Exploration of the functional significance of the stilbene disulfonate binding site in mouse band 3 by site-directed mutagenesis

Abstract

In 1985, it was shown that microinjection into Xenopus oocytes of mRNA prepared from the spleens of anemic mice leads to biosynthesis of band 3 protein (as shown by immunoprecipitation) and induction of a CIflux, which is not normally seen in the oocytes (Morgan et aI., 1985). This flux can be inhibited by typical inhibitors of band 3-mediated anion exchange, including 4,4' -dinitro stilbene-2,2'-disulfonate (DNDS), dipyridamole, phloretin and H+. These results encouraged us to microinject band 3 (AEl)-encoding cRNA derived from mouse band 3 cDNA (Hanke-Baier et aI., 1988). After biosynthesis of band 3, we microinjected 36CIinto oocytes and placed them individually into a perfusion chamber the bottom of which is formed by the mica window of a Geiger Muller tube. Perfusion of the chamber with a modified Amphibian Ringer solution removes 36CIescaping from the oocyte. The radioactivity inside the oocyte is recorded as a function of time. On a semilog scale, a plot of radioactivity vs. time yields a straight line relationship which persists until 80% of the initial radioactivity has left the oocyte. The slope of the straight line yields the rate constant for CIefflux (Grygorczyk et aI., 1987, 1989; Kietz et aI., 1991a). This efflux is inhibited when the CIof the Ringers solution is replaced by slowly penetrating SO/ions or nonpenetrating gluconate ions, indicating that, as in the red cell, the 36C1efflux can only take place by anion exchange. The K] values for H 2DIDS and DNDS are of the same order of magnitude as in the red cell (Kietz et aI., 1991a; Morgan et aI., 1985). Moreover, again as in the red cell, the hydrophobic domain suffices for the mediation of anion exchange after expression in the oocyte and remains susceptible to inhibition by H 2DIDS or DNDS (Lepke et aI., 1991). The agreement of important features of mouse band 3-mediated anion transport in red cells and oocytes encouraged us to use oligonucleotide-directed mutagenesis of a number of (conserved) lysine residues, Lys 449,558,649,869, to localize the sites of action of a range of covalently binding, inhibitory amino group reactive agents in the known primary structure of band 3 (see Fig 1). Regardless of the mutation, anion exchange is still accomplished. Thus, none of these residues is directly involved in anion binding and translocation. However, the susceptibility to inhibition by covalently and noncovalently binding inhibitors is drastically altered. Substitution by Asn of Lys 558 and Lys 561 (Bartel et aI., 1989b), or of Lys 558 alone (Bartel, 1989a), does not prevent reversible inhibition of CI/CIexchange by H 2DIDS, but prevents the subsequent irreversible reaction. Each of the two isothiocynate (NCS) groups of H 2DIDS is able to react with a different lysine residue in wild type band 3, to form an intramolecular cross-link. Because the NCS groups only react with the deprotonated forms of the lysine NH2 group, the reaction rate depends on pH. One of the NCS groups reacts with a lysine residue with pK ::II: 8.2, the other with a residue with pK '' 10.8 (20°C). In the wild type, the rate of covalent H2DIDS binding varies with pH in a manner which suggests reaction with the lysine residue with the lower pK value. After the mutation K558N, the pH dependence changes to a pattern compatible with a reaction with the lysine residue with the higher pK value (Kietz, 1991b). Pyridoxal phosphate (P-5P; 5 mM, pH 7.6) produces irreversible inhibition in the wild type (KK) and in the mutant in which Lys 558 (NK) or Lys 869 (KM) had been replaced by asparagine (N) or methionine (M), respectively. However, when both residues are replaced (NM), irreversible inhibition can no longer be achieved. This shows that P5-P is capable of producing inhibition with either one of the lysine residues 558 or 869 (Wood et aI., 1991; Kawano et aI., 1988). Inhibition by DNDS changes dramatically upon mutation. The Kial'P increases from 6.0 J.LM in the wild type (KK) to 23 J.LM in the mutant NK, to 73 J.LM in the mutant KM, and to 473 J.LM in the mutant NM. The K, value for activation of the transport system is 11 mM both in the wild type (KK) and in the mutant (NM) if measured by isosmotic substitution of CIby gluconate, which is not a substrate of band 3 (Passow et aI., 1992). The results show that both Lys 558 and Lys 869 are involved in the maintenance of the structure of the overlapping binding sites for stilbene disulfonates and the substrate CIand that at last one of the two is allosterically linked to the substrate binding site. In the mutant NM, the K( value for reversible H2DIDS binding is considerably increased. Nevertheless, at alka-