Identification of Residues Lining the Translocation Pore of Human AE1, Plasma Membrane Anion Exchange Protein
1999; Elsevier BV; Volume: 274; Issue: 6 Linguagem: Inglês
10.1074/jbc.274.6.3557
ISSN1083-351X
AutoresX. Charlene Tang, Miklós Kovács, Deborah Sterling, Joseph R. Casey,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoAE1 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 reagentspara-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. 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 reagentspara-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. AE1, also called Band 3, is the plasma membrane chloride bicarbonate anion exchange protein of the erythrocyte (1Kopito R.R. Lodish H.L. J. Cell. Biochem. 1985; 29: 1-17Crossref PubMed Scopus (42) Google Scholar) and kidney (2Brosius III, F.C. Alper S.L. Garcia A.M. Lodish H.F. J. Biol. Chem. 1989; 264: 7784-7787Abstract Full Text PDF PubMed Google Scholar). AE1 is a member of a multigene family (3Kopito R.R. Int. Rev. Cytol. 1990; 123: 177-199Crossref PubMed Scopus (166) Google Scholar); AE2 is most broadly expressed in tissues such as gastric parietal cells (4Jöns T. Warrings B. Jöns A. Drenckhahn D. Histochemistry. 1994; 102: 255-263Crossref PubMed Scopus (41) Google Scholar) and choroid plexus (5Lindsey A.E. Schneider K. Simmons D.M. Baron R. Lee B.S. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5278-5282Crossref PubMed Scopus (140) Google Scholar); AE3 is expressed in excitable tissues: heart (6Linn S.C. Kudrycki K.E. Schull G.E. J. Biol. Chem. 1992; 267: 7927-7935Abstract Full Text PDF PubMed Google Scholar), brain (7Kopito R.R. Lee B.S. Simmons D.M. Lindsey A.E. Morgans C.W. Schneider K. Cell. 1989; 59: 927-937Abstract Full Text PDF PubMed Scopus (204) Google Scholar), and retina (8Kobayashi S. Morgans C.W. Casey J.R. Kopito R.R. J. Neurosci. 1994; 14: 6266-6279Crossref PubMed Google Scholar). All of the anion exchangers have a two-domain structure: a 55-kDa membrane domain responsible for anion exchange and a 43–77-kDa cytoplasmic domain involved in cytoskeletal interactions. Membrane domains are highly conserved (80% homology in the whole domain and close to 90% in the transmembrane segments). All anion exchangers function by an electroneutral "Ping-Pong" mechanism of Cl−/HCO3− exchange. The high abundance of AE1 in the erythrocyte membrane (50% of integral protein (9Fairbanks G. Steck T.L. Wallach D.F.H. Biochemistry. 1971; 10: 2606-2617Crossref PubMed Scopus (6158) Google Scholar)) has made the protein a model for the study of transport protein structure and function (10Reithmeier R.A.F. Chan S.L. Popov M. Konings W.N. Kaback H.R. Lolkema J.S. Transport Processes in Eukaryotic and Prokaryotic Organisms. 2. Elsevier Science Publishing Co., Inc., New York1996: 281-309Google Scholar, 11Reithmeier R.A.F. Curr. Opin. Struct. Biol. 1993; 3: 515-523Crossref Scopus (94) Google Scholar).Several residues have been implicated as part of the anion exchange mechanism of AE1. On the basis of indirect evidence, Passow has proposed a model for anion translocation that involves residues from TMs 1The abbreviations used are: TM, transmembrane segment; AE1C−, cysteineless AE1; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-(5 and 6)-carboxyfluorescein, acetoxymethyl ester; biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; MTSEA, 2-(aminoethyl)methanethiosulfonate, hydrobromide; pCMBS, para-chloromercuribenzene sulfonate; CFTR, cystic fibrosis transmembrane regulator.1The abbreviations used are: TM, transmembrane segment; AE1C−, cysteineless AE1; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-(5 and 6)-carboxyfluorescein, acetoxymethyl ester; biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; MTSEA, 2-(aminoethyl)methanethiosulfonate, hydrobromide; pCMBS, para-chloromercuribenzene sulfonate; CFTR, cystic fibrosis transmembrane regulator. 5, 8, 10, 12, and 13, including mouse residues Glu472, Glu535, Lys539, Glu681, His703, Arg731, His735, His816, and His834 (12Müller-Berger S. Karbach D. Konig J. Lepke S. Wood P.G. Appelhans H. Passow H. Biochemistry. 1995; 34: 9315-9324Crossref PubMed Scopus (50) Google Scholar). Jennings and co-workers (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar) have strong evidence to implicate the TM8 residue, Glu681, in the transport process, since labeling this residue with Woodward's reagent K and reduction with sodium borohydride resulted in altered anion exchange kinetics. The functional role of Glu681 in AE1 was confirmed in mutagenesis experiments of mouse AE1 (16Chernova M.N. Jiang L. Crest M. Hand M. Vandorpe D.H. