Abstract
AE1 is the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane. We have used scanning cysteine mutagenesis and sulfhydryl-specific chemistry to identify pore-lining residues in the Ser643-Ser690 region of the protein. The Ser643-Ser690 region spans transmembrane segment 8 of AE1 and surrounds Glu681, which may reside at the transmembrane permeability barrier. Glu681 also directly interacts with some anions during anion transport. The introduced cysteine mutants were expressed by transient transfection of HEK293 cells. Anion exchange activity was assessed by measurement of changes of intracellular pH, which follow transmembrane bicarbonate movement mediated by AE1. To identify residues that might form part of an aqueous transmembrane pore, we measured anion exchange activity of each introduced cysteine mutant before and after incubation with the sulfhydryl reagents para-chloromercuribenzene sulfonate and 2-(aminoethyl)methanethiosulfonate hydrobromide. Our data identified transmembrane mutants A666C, S667C, L669C, L673C, L677C, and L680C and intracellular mutants I684C and I688C that could be inhibited by sulfhydryl reagents and may therefore form a part of a transmembrane pore. These residues map to one face of a helical wheel plot. The ability to inhibit two intracellular mutants suggests that transmembrane helix 8 extends at least two helical turns beyond the intracellular membrane surface. The identified hydrophobic pore-lining residues (leucine, isoleucine, and alanine) may limit interactions with substrate anions.
Highlights
AE1 in the erythrocyte membrane (50% of integral protein [9]) has made the protein a model for the study of transport protein structure and function [10, 11]
We have identified a sequence of leucine, isoleucine, and alanine residues that lie on one face of an ␣-helix and, when mutated to cysteine, are susceptible to inhibition by pCMBS and MTSEA
Because Glu681 is accessible to Woodward’s reagent K and has been implicated as one residue that interacts with anions, at least during sulfate transport, we decided to examine the region surrounding Glu681 to find other residues that might form the anion translocation channel
Summary
Materials—Restriction endonucleases were from New England Biolabs. ECL chemiluminescent reagent, horseradish peroxidase conjugated to sheep anti-mouse IgG, Hyperfilm, and Immobilon-P membrane were from Amersham Pharmacia Biotech. Polymerase chain reaction was performed using an ERICOMP thermal cycler and either Vent DNA polymerase (New England Biolabs) or Pwo polymerase (Boehringer Mannheim). Anion Exchange Assays—HEK293 cells were grown on top of 7 ϫ 11-mm glass coverslips in 60-mm tissue culture dishes and transfected as described. Uninhibited rates of anion exchange were determined by perfusion with chloride Ringer’s followed by chloridefree Ringer’s, chloride Ringer’s, and chloride-free Ringer’s. The cuvette was perfused with 10 ml of chloride-free Ringer’s containing either: 5 mM MTSEA or 0.2 mM pCMBS. Cells were incubated with the inhibitor for a total of 8 min, followed by washing with chloride-free Ringer’s until the fluorescence base line was stable (350 –550 s). All other procedures followed standard protocols [42]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
