Development of an spFRET method to measure structure changes in ion exchange proteins.

AIM: Understanding the mechanism of tightly coupled ion exchange proteins, important effectors of cell volume regulation and other physiologically important transport processes requires means to observe dynamic changes in structure during the transport cycle. As a step towards this goal, we have applied single-pair fluorescence resonance energy transfer to a monomeric bacterial oxalate-formate exchanger (OxlT). METHODS: A His-9 tagged OxlT mutant containing two cysteines at positions 17 and 224 was labelled with cyanine dye maleimides (Cy3 donor and Cy5 acceptor) and attached to glass coverslips for measurements of donor and acceptor emission from single molecules, as described (P. Pal et al. Biophys J89, L11, 2005). RESULTS: Time-series data from 20 spots containing donor and acceptor provided evidence for single-pair energy transfer. From the efficiency of energy transfer, the mean donor-acceptor distance was determined to be 44.2 A. Considering the size of the probes, this is in good agreement with the Calpha distance of 39.6 A for the corresponding sites found in the OxlT structural (homology) model (Q. Yang et al. Proc Natl Acad Sci102, 8513, 2005). CONCLUSION: These results demonstrate the feasibility of single-pair fluorescence resonance energy transfer to measure distances between known sites in single OxlT molecules. This technique provides a potential means to test models for transport-related conformational changes, as well as to detect real-time structure alterations during the catalytic transport cycle.

Hypotonicity induces L-selectin shedding in human neutrophils.

Expression levels of adhesion molecules on neutrophils are affected under various conditions, including ischemia, possibly because of associated increases in cell volume. We examined the effects of cell swelling in hypotonic media on the level of L-selectin (CD62L) and beta(2)-integrin (CD18) on human neutrophils. In hypotonic media, neutrophils shed L-selectin. The shedding was greatly reduced by 30 microM RO31-9790, the metalloprotease (sheddase) inhibitor. Hypotonicity-induced L-selectin shedding was also time and tonicity dependent. Decreasing tonicity caused increased shedding. In 0.6x medium (0.6x the normal tonicity of 300 mosmol/kgH(2)O), shedding increased over a 2-h period, after which >70% of the neutrophils had lost L-selectin. In contrast to L-selectin, the level of beta(2)-integrin on the neutrophil surface was not significantly affected. Thus L-selectin shedding, which occurs on neutrophil activation and is usually accompanied by beta(2)-integrin upregulation, was selectively induced by hypotonicity without a corresponding effect on beta(2)-integrin.

The noncompetitive inhibitor WW781 senses changes in erythrocyte anion exchanger (AE1) transport site conformation and substrate binding.

WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl(-). Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl(-) seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl(-) does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl(-) binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl(-)-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl(-)-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure-function relationships.

Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3.

Although its primary function is monovalent anion exchange, the band 3 protein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conserved throughout the anion exchanger family (AE family). To determine whether or not the monovalent anion binding site is located near Glu-681, we modified this residue with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate; WRK). Measurements of Cl(-) binding by (35)Cl-NMR show that external Cl(-) binds to band 3 even when Cl(-) transport is inhibited approximately 95% by WRK modification of Glu-681. This indicates that the external Cl(-) binding site is not located near Glu-681 and thus presumably is distant from the proton binding site. DIDS inhibits Cl(-) binding even when WRK is bound to Glu-681, indicating that the DIDS binding site is also distant from Glu-681. Our data suggest that the DIDS site and probably also the externally facing Cl(-) transport site are located nearer to the external surface of the membrane than Glu-681.

Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine.

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 microM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 microM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.

Source of transport site asymmetry in the band 3 anion exchange protein determined by NMR measurements of external Cl- affinity.

Flux measurements indicate that a far greater number of unloaded band 3 anion transport sites face the cytoplasm than face the external medium, but the reason for this striking asymmetry has remained obscure. To resolve this question, we have measured the apparent Cl- affinity of the transport site of human red blood cell band 3 protein under various conditions by analyzing the 35Cl NMR free induction decay (FID). The [Cl-] that half-saturates the transport sites with [Cli] = [Clo] (K1/2) in RBC membranes (ghosts) is 46 +/- 5 mM at 0 degree C, while the Ko1/2 (for half-saturation with [Clo] at constant [Cli]) of intact cells is 3.2 +/- 2.1 mM. When cells were pretreated with EM, an inhibitor of band 3 anion exchange that does not prevent Cl- binding to the external transport site, K1/2 and Ko1/2 are 41 +/- 14 and 46 +/- 12 mM, respectively. The EM-induced increase in Ko1/2 with little change in K1/2 can be most simply interpreted as meaning that EM abolishes the effects of the translocation rate constants on Ko1/2 so that Ko1/2 and K1/2 of EM-treated cells now both reflect the true dissociation constant for binding of Cl- to the external transport site, Ko. The fact that Ko for a slowly transported anion, iodide, is nearly the same in EM-treated as in control cells indicates that EM does not significantly affect Ko for chloride. Our results indicate that the true dissociation constants for Cl- at the inside and outside are very similar but that the rate constant for inward translocation is much larger than that for outward translocation. For this reason, both unloaded and Cl-loaded transport sites are asymmetrically oriented toward the inside, and Ko1/2 (in untreated cells) is much lower than Ko.

