Internal magnesium, 2,3-diphosphoglycerate, and the regulation of the steady-state volume of human red blood cells by the Na/K/2Cl cotransport system.

This study is concerned with the relationship between the Na/K/Cl cotransport system and the steady-state volume (MCV) of red blood cells. Cotransport rate was determined in unfractionated and density-separated red cells of different MCV from different donors to see whether cotransport differences contribute to the difference in the distribution of MCVs. Cotransport, studied in cells at their original MCVs, was determined as the bumetanide (10-mu-M)-sensitive Na-22 efflux in the presence of ouabain (50-mu-M) after adjusting cellular Na (Na(i)) and K(i) to achieve near maximal transport rates. This condition was chosen to rule out MCV-related differences in Na(i) and K(i) that might contribute to differences in the net chemical driving force for cotransport. We found that in both unfractionated and density-separated red cells the cotransport rate was inversely correlated with MCV. MCV was correlated directly with red cell 2,3-diphosphoglycerate (DPG), whereas total red cell Mg was only slightly elevated in cells with high MCV. Thus intracellular free Mg (Mg(i)free) is evidently lower in red cells with high 2,3-DPG (i.e., high MCV) and vice versa. Results from flux measurements at their original MCVs, after altering Mg(i)free with the ionophore A23187, indicated a high Mg(i) sensitivity of cotransport: depletion of Mg(i)free inhibited and an elevation of Mg(i)free increased the cotransport rate. The apparent K0.5 for Mg(i)free was approximately 0.4 mM. Maximizing Mg(i)free at optimum Na(i) and K(i) minimized the differences in cotransport rates among the different donors. It is concluded that the relative cotransport rate is regulated for cells in the steady state at their original cell volume, not by the number of copies of the cotransporter but by differences in Mg(i)free. The interindividual differences in Mg(i)free, determined primarily by differences in the 2,3-DPG content, are responsible for the differences in the relative cotransport activity that results in an inverse relationship with in vivo differences in MCV. Indirect evidence indicates that the relative cotransport rate, as indexed by Mg(i)free, is determined by the phosphorylated level of the cotransport system.