H. Passow

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4′-diisothiocyano stilbene-2,2′-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS)

SummaryDIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid) and H2DIDS (4,4′-diisothiocyano-1,2-diphenyl ethane-2,2′-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974.J. Cell Biol.62∶1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and >60% of DIDS binding were rapid, independent of temperature. Slow H2DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1–1.2, concentration-dependent. Under the conditions studied (0–50 μm, hematocrit 10%, 5–37°C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS.Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases. H2DIDS inhibition was complete at 1.9×106 or 1.2×106 molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3×106 and 1.1×106.It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.

Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis

SummaryIn accordance with former observations of Hoffman (1962a), ghost populations obtained by hypotonic hemolysis and subsequent restoration of isotonicity by the addition of alkali salts, were found to be composed of 3 types of ghosts. For our purposes it was useful to distinguish between: (1) ghosts which reseal immediately after hemolysis (type I); these ghosts are incapable of incorporating alkali ions which are added after hemolysis; (2) ghosts which reseal after the addition of alkali ions (type II); salt added to the hemolysate becomes trapped inside these ghosts in the course of the resealing process at temperatures above 0°C; and (3) ghosts which remain leaky regardless of the experimental condition (type III). The discrimination between the various types of ghosts was partly achieved by a kinetic method first devised by Hoffman (1962a), and partly by sucrose density gradient centrifugation.The relative sizes of the 3 fractions depend on the temperature at which hemolysis took place and on the time interval which elapsed between hemolysis and the addition of salt. At 37°C the resealing process is fast. Many of the ghosts reseal before salt can be added to the hemolysate. Hence, the fraction of type I ghosts is high after hemolysis at that temperature. At 0°C resealing is extremely slow. Hence, salt which has been added to the hemolysate at that temperature will enter the ghosts and become trapped during subsequent incubation at 37°C. There are no ghosts of type I and many ghosts of type II (about 60%). Regardless of the temperature at hemolysis, there are always ghosts which do not reseal even after prolonged incubation at 37°C. A method has been designed which permits the preparation of homogeneous populations of type II ghosts.Complexing agents (ATP, EDTA, 2,3-DPG) may prevent the resealing of the ghost membrane. However, they exert this effect only at elevated temperatures and when present in the medium at the instant of hemolysis. At 0°C, the presence of complexing agents in the medium at the instant of hemolysis has no effect on the subsequent resealing at 37°C. The recovery of the ghost membrane takes place in spite of the continued presence of the agents and eventually leads to trapping of these agents inside the resealed ghosts.The experiments support the contention that the complexing agents interact with a membrane constituent which is neither accessible from the inner nor from the outer surface of the cell membrane but becomes exposed during the hemolytic event when the complexing agents penetrate across the membrane. Apparently, at low tempertrures membrane ligands are more successful in competing with the added complexing agents for this constituent than at higher temperatures.Extending former observations of Hoffman, we found that not only Mg++ but also Ca++ facilitates the resealing process. Perhaps one or the other of the two alkaline earth ions is the membrane constituent which normally participates in the maintenance of the integrity of the red blood cell membrane.

Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes

Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS). Using appropriate values for the probability of a channel in the open state, we estimated that 25% of the cells in the population contain 11-55, and 75% of the cells 1-5 channels per cell that are activated in the time average (20 degrees C, pH 7.6).

The effect of pH at hemolysis on the reconstitution of low cation permeability in human erythrocyte ghosts

Abstract Human red blood cells were hemolysed at 0°C in hypotonic media of varying pH (range 4.5–10.0). Subsequently the pH in the hemolysate was readjusted to 7.2 and the ghosts were incubated at 37°C for 45 min. The curve relating the yield of resealed ghosts to pH showed a maximum after hemolysis at pH 6 and a broad shoulder in the range between pH 7 and 9. The experiments suggest that during hemolysis buried ionizable groups become exposed to the ambient medium. The ability of these groups to return to their original locations within the hydrophobic interior of the membrane seems to depend on the charge which they assume during their exposure to the aqueous environment. However, alternative explanations cannot be excluded.

Role of lys 558 and lys 869 in substrate and inhibitor binding to the murine band 3 protein : a study of the effects of site-directed mutagenesis of the band 3 protein expressed in the oocytes of Xenopus laevis

SummaryThe effect of mutation of either Lys 558 or Lys 869 or both on mouse erythroid band 3 protein (AE1)-mediated 36Cl− efflux and its inhibition by pyridoxal 5-phosphate (P5-P), DNDS and H2DIDS were studied. Regardless of the mutation, band 3 was always capable of executing Cl− self-exchange. P5-P (5 mm, pH 7.6) produced irreversible inhibition in the wild type (KK) and in the mutant in which Lys 558 (NK) or Lys 869 (KM) had been replaced by asparagine (N) or methionine (M), respectively. However, when both residues were replaced, mutant (NM), irreversible inhibition could no longer be achieved. This shows that P5-P is capable of producing inhibition with either one of the lysine residues, 558 or 869.Inhibition by DNDS changed dramatically upon mutation. The Kiapp increased from 6.0 μm in the wild type (KK) to 23 μm in the mutant NK, to 73 μm in the mutant KM and to 474 μm in the double mutant NM. The Km value for activation of the transport system by varying the substrate concentration by isosmotic substitution of Cl− with SO42−decreased from 42 mm in the wild type (KK) to 11.3 mm in the mutant NM. The results show that both Lys 558 and Lys 869 are involved in the maintenance of the structure of the overlapping binding sites for stilbene disulfonates and the substrate Cl−.In the double mutant NM, H2DIDS is no longer able to produce irreversible inhibition at pH 7.6. This is evidently related to the replacement of Lys 558 (pK 8.2) by Asn 558 in this mutant (see Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B., Passow, H., 1989. EMBOJ. 8:3601-3609). However, at pH 9.5, some irreversible inhibition could still be observed. This suggests that the other lysine residue (pK 10.8) that is known to be involved in covalent binding with the second isothiocyanate group of H2DIDS is still present, and hence, not identical to Lys 869, which had been substituted by a methionine residue. However, this result remains inconclusive since after mutagenesis, the H2DIDS may produce inhibition at a site that is not normally involved in H2DIDS binding.

