Anion transport: The ping-pong model for the mechanism whereby the band 3 protein mediates anion exchange in human erythrocytes postulates that a single transport site can oscillate between two conformations, one facing the inside of the cell and the other facing the outside. To test this model, chloride gradients will be imposed across the membrane to determine if they have the predicted effects on the inhibitory potency of chemical probes, which bind to specific functional sites of the transport system. These results will reveal the nature of any intrinsic asymmetry in the orientation of the transport sites, as well as possible effects of the conformational change on other band 3 sites and on band 3-associated lipids. The relationship of net anion flow to the anion exchange system will also be investigated, and a model for the mechanism of net anion flow will be tested. Band 3 biosynthesis: We plan to look for synthesis of the band 3 protein in late-stage erythroleukemic cells and early stage reticulocytes, using transport and chemical labelling techniques, with the aim of setting-up an experimental system for studying band 3 synthesis. Erythroleukemic cell induction: We plan to investigate the relationship between changes in membrane cation transport and the alterations of cell volume and hemoglobin gene expression which occur when either Friend mouse or human K562 erythroleukemic cells are exposed to various inducing agents.
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