Inhibition of Mouse Erythroid Band 3-Mediated Chloride Transport by Site-Directed Mutagenesis of Histidine Residues and Its Reversal by Second Site Mutation of Lys 558, the Locus of Covalent H2DIDS Binding

Abstract

Substitution by site-directed mutagenesis of any one of the histidine residues H721, H837, and H852 by glutamine, or of H752 by serine, inhibits Cl- flux mediated by band 3 expressed in Xenopus oocytes. Mutation of Lys 558 (K558N), the site of covalent binding of H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) in the outer membrane surface, in combination with any one of the His/Gln mutations leads to partial (H721Q; H837Q) or complete (H852Q) restoration of Cl- flux. In contrast, inhibition of Cl- flux by mutation of proline or lysine residues in the vicinity of His 837 at the inner membrane surface cannot be reversed by the second-site mutation K558N, indicating specificity of interaction between Lys 558 and His 837. The histidine-specific reagent diethyl pyrocarbonate (DEPC) is known to inhibit band 3-mediated anion exchange in red blood cells [Izuhara, K., Okubo, K., & Hamasaki, N. (1989) Biochemistry 28, 4725-4728]. It was also found to inhibit transport after expression in the oocyte of wild-type band 3, of the double mutants of the histidines listed above, and of the single mutant H752S. The effects on the wild type and the double mutants were indistinguishable, while the mutant H752S exhibited a considerably reduced sensitivity to inhibition, suggesting that His 752 is the most prominent site of action of DEPC. According to a hydrophobicity plot of band 3 and further independent evidence, Lys 558, the mutated histidines, and Glu 699, the mutation of which was also found to inhibit Cl- flux [Müller-Berger, S., Karbach, D., Kang, D., Aranibar, N., Wood, P. G., Rüterjans, H., & Passow, H. (1995) Biochemistry 34, 9325-9332], are most likely located in five different transmembrane helices. The interactions between Lys 558 and the various histidines suggest that these helices reside in close proximity. Together with the helix carrying Glu 699, they could form an access channel lined with an array of alternating histidine and glutamate residues. Together with a chloride ion bridging the gap between His 852 and His 837, they could have the potential to form, at low pH, a transmembrane chain of hydrogen bonds. The possible functional significance of such channel is discussed.

Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6

“…Although highly speculative, such a mechanism appears to account for the pH dependence of anion transport by the band 3 transporter. 48,49 …”

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

“…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 (12).…”

“…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 1 (12). Jennings and co-workers (13)(14)(15) have strong evidence to implicate the TM8 residue, Glu 681 , in the transport process, since labeling this residue with Woodward's reagent K and reduction with sodium borohydride resulted in altered anion exchange kinetics.…”

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

“…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]. Complementation studies suggest further that TMs 6 and 7 are not required for anion transport [12].…”