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (81) Google Scholar) and extended to the homologous position of mouse AE2, suggesting that the mechanistic role of Glu681 is conserved among anion exchange proteins (17Sekler I. Lo R.S. Kopito R.R. J. Biol. Chem. 1995; 270: 28751-28758Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Woodward's reagent K chemical modification of AE1 abolishes chloride transport, yet relieves the requirement for proton cotransport during sulfate transport. During sulfate transport in unmodified AE1, a proton, supplied by Glu681 is cotransported. Sulfate/proton cotransport takes place in both inward and outward directions, which implies that Glu681 has access to both the intracellular and extracellular sides of the membrane. Taken together, Glu681 is functionally involved in anion exchange events and may reside at the permeability barrier of AE1.Glu681 of TM8 is the best characterized residue that interacts with anions during translocation event. We have therefore focused on TM8 in efforts to identify residues involved in anion translocation. In a previous report we analyzed a panel of introduced cysteine residues spanning the TM8 region from Ser643 to Ser690. Using accessibility to chemical modification by 3-(N-maleimidylpropionyl)biocytin and lucifer yellow iodoacetamide we identified the bilayer spanning residues as the sequence Met664–Gln683 (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).Substituted cysteine mutagenesis and sulfhydryl chemistry have proved a fruitful approach to identify residues of the transport pathway. The method is to mutate all native cysteine residues in a protein and to systematically re-introduce unique cysteine residues into the cysteineless background. Inhibition of anion transport by sulfhydryl-specific reagents is then assessed for each mutant. The approach has been useful for studies of the bacterial transport proteins, lactose permease (19He M.M. Sun J. Kaback H.R. Biochemistry. 1996; 35: 12909-12914Crossref PubMed Scopus (35) Google Scholar, 20Frillingos S. Sun J. Gonzalez A. Kaback H.R. Biochemistry. 1997; 36: 269-273Crossref PubMed Scopus (45) Google Scholar, 21Dunten R.L. Sahin-Tóth M. Kaback R.H. Biochemistry. 1993; 32: 12644-12650Crossref PubMed Scopus (60) Google Scholar) and UhpT (22Yan R.T. Maloney P.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5973-5976Crossref PubMed Scopus (79) Google Scholar). Among mammalian membrane proteins, this approach has proved very successful for ion channels. Derivatization of a cysteine with a methanethiosulfonate will impair ion conductance through steric blockage of a confined ion translocation pore. Pore-lining residues in the cystic fibrosis chloride channel, CFTR (23Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 25Cheung M. Akabas M.H. J. Gen. Physiol. 1997; 109: 289-299Crossref PubMed Scopus (85) Google Scholar), γ-aminobutyric acid receptor chloride channel (26Xu M. Akabas M.H. J. Biol. Chem. 1993; 268: 21505-21508Abstract Full Text PDF PubMed Google Scholar) and acetylcholine receptor sodium channel (27Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar,28Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar) have been identified in this way. Differential sensitivity to inhibition by methanethiosulfonate compounds has also identified residues of the transport pathway in the glutamate transport protein GLT-1 (29Zarbiv R. Grunewald M. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 1998; 273: 14231-14237Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), sodium glucose cotransporter (30Loo D.D.F. Hirayama B.A. Gallardo E.M. Lam J.T. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7789-7794Crossref PubMed Scopus (140) Google Scholar), and sodium/calcium exchanger (31Doering A.E. Nicoll D.A. Lu Y. Lu L. Weiss J.N. Philipson K.D. J. Biol. Chem. 1998; 273: 778-783Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar).We used substituted cysteine mutagenesis and sulfhydryl chemistry to identify pore-lining residues in the TM8 region of human AE1 chloride/bicarbonate exchange protein. Previously we constructed a cysteineless form of human AE1, called AE1C−, and characterized the protein as fully functional (32Casey J.R. Ding Y. Kopito R.R. J. Biol. Chem. 1995; 270: 8521-8527Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We have systematically replaced the residues of AE1 from the glycosylation site at Asn642 through TM8 into the cytoplasmic region and identified the sequence Met664–Gln683 as spanning the bilayer (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In this report we measure the effect of two sulfhydryl reagents upon AE1 anion transport function, using BCECF fluorescence to monitor intracellular pH shifts associated with Cl−/HCO3− exchange in transfected HEK293 cells. Of the two sulfhydryl reagents, pCMBS is membrane-impermeant but slowly transported by AE1, while MTSEA is membrane-permeant in its unprotonated form (33Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (191) Google Scholar). We have identified a sequence of leucine, isoleucine, and alanine residues that lie on one face of an α-helix and, when mutated to cysteine, are susceptible to inhibition by pCMBS and MTSEA.DISCUSSIONIn this paper we have examined the amino acid sequence from SER643–SER690 of human AE1, to identify residues that line a transmembrane anion translocation pore. We have focused on this region because it contains Glu681, shown previously to be accessible to membrane impermeant Woodward's reagent K, from either side of the membrane (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar). This residue may therefore reside at the transmembrane permeability barrier. 