The long-term goal of this project is to understand the molecular mechanism of coupled transport across cell membranes. The focus of the project is on the red blood cell band 3 protein (also known as AE1), which is a major component of the red cell membrane and is a well-characterized model system fo mechanistic studies of coupled transport. In addition to its value as a model system, band 3-mediated chloride-sulfate exchange (the main emphasis of this project) is of potential relevance to the study of sulfate transport mediated by the DRA (down-regulated in adenoma) protein and the protein responsible for diastrophic displasia, an inherited disorder of cartilage formation. The general approach is to take advantage of the abundance of band 3 to study structure-function relations by biochemical methods, and to compare the result of biochemical studies with those obtained by site-directed mutagenesis.
The first aim concerns a particular glutamate residue (human E681; mouse E699), which has a role in the anion translocation event. The working hypothesis, based on chemical modification experiments, is that the negative charge on thi residue normally moves across much of the transmembrane electric field along with chloride and two protein-bound positive charges, resulting in an electroneutral translocation event. This idea will be tested in several ways, by using both chemically modified human band 3 and transgenic mice expressing band 3 mutated at this residue. The methods to be used include substrate binding measurements, pre-steady state and steady state tracer flux measurements, and patch-clamp electrical recordings.
The second aim i s to use improved chromatographic methods for analyzing hydrophobic peptides to finish full topological map of band 3, based on the positions of lysine residues. These studies should resolve current controversies regarding band 3 topology.
The third aim i s to use published methods to express the band 3 membrane domai in yeast (Saccharomyces cerevisiae), for the purpose of functional analysis of the site-directed mutations.
The fourth aim i s to identify, using both biochemical methods and mutagenesis, a second carboxyl group (other than E681) associated with the transport pathway and to localize exofacial lysine residue involved in subunit contacts. The mutagenesis will be performed in band 3 expressed in both Xenopus oocytes and in yeast.
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