The long-term goal of the proposed studies is to understand the molecular mechanisms and cellular functions of coupled Cl-HCO3 exchange across the plasma membranes of mammalian cells. This transport process is related to the larger problem f acid-base regulation, both on the cellular and whole-organism level. In the human red blood cell, Cl-HCO3 exchange is catalyzed by the major integrated membrane protein known as band 3. The topographical arrangement of the band 3 polypeptide in the membrane will be investigated by in situ proteolysis; the studies will be made feasible by the use of monoclonal antibodies directed against defined portions of the sequence. We recently developed a method for the labeling and conversion of functionally important extracellular glutamate side chains in band 3 to primary alcohols. A major goal of the proposed work is to localize these glutamate residues in the (known) band 3 sequence and to characterize the role of these residues in the anion translocation event, proton-anion cotransport, and the permeability barrier to conductive anion flux through the protein. The extracellular substrate anion binding site on band 3 is believed to be an arginine side chain. This side chain will be chemically modified with cyclohexanedione, and the labeled residue will be localized in the sequence by isolating the modified protein, digesting with protease to small peptides, and affinity- purifying the peptide of interest on immobilized borate derivative. Mammalian renal medullary collecting duct intercalated cells have a membrane protein that is immunologically related to red cell band 3. We will use monoclonal antibodies to isolate the homologous protein from bovine kidney. The protein will be reconstituted into lipid vesicles, and its possible function as a Cl- HCO3 exchanger will be investigated by a pH equilibration method. Other than for red cells and epithelia involved in acid or base secretion, it is not known whether Cl-HCO3 exchange has a general cellular function. Two human cell lines, K562 and HL60, are both known to have coupled Cl exchange; both lines also express the gene for a non-erythroid band 3 homolog, function of which is unknown. We will determine whether or not the Cl-Cl exchanger in these cells is likely to function physiologically as a Cl=HCO3 exchanger.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026861-11
Application #
3274302
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1987-07-01
Project End
1992-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
11
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
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

Showing the most recent 10 out of 29 publications