Download K -ATP-channel-related protein complexes

Research Article
3087
K+-ATP-channel-related protein complexes: potential
transducers in the regulation of epithelial tight
junction permeability
Thomas Jöns*,‡, Daniel Wittschieber*, Anja Beyer, Carola Meier, Andreas Brune, Achim Thomzig,
Gudrun Ahnert-Hilger and Rüdiger W. Veh
Charité-Universitätsmedizin Berlin, Centrum für Anatomie, Institut für Integrative Neuroanatomie, Philippstr. 12, 10115 Berlin, Germany
*These authors contributed equally
‡
Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 9 May 2006
Journal of Cell Science 119, 3087-3097 Published by The Company of Biologists 2006
doi:10.1242/jcs.03041
Summary
K+-ATP channels are composed of an inwardly rectifying
Kir6 subunit and an auxiliary sulfonylurea receptor (SUR)
protein. The SUR subunits of Kir6 channels have been
recognized as an ATPase, which appears to work as a
mechanochemical device like other members of the ABC
protein family. Thus, in spite of just gating ions, Kir6/Sur
might, in addition, regulate completely different cellular
systems. However, so far no model system was available to
directly investigate this possibility. Using highly specific
antibodies against Kir6.1-SUR2A and an in vitro model
system of the rat small intestine, we describe a new function
of the Kir6.1-SUR2A complex, namely the regulation of
paracellular permeability. The Kir6.1-SUR2A complex
localizes to regulated tight junctions in a variety of
gastrointestinal, renal and liver tissues of rat, pig and
Key words: SUR2A, Kir6.1, K+-ATP channels, Tight junction,
Permeability barrier, Gastrointestinal tract
Introduction
ATP-sensitive K+ channels (K+-ATP channels) are widely
distributed throughout the tissues of the mammalian body.
They contribute to important cellular actions by coupling the
metabolic stage of a given cell to its electrical activity,
including the regulation of vital functions, such as
cytoprotection or release of hormones (Inagaki and Seino,
1998). K+-ATP channels are protein complexes composed of
four pore-forming subunits and are members of the Kir6 family
of inwardly rectifying K+ channels. They are supplemented
by four auxiliary subunits that contribute sensitivity to
sulfonylurea-type drugs and participate in the regulation of
channel activity. These subunits are members of the
sulfonylurea receptor (SUR) subfamily, which belongs to the
superfamily of the ATP-binding cassette (ABC) proteins
(Zingman et al., 2001).
So far, K+-ATP channels, like other ion channels, were
considered to be passive transducers for ions, regulated by
membrane potential, ligand binding or covalent modification
(Zingman et al., 2001). The recent discovery of bifunctional
protein complexes that combine enzymatic and ion
conductance properties raises the possibility of alternative
principles for the biological function of Kir6/SUR complexes
(Banfi et al., 2000; Bienengraeber et al., 2000; Gadsby and
Nairn, 1999; Gulbis et al., 2000; Runnels et al., 2001). Thus,
the SUR subunit of the K+-ATP channel has been recognized
as an ATPase that binds and hydrolyzes the Mg2+-ATP
complex. Concomitantly, the confirmation of the SUR protein
changes from prehydrolytic to posthydrolytic (Zingman et al.,
2001). The chemical energy derived from ATP hydrolysis is
apparently used to drive the SUR protein from its inactive
(prehydrolytic, Kir6-pore closed as in resting conditions) to its
active (posthydrolytic, Kir6-pore open) conformation. Similar
mechanisms in other ABC proteins have led to the suggestion
that SUR proteins change their conformation following
changes in the ATP content and, therefore, were considered to
be mechanochemical devices (Karpowich et al., 2001;
Zingman et al., 2002b). Following this line of evidence, there
might be additional SUR-based mechanochemical actions
beyond channel gating.
Indeed, Kir6/SUR complexes are known to be involved
in the regulation of gap junction permeability in astrocyte
cultures (Velasco et al., 2000). The Ca2+-induced decrease in
permeability between neighbouring cells is partially inhibited
by drugs such as tolbutamide or glybenclamide, well-known
blockers of the SUR-component of K+-ATP channel activity,
which directly interact with the SUR subunit (Granda et al.,
1998). Moreover, a comparable inhibition has been observed
when using other gap junction uncouplers, such as oleic acid,
arachidonic acid, endothelin-1, octanol and ␣-glycyrrhenic
human, whereas it is absent in the urothelium. Changes in
paracellular permeability following food intake was
investigated by incubating the lumen of morphological
well-defined segments of rat small intestine with various
amounts of glucose. Variations in the lumenal glucose
concentrations and regulators of Kir6.1/SUR2A activity,
such as tolbutamide or diazoxide, specifically modulate
paracellular permeability. The data presented here shed
new light on the physiological and pathophysiological role
K+-ATP channels might have for the regulation of tight
junctions.
Journal of Cell Science
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Journal of Cell Science 119 (15)
acid, suggesting a direct interaction of the SUR
subunit with gap junction proteins (Granda et al.,
1998).
Irrespective of the drug, the inhibitory effects
require the presence of both, SUR and Kir6
proteins, since K+-channel openers such as
diazoxide and pinacidil, which directly interact
with the Kir6 subunit, are able to counteract
the SUR-mediated inhibition of gap junction
permeability. Interestingly, the Kir6 subunit of
astrocytes Kir6.1, localizes predominantly to the
very distal parts of astrocyte extensions, where
astrocyte gap junctions are found (Thomzig et al.,
2001).
Together, these data support the idea that
Kir6/SUR protein complexes function as
mechanochemical devices (Karpowich et al.,
2001; Zingman et al., 2002b). So far, however, no
direct data are available that support the physical
interaction of the Kir6/SUR protein pair with
other mechanochemical systems besides gap
junctions. Epithelial cells are characterized by the
formation of tight junctions that prevent
uncontrolled exchange between the lumen of, for
example, the gut and the subepithelial tissue.
However, paracellelar permeability may occur and
tight junctions are probably involved in its
regulation.
