Dibenzamido-2,2'-stilbenedisulfonate Research Articles

Studies on the DIDS-binding Site of Monocarboxylate Transporter 1 Suggest a Homology Model of the Open Conformation and a Plausible Translocation Cycle

Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys38, Lys45, Lys282, and Lys413. K38Q-MCT1 and K38R-MCT1 were inactive when expressed at the plasma membrane of Xenopus laevis oocytes, whereas K45R/K282R/K413R-MCT1 and K45Q/K282Q/K413Q-MCT1 were active. The former exhibited normal reversible and irreversible inhibition by DIDS, whereas the latter showed less reversible and no irreversible inhibition. K45Q/K413Q-MCT1 retained some irreversible inhibition, whereas K45Q/K282Q-MCT1 and K282Q/K413Q-MCT1 did not. These data suggest that the two DIDS SO3− groups interact with positively charged Lys282 together with Lys45 and/or Lys413. This positions one DIDS isothiocyanate group close to Lys38, leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys38 or Lys290 of MCT1 and Lys160 or Lys164 of embigin. We have modeled a possible structure for the outward facing (open) conformation of MCT1 by employing modest rotations of the C-terminal domain of the inner facing conformation modeled previously. The resulting model structure has a DIDS-binding site consistent with experimental data and locates Lys38 in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys38 accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp302− and Asp306+, an ion pair already identified as important for transport and located adjacent to Phe360, which controls channel selectivity. The cross-linking data have also been used to model a structure of MCT1 bound to embigin that is consistent with published data.

EFFECTS OF INDOMETHACIN ON INTRACELLULAR CALCIUM AND PROTON REGULATORY MECHANISMS

Stilbenedisulfonates (SD) bind to a “primary” SD (PSD) site on the outer membrane surface of band 3, and inhibit anion exchange (AE) allosterically. Yet, evidence [Membr. Biochem. 2 (1979) 297] suggests that SD can be transported by band 3, thus raising questions about the relative locations of the transport and SD binding sites. A “second” class of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) binding sites has been discovered, which is activated by protons (pK ∼ 5.0), and is located on the membrane domain of band 3 [Biochem. J. 388 (2005) 343]. Here we show that the “second” class of DBDS binding sites, not the PSD site, lies on the SD transport pathway. We compare the pH dependence of DBDS influx to DBDS binding using: (a) control cells, (b) cells selectively crosslinked at the PSD site by treatment with 300 μM BS3 (bis(sulfosuccinimidyl)suberate), and (c) cells with DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) bound covalently to the PSD site. DBDS binds to the “second” class of sites on band 3 in all three types of cells. DBDS does not bind to the PSD sites of BS3- or DIDS-modified cells. Proton-activated DBDS influx was observed using control and BS3-modified cells, but not when using DIDS-modified cells. The results with DIDS suggest that the PSD site and the transport site overlap. However, this interpretation is disproved by experiments with BS3-modified cells, where the PSD site is blocked, yet DBDS transport and binding to the “second” class of sites both take place.

Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. I: Functional identification of the proton-activated stilbenedisulfonate influx site

Stilbenedisulfonates (SD) bind to a “primary” SD (PSD) site on the outer membrane surface of band 3, and inhibit anion exchange (AE) allosterically. Yet, evidence [Membr. Biochem. 2 (1979) 297] suggests that SD can be transported by band 3, thus raising questions about the relative locations of the transport and SD binding sites. A “second” class of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) binding sites has been discovered, which is activated by protons (p K ∼ 5.0), and is located on the membrane domain of band 3 [Biochem. J. 388 (2005) 343]. Here we show that the “second” class of DBDS binding sites, not the PSD site, lies on the SD transport pathway. We compare the pH dependence of DBDS influx to DBDS binding using: (a) control cells, (b) cells selectively crosslinked at the PSD site by treatment with 300 μM BS 3 (bis(sulfosuccinimidyl)suberate), and (c) cells with DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) bound covalently to the PSD site. DBDS binds to the “second” class of sites on band 3 in all three types of cells. DBDS does not bind to the PSD sites of BS 3- or DIDS-modified cells. Proton-activated DBDS influx was observed using control and BS 3-modified cells, but not when using DIDS-modified cells. The results with DIDS suggest that the PSD site and the transport site overlap. However, this interpretation is disproved by experiments with BS 3-modified cells, where the PSD site is blocked, yet DBDS transport and binding to the “second” class of sites both take place.

