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Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Published on: March 10, 2012
ATPase activity of the cystic fibrosis transmembrane conductance regulator
1Divisions of Cell Biology, Research Institute, Hospital for Sick Children, Toronto, Canada M5G 1X8. bear@sickkids.on.ca
Abstract:
The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.
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