Here, we used highly specific, affinity-purified
antibodies against both pore-forming Kir6 and
auxiliary SUR subunits to screen non-neuronal
tissues for new and unexpected locations, which
might provide new insights concerning the
presumed mechanochemical role of Kir6 and SUR
proteins. We found exclusive positioning of the
Kir6.1-SUR2A protein pair at regulated but not at
unregulated tight junctions, suggesting a new role
for these proteins. In addition, we developed an in
vitro model that allows us to study in detail the
regulation of tight junction permeabilities through
Kir6.1-SUR2A protein complexes.
Fig. 1. Cellular and subcellular distribution of Kir6.1- and SUR2Aimmunoreactivities in the human gastric mucosa. Antibodies against SUR2A (a
and b, red; e, green; g, yellow when colocalized with ZO-1) or against Kir6.1 (c
and d, green or yellow – when colocalized with E-cadherin) yield a distinct dotlike or string-like staining pattern of the plasma membrane. (a) SUR2A
immunoreactivity is completely abolished by preabsorption with its cognate
recombinant protein (inset). (b) Localization of the gastric anion exchanger AE2
(green), a marker of parietal cell basolateral plasma membrane, and SUR2A does
not overlap. Red dots correspond to the SUR2A protein (both insets); they are
restricted to the apical edge of the basolateral plasma membrane. (c,d) Adherens
points can be visualized by staining for E-cadherin (red). The Kir6.1 protein
(green) is restricted to the apical edge of the basolateral surface, presumably
owing to the close spacing of tight junctions and adherens points at the lumenal
border of the cells (d, arrows). (e,f) By contrast, SUR2A (green) colocalizes
perfectly with the tight junction marker ZO-1 (red). (g) Merged image of e and f,
highlighting the entire distribution of the tight junctions (g). Bars, 30 ␮m.
Results
Kir6.1-SUR2A complexes are restricted to
tight junctions at gastrointestinal epithelial
barriers
To study the cellular and subcellular distributions
of the Kir6.1 and SUR2A proteins, appropriate
rat, pig and human tissues (mucosa of stomach,
small and large intestine, and liver) were selected.
The investigation was focused first on rat and
human gastric glands. Antisera against SUR2A
yielded a distinct spot- or string-like decoration
of the surface of the gastric mucosa (Fig. 1a,b,e).
The pattern suggests an antigen distribution restricted to small
areas of the plasma membrane between neighbouring
epithelial cells (Fig. 1a,b,e). Adjacent sections stained for
Kir6.1 immunoreactivities showed identical patterns,
suggesting colocalization or complex formation of Kir6.1 and
SUR2A proteins (data not shown). Immunostaining for
SUR2A and Kir6.1 was abolished by preincubation of
antibodies with recombinant protein containing the cognate
sequences (Fig. 1a insert and data not shown).
Immunoreactivities were found at the intercellular junctional
area of all epithelial cells in the gastric glands (Fig. 1b insert
1, see also Fig. 1a-e). Simultaneous incubation of the SUR2A
antiserum with an antiserum against the gastric anion
exchanger 2 (AE2), a highly specific marker of the basolateral
K+-ATP channels and tight junctions
Journal of Cell Science
plasma membrane of gastric parietal cells (Jöns et al., 1994),
revealed no overlap (Fig. 1b). The SUR2A immunoreactivity
was clearly separated from the AE2-positive lateral plasma
membrane (Fig. 1b, insert 2). The free apical plasma
membranes of the glandular epithelial cells and also the apical
and canalicular membranes of the parietal cells (Jöns et al.,
1994) were devoid of SUR2A (Fig. 1a,b,e) or Kir6.1
immunoreactivities (Fig. 1c,d). The distinct immunological
distribution suggested a specific localization of Kir6.1 and
SUR2A at epithelial cell-cell junctions. Simultaneous
incubations with antisera against E-cadherin (Fig. 1c,d), p120
(data not shown), ZO-1 (zonula occludens protein 1) (Fig.
1f,g), or occludin (data not shown) further support this
assumption. Double staining for Kir6.1 and E-cadherin
revealed, besides the typical dot-like appearance of the Ecadherin-containing puncta adherentia at the basolateral
plasma membrane, some overlap of both proteins within the
zonula adherens (Fig. 1c,d). No E-cadherin staining was found
at the most apical segment of the lateral plasma membrane,
representing the tight junction area, whereas this subdomain
3089
was brightly decorated by the Kir6.1-specific antiserum
(Fig. 1d, arrows). In addition, SUR2A immunoreactivity
completely colocalized with ZO-1, a marker protein of
epithelial tight junctions (Fig. 1e,f,g). Collectively, these data
suggest a specific association of Kir6.1-SUR2A with tight
junctions. Similar data were obtained when using tissue from
the small intestine (human and rat) and from the large intestine
(rat, data not shown). No obvious differences between the
various species were observed.
The villi of the small intestine are lined by a simple
columnar epithelium. The predominant cell type, the
absorptive enterocytes are tall columnar cells with a brush
border at their lumenal surface representing the microvilli. The
mucus-secreting goblet cells are scattered among the
enterocytes (Fig. 2a). The surface of the intestinal villi shows
invaginations (Fig. 2a, large arrows), which are also visible in
the immunofluorescence micrographs of the consecutive
section (Fig. 2b-d, large arrows). The junctional complexes
between adjacent epithelial cells of the small intestine are
located in the most apical part of the lateral membrane, just
beneath the microvilli (Fig. 2a, small arrows). A
double immunolabeling of the consecutive section
with Kir6.1 and ZO-1 antibodies revealed a
perfect colocalization of both proteins, most
probably highlighting the tight junctions (Fig. 2bd, small arrows). Similar results were obtained
with antibodies directed against SUR2A and ZO1 (Fig. 2e-g), the arrows indicate the invaginations
of the intestinal villi. Comparable to the
gastric mucosa, Kir6.1-SUR2A complexes are
exclusively localized to tight junctions in the small
intestine.
