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Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the alpha(M) and beta(2) subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals.

Substituting the medium chloride with glucuronate or glutamate causes a rapid, 10 to 30-fold, increase in the binding of the monoclonal antibody, CBRM1/5, which recognizes the high-affinity conformation of the Mac-1 integrin. This change is reflected in functional adhesion assays that show increased adhesion to ICAM-1 coated beads. Blocking antibodies indicate that the increased adhesion is almost entirely due to Mac-1. The inhibitor NPPB (100 microM) reduces Cl(-) efflux into low Cl(-) medium by 75%, and blocks increased CBRM1/5 binding after stimulation with fMLP or TNF-alpha, but has no effect on the anion substitution induced increase in CBRM1/5 binding or adhesion to immobilized ICAM-1. Thus, changes in external anion composition, not internal chloride or increases in Cl(-) efflux, are responsible for Mac-1 activation. This effect is substantial. The percentage of Mac-1 in the high affinity state approaches 100% in glutamate and 50% in glucuronate, a far greater response than what is observed after stimulation with fMLP.

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na(+)/H(+) exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH(i) was reduced while maintaining normal extracellular pH, by using an NH(4)Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH(4)Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, beta(2)-integrin (CD11b and CD18) was only slightly upregulated in the low-pH(i) condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH(i). Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.

Cell shrinkage is a hallmark of apoptosis. Potassium efflux, which is involved in cell shrinkage, has been previously described as an essential event of apoptosis. This study was designed to address the importance of potassium efflux in hypertonicity (450 mOsm and 600 mOsm) induced apoptosis. We initiated apoptosis in HL-60 cells in hypertonic medium consisting of either high concentrations of NaCl, mannitol or KCl. Apoptotic activity was evaluated based on the DNA content of the cells, annexin-V staining and calcium content. Apoptosis was initiated in hypertonic conditions consisting of high intracellular K(+). We demonstrate that apoptosis can occur in the presence of high intracellular potassium contrary to previous predictions.

The interaction of L-selectin expressed on leukocytes with endothelial cells leads to capture and rolling and is critical for the recruitment of leukocytes into sites of inflammation. It is known that leukocyte activation by chemoattractants, the change of osmotic pressure in cell media, or cross-linking of L-selectin all result in rapid shedding of L-selectin. Here we present a novel mechanism for surface cleavage of L-selectin on neutrophils during rolling on a sialyl Lewis x-coated surface that involves mechanical force. Flow cytometry and rolling of neutrophils labeled with Qdot(R)-L-selectin antibodies in an in vitro flow chamber showed that the mechanical shedding of L-selectin occurs during rolling and depends on the amount of shear applied. In addition, the mechanical L-selectin shedding causes an increase in cell rolling velocity with rolling duration, suggesting a gradual loss of L-selectin and is mediated by p38 mitogen-activated protein kinase activation. Thus, these data show that mechanical force induces the cleavage of L-selectin from the neutrophil surface during rolling and therefore decreases the adhesion of cells to a ligand-presenting surface in flow.

The one-for-one exchange of Cl(-) and HCO(3)(-) ions is catalyzed by human erythrocyte anion exchanger 1 (hAE1) through a ping-pong mechanism whereby the protein exists in two main conformations, with the single anion-binding site exposed at either the cytoplasmic (inner) side (E(i)) or the extracellular side (E(o)), with interconversion between the two states being possible only after anion binding. Steady-state and time-resolved resonance energy transfer (FRET) techniques were used to determine the distance of the binding site for diTBA (bis-(1,3-diethylthiobarbituric acid)trimethine oxonol), a high affinity fluorescent oxonol inhibitor of hAE1, from a benchmark site (probably Lys-430) labeled by external fluorescein maleimide (FM). Using red cell ghost membranes, energy transfer distances were measured in media containing different anions between FM as the donor, covalently attached to one monomer, and diTBA as the acceptor, reversibly bound to the adjacent monomer of a hAE1 dimer. Energy transfer increased significantly in chloride or bicarbonate buffers relative to conditions where no transportable anions were present, that is, in citrate buffer. These differences in transfer efficiencies were interpreted in light of the conformational distributions of hAE1 in various buffers and the possible effects of diTBA itself on the distribution. The analysis indicates that the diTBA binding site comes closer to the FM site by approximately 7 A in chloride buffer as compared to that in citrate (or equivalent changes in diTBA orientation occur) because of the effects of anion binding. This provides the first direct physical evidence for structural changes in hAE1 induced by substrates.