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. We recently determined the topology of the Ser643–Ser690 region and localized Glu681 to within four residues of the cytosolic surface of the protein (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).Our results have identified a sequence of introduced cysteine mutants (Ala666, Ser667, Leu669, Leu673, Leu677, Leu680, Ile684, and Ile688) in the transmembrane segment 8 region of human AE1 that can be inhibited either by the sulfhydryl reagents pCMBS or MTSEA. The locations of the mutants have a helical periodicity and reside on one face of a predicted α-helix, which extends at least one turn beyond the surface of the membrane. Seven of the eight identified residues are found within a 100° arc of a TM8 helical wheel plot, indicating that the region forms an α-helix. The sensitive helical face is directly opposite the most hydrophobic face of TM8 and is adjacent to Glu681, which was shown previously to interact with anions during transmembrane translocation. The simplest explanation for our data is a covalent interaction of introduced cysteine residues with the sulfhydryl compounds; at sites that line the pore, these reagents block the pore and inhibit anion transport. On this basis we propose that Ala666, Ser667, Leu669, Leu673, Leu677, Leu680, Ile684, and Ile688 line the transmembrane translocation channel and interact with substrate anions as they cross the bilayer. An alternate possibility is that the inhibited residues form a conformationally active surface of AE1, part of a cleft where two helices meet and whose movements are blocked by sulfhydryl modification. However, this model is unlikely since such a cleft would not be likely to have access to pCMBS and MTSEA, two hydrophilic reagents.Our observation that the hydrophobic amino acids leucine, isoleucine, and alanine line the TM8 portion of the anion translocation channel at first seems surprising. The alkyl side chains of these amino acids are not hydrophilic and cannot contribute hydrogen bonds to line an aqueous channel. However, hydrophobic residues have been found to form part of the lining of the cystic fibrosis chloride channel, CFTR (23Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar), the γ-aminobutyric acid receptor channel (26Xu M. Akabas M.H. J. Biol. Chem. 1993; 268: 21505-21508Abstract Full Text PDF PubMed Google Scholar), and the acetylcholine receptor Na+ channel (28Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar). The transmembrane carbohydrate translocation channel of the maltoporin protein is lined with hydrophobic residues (44Dutzler R. Wang Y.F. Rizkallah P. Rosenbusch J.P. Schirmer T. Structure (Lond.). 1996; 4: 127-134Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Similarly, the crystal structure of a bacterial K+ channel illustrated that the ion translocation pore is lined with hydrophobic amino acids (45Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5686) Google Scholar). The authors speculated that the hydrophobic lining provides the most energetically favorable surface for movement of ions. Interactions with the wall of the pore would only impede ion transit through the pore. A hydrophobic lining of the AE1 anion translocation pore could be important to facilitate the high turnover rate (105 anions·s−1 (46Jay D. Cantley L. Annu. Rev. Biochem. 1986; 55: 511-538Crossref PubMed Scopus (110) Google Scholar)), which is an order of magnitude slower than ion channel fluxes, but still very fast. The observation that the pore-lining face of the helix is not centered on Glu681 may also seem surprising, because Glu681 interacts with sulfate anions during transport (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar). However, Glu681 is on the edge of the pore in our model (Fig. 6) and could extend its side chain into the pore. Interestingly, Passow has presented a model of the AE1 anion translocation pathway in which TM8 interacts with TM9 and TM10, but E681 is predicted to be on the edge of the pore (12Müller-Berger S. Karbach D. Konig J. Lepke S. Wood P.G. Appelhans H. Passow H. Biochemistry. 1995; 34: 9315-9324Crossref PubMed Scopus (50) Google Scholar).Mutant S667C stands apart from the other sulfhydryl reagent-sensitive sites. This is the only hydrophilic site observed among the pore-lining residues. All other sites cluster on one face of the helix, while S667C is 60° removed from the rest of the mutants, on a helical wheel plot. S667C is close to Pro670, which may kink and twist the TM8 helix. This would result in exposure of a discontinuous helical face to line the pore. That is the TM8 helical region above and below Pro680 may not be directly aligned.Our results may provide insight into the structure of the anion translocation pore. We observed that AE1 introduced cysteine mutants are only partially (<40%) inhibited by pCMBS. Similarly, in studies of the CFTR chloride channel, pCMBS was only able to inhibit to 25–75% (24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar). In contrast translocation pathway mutants of the bacterial glucose 6-phosphate transporter, UhpT, were nearly fully inhibited by pCMBS (22Yan R.T. Maloney P.