This perfect colocalization of Kir6.1-SUR2A
with ZO-1 at tight junctions was confirmed on the
ultrastructural level (Fig. 3). The tight junctions of
the intestinal columnar epithelium are located
directly beneath the lumenal cell-surface, showing
the multitude of the microvilli in cross section
(Fig. 3a) and in full length (Fig. 3b). The Kir6.1
and ZO-1 immunodecoration, visualized by the
3,3⬘-diaminobenzidine-precipitation product is
precisely located at the plasma membrane area of
Fig. 2. Subcellular distribution of Kir6.1-SUR2A
complexes in the intestinal epithelial barrier. (a) The
intestinal mucosa of humans is composed of two types
of epithelial cells, the tall columnar absorptive cells
(enterocytes) bearing a multitude of microvilli at the
apical pole, representing the predominant cell type and
the goblet cells (note the mucous droplet produced by
a secreting goblet cell, arrowhead). The inner surface
of the intestinal villi shows invaginations (large
arrows). (b-g) These invaginations can also be
identified in immunofluorescence micrographs
(arrows). Tight junctions connect adjacent epithelial
cells at labelled positions (small arrows in a) and
appear as immunolabelled dots in fluorescence
microscopy (small arrows in b-d,). Note the precise
colocalization of Kir6.1 (green in b) and SUR2A
(green in e) with the ZO-1-epitopes (red in c and f,) as
identified in the merged photographs (d,g) in orange.
Bars, 20 ␮m.
Journal of Cell Science
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Journal of Cell Science 119 (15)
Fig. 3. (a,b) Ultrastructural localization of Ki6.1- and ZO-1-like
immunoreactivities at positions corresponding to those of tight
junctions between intestinal epithelial cells. Analysis at the electron
microscopic level indicates the presence of the Kir6.1-SUR2A
complex at epithelial tight junctions. Kir6.1 and ZO-1
immunoreactivities are restricted to the area of tight junctions in
electron micrographs of the apical region of the rat intestinal
epithelial border. Note that, the size of the immunoreactive area
corresponds to approximately one quarter of the height of the
intestinal brush border, which fits to the dimensions of intestinal tight
junctions. Bar, 0.5 ␮m.
adjacent epithelial cells that represent the tight junction (Fig.
3a,b).
Kir6.1 and SUR2A coimmunoprecipitate and colocalize
with occludin in liver
In the liver, bile acids conjugated to bilirubin and to
biotransformed xenogens (drugs) are secreted into small,
specialized spaces between adjacent hepatocytes, the bile
canaliculi. They are sealed by tight junctions to prevent leakage
of the canalicular contents into the blood. Cell fractionation
techniques allow for the isolation of liver bile canaliculi and
tight junctions at high biochemical purity (Tsukita and Tsukita,
1989).
Immunocytochemical staining for Kir6.1 and ZO-1, a
marker for canalicular tight junctions, resulted in an identical
staining pattern, representing the distribution of hepatocyte
tight junctions sealings (Fig. 4a). A similar colocalization was
obtained with antibodies against Kir6.1 and SUR2A (data not
shown). To provide further information on a direct molecular
interaction between Kir6.1-SUR2A and occludin, immunoblot
analysis and coimmunoprecipitations were performed using
purified canalicular tight junctions and antibodies against
Kir6.1, SUR2A and occludin. The antibodies against
occludin, Kir6.1 and SUR2A gave signals at the expected 65
kDa, 43 kDa and 150 kDa, respectively (Fig. 4b). Occludin was
also detected in precipitates obtained with Kir6.1 or SUR2A
antibody (Fig. 4c). Precipitation with an antiserum against
occludin or with a non-immune serum was used for positive
or negative controls, respectively (Fig. 4c). In an additional
immunoprecipitation experiment with antibodies against
occludin, the ratio of precipitated or co-precipitated protein in
comparison with the molecules still present in the supernatant
after the immunoprecipitation were investigated (Fig. 4d).
We found that the Kir6.1 and SUR2A subunit pair coprecipitated in almost equal amounts. The concentration of
Kir6.1-SUR2A molecules remaining in the supernatant after
the immunprecipitation were also similar to each other. The
immunoprecipitated occludin molecules and the molecules in
the corresponding supernatant were at the same ratio as coprecipitated Kir6.1-SUR2A molecules. These data suggest
that, in rat liver the Kir6.1-SUR2A pair is associated with the
tight junctional protein complexes. In addition, the presence
of Kir6.1 message in the liver was confirmed by reverse
transcriptase (RT)-PCR (Fig. 4e).
The Kir6.1-SUR2A complex is absent in tight junctions
of collecting duct and urothelial epithelia
To address the question whether the Kir6.1-SUR2A complex
is present in both tight junctions with regulated and also in
those with non-regulated permeability, we studied their
distribution in rat and human kidney epithelia and urinary
bladder epithelia.
Kidney epithelial cells of proximal and distal tubules express
regulated tight junctions that control the paracellular flux of
water and ions. Prominent immunostaining was obtained for
Kir6.1 (Fig. 5a) and SUR2A (data not shown), at positions
characteristic for tight junctions in proximal and distal tubules
in the rat kidney. However, collecting ducts clearly identified
by aquaporin-2-positive chief cells, which are known to
contain impermeable and non-regulated tight junctions, were
devoid of Kir6.1 and SUR2A immunoreactivities (compare the
consecutive sections Fig. 5a,b). The presence of tight junctions
in tubules and in collecting ducts is confirmed by the presence
Table 1. Immunoreactivities for Kir6.1, SUR2A and several tight junction proteins in selected rat, pig and human tissues
SUR2A
+
+
+
+
+
+
+
+
–
+
–
–
Kir6.1
ZO-1
Occludin
E-cadherin
p-120
+
+
+
+
+
+
+
+
–
+
–
–
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
nd
+
nd
+
nd
nd
nd
nd
nd
nd
nd
+
nd
+
nd
+
nd
nd
nd
nd
nd
nd
nd
CDs, collecting ducts; nd, not determined.
Tissue
Stomach (human)
Stomach (pig)
Stomach (rat)
Small intestine (human)
Small intestine (rat)
Large intestine (rat)
Liver (rat)
Kidney, tubules (human)
Kidney, CDs (human)
Kidney, tubules (rat)
Kidney, CDs (rat)
Urinary bladder (rat)
Journal of Cell Science
K+-ATP channels and tight junctions
3091
Fig. 4. Kir6.1 mRNA and protein are found in rat liver,
where the Kir6.1-SUR2A complex physically interacts
with components of tight junctions.
