The transmembrane anion exchange protein band 3 (B3) and band 3-related proteins (B3RP) are widely distributed in cells ranging from red blood cells (RBC), where B3 catalyzes the C1/HCO3, exchange that improves blood CO2 transport, to HL60 promyelocytic leukemic cells and brain neurons, where B3RP's probably play central roles in cell pH and volume regulation. B3 and B3RP's are also important in epithelia, particularly those of the kidney and digestive system that are involved in secretion of acid or base. B3 operates by a ping-pong mechanism, in which the transport site has two conformations: E1, with the transport site facing inward, and E0 with it facing outward. When a suitable anion such as C1- is bound to form ECli or EClo, the transport site can reorient to face the opposite side of the membrane, thus transporting the anion. To understand the structural changes that B3 undergoes during substrate binding and transport site reorientation, it is necessary to know what fraction of B3 is in each conformation, and to be able to alter the conformational distribution experimentally. 35Cl NMR techniques will be used to define for the first time the conformational distribution of B3 in C1- media. Inhibitory probes, including a newly discovered compound, diBA, that inhibits at 10/9 M, will be used to sense the conformational changes and to fix the protein in defined conformations for structural analysis. AE2, a B3RP that is expressed in HL60 cells and many other cell types, is structurally very similar to B3, but differs greatly from B3 in inhibitor sensitivity (e.g, 1000-fold less for diBA) and apparently even in its basic kinetic mechanism, which seems not to be a simple ping-pong mechanism based on transport measurements in HL60 cells. A recently- developed system for expressing human AE2 in insect cells, together with HL60 cells, will be used to compare more closely the kinetic mechanism of AE2 with that of B3. Site-directed mutants and AE2/B3 hybrid proteins will help to determine what portions of Be/AE2 are involved in inhibitor binding and in the transporting conformational change. Analysis of the structure and mechanism of these proteins is necessary for a better understanding of basic cellular processes, such as volume and pH regulation, as well as their modifications in disease. The very high-affinity probes investigated here, or their analogues, could also prove to be useful drugs for selectivity modifying these processes, thereby altering function in blood cells or kidney, stomach, and other epithelia.
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