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.

The cytoplasmic domain of the human erythrocyte anion exchanger 1 (cdAE1) serves as a center of organization for the red blood cell cytoskeleton as well as several metabolic enzymes and hemoglobin. The protein is known to undergo a reversible pH-dependent conformational change characterized by a 2-fold change in the intrinsic fluorescence and an 11 A change in the Stokes radius. While the exact changes in the molecular structure are unknown, on the basis of the crystal structure of the protein at pH 4.8 and site-directed mutagenesis studies, Zhou and Low (19) have proposed that the peripheral protein binding (PPB) domain of cdAE1 moves away from the dimerization domain in response to increasing alkalinity. To test this hypothesis, we have applied luminescence resonance energy transfer (LRET) to measure the intermonomer distance between donor and acceptor probes at the Cys201 site (located in the PPB domain) within the cdAE1 dimer. This distance was found to increase as the pH is increased from 5 to 10, in recombinant forms of both the wild type and a mutant (C317S) of cdAE1. Furthermore, LRET measurements in red blood cell inside-out vesicles indicate that when cdAE1 is linked to the membrane, the intermonomer distance is larger at pH 5, compared to that of the purified cdAE1 segments, and exhibits a different pH-dependent behavior. An increase in the distance was also observed on binding of a metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, to cdAE1. These data provide the first demonstration of a defined change in the molecular structure of cdAE1, and also indicate that the structure under physiological conditions is different from the crystal structure determined at low pH.

We have developed a new method for immobilization of single proteins by utilizing streptavidin-biotin and protein L-antibody interactions on glass coverslips coated with polyethylene glycol. The method is particularly well suited for single-molecule fluorescence studies. A monomeric, detergent-solubilized bacterial transport protein, GlpT, and the dimeric cytoplasmic region of a mammalian transporter, cdAE1, were immobilized by our method with a high degree of specificity. The fluorescence from single molecules attached to the immobilized proteins was detected with a high signal/noise ratio. Single-pair fluorescence resonance energy transfer (spFRET) measurements on cdAE1 dimers indicate that the structure of the protein is not compromised and provide evidence that the cdAE1 protein can exist in at least two conformations under physiological conditions.

Previous fluorescence resonance energy transfer (FRET) measurements, using BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) as a label for the disulfonic stilbene site and FM (fluorescein-5-maleimide) as a label for the cytoplasmic SH groups on band 3 (AE1), combined with data showing that the cytoplasmic SH groups lie about 40 A from the cytoplasmic surface of the lipid bilayer, would place the BIDS sites very near the membrane's inner surface, a location that seems to be inconsistent with current models of AE1 structure and mechanism. We reinvestigated the BIDS-FM distance, using laser single photon counting techniques as well as steady-state fluorescence of AE1, in its native membrane environment. Both techniques agree that there is very little energy transfer from BIDS to FM. The mean energy transfer (E), based on three-exponential fits to the fluorescence decay data, is 2.5 +/- 0.7% (SEM, N = 12). Steady-state fluorescence measurements also indicate <3% energy transfer from BIDS to FM. These data indicate that the BIDS sites are probably over 63 A from the cytoplasmic SH groups, placing them near the middle or the external half of the lipid bilayer. This relocation of the BIDS sites fits with other evidence that the disulfonic stilbene sites are located farther toward the external membrane surface than Glu-681, a residue near the inner membrane surface whose modification affects the pH dependence and anion selectivity of band 3. The involvement of two relatively distant parts of the AE1 protein in transport function suggests that the transport mechanism requires coordinated large-scale conformational changes in the band 3 protein.