(a) Immunocytochemical staining of bile canaliculi of
the liver with antibodies against Kir6.1 and ZO-1. The
bile canaliculi are sealed by tight junctions, which are
localized between adjacent hepatocytes. Note the
precise colocalization of Kir6.1 (left panel, green) and
ZO-1 (middle panel, red), orange in the merged image
(right panel, orange). The identical pattern of the
immunoreactivities suggest the localization of Kir6.1
and SUR2A (data not shown) in bile canalicular
membranes of the rat liver. Bar, 30 ␮m. (b) Bilecanalicular membranes were prepared from rat liver.
The proteins were separated on SDS-PAGE, transferred
to nitrocellulose and incubated with antisera against
occludin, Kir6.1 and SUR2A.
(c) Immunoprecipitations with antisera against SUR2A
and Kir6.1 led to a co-precipitation of occludin,
indicating that the Kir6.1-SUR2A complex physically
interacts with the tight-junction-protein complex.
Negative control without primary antibody (first lane);
immunoprecipitation with anti-occludin used as a
positive control (third lane). (d) Additional
immunprecipitation with anti-occludin antiserum
shows the ratio of the precipitated or co-precipitated
protein (Ip) in comparison with the amount of protein
still present in the supernatant (Sup). Please note that
occludin and the Kir6.1 and SUR2A protein pair
subunits were co-precipitated in almost equal amounts.
Analysed as a negative control was AE2, which does
not associate with tight junctions. As expected, AE2 is
only present in the supernatant. (e) Expression of
Kir6.1 mRNA in rat liver is also proven by RT-PCR.
of the ZO-1 protein in all types of renal epithelia
(Fig. 5c).
In the urinary bladder, the tight junctions
between the facet cells of the urothelium generate
one of the tightest barriers of the body (Fig. 6a,b).
To estimate the whole tissue section, the
immunofluorescence pictures were electronically
highlighted, by using the weak autofluorescence of
the tissue (Fig. 6c,d). The tight junctions can be
visualized by the presence of occludin (Fig. 6b).
In accordance with the differential expression in
kidney epithelia, tight junctions in bladder
epithelia were devoid of Kir6.1 immunoreactivity
(Fig. 6a; Table 1).
The paracellular permeability of the rat small intestine is
influenced by tolbutamide and diazoxide
Food-intake-dependent increase in the glucose concentration in
the intestinal lumen caused a coordinated alteration of the tight
junction structure and consequently results in an upregulation
of paracellular permeability (Pappenheimer et al., 1987;
Madara, 1994; Tomita et al., 2004). An increase in paracellular
flux is initiated when D-glucose is taken up by the sodium
glucose transporter 1 (SGLT1) leading to an increase in the
intracellular D-glucose concentration. The increased amount of
D-glucose may initiate an ATP-dependent change in the
conformation of the Kir6.1-SUR2A complex, probably leading
to the increased permeability.
To investigate these D-glucose-dependent modulatory
changes of tight junction permeability and to analyse the role
of the Kir6.1-SUR2A complex in this regulatory process, an in
vitro model system of the rat small intestine was developed
(see Materials and Methods). To measure permeability, we
used radioactive L-glucose, which is not transported by
enantiomer-specific SLGT-1 (Rummel and Stupp, 1960). A
rise in the D-glucose concentration in the intestinal lumen
caused an increase in the accumulation of L-[14C]glucose in
the lamina propria and tela submucosa, and was taken as an
indicator of an increase in paracellular permeability.
Generally, only one defined segment of the small intestine
was used per rat. First we established that the paracellular
uptake of L-[14C]glucose was linear over 60 minutes (data not
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Journal of Cell Science 119 (15)
Journal of Cell Science
shown). For the analysis of paracellular permeability, each
experimental series (consisting of three rats) included a baseline measurement of L-[14C]glucose uptake taken after 45
seconds with or without the indicated additives. The segments
of the other rats were incubated for 60 minutes with or without
the indicated additives. For calculation, L-[14C]glucose
accumulation obtained after 45 seconds under the individual
conditions was set as one.
A 60-minute incubation of a small intestine segment in a
lumenal buffer (containing 25 mM D-glucose) increased the L[14C]glucose accumulation by ~60% compared with an
Fig. 5. Collecting ducts in the kidney, characterized by non-regulated
tight junctions with high paracellular resistances do not express
Kir6.1-SUR2A complexes. (a) Staining of consecutive sections of the
rat kidney cortex for Kir6.1 visualizes tight junctions in proximal and
distal tubules, leaving collecting ducts (CDs) negative. (b) CDs are
easily identified by the presence of aquaporin 2 in their principal
cells. Tight junctions in CDs are not regulated and, therefore, exhibit
high paracellular resistance. (c) They are identified together with
those in proximal and distal tubules by staining for ZO-1
immunoreactivity. Bar, 20 ␮m.
incubation in buffer A (containing only 5 mM D-glucose) (Fig.
7a, compare bar I with bar II). Tolbutamide, a blocker of the
SUR component of Kir6 ion channel permeability, increased
L-[14C]glucose accumulation independently from a rise in the
lumenal D-glucose concentration. This increase even exceeded
the one obtained by 25 mM D-glucose (Fig. 7a, bar III). As a
control, an incubation with the same lumenal buffer (5 mM Dglucose) but supplemented with 20 mM L-glucose instead of
D-glucose had no significant effect because L-glucose is not
taken up by the epithelial cells and, therefore, can not cause an
increase in the intracellular ATP-concentration (Fig. 7a, see bar
IV) (Rummel and Stupp, 1960). Thus, accumulation of L[14C]glucose in the subepithelial tissue was not due to a specific
transporter-regulated transcellular uptake but rather mediated
by paracellular flux caused by changes in the permeability of
tight junctions.
To substantiate the potential function of the Kir6.1-SUR2A
complex in the regulation of tight junctions, we investigated
whether the effect of 25 mM D-glucose stimulation can be
modified by tolbutamide (3 mM) or by the Kir6-SUR activator
diazoxide (3 mM). In this case the permeability increased after
stimulation with 25 mM D-glucose alone was set as 100% (see
the bracket in Fig. 7a, bars I and II). The stimulatory effect of
25 mM D-glucose is reduced by ~50% in the presence of 3
mM diazoxide, whereas tolbutamide provoked a more than
twofold increase in paracellular permeability (Fig. 7b, bars II
and III).
