Project: Research project

Project Details


DESCRIPTION (Taken directly from the application)

The long term goal of this research is to elucidate the structural basis of
ion conduction, selectivity and gating in the cystic fibrosis transmembrane
conductance regulator (CFTR). CFTR, the protein which is defective in
cystic fibrosis, is a chloride channel whose activity is regulated by
phosphorylation and ATP binding. Although the functions of CFTR have been
extensively studied, the structure of CFTR, like most other integral
membrane proteins, is not well determined. The anion channel is presumably
lined, at least in part, by residues from the 12 membrane-spanning segments.
The goal of this project is to identify systematically the residue that line
the CFTR channel using the scanning-cysteine accessibility method. In this
approach reporter cysteines are substituted, one at a time, into putative
channel-lining segments. Each cysteine-substitution mutant is expressed in
Xenopus oocytes and the water-surface exposure of the cysteine is determined
by its ability to react with small, negatively and positively charged,
sulfhydryl-specific reagents which are derivatives of methanethiosulfonate.
For residues in membrane- spanning segments, we infer that if an engineered
cysteine reacts with the reagents then the corresponding wild-type residue
is exposed in the channel lumen. By this approach we have already
identified 18 channel-lining residues in the M1 and M6 membrane-spanning
segments. We will systematically identify the other residues that line the
CFTR ion channel, and determine their secondary structure, and the position
of the gate, the charge-selectivity filter and the size- selectivity filter.
The success of this project will allow us to create a low resolution
structural model of the CFTR channel. A model of the channel will provide
new insights into the molecular mechanisms underlying ion conduction,
selectivity and gating and help to elucidate the mechanism(s) by which
disease causing mutations alter CFTR channel function.
Effective start/end date1/1/9712/31/00


  • Genetics(clinical)


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