Kinetics of bicarbonate transport in human red blood cell membranes at body temperature.

We studied unidirectional [14C]HCO3- efflux from human resealed red cell ghosts with 1 mM acetazolamide under self-exchange conditions at pH = pH(i = o) 7.4-9.0 and 0-38 degrees C by means of the Millipore-Swinnex and continuous flow tube filtering techniques. 14CO2 loss from cells to efflux medium and further to the atmosphere was insignificant. [14C]HCO3- efflux was determined at pH 7.8, 38 degrees C under symmetric variation of the HCO3- concentrations (C(i = o)), and asymmetric conditions: C(i) varied, C(o) constant, or C(o) varied, C(i) constant. MM-fit, Jeff = Jmaxeff x C x (C + K1/2)-1, used to describe the concentration dependence of Jeff,o when only C(o) varied, yields at C(i) = 50 mM: K1/2o = 3.8 mMJ, Jmaxeff.o = 20 nmol cm-2 s-1; at C(i) = 165 mM: K1/2o = 10 mM, Jmaxeff.o = 32 nmol cm-2 s-1. When C(i) varied, noncompetitive self inhibition by HCO3- binding (inhibitor constant K1) to an intracellular site was included (MS-fit). Under conditions of (a) symmetry: C(i = o) = 9-600 mM, K1/2s = 173 mM, K1 = 172 mM, and Jmaxeff,s = 120 nmol cm-2 s-1, (b) asymmetry: C(o) = 50 mM, K1/2i = 116 mM, K1 = 136 mM, and Jmaxeff,i = 92 nmol cm-2 s-1. All flux parameters accord with the ping-pong model for anion exchange. The data for C(i) < 200 mM also fit well to the MM equation, but K1/2 and Jmaxeff are different from the MS-fit and are inconsistent with the ping-pong model. Thus, self-inhibition (MS-fit) must be included even at low concentrations. As at 0 degree C, the system is asymmetric: 8-10 times more unloaded transport sites face inward than outward when C(i = o). Jeff,s was not mono-exponentially dependent on temperature at 0-38 degrees C, indicating that the transmembrane anion transport is controlled by several rate constants with different temperature dependencies. Jeff,s was not significantly affected by increasing pH(i = o) from 7.4 to 7.8, but it decreased by 50% when pH was raised to 9.0.

The asymmetry of chloride transport at 38 degrees C in human red blood cell membranes.

Band 3-mediated Cl- exchange in human red blood cells and resealed ghosts was measured at 38 degrees C by the continuous flow tube method. When external Cl- concentration, C(o), is varied with constant internal Cl- concentration, C(i), the flux fits a simple Michaelis-Menten saturation curve (MM fit), with K1/2o = 3.8 +/- 0.4 mM. When the Cl- concentration is varied simultaneously at both sides of the membrane in resealed ghosts (C(i) = C(o) = C(i = o)), the flux rises toward a flat maximum between 200 and 450 mM Cl-, and then decreases at very high C(i = o). An MM fit to the data with C(i = o) < 500 mM gives K1/2s of 106 +/- 13 mM; fits including modifier site inhibition (MS fit) give an over threefold higher K1/2s. Despite this uncertainty, the intrinsic asymmetry of unloaded transport sites, A (defined as E(o)/E(i) with C(i) = C(o), where E(i) is the fraction of unloaded inward-facing sites and E(o) is the fraction of unloaded outward-facing sites), calculated from K1/2s and K1/2o, ranges only from 0.046 to 0.107. A new method, which uses the initial slope of a plot of Cl- flux versus C(i = o), gives A values of 0.023 to 0.038. Flufenamic acid (FA) inhibits Cl- exchange by binding to an external site different from the transport site. At 38 degrees C, FA binds 24-36 times more tightly to E(o) than to E(i). Estimates of A from FA inhibitory potency range from 0.01 to 0.05. All methods, including bicarbonate data from the preceding paper, indicate that at 38 degrees C, like 0 degree C, far more band 3 molecules are in the E(i) than in the E(o) form. The agreement of various methods supports the ping-pong model for anion exchange, and demonstrates that the intrinsic asymmetry is very slightly, if at all, affected by temperature.