Discussion
Transepithelial fluxes follow transcellular or paracellular
pathways
The passage of small molecules, such as water, ions or
nutrients, across various epithelia occurs by two distinct
mechanisms. The transcellular pathway consists of membrane
pumps, transporters and ion channels, whereas the paracellular
pathway follows the intercellular space between two adjacent
epithelial cells. Both routes are strictly regulated and, in some
tissues, the transcellular pathway apparently controls the
paracellular route by modifying the permeability of its
dominant barrier, the tight junctions (Madara, 1987; Madara
and Pappenheimer, 1987; Madara, 1994; Tomita et al., 2004).
Tight junctions are macromolecular complexes of at least
twenty individual protein components. These include
membrane proteins such as occludin, claudins and the
junctional adhesion molecule (JAM), scaffolding proteins such
as ZO-1, ZO-2 and ZO-3, as well as elements of the
cytoskeleton and the vesicle trafficking machinery (Lapierre,
2000; Martin-Padura et al., 1998; D’Atri and Citi, 2002).
Furthermore, the tight junction complex interacts with
membrane lipids, generating an intramembrane barrier that
prevents mixing of apical and basolateral membrane lipids.
Intracellularly, tight junction complexes are linked to an apical
perijunctional actomyosin ring (Fanning et al., 1998; Hirokawa
and Tilney, 1982; Itoh et al., 1997).
Fluxes by paracellular pathways are controlled by tight
junction permeability
As estimated from paracellular resistances, tight junction
permeabilities vary in a wide range according to the functional
requirements of the respective epithelium. For example the
transepithelial resistance of proximal tubules (6-7 ⍀⫻cm2) in
3093
Fig. 6. (a) Tight junctions between urothelial
facet cells are devoid of Kir6.1
immunoreactivity. (c,d) The morphology of
the lumenal surface of the rat urinary bladder
is visualized by electronically intensifying its
weak autofluorescence. The lumenal surface is
covered by the urothelium, a specialized
epithelium with a very tight permeability
barrier to prevent paracellular fluxes of urine.
This barrier is formed by non-regulated tight
junctions between facet cells, the uppermost
cell layer easily identified by their large nuclei
(asterisks). Arrows in b and d indicate the tight
junctions that can be visualized by their
occludin content (b, red), but are devoid of
Kir6.1 protein (a). Bar, 50 ␮m.
Stimulation of paracellular permeability
Relative concentration
of L-14[C]glucose
a
9
8
**
**
Fig. 7. Kir6.1-SUR2A complexes are involved in
7
the regulation of the paracellular permeability of
6
I
- buffer A
100%
the rat small intestinal epithelial cells. (a) Bar
5
graph, showing the effect of D-glucose,
4
II - buffer A + 20 mM D-glucose
tolbutamide and L-glucose for the regulation of
3
the intestinal tight junction permeability.
III - buffer A + 3 mM tolbutamide
2
Permeability was measured by the paracellular
1
flux of L-[14C]glucose over a period of 60
IV - buffer A + 20 mM L-glucose
0
minutes. Data are shown in comparison with the
I
III
IV
II
initial value (incubation with the same buffer for
45 seconds) as the relative concentration of L[14C]glucose. The initial value of all experiments
was set to one (data not shown). Bar I, incubation
Regulation of paracellular permeability
of gut segments with buffer A, containing 5 mM
by tolbutamide and diazoxide
D-glucose. Bar II, an increase of paracellular flux
of ~60% was obtained when the incubation buffer
**
250
contained 25 mM D-glucose instead of 5 mM. Bar
225
III, like D-glucose, tolbutamide also provoked an
200
increase in paracellular permeability. Therefore
175
both D-glucose and tolbutamide have a
150
stimulatory effect in this in vitro model. Bar IV, L125
glucose had no significant effect in paracellular
- buffer A + 20 mM D-glucose
I
permeability, confirming the enantiomer100
specificity of the sodium glucose transporter 1
75
II - buffer A + 20 mM D-glucose,
**
(SGLT1) of the small intestine. (b) Bar graph,
3 mM diazoxide
50
showing the effect of tolbutamide and diazoxide
III - buffer A + 20 mM D-glucose,
25
3 mM tolbutamide
after D-glucose stimulation. Bar I, increase in
0
III
paracellular permeability, determined after the rise
I
II
of the lumenal D-glucose concentration to 25 mM
(bracket in a – I/ II) was set as a 100%. Bar II in b, addition of the K+-ATP channel activator diazoxide decreased tight junction permeability by
~50%. Bar III, K+-ATP channel inhibitor tolbutamide increased the paracellular flux further by ~125%.
b
L-14[C]glucose (%)
Journal of Cell Science
K+-ATP channels and tight junctions
Journal of Cell Science
3094
Journal of Cell Science 119 (15)
the kidney is about 5⫻104 times lower than that of the
urothelium (300,000 ⍀⫻cm2) (Powell, 1981). The proximal
tubule is the place of major ion- and water-reabsorption in the
kidney, requiring a low resistance of the paracellular pathway.
By contrast, the bladder is designed to store the excreted and
concentrated fluids of the kidney. Reabsorption of bladder fluid
with its high content of urea would result in increased and
potentially harmful plasma concentrations of urea (Born et al.,
2003).
Tight junction permeability can vary even in the same tissue
as known from the small intestine (Pappenheimer, 1987;
Madara, 1994; Tomita et al., 2004). When, after a meal,
glucose is taken up through the saturating Na+-dependent
glucose transporter of the intestinal brush border, the increased
intracellular D-glucose concentration – by a so far unknown
mechanism – also increases tight junction permeability and,
thereby, paracellular transepithelial fluxes of monomers, such
as sugars and amino acids, by solvent drag (Pappenheimer,
1987; Madara, 1994; Turner, 2000; Turner et al., 2000b; Tomita
et al., 2004). Apparently, the resistance of the paracellular
pathway is regulated depending on local demands.
Current hypotheses suggest that the paracellular resistance
depends on the intracellular perijunctional actomyosin ring.
Actomyosin contraction could alter ZO-1, and trigger
modification of occludin and claudin function, two processes
that may be related to paracellular fluid movements (Kojima
et al., 1999; Van Italie and Anderson, 2004). In addition, the
activation of myosin light-chain kinase results in the
contraction of the perijunctional actomyosin ring, as well as in
increased permeability of epithelial and endothelial tight
junctions (Goeckeler and Wysolmerski, 1995; Hecht et al.,
1996; Turner, 2000; Yamaguchi et al., 1991). These data gave
rise to the idea that, paracellular transport is controlled by a
‘purse-string’ mechanism (Florian et al., 2002; Bement et al.,
1993), emphasizing the suspected role of the cytoskeleton in
the control of tight junction permeability.
