The as yet undefined membrane defect leading to sickle RBC K+ loss and dehydration is arguably the most important defect of the sickle RBC membrane. The proposed studies will examine a specific hypothesis regarding the etiology of K+ leak in sickle RBC cells and they will provide an accurate biophysical description of the K+ leak pathway(s) in these cells. Specifically, I hypothesize that autoxidative membrane damage induces a defect which makes the sickle RBC membrane abnormally susceptible to the potentially adverse effects of cellular deformation accompanying deoxygenation. This could explain the apparently reversible nature of sickling-induced abnormalities of cation homeostasis. Four approaches will be used. (1) RBC from different sickle patients and from density-subpopulations of single patients will be examined to see if the dehydration abnormality (as defined by size of the abnormally dense subpopulation and by monovalent cation content) correlates with various """"""""footprints"""""""" of sickle RBC oxidation (radical generation, membrane-bound heme, thiol oxidation, lipid peroxidation). (2) It will be determined whether in vitro oxidative perturbation accurately models the K+ leak of unmanipulated sickle RBC. Oxidative insults will include intracellular generation of superoxide, thiol oxidation without lipid peroxidation and lipid peroxidation without thiol oxidation. (3) It will be determined whether minimal oxidative insult (which itself does not cause K+ leak in the absence of deformation) predisposes RBC to K+ leak during minimal deformation (which itself does not cause K+ leak in the absence of oxidation). In these studies deformation will be induced by quantitative application of shear stress in a concentric cylinder viscometer. (4) Finally, the K+ leak pathway of normal, sickle and minimally-oxidized normal RBC will be compared under oxygenated and deoxygenated conditions, and during and after deformation. Under these conditions, K+ leak will be described in terms of actual pore size and pore number per cell (using differential seiving of radio-probes), the possible role of calcium, the ion selectivity sequence of the K+ leak pore, and its flux kinetics and activation energy.
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