Small increases in the concentration of Hb S inside sickle cells lead to dramatic increases in the rate of polymerization of Hb S within the cell and consequent cell sickling. We propose to study two important mechanisms by which the concentration of HbS can be decreased in sickle cells. The first mechanism is the regulation of cell volume by two principle K efflux pathways: the KCL cotransport system and the Ca- activated K channel. Activation of these pathways has been shown to mediate KCL loss from and dehydration of sickle cells. Though considerable information about the kinetic properties of these """"""""leak"""""""" pathways has been published, their molecular identities remain unknown, and clinically useful antagonists are not available. The second mechanism is the decrease in Hb S concentration and coincident increase in Hb F concentration which follows hydroxyurea therapy of sickle cell disease. The possible interactions between these two mechanisms of inhibition of Hb S polymerization are unexplored. Our experiments presented in this proposal have three specific aims: 1. To clone and characterize cDNAs encoding the KCL cotransporter and the Ca-activated K channel. We propose to clone the Ca-activated K channel cDNA by the parallel approaches of hybridization and PCR cloning on the one hand and expression cloning of 125-L-Charybdotoxin binding proteins on the other. We propose to clone the KCL cotransporter cDNA by oocyte expression cloning. We will raise antibodies to the polypeptide sequences deduced from the cloned cDNAs, and test them for possible inhibition of transport or regulatory function. We will examine the biosynthesis and function of the polypeptides encoded by the cloned cDNAs in oocytes and in mammalian cell heterologous expression systems. 2. To characterize the functional properties of these two transport pathways in sickle and normal erythrocytes. We will examine the regulation of the KCL cotransport in red cells and the effect of positively charged hemoglobins on this system. We will examine the role and properties of the Ca-activated K channel in sickle cell dehydration. We will also study the effect of hydroxyurea therapy on the transport properties of sickle erythrocytes. 3. To define the intracellular determinants of Hb polymerization. To evaluate properly any therapeutic strategy designed to decrease Hb S concentration, precise information must be available about the concentrations of Hb S and Hb F in SS erythrocytes needed to achieve measurable changes in oxygen saturation and morphologic sickling. We propose to evaluate the role of cellular hemoglobin S and F concentrations on oxygen saturation, morphologic sickling, and Hb S polymerization kinetics in intact red cells of defined volume and Hb composition.
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