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5973-5976Crossref PubMed Scopus (79) Google Scholar). This difference in sensitivity to pCMBS may reflect the size of the anion translocation pore, since glucose 6-phosphate is larger than chloride. The CFTR channel is estimated to have a pore diameter of at least 6 Å (24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar), which provides at least an approximation for the size of the AE1 pore.One prevailing model of AE1 suggests that the anion translocation pore forms an outward facing funnel and narrows to a permeability barrier at Glu681 (47Jennings M.L. Hamasaki N. Jennings M.L. Anion Transport Protein of the Red Blood Cell Membrane. Elsevier Science Publishers B. V., Amsterdam1989: 59-72Google Scholar). Our data may provide some support for the model. The first sulfhydryl reagent-sensitive site (Ala666) is located after the start of the TM8 (Met664) (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Since both pCMBS and MTSEA are expected to inhibit anion exchange by steric blockage of a pore, the observation that the inner portion of TM8 is more sensitive to sulfhydryl reagents than the outer suggests a more open outer region and a more closed pore in the inner region. In line with this model, we found that L669C was sensitive to pCMBS, but not MTSEA, reflecting the larger steric bulk of pCMBS. Furthermore, A666C and S667C, the outermost sensitive mutants, both had lower pCMBS sensitivity than the inner mutants. We found that the sequence of pCMBS-inhibited residues ended at Leu680, which is consistent with the identification of Glu681 as the site of the permeability barrier. Since pCMBS can permeate the membrane only via AE1, it will not accumulate on the cytosolic surface to any extent. Conversely, MTSEA is membrane permeant in its deprotonated form (33Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (191) Google Scholar) and therefore can access the anion translocation pore from both the extracellular and intracellular surfaces. Finally, our data show that the greatest sensitivity to pCMBS is found with mutant L680C, which is adjacent to Glu681, the presumed permeability barrier. The high sensitivity at this site may reflect a smaller pore diameter. AE1, also called Band 3, is the plasma membrane chloride bicarbonate anion exchange protein of the erythrocyte (1Kopito R.R. Lodish H.L. J. Cell. Biochem. 1985; 29: 1-17Crossref PubMed Scopus (42) Google Scholar) and kidney (2Brosius III, F.C. Alper S.L. Garcia A.M. Lodish H.F. J. Biol. Chem. 1989; 264: 7784-7787Abstract Full Text PDF PubMed Google Scholar). AE1 is a member of a multigene family (3Kopito R.R. Int. Rev. Cytol. 1990; 123: 177-199Crossref PubMed Scopus (166) Google Scholar); AE2 is most broadly expressed in tissues such as gastric parietal cells (4Jöns T. Warrings B. Jöns A. Drenckhahn D. Histochemistry. 1994; 102: 255-263Crossref PubMed Scopus (41) Google Scholar) and choroid plexus (5Lindsey A.E. Schneider K. Simmons D.M. Baron R. Lee B.S. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5278-5282Crossref PubMed Scopus (140) Google Scholar); AE3 is expressed in excitable tissues: heart (6Linn S.C. Kudrycki K.E. Schull G.E. J. Biol. Chem. 1992; 267: 7927-7935Abstract Full Text PDF PubMed Google Scholar), brain (7Kopito R.R. Lee B.S. Simmons D.M. Lindsey A.E. Morgans C.W. Schneider K. Cell. 1989; 59: 927-937Abstract Full Text PDF PubMed Scopus (204) Google Scholar), and retina (8Kobayashi S. Morgans C.W. Casey J.R. Kopito R.R. J. Neurosci. 1994; 14: 6266-6279Crossref PubMed Google Scholar). All of the anion exchangers have a two-domain structure: a 55-kDa membrane domain responsible for anion exchange and a 43–77-kDa cytoplasmic domain involved in cytoskeletal interactions. Membrane domains are highly conserved (80% homology in the whole domain and close to 90% in the transmembrane segments). All anion exchangers function by an electroneutral "Ping-Pong" mechanism of Cl−/HCO3− exchange. The high abundance of AE1 in the erythrocyte membrane (50% of integral protein (9Fairbanks G. Steck T.L. Wallach D.F.H. Biochemistry. 1971; 10: 2606-2617Crossref PubMed Scopus (6158) Google Scholar)) has made the protein a model for the study of transport protein structure and function (10Reithmeier R.A.F. Chan S.L. Popov M. Konings W.N. Kaback H.R. Lolkema J.S. Transport Processes in Eukaryotic and Prokaryotic Organisms. 2. Elsevier Science Publishing Co., Inc., New York1996: 281-309Google Scholar, 11Reithmeier R.A.F. Curr. Opin. Struct. Biol. 1993; 3: 515-523Crossref Scopus (94) Google Scholar). Several residues have been implicated as part of the anion exchange mechanism of AE1. On the basis of indirect evidence, Passow has proposed a model for anion translocation that involves residues from TMs 1The abbreviations used are: TM, transmembrane segment; AE1C−, cysteineless AE1; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-(5 and 6)-carboxyfluorescein, acetoxymethyl ester; biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; MTSEA, 2-(aminoethyl)methanethiosulfonate, hydrobromide; pCMBS, para-chloromercuribenzene sulfonate; CFTR, cystic fibrosis transmembrane regulator.1The abbreviations used are: TM, transmembrane segment; AE1C−, cysteineless AE1; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-(5 and 6)-carboxyfluorescein, acetoxymethyl ester; biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; MTSEA, 2-(aminoethyl)methanethiosulfonate, hydrobromide; pCMBS, para-chloromercuribenzene sulfonate; CFTR, cystic fibrosis transmembrane regulator. 5, 8, 10, 12, and 13, including mouse residues Glu472, Glu535, Lys539, Glu681, His703, Arg731, His735, His816, and His834 (12Müller-Berger S. Karbach D. Konig J. Lepke S. Wood P.G. Appelhans H. Passow H. Biochemistry. 1995; 34: 9315-9324Crossref PubMed Scopus (50) Google Scholar). Jennings and co-workers (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar) have strong evidence to implicate the TM8 residue, Glu681, in the transport process, since labeling this residue with Woodward's reagent K and reduction with sodium borohydride resulted in altered anion exchange kinetics. The functional role of Glu681 in AE1 was confirmed in mutagenesis experiments of mouse AE1 (16Chernova M.N. Jiang L. Crest M. Hand M. Vandorpe D.H. