Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. III: Role of solute and protein structure in proton-activated stilbenedisulfonate influx

DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) influx into magnesium resealed ghosts (MRSG) occurs over the anion/proton co-transport pH range (pK 5.0). Here, factors are studied which may influence the pH dependence of DBDS transport. Accumulation of various stilbenedisulfonate (SD) molecules was studied and found to be correlated with the hydrophobicity of the R-groups (Hansch factor), not protonation of the sulfonates. The role of proton binding to glutamate 681 was found not to be part of the rate-limiting step in DBDS uptake by MRSG. Finally, the pH dependence of changes in quaternary structure/conformational state was investigated using an assay involving photo-crosslinking of band 3 subunits in the presence of DASD (4,4′-diazido-2,2′-stilbenedisulfonate). Lowering the pH promoted intersubunit crosslinking by DASD, with a pK value of 4.75 ± 1.0. This value is comparable to the pK for DBDS binding to the “second” class of sites on control band 3 (pK = 5.01 ± 0.16), and to DBDS influx into control MRSG (pK values between 4.57 ± 0.15 and 4.7 ± 0.1). Susceptibility to photo-crosslinking was reversed by raising the pH prior to initiation of the reaction. Significantly, no photo-crosslinking was observed between pH 6.0 and 8.0, where band 3 subunits are known to exist as stable dimers and tetramers. We conclude that intersubunit photo-crosslinking does not simply involve random collision between photo-activated DASD and band 3. Rather, proton binding to band 3 either alters the conformation at the interface between subunits of pre-existing tetramers, or it promotes self-association of stable dimers to a “novel” tetrameric conformational state.