Function and Regulation of the Cystic Fibrosis 
Transmembrane Conductance Regulator 
Michael J. Welsh, M.D. — Investigator 
Dr. Welsh is also Professor of Internal Medicine and of Physiology and Biophysics at the University of Iowa 
College of Medicine, Iowa City. He earned his M.D. degree from the University of Iowa. He completed his 
residency at the University of Iowa College of Medicine; held clinical and research fellowships in 
pulmonary diseases and cardiovascular research at the University of California, San Francisco; and did 
postgraduate research in physiology and cell biology at the University of Texas, Houston. He then returned 
to the University of Iowa as a faculty member. 
CYSTIC fibrosis (CF) is a common lethal ge- 
netic disease involving defective electrolyte 
transport by several epithelia. In normal epithelia 
of the airways, the intracellular second messen- 
ger cAMP regulates chloride (Cl~) channels in 
the apical membrane. These channels provide 
both a pathway through which Cl~ flows and a 
key point for regulation of its movement. When 
cAMP increases, the channels open and Cl~ flows 
from the cell into the airway lumen, drawing 
water with it. Secretion of salt and water is impor- 
tant in generating the respiratory tract fluid, a crit- 
ical component of the mucocilliary defense 
mechanism. In CF airway epithelia, cAMP fails to 
open the Cl~ channels. As a result secretion is 
defective and the respiratory tract fluid is abnor- 
mal. This defect is believed to be the major cause 
of morbidity and mortality in CF lung disease. 
CF is caused by mutations in the gene encoding 
the cystic fibrosis transmembrane conductance 
regulator (CFTR). When we expressed the nor- 
mal CFTR gene in CF airway epithelial cells, the 
channel defect was corrected. That result indi- 
cated that CFTR was somehow intimately asso- 
ciated with cr channels, but did not reveal its 
function. Toward further understanding, we ex- 
pressed CFTR in a number of nonepithelial mam- 
malian cells lacking both endogenous CFTR and 
cAMP-activated CP channels. In every case in 
which CFTR was expressed, the cell generated a 
unique CI" channel that was activated by cAMP. 
Such channels were not observed in cells lacking 
the CFTR gene. 
The cAMP-regulated CP channels displayed 
regulatory and biophysical properties that were 
identical to those observed in cells expressing en- 
dogenous CFTR, as well as those in the apical 
membrane of airway epithelia. We stress apical 
membrane because that is where the CF defect is 
observed. These results suggested that CFTR itself 
might be a CP channel. This conclusion was 
quite controversial because CFTR did not resem- 
ble any channels previously described. It seemed 
instead to resemble a family of proteins that in- 
clude membrane pumps. 
To test the hypothesis that CFTR is a CP chan 
nel, we changed specific amino acids within the 
CFTR sequence. When an ion crosses a cell mem- 
brane through a channel, it must interact with the 
amino acids in that channel. In changing some of 
the positively charged amino acids to negatively 
charged ones, we changed the anion selectivity 
sequence. The channels normally favor CP over 
iodide (P), but after two of the amino acids were 
mutated in CFTR, the channel favored P over 
CP. The ability to change the properties of the 
conduction mechanism by altering specific 
amino acids provided the most compelling evi- 
dence that CFTR is itself a cAMP-regulated CP 
channel. 
The evidence that CFTR forms a CP channel 
and that the electrolyte transport defect in CF is 
in the apical membrane suggested that CFTR 
would be located in the apical membrane of se- 
cretory epithelia. To test that hypothesis, we de- 
veloped antibodies to CFTR and localized it with 
immunofluorescence confocal microscopy. In 
several lines of intestinal epithelial cells that se- 
crete CP, we found that CFTR was located in the 
apical membrane. That result indicates that CFTR 
is in a position where it can directly mediate CP 
movement across the membrane. 
An increase in the cellular concentration of 
cAMP opens the CFTR CP channel. Several of our 
studies have shown that CFTR is phosphorylated 
and thus regulated by a cAMP-dependent protein 
kinase (PKA). (Kinases regulate cell proteins by 
attaching a phosphate group.) When we used 
cell-free patches of membrane containing CFTR, 
we found that addition of the catalytic subunit of 
PKA opened the CFTR CP channel. 
Moreover, in vitro biochemical studies dis- 
closed that PKA phosphorylates CFTR on seven 
residues. In vivo studies further showed that PKA 
phosphorylates four serine residues located 
within a portion of the protein called the R do- 
main. Evidence that these reactions are important 
for opening the channel came from the observa- 
tion that PKA failed to open the channel when the 
four serines had been changed to alanines. Fur- 
thermore, when the R domain was deleted from 
CFTR, the channel no longer required PKA to 
open: it was open even without phosphorylation. 
The CFFR CP channel also contains stretches 
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