ALBERT

Notes: Summary In the presence of 8mm external Ca++, the K+ permeability of human red cell ghosts increases provided K+ is also present in the medium. This increase does not represent K+/K+ exchange but a stimulation of net K+ efflux. The stimulation is halfmaximal at 0.7±0.15mm (n=5). At concentrations above 4.0mm, external K+ inhibits net K+ efflux. Similar stimulatory and inhibitory effects of external K+ were also observed in intact cells after exposure to Pb++ or to Ca++ in the presence of fluoride, iodoacetate plus adenosine, or propranolol, suggesting that a common K+-activated K+-specific transfer system may be involved under all of these various circumstances. Internal K+ also stimulates net K+ efflux from ghosts, but it is uncertain whether internal K+ is an absolute requirement for the K+ permeability increase. In contrast to external Na+ which slightly stimulates K+ efflux, internal Na+ inhibits. The inhibition by internal Na+ is abolished by sufficiently high concentrations of external K+, showing that K+ binding to the outer membrane surface and Na+ binding to the internal surface are mutually interdependent. In red cell ghosts the Ca++−K+-stimulated net K+ efflux increases with increasing pH until a plateau is reached between pH 7.2 and 8.0. In fluoride-poisoned intact cells, the Ca++−K+ stimulated flux passes through a maximum around pH 6.8. Neither internal nor external Mg++ interferes with the combined effects of Ca++ and K+. Similarly, external EDTA has no influence at concentrations which are far lower than the Ca++ concentration required to produce a maximal response. In contrast, low concentrations of internal EDTA prevent the permeability change.

Notes: Mono-, di-, and trisulfonic acids, including 4,4′-diacetamido stilbene-2,2′-disulfonic acid (DAS) and 2-(4′-amino phenyl)-6-methylbenzene thiazol-3′,7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability.The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluoro-benzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, 1974). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid.In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements.The effect of the irreversibly binding inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) on DNFB binding to the protein in band 3 was studied in an attempt to differentiate DNFB binding related to inhibition of anion permeability from DNFB binding which is not involved. At least three distinguishable populations of DNFB binding sites were found: (1) binding sites common for DNFB and SITS which are probably related to inhibition, (2) other common sites which are not related to inhibition and (3) different sites whose dinitrophenylation is not affected by SITS. The number of sites in population (1) was estimated to be 0.8-1.2 ± 106/cell. A study of the concentration dependence of the inhibition of anion equilibrium exchange with 4,4′-isothiocyanato-2,2′-stilbene disulfonic acid (DIDS) and APMB further suggests that among the sites in population (1) a major fraction is susceptible to modification by APMB and DIDS while the rest is only susceptible to DIDS. It remains undecided whether these differences of susceptibility reflect differences of accessibility or reactivity.