Glutamate 681 is thought to be located within the transport channel of band 3 (AE1, the chloride/bicarbonate exchanger), where it acts as a proton donor for the anion/proton cotransport function. Here we show that neutralization of the negative charge on glutamate 681 by chemically modifying band 3 with Woodward's reagent K plus sodium borohydride (i.e., the modification process) exposes a cryptic, conformationally active chloride-binding site which functions to modulate allosterically the conformational state of the band 3 dimer. Chloride binding was determined by measuring the effect of increasing chloride concentration on the rate of DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) release from band 3 using a stopped-flow fluorescence kinetic inhibitor replacement assay with DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) as the replacing inhibitor. The time course for DBDS release from unmodified, control band 3 was monophasic and exponential. Chloride binding to the transport site accelerated the rate of DBDS release, with the observed rate constant showing a hyperbolic dependence on chloride concentration, while the total change in reaction fluorescence remained constant. After modification of glutamate 681, DBDS release was monophasic in the absence of chloride, but the rapid addition of chloride at constant ionic strength induced a doubling in the fluorescence quantum yield for the bound DBDS molecules. This was associated with the development of 50:50 biphasic kinetics for DBDS release. Such changes were independent of the degree of modification of the band 3 subunit population between the 66% and 91% levels. Titration of the increase in total reaction fluorescence gave an apparent chloride binding K(d) of between 7 and 10 mM, which is 25-40-fold higher in affinity than chloride binding to the transport site. The dependence of the kinetic constants for both phases of the DBDS release reaction on chloride concentration was nonhyperbolic, which contrasts with unmodified band 3, and is indicative of the presence of two classes of chloride-binding sites on the modified transporter. We have also found that the fraction of subunits capable of binding DBDS reversibly, or DIDS covalently, decreased nonlinearly in the absence of chloride as the level of modification of the band 3 subunit population increased. In contrast, the same DBDS binding correlation plot showed a maximum in the presence of saturating chloride. The observation of such nonlinear correlation plots is consistent with a noncooperative dimer model for the modification process, where each dimeric species must possess different properties with respect to stilbenedisulfonate binding capacity and with respect to the spectral-kinetic response of bound stilbenedisulfonate molecules to the addition of chloride. Within the context of this model, the fractions of the three molecular dimeric species (i.e., the unmodified dimer, the dimer with one subunit modified, and the fully modified band 3 dimer) are calculated as a function of the level of modif...

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.

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Band 3 mediates both electroneutral AE (anion exchange) and APCT (anion/proton co-transport). Protons activate APCT and inhibit AE with the same pK (approximately 5.0). SDs (stilbenedisulphonates) bind to a primary, high-affinity site on band 3 and inhibit both AE and APCT functions. In this study, we present fluorescence and kinetic evidence showing that lowering the pH activates a second site on band 3, which binds DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) independently of chloride concentration, and that DBDS binding to the second site inhibits the APCT function of band 3. Activation of the second site correlated with loss of chloride binding to the transport site, thus explaining the lack of competition. The kinetics of DBDS binding at the second site could be simulated by a slow-transition, two-state exclusive binding mechanism (R0<-->T0+D<-->TD<-->RD, where D represents DBDS, R0 and T0 represent alternate conformational states at the second DBDS-binding site, and TD and RD are the same two states with ligand DBDS bound), with a calculated overall Kd of 3.9 microM and a T0+D<-->TD dissociation constant of 55 nM. DBDS binding to the primary SD site inhibited approx. 94% of the proton transport at low pH (KI=68.5+/-11.8 nM). DBDS binding to the second site inhibited approx. 68% of the proton transport (KI=7.27+/-1.27 microM) in a band 3 construct with all primary SD sites blocked through selective cross-linking by bis(sulphosuccinimidyl)suberate. DBDS inhibition of proton transport at the second site could be simulated quantitatively within the context of the slow-transition, two-state exclusive binding mechanism. We conclude that band 3 contains two DBDS-binding sites that can be occupied simultaneously at low pH. The binding kinetic and transport inhibition characteristics of DBDS interaction with the second site suggest that it may be located within a gated access channel leading to the transport site.