INHIBITION OF MOUSE ERYTHROID BAND 3-MEDIATED CHLORIDE TRANSPORT BY SITE-DIRECTED MUTAGENESIS OF HISTIDINE-RESIDUES AND ITS REVERSAL BY 2NDSITE MUTATION OF LYS-558, THE LOCUS OF COVALENT H2DIDS BINDING

Substitution by site-directed mutagenesis of any one of the histidine residues H721, H837, and H852 by glutamine, or of H752 by serine, inhi bits Cl- flux mediated by band 3 expressed in Xenopus oocytes. Mutatio n of Lys 558 (K558N), the site of covalent binding of H2DIDS (4,4'-dii sothiocyanostilbene-2,2'-disulfonate) in the outer membrane surface, i n combination with any one of the His/Gin mutations leads to partial ( H721Q; H8374) 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 revers ed by the second-site mutation K558N, indicating specificity of intera ction between Lys 558 and His 837. The histidine-specific reagent diet hyl pyrocarbonate (DEPC) is known to inhibit band 3-mediated anion exc hange in red blood cells [Izuhara, K., Okubo, K., and Hamasaki, N. (19 89) Biochemistry 28, 4725-4728]. It was also found to inhibit transpor t after expression in the oocyte of wild-type band 3, of the double mu tants of the histidines listed above, and of the single mutant H752S. The effects on the wild type and the double mutants were indistinguish able,; while the mutant H752S exhibited a considerably reduced sensiti vity to inhibition, suggesting that His 752 is the most prominent site of action of DEPC. According to a hydrophobicity plot of band 3 and f urther independent evidence, Lys 558, the mutated histidines, and Glu 699, the mutation of which was also found to inhibit Cl- flux [Muller- Berger, S., Karbach, D., Kang, D., Aranibar, N., Wood, P. G., Ruterjan s, H., gr Passov, H. (1995) Biochemistry 34, 9325-9332], are most like ly located in five different transmembrane helices. The interactions b etween Lys 558 and the various histidines suggest that these helices r eside in close proximity. Together with the helix carrying Glu 699, th ey could form an access channel lined with an array of alternating his tidine and glutamate residues. Together with a chloride ion bridging t he gap between His 852 and His 837, they could have the potential to f orm, at low pH, a transmembrane chain of hydrogen bonds. The possible functional significance of such channel is discussed.