K+-ATP channel proteins are localized at tight junctions
in a variety of gastrointestinal and urogenital tissues
Here, we report that the Kir6.1-SUR2A protein pair is found
in the epithelial cells of stomach, small and large intestine, liver
and proximal and distal tubules of the kidney (Table 1). In
all these tissues Kir6.1-SUR2A is exclusively localized at
positions characteristic of tight junctions. This focal
localization is surprising for Kir6-SUR pairs, which are widely
accepted to subserve channel function. In general, ion channels
are distributed to more or less extended areas of the cellular
plasma membrane, which is a reasonable localization to
depolarize or hyperpolarize the corresponding cell.
However, there are cases where a highly distinct distribution
of K+ channels apparently subserves an unconventional
function. One example is the cerebellar pinceaux. Here, the
channels are believed to attribute a positive charge to a
restricted extracellular compartment (Laube et al., 1996),
thereby locally increasing the transmembrane potential of the
Purkinje-cell axon hillock. In this case, the septate junctions,
a distinct intercellular barrier system, are supposed to prevent
uncontrolled charge spread.
Tight junctions represent a similar system. They are
composed of several interconnected rows of proteins or
protein complexes, best visualized in freeze-fracture electron
micrographs. Usually they are considered as mechanical
barriers, preventing the uncontrolled leakage of fluids and their
components from one compartment (vessel or gut lumen)
to another (interstitium). However, they could also act as
electrical barriers, building up a potential between the
interstitium and the gut lumen. A possible role of the Kir6.1SUR2A protein pair in tight junctions could be to control the
charge of an extracellular microdomain defined by the outer
protein rows of the tight junction complex. Following this idea,
a modified charge concentration within the extracellular area
of the tight junction could be involved in their opening.
However, modified charge concentration within the
extracellular area of the tight junction can not explain the
regulation of paracellular fluxes. Such a regulation might
involve a coupling between extracellular conditions (i.e.
increase in nutrients) and changes in intracellular metabolic
processes.
The Kir6.1-SUR2A protein pair represents an
unconventional combination of K+-ATP channel subunits
So far, the molecular composition of epithelial K+-ATP
channels in the gastrointestinal tract is not completely
understood (Warth and Barhanin, 2003). Other Kir6-SURbased K+-ATP channels, such as the Kir6.1-SUR2B
complexes, are widely accepted as the molecular constituents
of endothelial and smooth muscle K+-ATP channels, whereas
the SUR2A subunit is mostly expressed in heart and skeletal
muscle. The Kir6.1-SUR2A protein pair, therefore, represents
a so far unconventional combination of K+-ATP channel
subunits. Interestingly, co-expression experiments of this pair
of subunits have failed to induce K+-conductance (AguilarBryan et al., 1998). This finding is consistent with the fact that
Kir6 and SUR messages and proteins are found in epithelial
cells of the ileal mucosa without corresponding channel
activity (Burleigh, 2003). These data, together with its
localization restricted to tight junctions, support the idea that
the Kir6.1-SUR2A pair fulfils a biological function quite
distinct from just channelling ions through membranes.
This is further supported by the fact that, although expressed
in several epithelial cell types, the colocalization of Kir6.1 and
SUR2A subunits with tight junction proteins does not represent
a general feature. Indeed, this pair is only found in tight
junctions with regulated permeability – not in constitutively
impermeable ones such as in the collecting duct of the kidney
or the facet cells of the bladder urothelium. This indicates a
direct involvement of the Kir6.1-SUR2A complexes in the
molecular mechanisms regulating tight junction permeability.
The Kir6.1-SUR2A protein pair might directly regulate
tight junction permeability
In line with the above considerations, SUR proteins belong to
the ABC family, whose members act as mechanochemical
devices (Karpowich et al., 2001; Zingman et al., 2001).
Binding and hydrolysis of ATP by the SUR protein results in
an altered conformation, which usually induces channelgating. In regulated tight junctions, the Kir6.1-SUR2A protein
pair could influence the paracellular resistance through
direct interaction with members of the tight-junction-protein
complex, as deduced from our coimmunoprecipitation
experiments. The altered conformation of the SUR protein may
be transferred to other tight junction proteins, finally regulating
Journal of Cell Science
K+-ATP channels and tight junctions
paracellular permeability (Karpowich et al., 2001; Zingman et
al., 2002a).
To provide further support, we established an in vitro model,
using morphologically defined segments of the rat small
intestine. This model system enables us to work with the beststudied and most plausible example of an acute physiological
regulation of tight junctions, the intestinal response to lumenal
D-glucose (Pappenheimer, 1987; Madara, 1994; Tomita et al.,
2004).
In general, an increase in intracellular glucose with a
concomitant elevation of ATP concentration favours the
prehydrolytic conformation of the SUR protein with closed
K+-ATP channels (see Introduction). Intestinal brush-border
cells contain Kir6 and SUR proteins, but never show cannel
activity (Burleigh, 2003). In these cells, lumenal glucose
import through the sodium-dependent transporter also results
in an increased ATP and a correspondingly decreased
ADP concentration. Again, this constellation favours the
prehydrolytic conformation of the SUR protein. But instead of
keeping K+-ATP channels closed, in this case, apparently the
SUR protein mechanically targets the tight-junction-protein
complex, increasing its permeability and thereby elevating
paracellular fluxes of sugars and other small nutrients (Turner
et al., 2000a). This might allow the organism after an
occasionally carbohydrate-rich meal to recruit nutrients even
when the transcellular pathways are already saturated.
According to our new hypothesis, Kir6.1-SUR2A complexes
are directly involved in the regulation of tight junction
permeability. Consequently, this permeability can be
influenced by drugs known to alter Kir6.1 and SUR2A function
specifically.
