Cl/HCO3 exchange in human red blood cells (RBC), mediated by the transmembrane protein band 3 (B3), is important for blood CO2 transport. B3 and B3-related proteins (B3RP) are widely distributed in cells ranging from HL60 promyelocytic leukemic cells and hepatoma cells to brain neurons and cells of the choroid plexus, where they probably play important roles in cell pH and volume regulation. B3RP's are also important to the function of epithelia, particularly those such as kidney and gastric mucosa which are involved in secretion of acid or base. Analysis of the structure and transport mechanism of these proteins is thus essential to a better understanding of these normal cellular processes and their possible alterations in disease. B3 in RBC functions by a ping-pong mechanism, in which the protein has two conformations, Ei, with the transport site facing the cytoplasm, and E0, with it facing the outside. To understand the anion transport mechanism at the molecular level it is necessary to know what structural changes B3 undergoes during substrate binding and transport site reorientation. Rapid-flow and NMR techniques will be used to see what fraction of B3 is in each form (and the corresponding Cl- or I-loaded forms) at 38degreesC, and to test distributions at OdegreesC predicted from inhibitor studies. Newly-developed kinetic techniques will be used to examine the effects of pH and membrane potential on affinities of B3 for substrates and inhibitors, to test hypotheses regarding the nature of these binding sites. Proteolytic enzymes, reactive chemical probes, and fluorescent probes will be used to detect structural changes resulting from experimentally-induced changes in B3 conformation. Newly-synthesized reactive analogues will be used to label inhibitor binding sites which are involved in the transport-related conformational changes. A B3 expression system will be developed in yeast to assess the functional consequences of specific site-directed modifications in B3 structure. The AE2 protein, a B3 analogue expressed in HL60 cells (and many other cell types, including liver and kidney), differs only slightly in structure from B3, but transports anions by a fundamentally different kinetic mechanism. The proposed mechanism will be tested by using competitive inhibitors. The structure of this protein and the functional significance of particular regions will be probed by proteolytic cleavage, reactive inhibitors, and antibodies against regions unique to AE2, such as the outside-facing Z-loop. The properties of this transport system will be compared with those of RBC and of neutrophils. Changes in transport characteristics and AE2 expression during differentiation of HL60 cells toward neutrophils will also be measured, to test the hypotheses that AE2 catalyzes Cl/HCO-3 exchange in these cells and that a new anion transport system appears during differentiation. The mechanism of """"""""active"""""""" net Cl uptake in these cells, a centrally important factor in pH regulation, will also be examined. Such studies should provide significant clues to structure-function relationships, and should also help to define the importance of AE2 and similar proteins in regulation of cell volume and pH.
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