Age decline in the activity of the Ca2+-sensitive K+ channel of human red blood cells.

The Ca(2+)-sensitive K(+) channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca(2+) loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age-activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K(+) permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age-density distribution pattern during dehydration. However, when Ca(2+) loads were used to induce maximal K(+) fluxes via Gardos channels in all RBCs (F(max)), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F(max) was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age-activity relation revealed a monotonic decline in F(max) with cell age, with a broad quasi-Gaussian F(max) distribution among the RBCs.

Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells.

Little is known about age-related changes in red blood cell (RBC) membrane transport and homeostasis. We investigated first whether the known large variation in plasma membrane Ca(2+) (PMCA) pump activity was correlated with RBC age. Glycated hemoglobin, Hb A1c, was used as a reliable age marker for normal RBCs. We found an inverse correlation between PMCA strength and Hb A1c content, indicating that PMCA activity declines monotonically with RBC age. The previously described subpopulation of high-Na(+), low-density RBCs had the highest Hb A1c levels, suggesting it represents a late homeostatic condition of senescent RBCs. Thus, the normal densification process of RBCs with age must undergo late reversal, requiring a membrane permeability increase with net NaCl gain exceeding KCl loss. Activation of a nonselective cation channel, Pcat, was considered the key link in this density reversal. Investigation of Pcat properties showed that its most powerful activator was increased intracellular Ca(2+). Pcat was comparably selective to Na(+), K(+), choline, and N-methyl-D-glucamine, indicating a fairly large, poorly selective cation permeability pathway. Based on these observations, a working hypothesis is proposed to explain the mechanism of progressive RBC densification with age and of the late reversal to a low-density condition with altered ionic gradients.

Distribution of dehydration rates generated by maximal Gardos-channel activation in normal and sickle red blood cells.

The Ca(2+)-activated K+ channels of human red blood cells (RBCs) (Gardos channels, hIK1, hSK4) can mediate rapid cell dehydration, of particular relevance to the pathophysiology of sickle cell disease. Previous investigations gave widely discrepant estimates of the number of Gardos channels per RBC, from as few as 1 to 3 to as many as 300, with large cell-to-cell differences, suggesting that RBCs could differ extensively in their susceptibility to dehydration by elevated Ca2+. Here we investigated the distribution of dehydration rates induced by maximal and uniform Ca2+ loads in normal (AA) and sickle (SS) RBCs by measuring the time-dependent changes in osmotic fragility and RBC volume distributions. We found a remarkable conservation of osmotic lysis and volume distribution profiles during Ca(2+)-induced dehydration, indicating overall uniformity of dehydration rates among AA and SS RBCs. In light of these results, alternative interpretations were suggested for the previously proposed low estimates and heterogeneity of channel numbers per cell. The results support the view that stochastic Ca2+ permeabilization rather than Gardos-channel variation is the main determinant selecting which SS cells dehydrate through Gardos channels in each sickling episode.

Distribution of plasma membrane Ca2+ pump activity in normal human red blood cells.

The plasma membrane calcium pump (PMCA) is the only active Ca2+ transporter in human red blood cells (RBCs). Previous measurements of maximal Ca2+ extrusion rates (Vmax) reported only mean values in the RBC population. Despite early evidence for differences in Ca2+ extrusion capacity among RBCs, the precise Vmax distribution remained unknown. It was important to characterize this distribution to assess the range and modality (uni- or multimodal) of PMCA Vmax variation and the likelihood of RBCs with elevated [Ca2+]i in the circulation participating in physiologic and pathologic processes. We report here the application of a new method to investigate the detailed distribution of PMCA Vmax activity in RBCs. The migrating profile of osmotic lysis curves was used to identify and quantify the fraction of cells that extrude a uniform Ca2+ load at different rates. The results revealed that RBCs from single donors have large variations in PMCA activity that follow a unimodal, broad distribution pattern consistently skewed toward higher Vmax values, suggesting an excess of cells with Vmax higher than the mean value. The method applied may provide a way of evaluating whether the observed variation in PMCA Vmax is related to cell age.