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.