The sulfonylurea compound tolbutamide and the K+-ATP
channel activator diazoxide were shown to provoke significant
effects. The increase in tight junction permeability observed
after physiological stimulation with 25 mM D-glucose alone
was enhanced more than two fold with tolbutamide and was
decreased by approximately 50% with diazoxide. In addition,
tolbutamide alone – without increase of the lumenal D-glucose
concentration – showed a strong stimulatory effect on the
paracellular flux. Thus, pharmacological modulation of the
Kir6.1-SUR2A protein pair, indeed, results in an altered
paracellular permeability of the intestinal epithelial barrier.
The specific effects demonstrated in our model system shed
some light on the possible mechanism of a well-known but often
neglected side effect of anti-diabetic therapy. Shortly after
starting treatment with sulfonylureas, some patients suffer from
severe diarrhoea (Groop and Neugebauer, 1996), which strongly
affects their quality of life. Fortunately, diarrhoea often stops
immediately when the therapy is stopped. This side effect could
be explained if Kir6.1-SUR2a complexes were involved in the
regulation of intestinal tight junction permeability. Acting
agonistic to an increased D-glucose concentration, sulfonylureas
usually provoke channel closure. In intestinal epithelial cells
sulfonylureas would increase tight junction permeability,
thereby mediating sulfonylurea-induced diarrhoea.
Taking together these information, the intestinal mucosa
might become a suitable and promising model to analyse
potentially new functions of Kir6-SUR complexes apart from
just forming K+-ATP channels. It should allow further
investigations of the molecular mechanisms underlying the
controlled regulation of paracellular permeability and the
3095
design of anti-diabetic drugs without deleterious side effects,
such as long-lasting diarrhoea.
Materials and Methods
Antibodies
Monoclonal antibodies against ZO-1 and E-cadherin were purchased from BioTrend
(Köln, Germany) or Transduction Laboratories BD Biosciences (Heidelberg,
Germany). The polyclonal antiserum against occludin was obtained from Zymed
Laboratories (San Francisco, CA). Antisera against aquaporin-2 (Klussmann et al.,
1999) (kindly provided by E. Klussmann, Forshungs Institute of Molecular
Pharmacology, Berlin, Germany), Kir6.1 (Thomzig et al., 2001), and AE2 (Jöns et
al., 1994) have been described elsewhere. An antiserum against a fusion protein of
the less conserved C-terminus of SUR2A (4507-4638 bp, presented as recombinant
fusion protein) was raised in rabbits. Antibodies were made monospecific by
crossabsorption and affinity-purification.
Specificity of antibodies was verified in ELISA assays, western blots and
immunoflorescence experiments by antigen blocking (Fig. 1a) and immunostaining
of SUR2A-transfected COS cells (Brune et al., 2000). Secondary anti-rabbit or antimouse antibodies from goat or donkey, labelled with either Oregon-Green or TexasRed, were purchased from Molecular Probes, Europe BV (Leiden, Netherlands).
For electron microscopy, biotinylated goat anti-rabbit and anti-mouse secondary
antibodies were visualized with DAB using the Elite ABC system (Vector
Laboratories, Burlington, Canada).
Other chemicals
A mixture of protein A/G sepharose was purchased from Santa Cruz Biotechnology,
IC Chemikalien, (Ismaning, Germany). L-[14C]glucose was obtained from Perkin
Elmer (Boston, MA).
Preparation of bile canalicular membranes
Bile canalicular membranes were isolated as described (Tsukita and Tsukita, 1989),
with minor modifications. In short, 12-week-old Wistar rats were anaesthetized with
diethylether and immediately decapitated. The liver was quickly removed and
soaked in ice-chilled phosphate-buffered saline (PBS). All subsequent steps were
carried out at 4°C. The liver was minced and treated with hypotonic buffer (1 mM
NaHCO3 and 2 ␮g/ml leupeptin, pH 7.5) for 30 minutes. The samples were
homogenized in two volumes of hypotonic buffer with a dounce homogenizer. The
homogenate was diluted with hypotonic buffer to 400 ml, filtered through four layers
of gauze and centrifuged at 1500 g for 10 minutes. The pellets were resuspended
in hypotonic buffer, filled up to 13.1 ml and mixed with 96.3 ml of a 55%
(weight/volume) sucrose solution, resulting in a final sucrose concentration of
48.5%. This solution was divided into six tubes and overlaied with 2 ml of a 42.9%
sucrose solution and centrifuged for 60 minutes at 100,000 g. Bile canaliculi were
recovered at the interface between 42.9% and 48.5%. The bile-canaliculi-enriched
membrane fraction was diluted with ten volumes of hypotonic buffer and
centrifuged at 4000 g for 30 minutes.
For solubilization, the pellet was homogenized again in immunoprecipitation
buffer, consisting of 1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM
EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF and 0.5% Nonidet
P-40 and recentrifuged at 20,000 g for 30 minutes.
Immunoprecipitation
Of the respective antibody, 3 ␮l were added to 1 ml of membrane extract,
corresponding to 300 ␮g of the total amount of membrane protein. After incubation
with antibody for 16 hours at 4°C, 20 ␮l of a protein A/G-sepharose mixture (50%
in immunoprecipitation buffer) was added and the suspension incubated for an
additional 4 hours, followed by three washing steps with the same buffer. The
resulting pellet was resuspended in electrophoresis sample buffer, boiled for 10
minutes and subjected to SDS-PAGE analysis.
Electrophoretically separated proteins were transferred to nitrocellulose
membranes and incubated in PBS containing 10% milk powder to block unspecific
protein-binding sites. Membrane strips were incubated with antisera in an
appropriate dilution of PBS-10% milk powder. Washing steps were performed with
PBS containing 0.05% Tween 20. The bound immunoglobulins were incubated with
horseradish-peroxidase (HRP)-labelled antisera raised against rabbit or mouse IgG
and chemoluminescence was visualized by using the ECL detection system
(Amersham Braunschweig, Germany).
Immunofluorescence microscopy
Immunofluorescence microscopical analysis followed protocols described earlier
(Jöns et al., 1994). Briefly, small pieces of rat, pig and human tissue
(approximately 1-2 mm3 in volume) were rapidly frozen in melting isopentane
(cooled with liquid nitrogen), freeze-dried and then vacuum-embedded in Epon
(Drenckhahn and Franz, 1986). Indirect immunofluorescence was performed with
1-␮m-sections. Epon was removed with sodium methoxide solution (Drenckhahn
and Franz, 1986) and sections were incubated with monoclonal or polyclonal
primary antibodies. Bound immunoglobulins were visualized with anti-mouse or
3096
Journal of Cell Science 119 (15)
anti-rabbit secondary antibodies labelled with Oregon-Green or Texas-Red, as
indicated.
analysed for radioactivity with a scintillation counter (LS 6500 Multi-Purpose
Scintillation Counter Beckman Coulter).
