Identification of Residues Lining the Translocation Pore of Human AE1, Plasma Membrane Anion Exchange Protein

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 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 permeability barrier. Glu 681 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.

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

“…We examined if these mutations affected the surface targeting of the protein. Our findings that many of the mutations of TM IV affected targeting, expression, or activity are similar to the results found with the human anion exchanger (35) and with TM XI of lactose permease (33). In contrast, for the tetracycline-resistant transporter of E. coli (39) and in P-glycoprotein (40), it was possible …”

“…Cysteine-scanning mutagenesis takes advantage of the fact that the sulfhydryl moiety is the most reactive functional group in a protein (29). It has been used to determine pore-lining residues in numerous membrane proteins (30 -32), including the lactose permease of E. coli (33), the mouse acetylcholine receptor (34), the human glucose transporter Glut1, and the human anion exchanger isoform 1 (AE1) (35). Cysteine-scanning mutagenesis uses the highly reactive sulfhydryl moiety to determine the accessibility of side chains of amino acids.…”

“…The most likely explanation for this reactivity is an interaction at a site that lines the ion translocation pore. The reagents then at least partially block the pore and inhibit ion transport (35). Thus, Phe 161 in TM IV is the first residue to be unambiguously identified as lining the ion transport pore of NHE1.…”

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

“…R656C formed a disulphide cross-linked product, although no maleimidemediated cross-linked product was observed. Previously, residues 656-663 in EC4 were suggested to provide an extramembraneous ' funnel ' guiding substrate to the anion channel [9]. Flexibility in this funnel region would presumably be undesirable by slowing both the rates of anion binding and release from AE, with concomitant reduction in the optimal rate of AE function.…”

“…The topology of the first eight transmembrane segments (TMs ; these are numbered according to the folding model presented in Figure 1) is well defined [5][6][7], whereas controversial topology models containing non-helical membrane-spanning segments have been proposed for the C-terminal region of the TM domain [6,8]. Mutagenesis studies indicate that TM8 of AE1 may form part of the aniontranslocation channel [9]. Histidine residues 703 [intracellular loop (IC) preceding TM9], 734 [extracellular loop (EC) 5, preceding TM10], 819 (region with undetermined topology) and 834 (TM12) may also be required for anion translocation [10,11].…”