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.

Slow transitions between two conformational states of band 3 (AE1) modulate divalent anion transport and DBDS binding to a second site on band 3 which is activated by lowering the pH (pK ∼5.0)

Evidence is emerging which indicates that the anion transport activity of band 3 may be regulated. I review the molecular basis for regulation of the anion transport function of band 3 in terms of evidence showing that divalent anion transport involves a slow “hysteretic” transition between two functional states, mediated by interactions between subunits within band 3 oligomers. In addition, I briefly describe recent work from my laboratory where we have discovered a novel manifestation of slow conformational changes in band 3. This involves 4,4′-dibenzamido-2,2′-stilbenedisulfonate (DBDS) binding to a second, proton-activated site distinct from the primary stilbenedisulfonate site. This site is exposed on the outer aspect of band 3 when the pH is lowered (pK ∼5.0). This is the same pK as the protonation site on band 3 involved in divalent anion–proton co-transport (APCT) [J. Gen. Physiol. 79 (1982) 87]. Significantly, we have found that DBDS binding to this proton-activated site has unusually slow kinetics, and increasing DBDS concentration causes a decrease in the apparent pseudo-first-order rate constant. These results suggest that a slow conformational pre-equilibrium is the rate limiting step in DBDS binding to the proton-activated site on band 3 observed at low pH. Our results support an allosteric two-state model for regulation of divalent anion transport by band 3 oligomers involving a slow conformational transition and interactions between subunits [Biochemistry 31 (1992) 7301].

Kinetic Mechanism of DIDS Binding to Band 3 (AE1) in Human Erythrocyte Membranes

ABSTRACTStilbenedisulfonates (S) are used widely in cell biology as competitive inhibitors of anion exchange, but the mechanism of competition is not resolved. Resolution requires understanding the detailed steps in the reaction of stilbenedisulfonates with various anion-exchange proteins. Studies on the reversible binding of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) and H2DIDS (4,4′-diisothiocyanatodihydro-2,2′-stilbenedisulfonate) to erythrocyte band 3 (B) have shown biphasic kinetic time courses at 25°C. Yet, results for the reversible binding of DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) are controversial. One recent report has shown monophasic kinetics, in experiments performed at 0°C, and at a single, very low concentration of DIDS (0.1 μM). Studies are presented which attempt to reconcile these recent findings with the other kinetic data in the literature. We measure the kinetics of DIDS reversible binding to band 3, over a wide DIDS concentration range. In addition, the time course for DIDS binding to band 3 at 0°C is compared with that at 25°C. The results show biphasic binding kinetics at both 0 and 25°C, and they are consistent with expectations for a two-step binding mechanism (S + B ⇄ SB ⇄ SB*). Furthermore, computer-assisted model simulation studies reveal that monophasic DIDS binding kinetics are generated by a two-step mechanism, when calculations are performed at 0.1 μM DIDS and 0°C. Under these conditions the initial binding step in the two-step reaction becomes rate limiting. We conclude that the two-step binding mechanism best describes stilbenedisulfonate binding to band 3 and that the observation of monophasic kinetics at low concentrations of DIDS, while valid, is not mechanistically discriminating, since both one-step and two-step mechanisms can yield the same result.

Anion binding characteristics of the band 3 / 4,4'-dibenzamidostilbene-2,2'-disulfonate binary complex: Evidence for both steric and allosteric interactions

A novel kinetic approach was used to measure monovalent anion binding to better define the mechanistic basis for competition between stilbenedisulfonates and transportable anions on band 3. An anion-induced acceleration in the release of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) from its complex with band 3 was measured using monovalent anions of various size and relative affinity for the transport site. The K1/2 values for anion binding were determined and correlated with transport site affinity constants obtained from the literature and the dehydrated radius of each anion. The results show that anions with ionic radii of 120-200 pm fall on a well-defined correlation line where the ranking of the K1/2 values matched the ranking of the transport site affinity constants (thiocyanate < nitrate approximately bromide < chloride < fluoride). The K1/2 values for the anions on this line were about 4-fold larger than expected for anion binding to inhibitor-free band 3. Such a lowered affinity can be explained in terms of allosteric site-site interactions, since the K1/2 values decreased with increasing anionic size. In contrast, iodide, with an ionic radius of about 212 pm, had a 10-fold lower affinity than predicted by the correlation line established by the smaller monovalent anions. These results indicate that smaller monovalent anions have unobstructed access to the transport site within the band 3 / DBDS binary complex, while iodide experiences significant steric hindrance when binding. The observation of steric hindrance in iodide binding to the band 3 / DBDS binary complex, but not in the binding of smaller monovalent anions, suggests that the stilbenedisulfonate binding site is located at the outer surface of an access channel leading to the transport site.

