Department of Physiology and Biophysics and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
A MAJOR GOAL in cystic fibrosis (CF) research is the
identification of alternate chloride channels that might substitute for the cystic fibrosis transmembrane conductance regulator (CFTR), the
anion channel that is encoded by the CF gene (2, 10). CFTR
localizes primarily to the luminal surfaces of epithelial cells, where
it mediates transcellular chloride and bicarbonate transport (1,
5). Activation of CFTR by cyclic nucleotide-dependent phosphorylation leads to the secretion of salt and water in intestine and exocrine glands. Excessive CFTR activity causes secretory diarrhea
(induced, for example, by bacterial toxins that elevate cyclic
nucleotide production in the gut). Reduced CFTR activity causes CF. The
severest cases of CF are characterized by airway infection and
inflammation, pancreatic insufficiency, and intestinal obstruction
(4). Whether all of these pathologies are attributable to
a loss of chloride channel activity vs. other possible functions of
CFTR is not clear (8). However, it seems reasonable to
search for additional epithelial chloride channels that might serve as proxies for CFTR, since the anion channel activity of CFTR is its most
well-accepted functional property.
The study by Gyömörey et al., the current article in focus
(Ref. 7, see p. C1787 in this issue),
provides evidence for a chloride channel other than CFTR that can
mediate chloride secretion in the mouse small intestine. The authors
show that segments of ileum isolated from CFTR knockout mice are
capable of exhibiting chloride-dependent secretion and that the
secretory rate of this tissue is increased by modest dilution of the
mucosal (i.e., luminal) bath. The activation of this pathway by
hypotonic shock, as well as its pharmacological profile [inhibited by
the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic
acid (NPPB), but not by DIDS], are characteristics of ClC-2, a member
of the ClC family of chloride channels (9, 12, 13). In
support of this interpretation, the authors provide solid evidence for
the expression of ClC-2 message and protein in mouse small intestine.
ClC-2 is an interesting candidate for an alternate chloride channel
because of its relatively broad tissue distribution and its capacity to
be activated by several factors including hypotonic shock, low pH, and
protein kinases (9, 12, 13). However, the most surprising
finding of the study by Gyömörey et al. is the predominant
localization of ClC-2 protein to the tight junctions between epithelial
cells in the surface villi of the small intestine. CFTR is most
abundant in the intestinal crypts, which are the major sites of fluid
secretion in this tissue (14, 16). The localization of
ClC-2 to the villi rather than to the crypts does not necessarily
exclude the possibility that activation of this channel could
substitute for the missing CFTR activity in the CF intestine. For
example, Zhou et al. (17) showed that the expression of
human CFTR in the intestines of CFTR knockout mice reversed the
intestinal pathology exhibited by these animals even though the human
CFTR protein was localized primarily to the villus epithelial cells.
What is more surprising is the apparent localization of ClC-2 to the
tight junctions, which form continuous belts between adjacent
epithelial cells. This localization of ClC-2 to tight junctions may be
unique to the small intestine, since it has not been observed for fetal
airway epithelial cells (which express ClC-2 along the luminal
membrane, Ref. 9) or for the large intestine
(Gyömörey K and Bear CE, unpublished observations). Thus
ClC-2 may serve a specialized function at epithelial tight junctions in
the small intestine.
What are the possible implications of localizing a chloride channel to
the region of the tight junctions between epithelial cells? Two
possibilities seem worth considering. First, ClC-2 channels may be
localized to the luminal aspect of the tight junction by
protein-protein interactions as a means to facilitate regulatory interactions between these channels and signaling molecules. Junctional complexes are "hot spots" for cell signaling molecules and PDZ domain-mediated protein interactions (reviewed in Ref.
15). Although ClC-2 appears to lack a canonical PDZ
recognition signal, it could be localized to junctions by another
mechanism and thereby interact with kinases and phosphatases that are
concentrated in this region of the cell. According to this scenario,
ClC-2 could still mediate chloride flow across the luminal membrane;
its localization near the tight junction would simply increase its
potential for regulation by signaling molecules.
A second possibility is that ClC-2 channels mediate chloride transport
across epithelial tight junctions in the small intestine. Tight
junctions are permselective, regulatable barriers to the flow of
solutes between epithelial cells (i.e., the paracellular pathway).
Although there has been considerable progress in our understanding of
the molecular architecture of epithelial tight junctions
(15), the mechanisms by which specific ions permeate this
barrier are largely unknown. One exception is the recent identification
of paracellin-1 as a mediator of magnesium reabsorption across tight
junctions in the thick ascending limb of Henle (11). In an
exciting convergence of genetics and epithelial physiology, the
paracellin-1 gene was identified in patients with hereditary hypomagnesemia by positional cloning and was shown to encode a member
of the claudin family of tight junction-associated proteins (11,
15). Paracellin-1 localizes specifically to the tight junctions
in the thick ascending limb of the nephron, where it is essential for
transepithelial magnesium reabsorption (11). Conceivably,
ClC-2 channels that are localized to the tight junctions in small
intestine could serve as conduits for paracellular chloride flow across
this tissue. In this regard, one of the major functions of the villi of
the small intestine is to reabsorb large quantities of sodium,
chloride, and fluid (6). Sodium reabsorption occurs across
the cells and is driven by the Na+-K+-ATPase at
the basolateral membrane. Chloride reabsorption occurs at least in part
via the paracellular pathway and is driven by the electrical gradient
generated by active sodium reabsorption. Factors that increase the
paracellular chloride permeability would increase the reabsorption of
sodium as well as chloride by shunting the transepithelial voltage
generated by sodium transport. It may be relevant that the chloride
permeability of the paracellular pathway in mouse ileum is stimulated
by agonists that also stimulate ClC-2 channel activity in heterologous
expression systems (3, 12). Perhaps ClC-2 provides a
regulated pathway for the paracellular flow of chloride in small
intestine as a means to modulate the rates of salt (and fluid)
reabsorption across this tissue.
