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This laboratory studies the physiology and
pathophysiology of ion channels in non-excitable cells.
A major focus is the study of
sodium and chloride channels controlling fluid balance.
It is well known that fluid balance in the lung and
intestine is maintained by a combination of both
chloride secretory and sodium absorptive mechanisms.
Abnormalities in fluid balance involved in cystic
fibrosis are the result of inhibited chloride secretion
and enhanced sodium absorption. In diarrhea, abnormally
high fluid secretion is caused by enhanced chloride
secretion (Trends
Microbiol Mar, 1994 pp 91-4).
We showed that E. coli heat stable enterotoxin,
STa, which is known to cause intestinal diarrhea,
activates the cystic fibrosis transmembrane regulator,
CFTR, the chloride channel which is defective in cystic
fibrosis (Am.
J. Physiol. 262 (31):C1304-1312,1992.,
Cell Physiol Biochem. 1995, Vol. 5, pp 23-32).
The
most common defect in cystic fibrosis, the deletion of
the phenyalanine in the first nucleotide-binding domain
of CFTR, causes decreased trafficking of CFTR to the
apical membrane in some tissues and decreased
functioning of CFTR in other tissues. This laboratory is
investigating the use of "chemical chaperones" to repair
the structural defect in D F508 CFTR. This approach has
received some recent interest because high
concentrations of glycerol have been shown to increase
the surface expression of D F508 CFTR by increasing the
processing of the endoplasmic reticulum-arrested CFTR
into the mature form of CFTR that inserts normally into
the apical membrane of cells.
The
endoplasmic reticulum is responsible for monitoring the
proper folding of CFTR (and many other proteins) before
delivery to the plasma membrane. This system proof-reads
protein structures to detect misfolding and subsequent
aggregation. It is thought that a helix around the D
F508 deletion is misfolded when this amino acid is
deleted. This results in either proteolysis of D F508
CFTR or a reduced channel open probability. Heat shock
proteins are well known to be part of the folding,
processing and trafficking of CFTR. When glycerol
surrounds D F508 CFTR it helps to refold the protein to
its normal structure.
In
the last few years the processing defect of D F508 CFTR
has been "cured" in cell cultures (in vitro) by either
cold temperatures or the addition of very high
concentrations of glycerol (1 molar). But these
approaches cannot be used in patients. In addition, CFTR
is expressed in great abundance in the kidney, yet the
kidney functions normally in cystic fibrosis. While it
is possible that CFTR does not perform an essential
renal function, another hypothesis is that organic
solutes such as myo-inositol, betaine, sorbitol, taurine,
and glycerophosphocholine that are accumulated in the
kidney can act like glycerol to protect D F508 CFTR from
assuming the partially unfolded conformation. These
solutes are accumulated in kidney cells of the renal
medulla when urine is concentrated as a protective
response against the high concentrations of urea that
denature proteins. The organic solutes are taken into
renal cells by organic solute cotransporters such as the
sodium myo-inositol cotransporter (SMIT), the taurine
cotransporter (TAUT) or the betaine/g -amino butyric
acid cotransporter (BGAT-1). We found that the mRNA for
SMIT, TAUT and BGAT-1 is also expressed in CF and normal
airway cell lines and that the mRNA in IB3 cells is
increased by hyperosmolality as it is in renal cells.
Immunoblotting and immunohistochemistry showed that
BGAT-1, SMIT and TAUT proteins are present in normal and
CF airway epithelial cell lines, and in rat airway
epithelium, particularly in the trachea.
Immuno-precipitation of CFTR from airway cells treated
with organic solutes showed increased processing of the
immature D F508 CFTR into the mature plasma membrane
protein. When the CF airway epithelial cell line IB3 is
incubated with myo-inositol alone, or myo-inositol given
sequentially with taurine and betaine, the processing of
D F508 CFTR to the plasma membrane was enhanced as
assessed by single channel recordings which show
increased open probability of CFTR on the apical
membrane. In addition the activity of the outwardly
rectifying chloride channel (ORCC) was detected in cells
given organic solutes, whereas control cells did not
have ORCC single channel fluctuations. We speculate that
organic solutes can repair the processing defect and
interaction with ORCC because organic solutes are
transported into airway epithelial cells and accumulated
in high enough concentrations to repair the misfolding
of the nucleotide binding domain around the deleted
phenylalanine of D 508F CFTR.
The amiloride- sensitive sodium
channel, ENaC, is well known to be the ion channel
involved increasing sodium absorption across epithelial
tissues. The activity of the amiloride-sensitive sodium
channel is increased in cystic fibrosis causing a
further exacerbation of the compromised airway balance
in this disease. Using ribonuclease protection assay,
in situ hybridization, and RT-PCR we found that
another cation channel is also present in epithelial
tissues. The nucleotide-gated cation channel, CNGC, is
expressed in rat airway (alveoli, trachea, bronchi and
bronchioles) and gut (duodenum, jejunum, ileum and
colon), two organs involved in cystic fibrosis (Cloning
and Widespread Distribution of the Rat Rod-type Cyclic
Nucleotide-gated Cation Channel. Am. J. Physiol.
272:C1335-1344, 1997). This channel that is stimulated
by cGMP contributes to sodium-mediated short circuit
current and net transepithelial sodium flux in rat
tracheal airway epithelial cells (Am.
J. Physiol. 272: C911-922, 1997).
Blockers of cyclic nucleotide gated cation channels (l-cis-diltiazem
and dichlorobenzamil) inhibit sodium and fluid
absorption from whole perfused rat or sheep lung where
their effects are additive to that of amiloride. We have
used the whole cell patch clamp technique in the airway
alveolar cell line A549 to show that cyclic nucleotide
gated cation channels expressed in these cells have the
same pharmacology as transfected CNGC1 expressed in HEK
293 cells. The sodium-mediated whole cell currents in
both these systems are stimulated by 8-Br-cGMP, and
inhibited by the blockers of the cyclic nucleotide gated
cation channels, dichlorobenzamil, l-cis-diltiazem.
These results suggest that amiloride in combination with
diltiazem may be a more effective way to inhibit sodium
absorption in the CF lung.
The
mRNA of both ENaC and CNGC1 in lung are increased by
glucocorticoids. This suggests that stress, or
glucocorticoid therapy given for lung inflammation would
exacerbate the already compromised fluid balance in a
cystic fibrosis lung. The identification of a new ion
channel in lung and intestinal epithelia has important
consequences for understanding sodium and fluid
absorption in these tissues. Until now it was thought
that sodium absorption was mediated only via ENaC. Our
experiments suggest that cyclic nucleotide gated
channels mediate a component of sodium-mediated fluid
absorption in the adult lung. |
