BIOCHEMICAL STUDIES OF ERYTHROCYTE ANION TRANSPORT

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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.