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (81) Google Scholar) and extended to the homologous position of mouse AE2, suggesting that the mechanistic role of Glu681 is conserved among anion exchange proteins (17Sekler I. Lo R.S. Kopito R.R. J. Biol. Chem. 1995; 270: 28751-28758Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Woodward's reagent K chemical modification of AE1 abolishes chloride transport, yet relieves the requirement for proton cotransport during sulfate transport. During sulfate transport in unmodified AE1, a proton, supplied by Glu681 is cotransported. Sulfate/proton cotransport takes place in both inward and outward directions, which implies that Glu681 has access to both the intracellular and extracellular sides of the membrane. Taken together, Glu681 is functionally involved in anion exchange events and may reside at the permeability barrier of AE1. Glu681 of TM8 is the best characterized residue that interacts with anions during translocation event. We have therefore focused on TM8 in efforts to identify residues involved in anion translocation. In a previous report we analyzed a panel of introduced cysteine residues spanning the TM8 region from Ser643 to Ser690. Using accessibility to chemical modification by 3-(N-maleimidylpropionyl)biocytin and lucifer yellow iodoacetamide we identified the bilayer spanning residues as the sequence Met664–Gln683 (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Substituted cysteine mutagenesis and sulfhydryl chemistry have proved a fruitful approach to identify residues of the transport pathway. The method is to mutate all native cysteine residues in a protein and to systematically re-introduce unique cysteine residues into the cysteineless background. Inhibition of anion transport by sulfhydryl-specific reagents is then assessed for each mutant. The approach has been useful for studies of the bacterial transport proteins, lactose permease (19He M.M. Sun J. Kaback H.R. Biochemistry. 1996; 35: 12909-12914Crossref PubMed Scopus (35) Google Scholar, 20Frillingos S. Sun J. Gonzalez A. Kaback H.R. Biochemistry. 1997; 36: 269-273Crossref PubMed Scopus (45) Google Scholar, 21Dunten R.L. Sahin-Tóth M. Kaback R.H. Biochemistry. 1993; 32: 12644-12650Crossref PubMed Scopus (60) Google Scholar) and UhpT (22Yan R.T. Maloney P.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5973-5976Crossref PubMed Scopus (79) Google Scholar). Among mammalian membrane proteins, this approach has proved very successful for ion channels. Derivatization of a cysteine with a methanethiosulfonate will impair ion conductance through steric blockage of a confined ion translocation pore. Pore-lining residues in the cystic fibrosis chloride channel, CFTR (23Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 25Cheung M. Akabas M.H. J. Gen. Physiol. 1997; 109: 289-299Crossref PubMed Scopus (85) Google Scholar), γ-aminobutyric acid receptor chloride channel (26Xu M. Akabas M.H. J. Biol. Chem. 1993; 268: 21505-21508Abstract Full Text PDF PubMed Google Scholar) and acetylcholine receptor sodium channel (27Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar,28Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar) have been identified in this way. Differential sensitivity to inhibition by methanethiosulfonate compounds has also identified residues of the transport pathway in the glutamate transport protein GLT-1 (29Zarbiv R. Grunewald M. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 1998; 273: 14231-14237Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), sodium glucose cotransporter (30Loo D.D.F. Hirayama B.A. Gallardo E.M. Lam J.T. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7789-7794Crossref PubMed Scopus (140) Google Scholar), and sodium/calcium exchanger (31Doering A.E. Nicoll D.A. Lu Y. Lu L. Weiss J.N. Philipson K.D. J. Biol. Chem. 1998; 273: 778-783Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We used substituted cysteine mutagenesis and sulfhydryl chemistry to identify pore-lining residues in the TM8 region of human AE1 chloride/bicarbonate exchange protein. Previously we constructed a cysteineless form of human AE1, called AE1C−, and characterized the protein as fully functional (32Casey J.R. Ding Y. Kopito R.R. J. Biol. Chem. 1995; 270: 8521-8527Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We have systematically replaced the residues of AE1 from the glycosylation site at Asn642 through TM8 into the cytoplasmic region and identified the sequence Met664–Gln683 as spanning the bilayer (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In this report we measure the effect of two sulfhydryl reagents upon AE1 anion transport function, using BCECF fluorescence to monitor intracellular pH shifts associated with Cl−/HCO3− exchange in transfected HEK293 cells. Of the two sulfhydryl reagents, pCMBS is membrane-impermeant but slowly transported by AE1, while MTSEA is membrane-permeant in its unprotonated form (33Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (191) Google Scholar). We have identified a sequence of leucine, isoleucine, and alanine residues that lie on one face of an α-helix and, when mutated to cysteine, are susceptible to inhibition by pCMBS and MTSEA. DISCUSSIONIn this paper we have examined the amino acid sequence from SER643–SER690 of human AE1, to identify residues that line a transmembrane anion translocation pore. We have focused on this region because it contains Glu681, shown previously to be accessible to membrane impermeant Woodward's reagent K, from either side of the membrane (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar). This residue may therefore reside at the transmembrane permeability barrier. 