The long-term goal of this project is to understand the molecular mechanisms of protein-mediated transport across biological membranes. The proposal is focused on three red blood cell membrane proteins: the C1--HCO3- exchanger (band 3 or AE1); a 43 kDa lactic acid transporter; and the water channel protein, CHIP 28. All three of these proteins are abundant enough to make it possible to study structure-function relations in the native membrane.
The first aim i s to use biochemical techniques to refine the topographic model of the arrangement of the band 3 polypeptide in the membrane and to define arginine, lysine and aspartate or glutamate residues of functional importance. Recent data indicate that red cell band 3 polypeptide may be heterogeneous as a result of alternately spliced RNA; the nature of this heterogeneity will be characterized in detail, using both biochemical and molecular biological approaches. Further advances in the understanding of band 3 will require higher resolution structure. One of the goals of the proposed work is to prepare two-dimensional crystals of band 3 in native membranes; these crystals will provide material for structural studies in other laboratories. Lactate transport is of significance in several pathophysiological situations, including diabetes, ischemic heart disease, and neoplasia. However, almost nothing is known about the transport protein itself. The cDNA for the protein will be cloned and sequenced from an erythroid library, using probes derived from the sequence of isolated protein. The cDNA sequence will then be used as a guide for further studies of structure-function relations in this protein. The function of the protein will also be studied in intact cells under pre-steady state conditions, to test a kinetic model for the catalytic cycle for transport. The water channel (CHIP 28) will be characterized with respect to the topography of the protein, using a monoclonal antibody recently raised in this laboratory. The goal of the studies is to test a simple structural model for the folding of the protein in the membrane. The locations of functionally important amino acid residues will also be determined, using methods that have been developed for band 3. Finally, the rate of proton or hydroxyl ion transport through the water channel will be quantified.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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University of Arkansas for Medical Sciences
Schools of Medicine
Little Rock
United States
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Jennings, Michael L (2013) Transport of H2S and HS(-) across the human red blood cell membrane: rapid H2S diffusion and AE1-mediated Cl(-)/HS(-) exchange. Am J Physiol Cell Physiol 305:C941-50
Jennings, Michael L; Cui, Jian (2012) Inactivation of Saccharomyces cerevisiae sulfate transporter Sul2p: use it and lose it. Biophys J 102:768-76
Chernova, Marina N; Stewart, Andrew K; Barry, Parul N et al. (2008) Mouse Ae1 E699Q mediates SO42-i/anion-o exchange with [SO42-]i-dependent reversal of wild-type pHo sensitivity. Am J Physiol Cell Physiol 295:C302-12
Jennings, Michael L; Cui, Jian (2008) Chloride homeostasis in Saccharomyces cerevisiae: high affinity influx, V-ATPase-dependent sequestration, and identification of a candidate Cl- sensor. J Gen Physiol 131:379-91
Jennings, Michael L; Howren, Todd R; Cui, Jian et al. (2007) Transport and regulatory characteristics of the yeast bicarbonate transporter homolog Bor1p. Am J Physiol Cell Physiol 293:C468-76
Kuma, Hiroyuki; Shinde, Anjali A; Howren, Todd R et al. (2002) Topology of the anion exchange protein AE1: the controversial sidedness of lysine 743. Biochemistry 41:3380-8
Jennings, M L; Adame, M F (2001) Direct estimate of 1:1 stoichiometry of K(+)-Cl(-) cotransport in rabbit erythrocytes. Am J Physiol Cell Physiol 281:C825-32
Jennings, M L (1999) Volume-sensitive K(+)/Cl(-) cotransport in rabbit erythrocytes. Analysis of the rate-limiting activation and inactivation events. J Gen Physiol 114:743-58
Jennings, M L; Whitlock, J; Shinde, A (1998) Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS. Biochem Cell Biol 76:807-13
Jennings, M L; Adame, M F (1996) Characterization of oxalate transport by the human erythrocyte band 3 protein. J Gen Physiol 107:145-59

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