Many agents that activate neutrophils, enabling them to adhere to venular walls at sites of inflammation, cause a rapid Cl(-) efflux. This Cl(-) efflux and the increase in the number and affinity of beta(2) integrin surface adhesion molecules (up-regulation) are all inhibited by ethacrynic acid and certain aminomethyl phenols. The effectiveness of the latter compounds correlates with their inhibition of swelling-activated Cl(-) channels (I(Clvol)), suggesting that I(Clvol) mediates the activator-induced Cl(-) efflux. To test this hypothesis, we used whole-cell patch clamp in hypotonic media to examine the effects of inhibitors of up-regulation on I(Clvol) in neutrophils and promyelocytic leukemic HL-60 cells. Both the channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid and [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol (WW781), a nonpenetrating oxonol, inhibited I(Clvol) at concentrations similar to those that inhibit beta(2) integrin up-regulation. However, ethacrynic acid, at the same concentration that inhibits activator-induced Cl(-) efflux and up-regulation, had no effect on I(Clvol) and swelling-activated Cl(-) efflux, providing evidence against the involvement of I(Clvol) in the activator-induced Cl(-) efflux.

To understand how red blood cell and other proteins carry out their functions, it is necessary not only to have high-resolution crystal structures, but also to have methods that can measure changes in position of parts of the protein on the scale of Angstroms. The method of luminescence resonance energy transfer (LRET) has considerable advantages for this purpose, particularly for proteins, such as the AE1 anion exchange protein in the red cell, that are homodimers. We have applied this method, using a terbium maleimide chelate (TbM) as donor and fluorescein maleimide (FM) as acceptor, to measure the distance between the C201 residues in adjacent dimerized cytoplasmic domains of AE1 (cdAE1). The distance measured by LRET (40.8 A) corresponds closely with that calculated from the crystal structure of the cdAE1, indicating that the method can provide useful information for testing hypotheses concerning motions in this and other blood cell proteins.

The tightly coupled, one-for-one exchange of anions mediated by the human red blood cell AE1 anion-exchange protein involves a ping-pong mechanism, in which AE1 alternates between a state with the anion-binding site facing inward toward the cytoplasm (Ei) and a state with the site facing outward toward the external medium (Eo). The conformational shift (Ei <--> Eo) is only permitted when a suitable substrate such as Cl(-) or HCO(3)(-) (B(-)) is bound. With no anions bound, or with Cl(-) bound, far more AE1 molecules are in the inward-facing than the outward-facing forms (Ei Eo, ECli EClo). We have constructed a model for CI(-)-B(-) exchange based on Cl(-)-Cl(-) and B(-)-B(-) exchange data, and have used it to predict the heteroexchange flux under extremely asymmetric conditions, with either all Cl(-) inside and all B(-) outside (Cli-Bo) or vice versa (Bi-Clo). The experimental values of the ratio of the exchange rate for Bi-Clo to that for Cli-Bo are only compatible with the model if the asymmetry of bicarbonate-loaded sites (A(B) = EBo/EBi) > 10, the opposite of the asymmetry for unloaded or Cl-loaded sites. Furthermore, the Eo form has a higher affinity for HCO(3)(-) than for Cl(-), whereas the Ei form has a higher affinity for Cl(-). The fact that this "passive" system exhibits changes in substrate selectivity with site orientation ("sidedness"), a characteristic usually associated with energy-coupled "active" pumps, suggests that changes in affinity with changes in sidedness are a more general property of transport proteins than previously thought.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 ([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.

Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.