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. We recently determined the topology of the Ser643–Ser690 region and localized Glu681 to within four residues of the cytosolic surface of the protein (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).Our results have identified a sequence of introduced cysteine mutants (Ala666, Ser667, Leu669, Leu673, Leu677, Leu680, Ile684, and Ile688) in the transmembrane segment 8 region of human AE1 that can be inhibited either by the sulfhydryl reagents pCMBS or MTSEA. The locations of the mutants have a helical periodicity and reside on one face of a predicted α-helix, which extends at least one turn beyond the surface of the membrane. Seven of the eight identified residues are found within a 100° arc of a TM8 helical wheel plot, indicating that the region forms an α-helix. The sensitive helical face is directly opposite the most hydrophobic face of TM8 and is adjacent to Glu681, which was shown previously to interact with anions during transmembrane translocation. The simplest explanation for our data is a covalent interaction of introduced cysteine residues with the sulfhydryl compounds; at sites that line the pore, these reagents block the pore and inhibit anion transport. On this basis we propose that Ala666, Ser667, Leu669, Leu673, Leu677, Leu680, Ile684, and Ile688 line the transmembrane translocation channel and interact with substrate anions as they cross the bilayer. An alternate possibility is that the inhibited residues form a conformationally active surface of AE1, part of a cleft where two helices meet and whose movements are blocked by sulfhydryl modification. However, this model is unlikely since such a cleft would not be likely to have access to pCMBS and MTSEA, two hydrophilic reagents.Our observation that the hydrophobic amino acids leucine, isoleucine, and alanine line the TM8 portion of the anion translocation channel at first seems surprising. The alkyl side chains of these amino acids are not hydrophilic and cannot contribute hydrogen bonds to line an aqueous channel. However, hydrophobic residues have been found to form part of the lining of the cystic fibrosis chloride channel, CFTR (23Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar), the γ-aminobutyric acid receptor channel (26Xu M. Akabas M.H. J. Biol. Chem. 1993; 268: 21505-21508Abstract Full Text PDF PubMed Google Scholar), and the acetylcholine receptor Na+ channel (28Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar). The transmembrane carbohydrate translocation channel of the maltoporin protein is lined with hydrophobic residues (44Dutzler R. Wang Y.F. Rizkallah P. Rosenbusch J.P. Schirmer T. Structure (Lond.). 1996; 4: 127-134Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Similarly, the crystal structure of a bacterial K+ channel illustrated that the ion translocation pore is lined with hydrophobic amino acids (45Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5686) Google Scholar). The authors speculated that the hydrophobic lining provides the most energetically favorable surface for movement of ions. Interactions with the wall of the pore would only impede ion transit through the pore. A hydrophobic lining of the AE1 anion translocation pore could be important to facilitate the high turnover rate (105 anions·s−1 (46Jay D. Cantley L. Annu. Rev. Biochem. 1986; 55: 511-538Crossref PubMed Scopus (110) Google Scholar)), which is an order of magnitude slower than ion channel fluxes, but still very fast. The observation that the pore-lining face of the helix is not centered on Glu681 may also seem surprising, because Glu681 interacts with sulfate anions during transport (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar). However, Glu681 is on the edge of the pore in our model (Fig. 6) and could extend its side chain into the pore. Interestingly, Passow has presented a model of the AE1 anion translocation pathway in which TM8 interacts with TM9 and TM10, but E681 is predicted to be on the edge of the pore (12Müller-Berger S. Karbach D. Konig J. Lepke S. Wood P.G. Appelhans H. Passow H. Biochemistry. 1995; 34: 9315-9324Crossref PubMed Scopus (50) Google Scholar).Mutant S667C stands apart from the other sulfhydryl reagent-sensitive sites. This is the only hydrophilic site observed among the pore-lining residues. All other sites cluster on one face of the helix, while S667C is 60° removed from the rest of the mutants, on a helical wheel plot. S667C is close to Pro670, which may kink and twist the TM8 helix. This would result in exposure of a discontinuous helical face to line the pore. That is the TM8 helical region above and below Pro680 may not be directly aligned.Our results may provide insight into the structure of the anion translocation pore. We observed that AE1 introduced cysteine mutants are only partially (<40%) inhibited by pCMBS. Similarly, in studies of the CFTR chloride channel, pCMBS was only able to inhibit to 25–75% (24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar). In contrast translocation pathway mutants of the bacterial glucose 6-phosphate transporter, UhpT, were nearly fully inhibited by pCMBS (22Yan R.T. Maloney P.