Suzuki,
H., Tani, K., Tamura, A., Tsukita, S. (2015) Model for the Architecture of
Claudin-Based Paracellular Ion Channels through Tight Junctions. J Mol Biol.
2015 Jan 30; 427(2):291-297
Epithelial cells form a border between the
inside and outside of the body. They
cover both the external surface of the body (skin) and the lining of internal
cavities such as the lungs or the gastrointestinal tract. These cells are sealed together by tight
junctions, which are just beneath the external surface.
The tight junctions form a cell to cell
adhesion network that simultaneously blocks the passage of some substances
across the epithelia and allows the highly regulated movement of specific ions
and solutes across the barrier and between cells.
The primary components of tight junctions
are claudins and recently there have been great advances in explaining their
structure and function.
The first crystal structure of a single
claudin monomer, solved in 2014, is shown below.
.
The claudin protomer (monomer) has two
distinct sections: a tight bundle of
four α helices, which are embedded in the membrane of an epithelial cell, and
five β strands, which form a curved sheet on the extracellular side. These extracellular β sheets contain several
negatively charged residues which have previously been shown to be necessary
for both the formation of a tight barrier between cells and its selectivity for
positive ions (cations). They are also
thought to form the paracellular channels that traverse the epithelium.
Two of these functions have been
demonstrated by elegant experiments that substituted the acidic residues on the
β sheet with positively charged ones, resulting in paracellular channels that
were selective for anions.
The monomer structure alone, however, could
not provide explanations for either the structure of tight junction strands,
which form the mesh that creates cell adhesion, or the transcellular channels
that allow interaction between cells.
A recent study by the same team that solved
the first claudin structure, (Suzuki,
H. et al., (2015)), uses cysteine crosslinking experiments to suggest an
arrangement of the claudin monomers which would produce paracellular channels
which run across the epithelium, transcellular channels which are perpendicular
to the paracellular channels, and linear tight junction strands.
The proposed model uses a basic building
block of two claudin monomers lying alongside each other but antiparallel so
that the outer edges of the β sheets connect via hydrogen bonds to form half of
a β barrel structure. These dimers then
associate to form long polymers of the double row of claudins (see figure).
Image adapted
from (Suzuki, H. et al., (2015)). The first image shows the association of claudin monomers and their
polymerization. The second image shows
the same structure rotated through 90˚ so that we are looking down the half β
barrel .
This model was tested by substituting Cys
residues for key Asn positions at the proposed interface between the outer β
strands. The mutant claudins were found
to form dimers that reverted to ther usual monomeric structure in reducing
conditions. This illustrates that the
molecules normally associate face to face, along the proposed β strands, so that once the substitutions were
made disulphide bridges could be formed.
More corroboration was obtained using
electron microscopy (EM), which is studied in the second-year TSMB
course. A form of EM known as freeze fracture
electron microscopy, which is often used to examine proteins embedded in
lipid membranes, was used to provide images of tight junction strands, which appear
as a network of linear strands whose width is consistent with the model.
In the epithelial sheet, a claudin double
row would be anchored in the cell membrane with its half β barrel projecting
out. By lining itself up against a
similarly projecting double row in the adjacent cell we can see how tight
junction strands with paracellular channels could be formed. The diameter of this channel would be less
than 10 Å, enough to allow the passage of hydrated ions but to restrict their
flow.
Image adapted
from (Suzuki, H. et al., (2015)). The image shows the joining of a row of half β barrels from adjacent
cells, forming a row of paracellular channels.
Epithelial cells maintain the separation of
several internal spaces within the body and each of the interfaces has its own
requirements for substances that are allowed to cross the border. This means that the paracellular channels
need different ion specificity depending on which boundary they maintain. This could be provided by the charges and
orientation of the residues in the channel lining and may also be influenced by
flexible loops that link the β strands.
It is also possible that these loops, which are not well conserved among
the 27 members of the claudin family in the mouse (or human) proteome, could
function as recognition regions so that only claudins of the same type can
associate.
Possibly the most remarkable aspect of this
model, however, can be seen by rotating it through 90˚.
Image adapted
from (Suzuki, H. et al., (2015)). In this image the paracellular channels are
seen in magenta whilst the α helices are aligned perpendicular to the
page.
Further gaps can now be seen; these could
provide transcellular channels allowing the flow of ions and solutes between
epithelial cells.
In short this model appears to provide
solutions to all of the functions required by claudins in tight junctions
between epithelial cells and it will be very interesting to see if it proves to
be correct as further crystal structures
are solved.
Do look at the original paper if you have
the chance as it includes an excellent short animation that shows the
association of single claudin monomers into tight junction strands and then rotates
the structure to show the different channels. (Follow the web link to the
Supplementary Data.)