Quantitative analysis of the kinetics of stilbenedisulfonate binding to band 3

Stilbenedisulfonates are potent inhibitors of erythrocyte band 3 chloride/bicarbonate exchange. Band 3 exists as dimers and tetramers in situ, and each monomer binds one stilbenedisulfonate molecule. We determine: (a) whether stilbenedisulfonates exhibit cooperativity in reversible binding to the Band 3 dimer, and (b) whether stilbenedisulfonates directly compete with chloride. Stopped-flow and static fluorescence spectroscopy were used to measure the kinetics and equilibrium of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) binding to isolated and membrane-bound Band 3. DBDS binding showed biphasic kinetic time courses which were consistent with a two step mechanism: ▪ Static binding studies showed no evidence for cooperativity, in agreement with the kinetic measurements. Chloride (150 mM) strongly affected the second step in the binding process by increasing k −2 about 20-fold, without significantly affecting k 1, k −1 or k 2. Our results indicate: (a) that DBDS binds independently to each monomer of the band 3 dimer, and (b) that DBDS is not competitive with chloride for binding to the transport site, but rather interacts with the transport site allosterically.

Conformational changes in human red cell membrane proteins induced by sugar binding

We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

Interaction among anion, cation and glucose transport proteins in the human red cell.

The time course of binding of the fluorescent stilbene anion exchange inhibitor. DBDS (4.4'-dibenzamido-2.2'-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant. tau DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3 +/- 0.1 microM) for H2-DIDS (4.4'-diisothiocyano-2.2'-dihydrostilbene disulfonate) inhibition of tau DBDS is virtually the same as the ID50 (0.47 +/- 0.04 microM) for H2-DIDS inhibition of red cell Cl- flux, thus relating tau DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in tau DBDS, which can be reversed with intracellular, but not extracellular, D-glucose, ID50 for cytochalasin B modulation of tau DBDS is 0.1 +/- 0.2 microM in good agreement with KD = 0.06 +/- 0.005 microM for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 microM), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl- exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport protein complex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.

Mechanism of coupling between Cl − and OH − transport in renal brush-border membranes

The coupling mechanism for Cl − and H +/OH − transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H + and Cl − gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H + gradients and Cl −, gluconate, or sulfate gradients, each with and without a K +/valinomycin voltage clamp. Parallel experiments were performed with vesicles equilibrated with 10 mM HCO 3 − or 5 mM formate. Rate of H +/OH − transport was determined from the initial rate of change in 6-carboxyfluorescein fluorescence, vesicle buffer capacity and the relationship between fluorescence and vesicle pH. In contrast to gluconate or sulfate, Cl − caused enhanced H +/OH − transport under all conditions. This difference was eliminated with voltage clamping in the presence of gluconate, SO 4 2−, or HCO 3 −, but not in the presence of formate. These findings were not affected by the method of preparation of the vesicles. Electrically coupled Cl −/OH − transport was not inhibited by 100 μM DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate) or 100 μM DBDS (4,4′-dibenzamidostilbene-2,2′-disulfonate). SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate) was found to be a protonphore at concentrations > 500 μM. As a control for the method, we demonstrated amiloride inhibitable, electroneutral Na +-H + exchange (H + flux = 107 ± 9 nmol/s per mg, 100 mM Na +) and electroneutral, DBDS inhibitable Cl −-HCO 3 − exchange in sealed human red blood cell ghosts. Therefore, electroneutral Cl −-OH − or HCO 3 − exchange does not measurably contribute to Cl − transport in the proximal tubule brush border. Cl −-formate exchange with formic acid recyling appears to be the only electroneutral coupling mechanism between Cl − and OH − transport demonstrable in renal brush-border membrane vesicles.

Control of red cell urea and water permeability by sulfhydryl reagents

The binding constant for pCMBS ( p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 ± 30 μM and that for urea transport inhibition to be 0.09 ± 0.06 μM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10 5 times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4′-dibenzamidostilbene-2,2′-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.