Swelling-activated amino acid efflux in the human neuroblastoma cell line CHP-100.

1. The effects of hypoosmotic stress on cell volume and amino acid efflux were evaluated in the human neuroblastoma cell line CHP-100 with the Coulter Counter Multisizer and radiolabeled amino acid efflux, respectively. 2. CHP-100 cells swelled by approximately 35 +/- 5% (means +/- SE) when the osmolarity of the solution was decreased from 290 to 190 mOsm/kg H2O. The rapid swelling was followed by a biphasic regulatory volume decrease (RVD). 3. In cells loaded with 14C-taurine, hypoosmotic stress induced a 300 +/- 22% (n = 23, P < 0.05) increase in taurine efflux compared with controls. This efflux was inhibited by the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS), niflumic acid and by the volume-activated anion channel blocker tamoxifen. In addition, the swelling-activated taurine efflux was dependent upon extracellular calcium. 4. Similarly, in cells loaded with 14C-glycine, hypoosmotic stress significantly increased glycine efflux, which was also sensitive to NPPB. In contrast, efflux of 3H-glutamate was not significantly altered after hypoosmotic stress. 5. With the use of patch clamp recording techniques, Cl- channels were activated in cell attached patches after exposure to hypoosmotic solutions. 6. In nystatin perforated patches, permeability of the hypoosmotically activated anion channel was observed to be SCN- > I- > Br- > Cl- >> Glutamate. 7. It is concluded that in CHP-100 cells, anion channels are activated during hypoosmotic stress and these channels represent a pathway for efflux of amino acids.

Changes in cytoskeletal actin content, F-actin distribution, and surface morphology during HL-60 cell volume regulation.

Cell volume regulation occurs via the regulated fluxes of ions and solutes across the cell membrane in response to cell volume perturbations under anisotonic conditions. Our earlier studies in human promyelocytic leukemic HL-60 cells showed that volume-dependent changes in total cellular F-actin content occur concomitantly as an inverse function of acute cell volume changes in anisotonic media (Hallows et al., 1991, Am. J. Physiol., 261:C1154-C1161). Although treatment with cytochalasin under anisotonic conditions significantly reduced total cellular F-actin levels, cytochalasin did not significantly affect the ability of cells to undergo normal volume regulation responses, which suggested that these volume-dependent changes in F-actin content may not play a critical role in HL-60 cell volume regulation. To examine more closely the possible role of the actin cytoskeleton in HL-60 cell volume regulation, we quantitated changes in Triton-insoluble cytoskeletal actin in the presence and absence of cytochalasin and also observed changes in F-actin distribution and surface morphology during volume regulation. The quantity of cytoskeletal-associated F-actin, like total F-actin, shifts inversely with initial cell volume changes in anisotonic media; however, subsequent changes in cytoskeletal actin levels during volume regulation are not significant. The soluble F-actin pool in HL-60 cells may thus be more susceptible to the physicochemical effects of shifts in cell volume than the insoluble (cytoskeletal) F-actin pool. Twenty-five micromolar dihydrocytochalasin B (DHB) treatment dramatically lowers cellular cytoskeletal actin levels by approximately 75% under resting (isotonic) conditions, but there are no significant further changes in cytoskeletal actin as cells undergo anisotonic volume regulation in the presence of DHB. These results suggest that volume-dependent changes in the absolute amounts of cytoskeletal-associated F-actin are not critical for HL-60 cell volume regulation. However, because some portions of the actin cytoskeleton are resistant to cytochalasin disruption during volume regulation, a role for the cytoskeleton in the sensing and signaling of HL-60 cell volume regulatory responses cannot be rigorously excluded. Particular F-actin distribution patterns, as observed using confocal fluorescent microscopy, were correlated with particular phases of volume regulation. Also, comparison of cellular F-actin distribution with surface morphology (observed by scanning electronic microscopy) of cells during volume regulation reveals a positive correlation between surface blebs and increased cortical F-actin staining intensity.

Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance.

We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site. Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions. In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential. The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange. This result supports previous reports that EM does not block the external chloride binding site. The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.

Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3.

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6-fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.

Effects of external pH on binding of external sulfate, 4.4-dinitro-stilbene-2,2'-disulfonate (DNDS), and chloride to the band 3 anion exchange protein.

A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'-disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.

Volume-activated chloride channels in HL-60 cells: potent inhibition by an oxonol dye.

When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K+ and Cl-. This is primarily caused by a dramatic increase in Cl- permeability, which may reflect the opening of volume-sensitive channels (11). To test this hypothesis, we measured volume-activated Cl- currents in HL-60 cells using the patch-clamp technique. The whole cell Cl- conductance (in nS/pF at 100 mV) increased from 0.09 +/- 0.06 to 1.15 +/- 0.19 to 1.64 +/- 0.40 as the tonicity (in mosmol/kgH2O) of the external medium was decreased from 334 to 263 to 164, respectively. Cl- currents showed no significant inactivation during 800-ms pulses. Current-voltage curves exhibited outward rectification and were identical at holding potentials of 0 or -50 mV, suggesting that the gating of the channels is voltage independent. The selectivity sequence, based on permeability ratios (PX/PCl) calculated from the shifts of the reversal potentials, was SCN- > I- approximately NO3- > Br- > Cl- >> gluconate. 4-Acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid (SITS; 0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with approximately 10-fold increased affinity at potentials greater than +40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20% of the membrane potential. These Cl- channels were also inhibited in a voltage-independent manner by the oxonol dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol [diBA-(5)-C4] with a concentration that gives half inhibition (IC50) of 1.8 microM at room temperature. A similar apparent IC50 value (1.2 microM) was observed for net 36Cl- efflux into a Cl(-)-free hypotonic medium at 21 degrees C. It seems likely, therefore, that the volume-activated Cl- channels are responsible for the net Cl- efflux during RVD. These Cl- channels have properties similar to the "mini-Cl-" channels described in lymphocytes and neutrophils and are strongly inhibited by low concentrations of diBA-(5)-C4.

An oxonol dye is the most potent known inhibitor of band 3-mediated anion exchange.

When cells are acutely exposed to the oxonol dye, bis(1,3-dibutylbarbituric acid)pentamethine oxonol (diBA), at 0 degrees C, the concentration that gives half inhibition of Cl- exchange (IC50) is 0.146 +/- 0.013 microM (n = 12) initially, but the inhibition increases with time. These characteristics indicate that a rapid initial binding is followed by a slow conformational change that makes the binding tighter. If diBA is allowed to equilibrate with band 3, the IC50 is only 1.05 +/- 0.13 nM (n = 5), making diBA a more potent inhibitor than 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), for which the IC50 under similar conditions is 31 +/- 6 nM [T. Janas, P. J. Bjerrum, J. Brahm, and J. O. Wieth. Am. J. Physiol. 257 (Cell Physiol. 26): C601-C606, 1989]. Inhibition by diBA is very slowly reversible at 0 degrees C (t1/2 > 50 h), but the effect is more readily reversible at higher temperatures. DiBA competes with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) for inhibition, suggesting an external site of action. In contrast to DIDS and DNDS, however, increasing Cl- concentrations do not decrease the inhibitory effect of diBA, indicating that the inhibition is not competitive. Thus diBA may be useful for investigating conformational changes during anion exchange and for stopping transport without preventing substrate binding. However, when diBA and other oxonols are used to sense membrane potential, they may have undesirable side effects on anion transport processes.

35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3.

It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.

NIP- and NAP-taurine bind to external modifier site of AE1 (band 3), at which iodide inhibits anion exchange.

External iodide (I-o) inhibits AE1 (band 3)-mediated anion exchange in human red blood cells by binding to a noncompetitive inhibitory site, the external halide modifier site. External N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) and N-(4-isothiocyano-2-nitrophenyl)-2-aminoethyl sulfonate (NIP-taurine) also inhibit Cl- exchange noncompetitively. Increasing I-o decreases the inhibitory potency of NIP-taurine in a competitive fashion; this effect is not due to I- binding to the transport site, which has little effect on the NIP-taurine affinity. Bis(sulfosuccinimidyl)-suberate (BSSS) abolishes the noncompetitive inhibitory effect of I-o and greatly reduces the inhibitory effect of NAP-taurine. Together with previous work, these data suggest that external halides, such as I-, Br-, and probably also Cl-, bind to the same noncompetitive inhibitory site as do NAP- and NIP-taurine and that these reagents can be used to label the halide modifier site. Lys-539, a probable reaction site of BSSS, lies within the same segment of AE1 that is labeled by NAP-taurine and thus may be part of the modifier site.

