The objective of this proposal is to elucidate the mechanisms of anion exchange and proton-anion cotransport by the band 3 transporter of human erythrocytes. The mediated exchange of chloride and bicarbonate is essential for efficient removal of CO2 and metabolic protons from tissues and appears to be rate-limiting for CO2 excretion in the lungs during severe exercise. Cotransport of small molecules is important in epithelial membranes and volume regulation by cells. We propose 1) to characterize the net flux of proton equivalents across red cells in order to test the relationships between these two functions on band 3; 2) to characterize inhibitor interactions with the anion transporter which will test the topology of the sites and determine the conformations of the transporter that permit binding; and 3) to develop and test a new working hypothesis for the mechanisms of anion and proton transport. In the newly proposed mechanism each band 3 monomer has a single proton transport site and a single anion transport site which do not always have access to the same side of the membrane at the same time. This asynchronous behavior of these two transport sites, for which there is already evidence, results in equations for the transport velocity that exhibit 1) self-inhibition on each side of the membrane at high anion concentrations; 2) sigmoid activation kinetics for monovalent anions at low pH; 3) inhibition of proton-sulfate cotransport self-exchange at low pH; 4) the relationships between anion exchange and proton-anion (H + SO4 and H + Cl) cotransport; and 5) an explanation for residual transport after irreversible inhibition with phenylglyoxal. None of these characteristics have been so simply explained before. These studies involve measurements of net fluxes and tracer exchange across human red cell membranes, development of computer methods for non-numerical functional analysis of reaction velocities, and ligand binding to functional transmembrane proteins as probes for conformational analysis. These studies will both extend our knowledge of chloride-bicarbonate exchange and develop new analytical tools for the study of cotransport mechanisms and functional protein conformations.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL028674-05
Application #
3340000
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1981-09-01
Project End
1989-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Emory University
Department
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Elmariah, Sammy; Gunn, Robert B (2003) Kinetic evidence that the Na-PO4 cotransporter is the molecular mechanism for Na/Li exchange in human red blood cells. Am J Physiol Cell Physiol 285:C446-56
Meng, X J; Timmer, R T; Gunn, R B et al. (2000) Separate entry pathways for phosphate and oxalate in rat brain microsomes. Am J Physiol Cell Physiol 278:C1183-90
King, P A; Gunn, R B (1991) Glycine transport by human red blood cells and ghosts: evidence for glycine anion and proton cotransport by band 3. Am J Physiol 261:C814-21
Frohlich, O; Macey, R I; Edwards-Moulds, J et al. (1991) Urea transport deficiency in Jk(a-b-) erythrocytes. Am J Physiol 260:C778-83
Joiner, C H; Gunn, R B; Frohlich, O (1990) Anion transport in sickle red blood cells. Pediatr Res 28:587-90
Gunn, R B; Gunn, H B (1989) Inhibition of erythrocyte anion exchange by tolrestat, an inhibitor of aldose reductase. Metabolism 38:801-4
Shoemaker, D G; Bender, C A; Gunn, R B (1988) Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism. J Gen Physiol 92:449-74
Frohlich, O; Gunn, R B (1987) Interactions of inhibitors on anion transporter of human erythrocyte. Am J Physiol 252:C153-62
Milanick, M A; Gunn, R B (1986) Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites? Am J Physiol 250:C955-69