In summary, the results of Gyömörey et al. remind us that
CFTR is not the only interesting chloride channel in epithelial cells.
Whether ClC-2 will become a useful target for drugs to circumvent the
CF defect depends on the extent to which this channel can mediate
chloride transport across the luminal membranes of airway and
intestinal epithelial cells and on the identification of specific
factors that increase its activity. However, irrespective of any
possible connection to CF, the localization of ClC-2 to epithelial
tight junctions raises interesting possibilities about the functional
role of this chloride channel in the small intestine.
ARTICLE
TOP
ARTICLE
REFERENCES
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
1.
Anderson, MP,
Sheppard DN,
Berger HA,
and
Welsh MJ.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am J Physiol Lung Cell Mol Physiol
263:
L1-L14,
1992
2.
Bear, CE,
Li C,
Kartner N,
Bridges RJ,
Jensen TJ,
Ramjeesingh M,
and
Riordan JR.
Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).
Cell
68:
809-818,
1992[ISI][Medline].
3.
Bijlsma, PB,
Bakker R,
and
Groot JA.
The chloride conductance of tight junctions of rat ileum can be increased by cAMP but not by carbachol.
J Membr Biol
157:
127-137,
1997[ISI][Medline].
4.
Boat, TF,
Welsh MJ,
and
Beaudet AL.
Cystic fibrosis.
In: The Metabolic Basis of Inherited Disease (6th ed.), edited by Shriver CL,
Beaudet AL,
Sly WS,
and Valles D.. New York: McGraw-Hill, 1989, p. 2649-2860.
5.
Cohn, JA,
Nairn AC,
Marino CR,
Melhus O,
and
Cole J.
Characterization of the cystic fibrosis transmembrane conductance regulator in a colonocyte cell line.
Proc Natl Acad Sci USA
89:
2340-2344,
1992[Abstract].
6.
Frizzell, RA,
and
Schultz SG.
Models of electrolyte absorption and secretion by gastrointestinal epithelia.
Int Rev Physiol
19:
205-225,
1979[Medline].
7.
Gyömörey, K,
Yeger H,
Ackerley C,
Garami E,
and
Bear CE.
Expression of chloride channel ClC-2 in the murine small intestine epithelium.
Am J Physiol Cell Physiol
279:
C1787-C1794,
2000
8.
Jilling, T,
and
Kirk KL.
The biogenesis, traffic and function of the cystic fibrosis transmembrane conductance regulator.
Int Rev Cytol
172:
193-241,
1997[ISI][Medline].
9.
Murray, CB,
Morales MM,
Flotte TR,
McGrath-Morrow SA,
Guggino WB,
and
Zeitlin PL.
ClC-2: a developmentally dependent chloride channel expressed in the fetal lung and downregulated after birth.
Am J Respir Cell Mol Biol
12:
597-604,
1995[Abstract].
10.
Riordan, JR,
Rommens JM,
Keren B,
Alon N,
Rozmahel R,
Grzelczak Z,
Zielenski J,
Lok S,
Plavsic N,
Chou J,
Drumm ML,
Iannuzzi MC,
Collins FS,
and
Tsui LC.
Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.
Science
245:
1066-1072,
1989[ISI][Medline].
11.
Simon, DB,
Lu Y,
Choate KA,
Velazquez H,
Al-Sabbon E,
Praga M,
Casari G,
Bettinelli A,
Colussi G,
Rodriguez-Soriano J,
McCredie J,
Milford D,
Sanjad S,
and
Lifton RP.
Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption.
Science
285:
103-106,
1999
12.
Tewari, KP,
Malinowska DH,
Sherry AM,
and
Cuppoletti J.
PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels.
Am J Physiol Cell Physiol
279:
C40-C50,
2000
13.
Thiemann, A,
Gründer S,
Pusch M,
and
Jentsch T.
A chloride channel widely expressed in epithelial and non-epithelial cells.
Nature
356:
57-60,
1992[ISI][Medline].
14.
Trezise, AE,
and
Buchwald M.
In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator.
Nature
353:
434-437,
1991[ISI][Medline].
15.
Tsukita, S,
Furuse M,
and
Itoh M.
Structural and signalling molecules come together at tight junctions.
Curr Opin Cell Biol
11:
628-633,
1999[ISI][Medline].
16.
Welsh, MJ,
Smith PL,
Fromm M,
and
Frizzell RA.
Crypts are the site of intestinal fluid and electrolyte secretion.
Science
218:
1219-1221,
1982[ISI][Medline].
17.
Zhou, L,
Dey CR,
Wert SE,
DuVall MD,
Frizzell RA,
and
Whitsett JA.
Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR.
Science
266:
1705-1708,
1994[ISI][Medline].