Foreign anion substitution for chloride in human red blood cells: effect on ionic and osmotic equilibria

Abstract

In human red blood cells, when chloride was replaced isosmotically with a permeant chaotropic anion of the lyotropic series (NO3, I, or SCN), an immediate and significant loss of cell water was observed. In contrast, replacement of chloride by a substituted monovalent sulfonate, such as methanesulfonate or sulfamate, had no significant effect on cell water. Cell water loss in the presence of lyotropic anions was not the result of hemolysis or cation loss but was associated with a significant fall in the distribution ratios of protons (out/in) and chloride (in/out), suggesting an increase in nondiffusible intracellular negative charges. This hypothesis was examined using the equilibrium dialysis technique of Freedman and Hoffman (J. Gen. Physiol. 74: 157-185, 1979) in which fixed charges are titrated in cells permeabilized by nystatin. The equilibrium concentration ratios (in/out) of potassium, sodium, and chloride were determined at various external pH (pHo) values. The point at which anion and cation ratios are equal is the effective isoelectric point for the intracellular charges. In normal chloride-containing medium at 24 degrees C, this point was found at a pHo of 6.93. When chloride was replaced by a chaotropic anion, the isoelectric point at 24 degrees C shifted to a lower pHo: NO3 (6.38), I (5.98), and SCN (5.70). The substituted monovalent sulfonates had little effect on isoelectric point: methyl sulfate (6.81), sulfamate (7.00), and methanesulfonate (7.07). Calculation of the intracellular charges from titration data, as well as equilibrium distribution studies with [14C]SCN, suggests that lyotropic anion binding to intracellular sites (mainly hemoglobin) is responsible for the observed changes in cell water, cell pH, and chloride distribution.