DIDS inhibition of deformation-induced cation flux in human erythrocytes.
The permeability of human erythrocytes to sodium, potassium and calcium increases when the cells are deformed by shear. We now report that the anion-exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) inhibited 55-60% of the deformation-induced flux with an apparent K1/2 of 1 microM. Covalently bound DIDS was also effective. In cells partially derivatized at 0 degrees C (pH 7.4), anion exchange and the deformation flux were inhibited in parallel, implying that lysine a is the site of inhibition for both fluxes. Ektacytometry showed that DIDS does not inhibit by lowering the cell's ability to deform. Crosslinking of lysines in Band 3 was not required for inhibition of the stress flux, as demonstrated by electrophoretic analysis of chymotrypsin-cleaved Band 3 after DIDS treatment. Chymotrypsin cleavage itself did not affect the cation flux rates. DNDS, an anion exchange inhibitor that binds to the chloride site on Band 3 but is unable to derivatize lysine a, is an ineffective inhibitor of the deformation flux. Other high-affinity inhibitors of anion exchange were also relatively ineffective against the deformation flux, and anion exchange itself was unchanged by shear. These results suggest that 55-60% of the deformation-induced cation movement traverses a route that includes Band 3, but is distinct from the pathway utilized by anion exchange. Chloride-dependent cation pathways do not participate in the stress induced cation flux, since complete exchange of intracellular chloride for sulfate had no effect on the rates. Deformation of erythrocytes by laminar shear appears to increase the non-specific cation permeability.
Department of Biochemistry, Wayne State Medical School, Detroit, MI 48201.
R M
RM
Johnson
K
K
Tang
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D002412:Cations; D002463:Cell Membrane Permeability / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism; D002918:Chymotrypsin; D004912:Erythrocytes / Q000187:drug effects*; D006801:Humans; D008239:Lysine / Q000737:chemistry; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
D002412:Cations; D002712:Chlorides; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012964:Sodium; D002918:Chymotrypsin; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium