The main long-term goal is a thorough understanding of the molecular and cellular pathophysiology of sickle cell (SS) disease. We now focus on the mechanisms by which Hb S interacts with the RBC membranes to alter cell functions, leading clinically to widespread microvascular occlusion and hemolytic anemia. Our immediate aims are: I. To characterize the functional properties and possible molecular nature of the sickling-induced ion permeability pathway(s), "P-sickle", generated by interaction of deoxy-Hb S polymers with the cytosolic membrane surface of SS cells. P-sickle -mediated Ca2+ influx is a critical step in SS cell dehydration. We address several fundamental questions about P-sickle: is there a single poorly selective permeability pathway or different pathways for the different cations? How does the extent of P-sickle vary among SS cells? How does it vary with pO2 and [Ca2+]0? Is the single pathway conductance high or low, constant or variable? Does a mean P-sickle value reflect tens or thousands of polymer-membrane contacts? Is the stochastic event the number of P-sickle units per cell or their unit conductance? Identifying agents that stimulate or inhibit P-sickle may point to membrane components involved and clues to its molecular nature. II. To investigate the mechanism(s) of generation of the high-Na+, low-K+, low-density, cation-leaky SS and normal RBCs we discovered, and test the hypotheses (i) that many of these cells are derived from dense irreversibly sickled cells (ISCs); (ii) that they comprise the very rapid-turnover K pool we found among low density SS cells; and (iii) that they represent a major final pathway of cell death for sickle cells, and perhaps for normal RBCs. III. To pursue our hypothesis that the marked heterogeneity of volume and density (or cell Hb concentration) among circulating SS and variant RBCs results from heterogeneity of transport systems in the reticulocytes, with the direct and indirect effects of Hb-membrane interactions. These studies employ our newly available flow cytometric technology (modified H*3) in an experimental design in which the transporter distribution among cells is expressed in their rates of volume change. Our integrated RBC and reticulocyte models will be used to test alternative mechanisms of reticulocyte volume control (and decontrol in SS cells). We aim to characterize in detail the transport heterogeneity of SS and normal retics/RBCs, with particular emphasis on the contribution of retics to cell dehydration. IV. To characterize the Hb-polymerization properties and RBC ion-transport functions of a variety of transgenic sickle mouse models to identify those most suitable to test various pathophysiological mechanisms and therapeutic maneuvers in sickle cell disease. Once characterized, these will serve investigations within the above three aims.