosis. Mutations in the gene encoding SLC4A4 (NBCe1), a protein expressed in the basolateral surface of the proximal tubule of the kidney, can give rise to proximal renal tubular acidosis and defective acid secretion by the kidney [2, 19] One of these mutations, R298S (human sequence), causes defective bicarbonate absorption across the basolateral membrane and metabolic acidosis due to an impairment in its functional expression (20 –22). By deleting the disordered acidic N-terminal and C-terminal linker regions, we were able to crystallize cd⌬54AE1 (residues 55–356) at neutral pH and to solve the structure to high resolution (2.1 Å), confirming the presence of a hydrogen-bonded network at physiological pH involving Arg283, Glu, and neighboring residues contained within a polar pocket Mutations in these residues affected the sensitivity of cdAE1 to structural changes induced by pH and by urea denaturation. We conclude that a substrate access tunnel is not present in the cytosolic domain of AE1 and is not an essential feature of the SLC4 family of bicarbonate transporters
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