Differential Sensitivity of Stilbenedisulfonates in their Reaction with Band 3 HT (Pro-868 → Leu)

• Published in: Proceedings of the National Academy of Sciences - PNAS 1995-12, Vol.92 (25), p.11844-11848
• Main Authors: Salhany, James M., Schopfer, Lawrence M., Marguerite M. B. Kay, Gamble, Debra N., Lawrence, Christine
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - analogs & derivatives; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - metabolism; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Adducts; Anion Exchange Protein 1, Erythrocyte - antagonists & inhibitors; Anion Exchange Protein 1, Erythrocyte - drug effects; Anion Exchange Protein 1, Erythrocyte - genetics; Anion Exchange Protein 1, Erythrocyte - metabolism; Anions - metabolism; Biochemistry; Biological Transport; Blood; Cellular biology; Chlorides; Fluorescence; Humans; Kinetics; Male; Mathematical constants; Models, Molecular; Phosphates; Point Mutation; Protein Conformation; Proteins; Replacement reactions; Sodium; Spectrometry, Fluorescence; Tanneries
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.25.11844

Band 3 HT (Pro-868$\longrightarrow$Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostillbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.