Regulatory volume decrease in HL-60 cells: importance of rapid changes in permeability of Cl- and organic solutes.

Results obtained through the use of inhibitors and isotope flux and equilibration techniques indicate that the regulatory volume decrease (RVD) response of human promyelocytic leukemic HL-60 cells occurs largely through the efflux of K+ and Cl- through separate conductive membrane pathways. These "channels" differ pharmacologically and in their modes of activation from those described in lymphocytes and Ehrlich ascites tumor cells. With use of measured 86Rb+ and 36Cl- fluxes, together with a diffusion kinetic model, the membrane potential (Em) and apparent K+ and Cl- permeabilities (PK and PCl) were estimated under various isotonic and hypotonic conditions. Under isotonic (300 mosM) conditions, Em is close to the Nernst potential for K+ and PCl is < 0.1 PK. Rapid and steeply graded increases in the measured Cl- efflux rate and calculated PCl occur with decreasing tonicity, with the largest increases at tonicities < 80% of isotonic. K+ efflux and the apparent PK increase only modestly with decreasing tonicity. At 50% tonicity, PCl rises to nearly 10 times PK, which should cause substantial membrane depolarization, with Em approaching the Nernst potential for Cl-. Gramicidin treatment markedly accelerates the rate of RVD and net 36Cl- efflux in hypotonic Na(+)-and Cl(-)-free media, providing further evidence that PK is rate limiting during RVD. K+ loss exceeds Cl- loss during RVD, and the total loss of K+ and Cl- is insufficient to account for the observed degree of volume recovery in 50% tonicity media, indicating that other (organic) osmolytes must take part in the HL-60 cell RVD response.

Control of intracellular pH during regulatory volume decrease in HL-60 cells.

Intracellular pH (pHi) homeostasis was investigated in human promyelocytic leukemic HL-60 cells as they undergo regulatory volume decrease (RVD) in hypotonic media to determine how well pHi is regulated and which transport systems are involved. Cells suspended in hypotonic (50-60% of isotonic) media undergo a small (< 0.2 pH units), but significant (P < 0.05), intracellular acidification within 5 min. However, after 30 min of RVD, pHi is not significantly different from the initial pHi in 20 mM HCO3- medium and is significantly higher in HCO3(-)-free medium. Experiments performed in media with or without 150 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3- demonstrate that the anion exchanger (AE) mediates a net Cl- influx, with compensating HCO3- efflux, during RVD. To determine which transport systems are involved in counteracting this tendency toward acidification, we measured transport rates and examined the effect of transport system inhibitors on pHi. We found that inhibition of Na+/H+ exchange (NHE) with 12.5 microM ethylisoproplamiloride (EIPA) causes pHi to fall significantly by the end of 30 min of RVD. As assessed by EIPA-sensitive 22Na+ uptake measurements, NHE, largely dormant under resting isotonic conditions, becomes significantly activated by the end of 30 min of RVD, despite recovery of pHi and cell volume to near-normal levels. Thus a shift in the normal pHi dependence and/or volume dependence of NHE activity must occur during RVD under hypotonic conditions. In contrast, H(+)-monocarboxylate cotransport appears to play only a supportive role in pH regulation during RVD, as indicated by lack of stimulation of [14C]lactate efflux during RVD.

WW-781, a potent reversible inhibitor of red cell Cl- flux, binds to band 3 by a two-step mechanism.

WW-781 ([3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbit uric acid]-pentamethine oxonol), a fluorescent dye that has been used for measuring membrane potentials by optical methods, inhibits human red blood cell Cl- exchange, which is mediated by the membrane protein known as band 3 or capnophorin. The inhibition is slowly reversible upon removal of WW-781 from the medium, with a half time of approximately 4.7 min in 150 mM Cl- medium at 0 degrees C. The mechanism of inhibition by WW-781 involves a two-step binding reaction. WW-781 binds rapidly to band 3 to form an initial complex, which can also rapidly dissociate. Formation of this initial complex is followed by the much slower formation of a second complex (with a rate constant of approximately 1.1 min-1), probably involving a protein conformational change, through which WW-781 is more tightly bound to band 3. At low concentrations, WW-781 inhibits Cl- exchange with a stoichiometry of 1 WW-781 molecule per band 3 monomer, suggesting that under these conditions the binding of WW-781 is highly selective for the band 3 protein.