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5973-5976Crossref PubMed Scopus (79) Google Scholar). This difference in sensitivity to pCMBS may reflect the size of the anion translocation pore, since glucose 6-phosphate is larger than chloride. The CFTR channel is estimated to have a pore diameter of at least 6 Å (24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar), which provides at least an approximation for the size of the AE1 pore.One prevailing model of AE1 suggests that the anion translocation pore forms an outward facing funnel and narrows to a permeability barrier at Glu681 (47Jennings M.L. Hamasaki N. Jennings M.L. Anion Transport Protein of the Red Blood Cell Membrane. Elsevier Science Publishers B. V., Amsterdam1989: 59-72Google Scholar). Our data may provide some support for the model. The first sulfhydryl reagent-sensitive site (Ala666) is located after the start of the TM8 (Met664) (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Since both pCMBS and MTSEA are expected to inhibit anion exchange by steric blockage of a pore, the observation that the inner portion of TM8 is more sensitive to sulfhydryl reagents than the outer suggests a more open outer region and a more closed pore in the inner region. In line with this model, we found that L669C was sensitive to pCMBS, but not MTSEA, reflecting the larger steric bulk of pCMBS. Furthermore, A666C and S667C, the outermost sensitive mutants, both had lower pCMBS sensitivity than the inner mutants. We found that the sequence of pCMBS-inhibited residues ended at Leu680, which is consistent with the identification of Glu681 as the site of the permeability barrier. Since pCMBS can permeate the membrane only via AE1, it will not accumulate on the cytosolic surface to any extent. Conversely, MTSEA is membrane permeant in its deprotonated form (33Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (191) Google Scholar) and therefore can access the anion translocation pore from both the extracellular and intracellular surfaces. Finally, our data show that the greatest sensitivity to pCMBS is found with mutant L680C, which is adjacent to Glu681, the presumed permeability barrier. The high sensitivity at this site may reflect a smaller pore diameter. In this paper we have examined the amino acid sequence from SER643–SER690 of human AE1, to identify residues that line a transmembrane anion translocation pore. We have focused on this region because it contains Glu681, shown previously to be accessible to membrane impermeant Woodward's reagent K, from either side of the membrane (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar). This residue may therefore reside at the transmembrane permeability barrier. 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. We recently determined the topology of the Ser643–Ser690 region and localized Glu681 to within four residues of the cytosolic surface of the protein (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Our results have identified a sequence of introduced cysteine mutants (Ala666, Ser667, Leu669, Leu673, Leu677, Leu680, Ile684, and Ile688) in the transmembrane segment 8 region of human AE1 that can be inhibited either by the sulfhydryl reagents pCMBS or MTSEA. The locations of the mutants have a helical periodicity and reside on one face of a predicted α-helix, which extends at least one turn beyond the surface of the membrane. Seven of the eight identified residues are found within a 100° arc of a TM8 helical wheel plot, indicating that the region forms an α-helix. The sensitive helical face is directly opposite the most hydrophobic face of TM8 and is adjacent to Glu681, which was shown previously to interact with anions during transmembrane translocation. The simplest explanation for our data is a covalent interaction of introduced cysteine residues with the sulfhydryl compounds; at sites that line the pore, these reagents block the pore and inhibit anion transport. On this basis we propose that Ala666, Ser667, Leu669, Leu673, Leu677, Leu680, Ile684, and Ile688 line the transmembrane translocation channel and interact with substrate anions as they cross the bilayer. An alternate possibility is that the inhibited residues form a conformationally active surface of AE1, part of a cleft where two helices meet and whose movements are blocked by sulfhydryl modification. However, this model is unlikely since such a cleft would not be likely to have access to pCMBS and MTSEA, two hydrophilic reagents. Our observation that the hydrophobic amino acids leucine, isoleucine, and alanine line the TM8 portion of the anion translocation channel at first seems surprising. The alkyl side chains of these amino acids are not hydrophilic and cannot contribute hydrogen bonds to line an aqueous channel. However, hydrophobic residues have been found to form part of the lining of the cystic fibrosis chloride channel, CFTR (23Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar), the γ-aminobutyric acid receptor channel (26Xu M. Akabas M.H. J. Biol. Chem. 1993; 268: 21505-21508Abstract Full Text PDF PubMed Google Scholar), and the acetylcholine receptor Na+ channel (28Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (356) Google Scholar). The transmembrane carbohydrate translocation channel of the maltoporin protein is lined with hydrophobic residues (44Dutzler R. Wang Y.F. Rizkallah P. Rosenbusch J.P. Schirmer T. Structure (Lond.). 