KCl cotransport activity in light versus dense transferrin receptor-positive sickle reticulocytes

A subset of sickle cells becomes K(+)-depleted and dehydrated before or soon after leaving the bone marrow. These young cells may be identified in blood as transferrin receptor-positive (TfR+) dense reticulocytes. KCl cotransport, which is normally active in young erythroid cells with a maximum at pH 6.8, is a candidate pathway for K+ depletion of sickle reticulocytes. In this investigation, KCl cotransport activity was evaluated in young, TfR+ cells which had become dense in vivo and in age-matched cells which had retained normal hydration. Sickle erythrocytes were first separated into three primary density fractions, with care taken to preserve the in vivo hydration state. After normalization of intracellular hemoglobin concentration with nystatin, the cells were incubated at 37 degrees C for 20 min at pH 6.8 and 7.4. Before and after incubation, each primary fraction was separated into four secondary density fractions. The percentage of TfR+ cells in each secondary fraction was measured and a density distribution for TfR+ cells was determined for each primary fraction before and after incubation. The density shift during incubation was a measure of KCl cotransport. TfR+ cells from the denser primary fractions II and III had significantly more density shift than TfR+ cells from the light fraction I. Although the shifts were larger at low pH, differences between primary fractions were also observed at pH 7.4. These data indicate that the cells which become dense quickly in vivo have more KCl cotransport activity than those which remain light in vivo, and support this pathway as a primary mechanism for dehydration of young sickle cells.