Zipora Etzion

Age Decline in the Activity of the Ca2+-sensitive K+ Channel of Human Red Blood Cells

The Ca2+-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 Ca2+ 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 Ca2+ 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.

The distribution of intracellular calcium chelator (fura-2) in a population of intact human red cells

Using quantitative fluorescence microscopy of red cells loaded non-disruptively with 1-2.5 mmol/l cells of fura-2, we examined the distribution of the incorporated free chelator among and within individual cells. Cytoplasmic hemoglobin quenched the effective fluorescence yield of fura-2 by a factor of about 100. All red cells were found to fluoresce upon excitation at 380 nm, and the fluorescence intensities they emitted at 510 nm were approximately +/- 20% about the mean intensity, indicating a fairly uniform distribution of incorporated chelator among the cells. Red cells loaded with these high levels of fura-2 retained their biconcave shape, and a comparison between their transmission images at 415 nm and their fura-2 fluorescence images suggests that the concentration of fura-2 was also uniform throughout the cytosol. These results validate assumptions made in earlier experiments with non-fluorescent incorporated Ca2+ chelators, and demonstrate the feasibility of fura-2 and Ca2+ imaging of intact red cells, despite considerable quenching of probe fluorescence by hemoglobin.

A TRANSGENIC MOUSE MODEL EXPRESSING EXCLUSIVELY HUMAN HEMOGLOBIN E: INDICATIONS OF A MILD OXIDATIVE STRESS

Hemoglobin (Hb) E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid β(E)-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of β(E) globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/β (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.