Topology of the Region Surrounding Glu681 of Human AE1 Protein, the Erythrocyte Anion Exchanger

doi:10.1074/jbc.273.35.22545

Topology of the Region Surrounding Glu⁶⁸¹ of Human AE1 Protein, the Erythrocyte Anion Exchanger*

From the ^‡Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and the ^§Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

Abstract

AE1 protein transports Cl⁻ and HCO₃ ⁻ across the erythrocyte membrane by an electroneutral exchange mechanism. Glu⁶⁸¹ of human AE1 may form part of the anion translocation apparatus and the permeability barrier. We have therefore studied the structure of the sequence surrounding Glu⁶⁸¹, using scanning cysteine mutagenesis. Residues of the Ser⁶⁴³ (adjacent to the glycosylation site) to Ser⁶⁹⁰ region of cysteineless mutant (AE1C⁻) were replaced individually with cysteine. The ability of mutants to mediate Cl⁻/ HCO₃ ⁻ exchange in transfected HEK293 cells revealed that extracellular mutants, W648C, I650C, P652C, L655C, and F659C have an important role in transport. By contrast, only transmembrane mutation E681C fully blocked anion exchange activity. The topology of the region was investigated by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent 3-(N-maleimidylpropionyl)biocytin, with or without prior labeling with membrane-impermeant lucifer yellow iodoacetamide (LYIA). Two regions readily label with 3-(N-maleimidylpropionyl)biocytin (Ser⁶⁴³–Met⁶⁶³ and Ile⁶⁸⁴–Ser⁶⁹⁰). We propose that poorly labeled Met⁶⁶⁴–Gln⁶⁸³ corresponds to transmembrane segment 8 of AE1. Regions Ser⁶⁴³–Met⁶⁶³ and Ile⁶⁸⁴–Ser⁶⁹⁰ localize, respectively, to extracellular and intracellular sites on the basis of accessibility to LYIA. On the basis of LYIA accessibility, we propose that the Arg⁶⁵⁶–Met⁶⁶³ region forms a “vestibule” that leads anions to the transport channel. Glu⁶⁸¹ is located 3 amino acids from the C terminus of transmembrane segment 8, which places the membrane permeability barrier within 5 Å of the intracellular surface of the membrane.

Footnotes

↵* A preliminary version of this work has been published in abstract form (17).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Dept. of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 403-492-7203; Fax: 403-492-8915; E-mail:joe.casey{at}ualberta.ca.
Abbreviations:

WRK

Woodward’s reagent K

AE1C⁻

cysteineless AE1

BCECF-AM

2′,7′-bis(2-carboxyethyl)-(5 and 6)-carboxyfluorescein, acetoxymethyl ester

biotin maleimide

3-(N-maleimidylpropionyl)biocytin

LYIA

lucifer yellow iodoacetamide, dipotassium salt

stilbene maleimide

4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt

TLCK

N-p-tosyl-l-lysine chloromethyl ketone

TPCK

N-tosyl-l-phenylalanine chloromethyl ketone

PBS

phosphate-buffered saline.
- Received May 4, 1998.
- Revision received June 16, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.