Cystic fibrosis (CF) is a disease of salt and water transport. The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel encoded by the gene defective in patients with CF. It is generally well- accepted that nucleotide binding and hydrolysis coordinates CFTR gating via long-range conformation changes. The molecular mechanism by which CFTR couples catalysis and transport, however, remains poorly characterized. It is essential to understand this link between nucleotide binding/hydrolysis and CI-transport. In working towards this goal, I will purify biochemical quantities of both wild-type CFTR and the delta508 derived, which is responsible for most instances of CF, using conditions that maximize recovery of active material. The adequacy of this protocol will be determined with parallel tests of two other ABC transporters, MDR1 (the human multi-drug exporter) and STE6 (the yeast pheromone exporter). With active material on hand, the ATPase activity of wild type and delta508 will be fully characterized in terms of kinetic parameters, pH dependence, requirements for divalent cations, nucleotide specificity, and modulation by known activators and inhibitions of ABC-type transporters. This will allow the specificity, and modulation by known activators and inhibitors of ABC- type transporters. This will allow the derivation of models concerning CFTR nucleotide binding domain function and to assess the step(s) that may be abnormal in the delta508 mutant. In parallel, we will devise an in vitro assay for C1-transport to study how catalysis and transport are coordinated under the same conditions. By completing these three primary steps, we will have established a model system for screening potential CFTR modulators, which may ultimately lead to identification of pharmacological agents that correct the fatal defects of salt and water transport in CF patients.
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|Ketchum, Christian J; Yue, Hongwen; Alessi, Karen A et al. (2002) Intracellular cysteines of the cystic fibrosis transmembrane conductance regulator (CFTR) modulate channel gating. Cell Physiol Biochem 12:1-8|
|Ketchum, C J; Schmidt, W K; Rajendrakumar, G V et al. (2001) The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export. J Biol Chem 276:29007-11|