Molecular Pathology Unit, Department of Pathology, Massachusetts General Hospital, Charlestown, Massachusetts 02129
THE ABILITY OF EPITHELIA to form a
diffusion barrier between cellular compartments of very different fluid
and solute composition is dependent not only on asymmetrically
distributed transcellular transport mechanisms (transcellular
pathway) but also on structures that regulate the diffusion
of ions and small, noncharged solutes through the paracellular pathway.
Although those involved in regulating solute and water transport across
the transcellular route are well understood, the mechanisms governing
the paracellular pathway have only recently begun to be examined. At
the apical end of the paracellular space, adjacent cell membranes are
in close apposition, a site that was termed by early anatomists as the
"terminal bar" (2) and was considered to be an
impermeable barrier in the paracellular space. It was not until the
elegant ultrastructural studies of Farquhar and Palade (8)
that the terminal bar was shown, in fact, to consist of a junctional
complex composed of an apical tight junction (zonula
occludens), an intermediate junction (zonula
adherens), and a desmosome (macula adherens).
Analysis of freeze-fracture replicas indicated that tight junctions
form a circumferential network of anastamosing strands of varying
complexity located in the plane of the plasma membrane
(20). Furthermore, it was suggested that the number of
parallel tight junction strands might correlate with the level of
measured transepithelial electrical resistance (TER) (5).
When the number of strands was plotted against TER, however, it was
found that the relationship between the increase in TER with each
additional strand was not a linear, but an exponential, one. This led
to the suggestion that tight junction strands contain pores that
flicker between an open and closed conformation (4).
Electrophysiological measurements had indicated that whereas the plasma
membrane contains pores of 0.4 nm radius (18), pores of
3-4 nm radius are present in the paracellular space
(13). In other words, the tight junction barrier in the
paracellular space is considerably more permeable to water and small
solutes than the plasma membrane. Furthermore, tight junctions appeared to be capable of discriminating between ions of similar charge and, in
general, to be predominantly cation selective (15). This
led to the prediction that tight junctions must contain aqueous pores
lined by proteins, the amino acid composition of which would determine
the charge selectivity of the tight junction pores.
Initial studies designed to identify integral tight junction proteins
resulted in the discovery of two important tight junction-associated cytoplasmic proteins, ZO-1 (22) and cingulin
(3). It was not until 1993, however, that the molecular
composition of the tight junction strands began to be clarified in a
series of groundbreaking studies by the Tsukita group. The first of
these was occludin (11), a ~58-kDa tetraspan
phosphoprotein, which, when overexpressed in MDCK cells, resulted in an
elevation of TER and a paradoxical increase in the flux of small,
water-soluble solutes (1, 14). However, when occludin-null
embryonic stem cells were found to differentiate into tight
junction-expressing cells (16), it became clear that other
protein(s) must contribute to the formation of tight junction strands
and that the precise function of occludin in the tight junction
remained to be established. This was further supported by the
observation that occludin-null mice survived to adulthood, although
they developed a complex phenotype (17).
In their quest to identify other integral tight junction proteins, the
Tsukita group re-examined the junction fraction from their original
chicken liver preparations. The search yielded two novel, ~23-kDa
integral tight junction proteins, claudins-1 and -2 (9).