Endogenousd-glucose transport in oocytes ofXenopus laevis

SummaryEndogenous glucose uptake by the oocytes ofXenopus laevis consists of two distinct components: one that is independent of extracellular Na+, and the other one that represents Na+-glucose cotransport. The latter shows similar characteristics as 2 Na+-1 glucose cotransport of epithelial cells: The similarities include the dependencies on external concentrations of Na+, glucose, and phlorizin, and on pH. As in epithelial cells, the glucose uptake in oocytes can also be stimulated by lanthanides. Both the electrogenic cotransport and the inhibition by phlorizin are voltage-dependent; the data are compatible with the assumption that the membrane potential acts as a driving force for the reaction cycle of the transport process. In particular, hyperpolarization seems to stimulat transport by recruitment of substrate binding sites to the outer membrane surface. The results described pertain to oocytes arrested in the prophase of the first meiotic division; maturation of the oocytes leads to a downregulation of both the Na+-independent and the Na+-dependent transport systems. The effect on the Na+-dependent cotransport is the consequence of a change of driving force due to membrane depolarization associated with the maturation process.

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation of Xenopus oocytes

SummaryDuring progesterone-stimulated maturation of defolliculated full-grownXenopus oocytes, the activities of the transport systems forl-alanine, thymidine, chloride, phosphate, and alkali ions decrease. Differences of the extent and time course of these changes suggest that they are controlled by at least partially independent mechanisms.A closer investigation of the Na-K ATPase has shown that in unstimulated oocytes, ouabain produces maximal inhibition when 8–12×109 molecules are bound per cell. This number is bound during the first phase of a diphasic uptake process. Since this phase can be suppressed by increasing the concentration of external K+ to 45 mmol/liter or more, it is concluded that it refers to binding to the Na−K pump in the plasma membrane. Ouabain bound prior to progesterone-induced germinal vesicle breakdown (GVBD) remains bound after the breakdown, although the Na−K pump loses the capacity to bind ouabain after GVBD in oocytes that had not been exposed to ouabain preceding GVBD. In the presence of Mg++ membranes isolated before regulatory inhibition of pumping and ouabain binding show a Na+-dependent incorporation of32P from γ-[32P]-ATP that can be reversed by the addition of K+. The phosphorylation site migrates on LiDS-polyacrylamide gel electropherograms at about 98,000 daltons and can be identified as a Commassie blue-stainable band.

Effects of calcium and lead on potassium permeability of human erythrocyte ghosts

K+ efflux from reconstituted erythrocyte ghosts was induced by Pb2+ or by Ca2+ in the presence or absence of F− or iodoacetic acid plus adenosine. Oligomycin completely inhibited Ca2+-induced K+ loss and partially inhibited loss caused by maximally effective doses of Pb2+. However, at submaximal Pb2+ concentrations oligomycin increased K+ exit. Incorporation into ghosts of ATP or EDTA prevented Ca2+-induced K+ efflux but increased Pb2+-induced K+ efflux. ATP also prevented oligomycin from inhibiting Pb2+-induced loss of K+. The kinetics of Ca2+-induced K+ loss are similar to those previously observed with Pb2+ and suggest an ‘all or none’ effect.

Identification by site-directed mutagenesis of Lys-558 as the covalent attachment site of H2DIDS in the mouse erythroid band 3 protein

After functional expression of mouse erythroid band 3 by cRNA microinjection into Xenopus oocytes, 36Cl- efflux is irreversibly inhibited by H2DIDS. When a cRNA is injected that is derived from a cDNA in which the nucleotides encoding for lysine-558 were replaced by nucleotides encoding for asparagine, transport and inhibition of transport by H2DIDS still occur. However, when measured under conditions where no intramolecular crosslinking takes place the inhibition by H2DIDS is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 takes place at Lys-558.

Sidedness of the inhibitory action of disulfonic acids on chloride equilibrium exchange and net transport across the human erythrocyte membrane

The characterization of membrane transport systems requires a study of the sidedness of the effects of modifying agents. Previous work with phlorizin showed that although this agent inhibits sugar transport at either surface of the red cell membrane, the effects on anion transport and acceleration of Clnet movements [l] and an inhibition of Clor SO;equilibrium exchange [2,3] can only be observed if the agent is added to the external medium. Intracellular phlorizin was without effect except for a slight acceleration of Cl-/Clequilibrium exchange at low pH [4]. Similar studies on the sidedness of the effects of two disulfonic acids, 4,4’-diacetamido stilbene-2,2’-disulfonic acid (DAS) and 2-(4’-aminophenyl)d-methylbenzene-thiazole-3’,7-disulfonic acid (APMB), revealed that the former compound inhibits SO:equilibriufixchange only at the outer membrane surface while APMB produced a strong inhibition at both surfaces [5]. The present work characterizes the actions of these two inhibitors on Cltransport as measured under conditions of Clequilibrium exchange, and net flow. The sidedness was the same under both experimental conditions. This indicates that, although Clequilibrium exchange proceeds about 10’ times faster than SOiequilib-