CFTR--ION CONDUCTION AND GATING MECHANISMS

The long-range goal of the proposed research is to investigate the
sequence of events which leads to the activation of Cl conductance in
cells expressing CFTR and to understand how disease-causing mutations
alter the sensitivity to activating conditions. Cystic Fibrosis is the
most common lethal, recessively inherited disease among caucasians,
affecting nearly 1 in 2500 newborns, and the disease is caused by mutations
in the gene coding for a membrane protein, the cystic fibrosis
transmembrane conductance regulator (CFTR). Wild type CFTR has been
associated with the expression of a cAMP activated, Cl-selective
conductance in a variety of cell types, and until recently it was thought
that this Cl conduction was absent in cells expressing disease-causing,
mutant CFTRs, particularly deltaF508, the most common mutation associated
with severe cystic fibrosis. We have shown, however, that in Xenopus
oocytes expression of deltaF508 and other mutant CFTRs is associated with
a cAMP activatable Cl conductance which exhibits a markedly reduced
sensitivity to an activating stimulus (forskolin + IBMX). Most
importantly, the reduction in sensitivity was highly correlated with the
severity of cystic fibrosis in patients carrying the corresponding
mutations. We propose to characterize in detail the activation of Cl
conductance in Xenopus oocytes expressing wild type and mutant CFTRs. A
quantitative description of the events leading to the activation of
chloride conductance should reveal the points at which mutations alter
activation. The specific aims are: 1. To characterize the sequential
reaction steps involved in the activation of Cl channels by cAMP in oocytes
expressing CFTR. 2. To characterize the conduction properties associated
with the expression of wild type CFTR in oocytes. 3. To use specific
mutations to evaluate the functional significance of the five putative
structural domains of CFTR. The results of these studies could provide a
mechanistic basis for the design of a rational drug therapy to ameliorate
the symptoms of C.F., which appear to be largely due to insufficient Cl
secretion. This proposal has been designed to interact closely with a
second R01 submitted by Mitchell Drumm which emphasizes cell-specific
expression of CFTR and the identification of therapeutic modalities in
different cell types.