Regulation of Cell Membrane Ion Channels 
and 4) an increase in both second messengers 
produces an additive increase in chloride 
current. These results also explain the previously 
puzzling observation that calcium-stimulated 
chloride secretion is defective in CF intestine but 
intact, in CF airway: the calcium-activated chlo- 
ride channels that could circumvent the chloride 
secretory defect in CF airway are missing from the 
apical membrane of intestinal epithelia. 
The gene that is defective in CF has recently 
been cloned by other investigators. This gene 
— the cystic fibrosis transmembrane conduc- 
tance regulator (CFTR) — was cloned based on 
a correct chromosomal location, an appropriate 
pattern of tissue expression, and a muta- 
tion (AF508) that was present on 70 percent 
of CF chromosomes and absent on non-CF 
chromosomes. 
We tested the hypothesis that expression of 
normal CFTR would complement the CF pheno- 
type, by expressing nonmutated CFTR in cul- 
tured CF airway epithelial cells. We assessed chlo- 
ride channel activation in single cells using a 
fluorescence microscopic assay and the patch- 
clamp technique. Expression of CFTR, but not 
the AF508 mutation, corrected the chloride 
channel defect. These results demonstrate a 
causal relationship between mutations in the 
CFTR gene and the CF phenotype. Because ex- 
pression of mutated CFTR did not complement 
the CF phenotype, other interpretations are un- 
likely. Demonstration that the CF phenotype can 
be corrected in cultured CF cells suggests the fea- 
sibility of a therapeutic approach based on cor- 
rection of the underlying defect. 
Although correction of defective chloride trans- 
port in CF epithelial cells strengthened the rela- 
tionship between CF and epithelial chloride 
channels, it did not demonstrate the function of 
CFTR. To evaluate its function, we expressed 
CFTR in several heterologous cells: HeLa cells, 
Chinese hamster ovary cells, and NIH 3T3 fibro- 
blasts. In each of these cell types, expression of 
CFTR, but not the AF508 mutant, causes an in- 
crease in cAMP-stimulated anion permeability 
and chloride currents. These data show that ex- 
pression of CFTR produces cAMP-activated chlo- 
ride currents in cells that do not normally express 
CFTR and do not normally have cAMP-activated 
chloride channels. When CFTR is overexpressed 
in cells that already express high levels of CFTR, 
the chloride permeability increases even further. 
These data suggest that CFTR is itself a cAMP-acti- 
vated chloride channel. Other alternatives have 
not yet been excluded, but all would require that 
this diverse group of cells must contain quiescent 
chloride channels that can become sensitive to 
cAMP only in the presence of CFTR. 
In addition to studies of the chloride channel, 
the laboratory also focuses on understanding the 
function and regulation of basolateral membrane 
potassium channels. The coordinated regulation 
of basolateral potassium channels and apical 
chloride channels is critical to effective chloride 
secretion. Our current work suggests the pres- 
ence of two potassium channels at the basolateral 
membrane — one regulated by calcium and one 
regulated by some other agent. The changes in 
the activation of the potassium channels are criti- 
cal to prevent changes in cell volume and to 
maintain an electrically negative intracellular 
voltage that drives chloride out of the cell across 
the apical membrane. 
A third aim of the laboratory focuses on the 
regulation of intracellular calcium concentra- 
tion, which controls many cellular processes. 
Stimulation of single cells with agonists often 
does not produce a steady increase in cell cal- 
cium; instead it causes oscillating, or pulsatile, 
elevations of calcium. Such oscillations have 
been observed in many cells, and it has been pro- 
posed that the frequency at which calcium oscil- 
lates determines the biologic response. We have 
turned to the Xenopus oocyte as a model system 
that allows us to manipulate the factors that con- 
trol intracellular calcium. Studies of the genesis 
of oscillations in cell calcium in Xenopus oo- 
cytes should be relevant to understanding mecha- 
nisms that underlie this important regulatory sys- 
tem in many cells. 
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