Although eosin-5-maleimide (EM) covalently labels band 3 and has been thought to react at the external-facing anion transport site, EM reversibly inhibits Cl- exchange at 0 degrees C in a noncompetitive fashion, indicating that under these conditions it does not bind to the transport site [Knauf, P.A., N.M. Strong, J. Penikas, R.B. Wheeler, Jr., and S.J. Liu. Am. J. Physiol. 264 (Cell Physiol. 33): C1144-C1154 1993]. To see whether or not the covalent labeling by EM takes place at the same noncompetitive site as the reversible binding, we examined the dependence of reaction rate on EM concentration. The reaction rate saturates with increasing EM concentration, indicating that reversible binding precedes covalent reaction and that EM therefore acts as an affinity label. A more complex model in which reversible binding prevents a bimolecular reaction at a different site cannot, however, be ruled out. Cl- gradients across the membrane affect EM reversible binding in a manner suggesting that EM binds preferentially to the Eo form of band 3, with the transport site unloaded and facing outward. Thus EM binds to and probably reacts covalently with a site that is different from the transport site, but whose conformation is affected by the orientation of the transport site. Lysine-430, the amino acid residue which is covalently labeled by EM (4), may be near the transport site but does not seem to be directly involved in the binding of transported substrates such as chloride. EM binding to one band 3 monomer decreases the reactivity of the adjacent monomer but does not decrease the affinity constant of the reversible binding step that precedes covalent reaction. Although a small fraction (approximately 1%) of band 3 monomers fail to react with EM, EM nearly completely inhibits transport in those monomers with which it reacts.

We used baculovirus to transiently express a rat anion exchanger (AE2) in Spodoptera frugiperda (Sf9) insect cells. No detectable Cl(-)-HCO3- exchange activity was observed in wild type or sham-infected Sf9 cells, monitored using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, a pH-sensitive fluorescent dye. Functional expression of anion exchange activity in the AE2 recombinant baculovirus-infected cells was observed within the first day after infection and sustained over the next 3 days. The expressed AE2 anion exchange activity was Na+ independent and could be reversibly and irreversibly inhibited by the specific anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The reversible inhibition was sensitive to the concentration of DIDS, with a half inhibition of 4 microM. These results indicate that the rat AE2 protein produced in the recombinant baculovirus-infected insect cells is inserted into the plasma membrane in a biologically active form that appears suitable for functional studies of AE2.

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.

To investigate the possible role of the cytoskeleton in volume regulatory responses of human promyelocytic leukemic (HL-60) cells, we monitored and modulated the F-actin content of these cells undergoing volume regulation in anisotonic media. Initial volume changes of HL-60 cells suspended in hypertonic media followed a Van't Hoff relationship, and intracellular F-actin content during volume regulatory responses in anisotonic media changed concomitantly as an inverse function of the volume shifts. These F-actin changes were shown to be an explicit function of cell volume and not tonicity of the medium. The data fit with the idea that changes in affinity of actin-binding proteins (ABPs) for actin and/or changes in the overall effective critical concentration of actin occur during acute cell volume changes, producing shifts in the relative amounts of G- and F-actin. Treatment of HL-60 cells with dihydrocytochalasin B (DHB), which perturbs cellular actin assembly, lowered resting levels of intracellular F-actin but did not prevent volume-associated F-actin changes in anisotonic media. Despite the lowered F-actin levels, HL-60 cells in the presence of DHB still undergo normal volume regulatory responses. Thus the absolute amount of intracellular F-actin does not appear to be critical for volume regulation in HL-60 cells.

According to the ping-pong model of band 3-mediated anion exchange, the transport protein has a single transport site, which can exist in either an inward-facing or an outward-facing conformation. Anions bind to these unloaded forms of the carrier, and translocation takes place only when a suitable anion is bound to the transport site. In a previous paper [Am. J. Physiol. 257 (Cell Physiol. 26): C520-C527, 1989], we had shown that the substrate kinetics of Cl-Cl exchange in the promyelocytic HL-60 cell cannot be explained by this simple ping-pong model of anion exchange but is consistent with a simultaneous model according to which both extracellular and intracellular anions must bind before simultaneous translocation can take place. In the present paper we show that external 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibits anion exchange in HL-60 cells by competing with Cl- for binding to the outward-facing transport site. Furthermore, there is a linear dependence of the slope of the Dixon plot for inhibition by DIDS on the reciprocal of the intracellular Cl- concentration. This result clearly rules out a simple ping-pong scheme. In addition, the data also rule out a ping-pong model in which some translocation of the unloaded carrier is allowed (ping-pong model with slippage). The observed inhibition kinetics can be modeled by a simultaneous model of Cl-Cl exchange with competitive inhibition by DIDS.

