Identification of residues lining the translocation pore of human AE1, plasma membrane anion exchange protein

AE1 is the chloride/bicarbonate anion exchanger of the erythrocyte plasma m embrane. We have used scanning cysteine mutagenesis and sulfhydryl-specific chemistry to identify pore-lining residues in the Ser(643)-Ser(690) region of the protein. The Ser(643)-Ser(690) region spans transmembrane segment 8 of AE1 and surrounds Glu(681), which may reside at the transmembrane perme ability barrier. Glu(681) also directly interacts with some anions during a nion transport. The introduced cysteine mutants were expressed by transient transfection of HEK293 cells. Anion exchange activity was assessed by meas urement of changes of intracellular pH, which follow transmembrane bicarbon ate 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)methanethios ulfonate hydrobromide, Our data identified transmembrane mutants A666C, S66 7C, L669C, L673C, L677C, and L680C and intracellular mutants I684C and I688 C that could be inhibited by sulfhydryl reagents and may therefore form a p art of a transmembrane pore. These residues map to one face of a helical wh eel plot. The ability to inhibit two intracellular mutants suggests that tr ansmembrane helix 8 extends at least two helical turns beyond the intracell ular membrane surface. The identified hydrophobic pore-lining residues (leu cine, isoleucine, and alanine) may limit interactions with substrate anions .