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The long-term objective of this work is to understand the mechanism of cell volume regulation in mammalian red blood cells. The studies focus on a KC1 cotransport system that is stimulated by cell swelling. An understanding of this transport system could lead to new therapies for sickle cell disease. Rabbit red cells are used as a model system in which the swelling-stimulated K+ flux is large and can be studied in detail. Two of the specific aims concern the mechanism of transport. Measurements of 86Rb+ will be used to test specific catalytic cycles. Fluxes of 36Cl-under conditions of complete inhibition of the anion exchanger will be measured to determine the stoichiometry of KC1 cotransport.

Most of the proposed work is designed to investigate the mechanism by which a 25% increase in cell volume can produce a 10-fold increase in the KC1 cotransport flux. We will use a new approach in which the rates of activation and inactivation of KC1 cotransport are measured and compared with the predictions of a simple mechanistic model. Previous work had shown that the rate constant for inactivation of transport is strongly volume dependent. Further experiments will determine whether the activation rate constant is detectably affected by cell volume. Besides cell swelling, two other interventions (low pH, low Mg++) also activate KC1 cotransport. The rates of change of transport following step changes in these parameters will be measured to determine whether activation is caused by a change in the activation rate constant, inactivation rate constant, or both.

The working hypothesis, based on recent work with the protein phosphatase inhibitor, okadaic acid, is that a net dephosphorylation catalyzed by either type 1 or (less likely) type 2a protein phosphatase is necessary for the activation of KC1 cotransport by cell swelling. Okadaic acid-sensitive protein phosphatases in rabbit red blood cells will be characterized to test the hypothesis that the rate constant for activation of the transporter is proportional to a particular phosphatase activity (in either membrane or cytoplasm). The working model proposes that transport is activated by net dephosphorylation caused mainly by protein kinase inhibition rather than phosphatase activation. Protein kinase activities will be determined in membrane and cytoplasm to test the hypothesis that a particular kinase activity has the characteristics of the inactivation rate constant for transport. In addition to studying the enzymes responsible for regulation of KC1 cotransport, 32P phosphorylation experiments will be conducted to try to identify the substrate that is dephosphorylated in parallel with activation of transport.

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