Treatment with adriamycin for 8-14 h irreversibly induces K562 human erythroleukemic cells to synthesize hemoglobin. With 16-h exposure, this effect is maximal at concentrations between 180 and 400 nM, yielding 70%-90% benzidine-positive (B+) cells and 24 pg/cell hemoglobin 4 days after the beginning of adriamycin treatment. This induction is accompanied by changes in ouabain-sensitive 86Rb influx opposite to those seen with murine erythroleukemic (MEL) cells. Amiloride and several amiloride analogues strongly inhibit adriamycin induction of hemoglobin synthesis as well as cell growth in the absence of adriamycin. The inhibition of induction is enhanced with the analogues bearing a benzyl or substituted benzyl group on the 5-amino or on a terminal guanidino nitrogen atom. The effect on growth was somewhat greater with the analogue bearing a 2-chlorobenzyl moiety on a terminal guanidino nitrogen atom and with the one bearing a 2-fluorobenzyl group on the 5-amino nitrogen atom. The structural features required for growth inhibition resemble those seen with MEL cells, but the features required for inhibition of induction of hemoglobin synthesis are completely different. These data suggest that different specific binding sites are involved in these two effects of amiloride and its analogues.

Niflumic acid is a noncompetitive inhibitor of chloride exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl-, the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the chloride gradient is reversed, with Clo greater than Cli , the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. These data suggest that the intrinsic dissociation constants for Cl- at the two sides of the membrane are nearly equal. Thus, the chloride-loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with approximately 15 times more sites facing the inside than the outside. The asymmetry reflects an approximately 1.5 kcal/mol free energy difference between the inward-facing and outward-facing chloride-loaded forms of band 3. High concentrations of chloride (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that chloride binds equally well to the modifier site regardless of the orientation of the transport site.

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.

N-(4-isothiocyano-2-nitrophenyl)-2-aminoethanesulfonate (NIP-taurine), a newly synthesized isothiocyano derivative of N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate (NAP-taurine), is a potent inhibitor of human erythrocyte chloride exchange. At 0 degrees C, the inhibition is reversible, but at 37 degrees C, NIP-taurine inhibits irreversibly, indicating that it may be a useful label for its binding site. When present at the outside of the cell, NIP-taurine binds with low affinity to a site that seems to be the Cl- transport site (on the basis of its affinity for Cl-) and with much higher affinity to a different site, MN, which has a much lower affinity for Cl-. In this respect, NIP-taurine resembles NAP-taurine, and an analysis of interactions between these two probes is consistent with the idea that they bind to the same two sites. The affinity of NIP-taurine for the high-affinity MN site is enhanced by about fourfold when the transport protein, band 3, is in the conformation with the transport site facing outward (Eo), as compared with the conformation with the transport site facing inward (Ei). External Cl-, but not cytoplasmic Cl-, competes with NIP-taurine for binding to the external, high affinity site. Thus NIP-taurine provides a label for an external site, at which Cl- and perhaps other anions bind, which is separate from both the transport site and the cytoplasmic modifier site at which high Cl- concentrations inhibit Cl- exchange.

The intra- and extracellular chloride concentration dependencies of the rate of Cl-Cl exchange in human promyelocytic leukemic HL-60 cells were studied by means of radioactive isotope (36Cl) efflux measurements. Efflux of isotope from cells follows an exponential time course. The Cl-Cl exchange flux follows Michaelis-Menten kinetics as a function of both intra- and extracellular chloride concentrations. The ratio of the maximum exchange velocity to the apparent Michaelis constant for both extracellular and intracellular substrate increases as a function of trans Cl concentration, indicating that Cl-Cl exchange in the HL-60 cell does not follow ping-pong kinetics. A kinetic scheme in which extracellular and intracellular chloride ions bind in random order to the transporter and are then translocated simultaneously can adequately model the experimental data.