They, like occludin, are tetraspan proteins; however, the claudins
share no sequence homology with occludin. In addition, in contrast to
occludin, when claudin-1 cDNA was transfected into fibroblasts, they
formed a network of tight junction strands in the plasma membrane,
indicating that claudin is necessary and sufficient to form tight
junction strands (12). To date, a >20-member family of
claudins has been recognized (23) and these, either singly
or in combination with several different claudins, are expressed in a
cell- and tissue-specific pattern. Although clearly indicating that
claudin(s) are the primary component of the tight junction strands,
their contribution to the formation of the predicted ion- selective
pores within the tight junction strands was unclear. A significant
breakthrough was achieved with the report that a cohort of kindreds
suffering from a rare, autosomal recessive form of hypomagnesemia
associated with a distal renal tubular defect had a variety of
mutations in the claudin-16 gene (19). Claudin-16 is
uniquely expressed by epithelial cells lining the thick ascending limb
of Henle, where it serves as a paracellular divalent cation-selective
channel. Mutations in the claudin-16 gene are associated with a defect
in magnesium reabsorption, which results in turn in the urinary loss of
this ion. From these data, it appears that claudin-16 forms a
Mg2+-selective channel in the epithelial tight junctions of
the thick ascending limb of Henle. Further evidence that claudins might be involved in forming ion-selective channels was obtained in experiments utilizing two types of Madin-Darby canine kidney (MDCK) cells. High-resistance MDCK I cells (TER >1,000
These experiments were a prelude to a series of elegant studies
conducted by the Anderson group (6, 7), including the current article in focus (Ref. 7, see p. C1336 in this
issue), which examined the role of the two extracellular domains of
selected claudins in the formation of ion- selective channels within
tight junction strands. In the first, site-directed mutagenesis was utilized to reverse the net charge on the first extracellular loop of
claudins-4 and -15 (6). These mutated claudins were then
expressed under an inducible promoter in MDCK cells. When a single
negative charge was substituted for a positive charge in the first
extracellular domain of claudin-4, paracellular Na+
permeability was increased. Conversely, when one or more negative charges on the first extracellular loop of claudin-15 were replaced singly or in combination with positive charges, the paracellular charge
selectivity changed from a Na+- to a
Cl The first of these concerns is neatly circumvented in the current
study, in which chimeric molecules are constructed by engrafting either
the first or second or both extracellular domains of claudin-4 onto
claudin-2 and vice versa (7). This approach accomplishes two things. 1) It circumvents the potential problems
inherent in site-directed mutagenesis, and 2) it provides a
means to examine the functional contribution of specified segments of
the claudin in question. Because these experiments were conducted in
cells expressing endogenous tight junction proteins, the authors
acknowledge that the expression system utilized may lead to altered
expression and/or localization of endogenous proteins. These potential
problems aside, the study clearly shows that expression of the first or both extracellular domains of claudin-4 on claudin-2 markedly elevates
TER and decreases the permeability of Na+ relative to
Cl
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ARTICLE
REFERENCES
.cm2) and low-resistance MDCK II cells (TER
<100
.cm2) are interesting in that analysis
of freeze-fracture replicas indicates that the number of tight junction
strands is the same in these two cell lines (21). However,
when MDCK I cells that lack endogenous claudin-2 were transfected with
claudin-2 cDNA, the TER fell to a level that was similar to that of
low-resistance MDCK II cells that are known to express endogenous
claudin-2 (10). This led to the speculation that the type
and combination of claudins determines the paracellular permeability of
a given epithelium.
-selective one. While these are convincing and
important observations, two notes of caution are raised. First,
site-directed mutagenesis resulting in amino acid substitutions may
lead not only to a change in net charge but also to an altered
molecular conformation of the protein in question. Second,
overexpression of a given protein can result in its disproportionate
incorporation into the tight junction with a concomitant replacement
and/or displacement of other endogenous tight junction proteins, a
result that may alter paracellular ion selectivity. Finally the
overexpressed protein may be expressed in an aberrant location.
. By contrast, engrafting the first or both
extracellular domains of claudin-2 onto claudin-4 has only a modest
effect on TER and charge selectivity. Furthermore, although these
studies indicate that the larger, first extracellular loop of
claudins-2 and -4 appears to confer many of the charge-selective
characteristics, the function of the shorter second loop is unclear,
because chimeric expression of the second loop alone produced
biologically inactive molecules. Perhaps one of the most novel
observations in the present study is that expression of the first or
both extracellular domains of claudin-4 on claudin-2 has a more marked
effect on TER and charge selectivity than does native claudin-4. This
suggests that other segments of the molecule, including the carboxy
terminus, may regulate, possibly by interactions with cytoskeletal
proteins, the barrier function of the tight junction. Clearly, further
studies addressing these questions will yield important new insights
into the molecular structure and function of the tight junction.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. E. Schneeberger, Molecular Pathology Unit, Rm. 7147, 149 13th St., Charlestown, MA 02129 (E-mail: eschneeberger{at}partners.org).
10.1152/ajpcell.00037.2003
Received 23 January 2003; accepted in final form 27 January 2003.
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