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Although urea transport across the human red cell membrane has been studied extensively, there is disagreement as to whether urea and water permeate the red cell by the same channel. We have suggested that the red cell anion transport protein, band 3, is responsible for both water and urea transport. Thiourea inhibits urea transport and also modulates the normal inhibition of water transport produced by the sulfhydryl reagent, pCMBS. In view of these interactions, we have looked for independent evidence of interaction between thiourea and band 3. Since the fluorescent stilbene anion transport inhibitor, DBDS, increases its fluorescence by two orders of magnitude when bound to band 3 we have used this fluorescence enhancement to study thiourea/band 3 interactions. Our experiments have shown that there is a thiourea binding site on band 3 and we have determined the kinetic and equilibrium constants describing this interaction. Furthermore, pCMBS has been found to modulate the thiourea/band 3 interaction and we have determined the kinetic and equilibrium constants of the interaction in the presence of pCMBS. These experiments indicate that there is an operational complex which transmits conformational signals among the thiourea, pCMBS and DBDS sites. This finding is consistent with the view that a single protein or protein complex is responsible for all the red cell transport functions in which urea is involved. [ABSTRACT FROM AUTHOR]

The transport of inorganic anions across human red blood cell membranes is accomplished by a carrier-like mechanism which involves an electroneutral and obligatory one-for-one anion exchange. The transport kinetics were described by models that involve alternation of single transport sites between the two membrane surfaces. These models predict that each carrier shows either an inward-facing E or an outward-facing E, conformation, each capable of binding either a monovalent anion or a divalent anion+a proton, to yield an electroneutral translocating complex. Unidirectional transport rates provide, therefore, a measure for the relative concentration of carriers at a given membrane surface. In the present work we assessed how modulation of the transmembrane distribution of carriers by the anion composition of cells and media, and by pH, affect the anion transport system. We have set the system in asymmetric conditions with respect to anions, so that a fast transportable anion (e.g., chloride) was present in one side of the membrane and slow transportable anions (e.g., sulfate, phosphate, oxalate, isethionate, gluconate, HEPES) were present on the other side of the membrane. The skewed distribution of carriers induced in these conditions were assessed by two methods: 1) NBD-taurine transfer which provided a measure for [ E], the monovalent inward-facing form of the carrier, and 2) inhibition of NBD-taurine transfer by the specific impermeant and competitive inhibitor 4,4′-dinitro-2,2′-stilbene disulfonic acid (DNDS), which provided a measure for the availability of the carrier at the outer membrane surface. In the various symmetric and asymmetric conditions, we found marked differences in transport rates and transport profiles as well as in the susceptibility of the system to inhibition by DNDS. Direct binding studies of DNDS to cells in the various asymmetric conditions supported the conclusion derived from transport studies that transport sites can be recruited towards the membrane surface facing the slow transportable anions. [ABSTRACT FROM AUTHOR]

The molecular mechanism of anion exchange across the human red blood cell membrane was assessed with the fluorescent substrate analog NBD-taurine and the method of continuous monitoring of transport by fluorescence. The efflux of NBD-taurine was studied under a variety of experimental conditions such as temperature, pH and anion composition of cells and media. The temperature profile of NBD-taurine transfer from Cl-loaded cells into Cl media resembled that of Cl self-exchange, whereas that of NBD-taurine transfer from sulfate-loaded cells into sulfate media resembled that of sulfate self-exchange. Although the pH profiles of NBD-taurine transfer from Cl-loaded cells into Cl media and that of Cl self-exchange resembled each other, the analogous transfer with sulfate replacing Cl was markedly different. These and other data were analyzed and found to be consistent with a model which comprises the following: (a) a H-titratable group in the carrier mechanism; (b) alteration of transport sites between the two sides of the membrane (i.e., ping-pong kinetics); and (c) transmembrane distribution of transport sites which is modulated by pH. It is shown that NBD-taurine transfer represents a tracer flux of a fluorescent substrate which gives a measure for the presence of monovalent transport sites at the inner surface of the membrane. The latter is markedly affected by the relative concentrations of anions and H on both sides of the red blood cell membrane. [ABSTRACT FROM AUTHOR]

The interaction between chloride and the anion transport inhibitor DNDS (4,4′-dinitro stilbene-2,2′-disulfonate) at the external anion binding site of the human erythrocyte anion transporter was examined by two techniques: a) chloride tracer flux experiments in the presence of varying concentrations of DNDS, and b) DNDS equilibrium binding experiments in the presence of varying concentrations of intracellular and extracellular chloride, Cl and Cl. DNDS inhibited competitively the Cl-stimulated chloride efflux from intact red cells at 0°C and pH 7.8 with an inhibitor constant of 90 nm. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5×10 molecules/cell. Cl competitively inhibited DNDS binding with an inhibitor constant of 6 mm. In the absence of Cl the DNDS binding constant was 84 mm. The competition between chloride and DNDS was also tested in nystatintreated cells in which Cl always equaled Cl. Under these conditions the values of the DNDS binding constant and the chloride inhibitor constant were significantly larger. All these data were in quantitative agreement with a single-site, alternating access kinetic scheme with ping-pong-type kinetics that we have previously developed for modeling chloride exchange transport. The data also served to rule out special cases of an alternative two-sited sequential-type kinetic scheme. DNDS binding experiments were also performed at 10 and 20°C. We found that neither the DNDS binding constant nor the Cl inhibitor constant were significantly changed compared to 0°C. [ABSTRACT FROM AUTHOR]

Up to a SCN- concentration of about 110 mM, the concentration dependence of SCN- equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits a trans acceleration by Cl- and can be inhibited by H2DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced in trans acceleration experiments with Cl- and insensitive to H2DIDS but susceptible to inhibition by phloretin. At SCN- concentrations exceeding 110 mM, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered. The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0 mM at pH 7.4 to 13.3 mM at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19 x 10(-12) to 22 x 10(-12) moles x cm-2 x sec-1. The nonsaturable flux component also increases slightly. In accordance with previous observations of Wieth (J. Physiol. (London) 207:563-580, 1970), we find that SCN- increases K+ and Na+ permeability. The induced cation-permeability is considerably smaller than the SCN- exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former. [ABSTRACT FROM AUTHOR]