1996; 4: 127-134Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Similarly, the crystal structure of a bacterial K+ channel illustrated that the ion translocation pore is lined with hydrophobic amino acids (45Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5686) Google Scholar). The authors speculated that the hydrophobic lining provides the most energetically favorable surface for movement of ions. Interactions with the wall of the pore would only impede ion transit through the pore. A hydrophobic lining of the AE1 anion translocation pore could be important to facilitate the high turnover rate (105 anions·s−1 (46Jay D. Cantley L. Annu. Rev. Biochem. 1986; 55: 511-538Crossref PubMed Scopus (110) Google Scholar)), which is an order of magnitude slower than ion channel fluxes, but still very fast. The observation that the pore-lining face of the helix is not centered on Glu681 may also seem surprising, because Glu681 interacts with sulfate anions during transport (13Jennings M.L. Al-Rhaiyel S. J. Gen. Physiol. 1988; 92: 161-178Crossref PubMed Scopus (50) Google Scholar, 14Jennings M.L. Smith J.S. J. Biol. Chem. 1992; 267: 13964-13971Abstract Full Text PDF PubMed Google Scholar, 15Jennings M.L. Anderson M.P. J. Biol. Chem. 1987; 262: 1691-1697Abstract Full Text PDF PubMed Google Scholar). However, Glu681 is on the edge of the pore in our model (Fig. 6) and could extend its side chain into the pore. Interestingly, Passow has presented a model of the AE1 anion translocation pathway in which TM8 interacts with TM9 and TM10, but E681 is predicted to be on the edge of the pore (12Müller-Berger S. Karbach D. Konig J. Lepke S. Wood P.G. Appelhans H. Passow H. Biochemistry. 1995; 34: 9315-9324Crossref PubMed Scopus (50) Google Scholar). Mutant S667C stands apart from the other sulfhydryl reagent-sensitive sites. This is the only hydrophilic site observed among the pore-lining residues. All other sites cluster on one face of the helix, while S667C is 60° removed from the rest of the mutants, on a helical wheel plot. S667C is close to Pro670, which may kink and twist the TM8 helix. This would result in exposure of a discontinuous helical face to line the pore. That is the TM8 helical region above and below Pro680 may not be directly aligned. Our results may provide insight into the structure of the anion translocation pore. We observed that AE1 introduced cysteine mutants are only partially (<40%) inhibited by pCMBS. Similarly, in studies of the CFTR chloride channel, pCMBS was only able to inhibit to 25–75% (24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar). In contrast translocation pathway mutants of the bacterial glucose 6-phosphate transporter, UhpT, were nearly fully inhibited by pCMBS (22Yan R.T. Maloney P.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5973-5976Crossref PubMed Scopus (79) Google Scholar). This difference in sensitivity to pCMBS may reflect the size of the anion translocation pore, since glucose 6-phosphate is larger than chloride. The CFTR channel is estimated to have a pore diameter of at least 6 Å (24Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar), which provides at least an approximation for the size of the AE1 pore. One prevailing model of AE1 suggests that the anion translocation pore forms an outward facing funnel and narrows to a permeability barrier at Glu681 (47Jennings M.L. Hamasaki N. Jennings M.L. Anion Transport Protein of the Red Blood Cell Membrane. Elsevier Science Publishers B. V., Amsterdam1989: 59-72Google Scholar). Our data may provide some support for the model. The first sulfhydryl reagent-sensitive site (Ala666) is located after the start of the TM8 (Met664) (18Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Since both pCMBS and MTSEA are expected to inhibit anion exchange by steric blockage of a pore, the observation that the inner portion of TM8 is more sensitive to sulfhydryl reagents than the outer suggests a more open outer region and a more closed pore in the inner region. In line with this model, we found that L669C was sensitive to pCMBS, but not MTSEA, reflecting the larger steric bulk of pCMBS. Furthermore, A666C and S667C, the outermost sensitive mutants, both had lower pCMBS sensitivity than the inner mutants. We found that the sequence of pCMBS-inhibited residues ended at Leu680, which is consistent with the identification of Glu681 as the site of the permeability barrier. Since pCMBS can permeate the membrane only via AE1, it will not accumulate on the cytosolic surface to any extent. Conversely, MTSEA is membrane permeant in its deprotonated form (33Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (191) Google Scholar) and therefore can access the anion translocation pore from both the extracellular and intracellular surfaces. Finally, our data show that the greatest sensitivity to pCMBS is found with mutant L680C, which is adjacent to Glu681, the presumed permeability barrier. The high sensitivity at this site may reflect a smaller pore diameter. We thank Dr. Myles Akabas for helpful advice. Dr. Andrew Taylor and Dr. Jocelyne Fujinaga provided helpful comments on the manuscript.
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