With Cl as substrate, the human red blood cell anion transport (band 3) protein can exist in four conformations: Ei, with the transport site facing the cytoplasm; Eo, with the transport site facing the external medium; and ECli and EClo, the corresponding forms loaded with Cl. Flufenamic acid (FA), an inhibitor that binds to an external site different from the transport site, binds to Eo with a dissociation constant of 0.0826 +/- 0.0049 (SE) microM. Binding of iodide or sulfate to the external-facing transport site reduces the affinity by 1.66 or 14.3-fold, respectively. Changing from Eo to Ei lowers the affinity by 3.7-fold, and binding of cytoplasmic iodide to Ei further decreases the affinity by 5.5-fold. Thus changes in orientation of the transport site and substrate binding, even at the opposite side of the membrane, cause sufficient conformational changes in band 3 to affect FA binding substantially. If the possible effects of Cl binding to the transport site on FA affinity are estimated from the iodide data, the dependence of FA inhibitory potency on Cl concentrations inside and outside the cell suggests that there are at least 6.5 times as many inward-facing as outward-facing Cl-loaded transport sites. This information can be used to calculate the distribution of capnophorin among the various conformations under different circumstances and to devise conditions for recruiting the transport molecules toward a particular conformation.

N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]sulfonate is employed as a photoreactive probe for the anion transport system in the human erythrocyte. In the dark and at 37 degrees C the probe penetrates the membrane via a pathway sensitive to specific inhibitors of anion permeability. It reversibly inhibits sulfate and chloride fluxes but the inhibition is reduced by higher concentrations of sulfate. Upon photolysis to produce a reactive nitrene (at 0 degrees C to minimize penetration), the probe inhibition of anion permeability. Under appropriate conditions the degree of inhibition after photoactivation (irreversible) is almost the same as that in the dark (reversible). The binding sites for the radioactive probe are largely found in proteins of 95 000 apparent molecular weight (band 3). After pronase treatment of the labelled cells, most of the probe is found in a 65 000 molecular weight segment derived from the 95 000 molecular weight protein. In this respect the photoreactive probe resembles another potent irreversible inhibitor of anion transport, 4, 4'-diisothiocyano-2, 2' stilbene disulfonate. In fact, most of the binding sites for each probe are common to both. Thus, in the dark, the azido derivative protects the anion system from inhibition by DIDS and substantially reduces the binding of DIDS to band 3 protein. Conversely, pretreatment with DIDS substantially reduces the binding of the photoreactive probe to the same protein. The fact that an apparent substrate for the anion permeation system competes for binding sites with a specific non-penetrating inhibitor of anion permeability suggests that the inhibitory and transport sites may be closely related and implicates the 95 000 molecular weight protein as the element of the anion transport system which contains the substrate binding site.

Friend erythroleukemic cells, which grow continuously in tissue culture, resemble in many respects early precursors of mouse erythrocytes. To determine whether or not the membranes of these cells exhibit the rapid and selective exchange of chloride, a specialized feature of the mature erythrocyte membrane, anion fluxes were compared in Friend cells and mouse erythrocytes. The chloride flux in Friend cells at 37 degrees C was about 800-fold lower than in mouse erythrocytes (extrapolated from data at lower temperatures). This difference could not be accounted for by the somewhat lower chloride concentration in Friend cells relative to erythrocytes. Comparison of chloride and sulfate fluxes revealed that the Friend cells had over a 1,000-fold lower selectivity for chloride versus sulphate than did the mouse red cells. The temperature dependence of chloride fluxes in Friend cells corresponded to an Arrhenius activation energy of 17.9 kcal/mol, in contrast to over 30 kcal/mol for mature red cells. The chloride flux in Friend cells was also 10-fold less sensitive to the inhibitor, furosemide, than was the flux in mature red cells. The selective chloride exchange system of the mature erythrocyte therefore does not seem to be functional at the stage represented by the Friend cell, and must appear at some later stage of erythroid maturation.

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.