CFTR ANION SELECTIVE CHANNEL--STRUCTURE AND FUNCTION

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.