Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease.

Erythrocytes from patients with homozygous hemoglobin C disease (CC cells) contain less K, Na, and water than do erythrocytes from normal subjects that contain only hemoglobin A (AA cells). In this paper, we provide evidence that the reduced K content and volume of CC cells are due to the activity in these but not in AA cells of a K transport system that is: (a) insensitive to ouabain and bumetanide, and (b) stimulated by increased cell volume, and dependent on internal pH (pHi). When the cation and water content of CC cells was increased (by making the membrane temporarily permeable to cations with nystatin) and the cells were then incubated in an isotonic medium containing 140 mM NaCl and 4 mM KCl, they lost K and shrunk back toward the original volume. This regulatory K and volume decrease was not inhibited by ouabain or bumetanide. When CC cells were incubated in a hypotonic medium, with ouabain and bumetanide, they also lost K and shrunk toward the original volume. This behavior was not observed in control AA cells. The ouabain- and bumetanide-resistant K efflux from CC cells was volume and pH dependent: K efflux from CC cells rose from 5-6 to 20-25 mmol/liter of cells X h, when cell volume was increased by increasing cell solute content (nystatin method) or by exposure to hypotonic media. In CC cells, the dependence of K efflux on pHo had a bell shape, with a maximal flux (20-25 mmol/liter of cells X h) at pHo 6.8-7.0. In contrast, the K efflux from control cells was minimal at pH 7.4 (1.2 mmol/liter of cells X h) and was slightly stimulated by both acid and alkaline pH. In order to study the effect of pHi and pHo on K efflux, CC cells were incubated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM) and acetazolamide (1 mM) at different pHi (6.7, 7.3, and 7.8), and resuspended in media with different pHo (6.75, 7.4, and 8): K efflux was stimulated by reducing pHi but was independent of pHo. The ouabain- and bumetanide-resistant K efflux from CC cells was not inhibited by some inhibitors of the Ca2+-activated K permeability. It seems likely that the genetically determined change in the primary structure of hemoglobin C directly or indirectly causes this modification in K transport. One possible mechanism could involve an electrostatic interaction between C hemoglobin and components of the erythrocyte membrane.