Electron microscopy
The authors thank J. Roeper for illuminating discussions, A. Bräuer
for help with the PCR-analysis, and I. Mitschke for helpful comments
on the manuscript. Large parts of this publication belong to the MD
thesis of D.W.
Journal of Cell Science
For transmission electron microscopy, frozen rat duodenal tissue was cut in 25␮m-sections on a cryostate and rinsed several times in PBS. Sections were treated
with 1% sodium borohydride in PBS to remove excessive aldehyde groups,
followed by washing steps with PBS containing 0.3% Triton X-100 and
0.05% phenylhydrazine to reduce the endogenous peroxidases. Sections were
subsequently washed three times in PBS, followed by incubation for 36 hours at
4°C with the primary antibody in a solution of 10% normal goat serum (NGS) in
PBS containing 0.1% sodium azide and 0.01% thimerosal. Sections were washed
in PBS for 20 and 40 minutes, preincubated for 1 hour in PBS-A (PBS
supplemented with 2 mg/ml BSA), and incubated for 16 hours at room temperature
in the secondary antibody solution (biotinylated goat anti-rabbit IgG or rabbit antimouse IgG in PBS-A containing 0.1% sodium azide). After another two washing
steps in PBS and incubation with avidin-biotin complex (Elite A+B, Vector
Laboratories; 1:1000 in PBS-A) for 6 hours at room temperature, sections were
washed three times in PBS, followed by preincubation in 3,3⬘-diaminobenzidine
(0.5 mg in 1 ml 50 mM Tris pH 7.6), containing 10 mM imidazole, for 15 minutes.
Bound antibodies were visualized by adding H2O2 (0.015% final concentration) for
10 minutes. Finally, sections were washed several times in PBS.
Sections were postfixed with 1% osmium tetroxide in PBS for 30 minutes,
dehydrated with graded ethanol [70%, 90%, 96%, 100% (including block staining
with 2% uranyl acetate in 70% ethanol for 10 minutes at 4°C)] and propylene oxide,
and embedded in araldite between two sheets of Aclar plastic (Ted Pella, Redding,
CA). Ultrathin tissue sections (60 nm) were cut with a diamond knife and collected
on copper grids. The sections were counterstained with uranyl acetate and lead
citrate and examined with a transmission electron microscope (EM 900; Zeiss,
Oberkochen, Germany).
Polymerase chain reaction
Total RNA was extracted from rat liver tissue using the guanidinium thiocyanate,
phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). DNA-free
RNA was obtained by treatment with RNase-free DNase I (Boehringer, Mannheim,
Germany) for 30 minutes at 37°C. After phenol-chloroform extraction and ethanol
precipitation the probes were stored at –80°C. The first-strand reactions contained
50 ng oligonucleotide dT15-18, 400 units of superscript II reverse transcriptase and
100 nM DTT in 20 ␮l 1⫻ transcription buffer (all from Gibco, Germany) and were
incubated with 2 ␮g RNA and 200 ␮M dNTPs (Pharmacia, Germany) for 1 hour
at 42°C. Following first-strand synthesis, two units of RNase H were added and the
reactions were incubated at 37°C for 30 minutes. The cDNA was purified with the
PCR Purification Kit (Qiagen).
PCR was performed with 2 ␮l of the RT reaction per 25 ␮l. All reactions were
performed in quadruplicates. The primer used anchor and arbitrary primer (Roth,
Karlsruhe, Germany) in different combinations. Reactions were performed in 25 ␮l
with 25 mM MgCl2, 2.5 ␮l 10⫻ reaction buffer, 2.5 units Ampli Taq Gold (all from
Perkin Elmer), 200 ␮M dNTPs (Pharmacia), 20 pmol per anchor-primer 5⬘-GGA
GCT GGA CGA GAA ACC TTC CAT C-3⬘ (spanning bases 1246-1271) and 10
pmol of arbitrary-primer 5⬘-GTC GTC CTG CCC TCA TGA TTC TGA TG 3⬘
(spanning bases 1440-1466). The thermal cycler was set for 1 cycle of 1 minute at
95°C, 90 seconds at 40°C and 45 seconds at 72°C, followed by 39 cycles of 30
seconds at 94°C, 90 seconds at 40°C, 45 seconds at 72°C. The cycling was followed
by 5 minutes at 72°C. For electrophoretic separation the whole PCR reaction
product was precipitated by ethanol. For the analysis of the PCR-products the
CleanGel DNA analysis Kit (Pharmacia Biotech, Germany) and the DNA silver
staining Kit (Pharmacia Biotech) were used according to the manufacturer’s
instructions.
Measurement of intestinal permeability
Adult Wistar rats (weighing ~300 g) were anaesthetized with diethylether and
immediately decapitated. A 15-cm-segment of jejunum, starting ~2 cm caudal to
the ligament of Treitz, was carefully separated from the mesenterium, connected to
a two-way valve and thoroughly rinsed with buffer A (130 mM NaCl, 3 mM
KH2PO4, 2 mM K2HPO4, 5 mM D-Glucose, 10 mM Hepes, 1 mM CaCl2, 1 mM
MgCl2, pH 7.4, 37°C). The intestinal segment was tightly closed with a dialysis
tubing-clip and filled with the appropriate buffer. As a starting point, the incubation
was performed for 45 seconds, the endpoint was determined after a 60-minute
incubation in a 37°C water bath containing buffer A.
All experiments were performed in triplicates. The incubation buffer contained
varying concentrations of D-glucose, L-glucose, diazoxide and tolbutamide, alone
or in combination. All incubation-buffers were saturated with O2 and supplemented
with 5.5 ␮Ci L-[14C]glucose/25 ml. The incubation was stopped by washing with
buffer A. The intestinal segment was cut into six identical parts that were
immediately homogenized in 2 ml H2O and centrifuged at 20,000 g for 30 minutes
at 4°C. The supernatant was collected and the protein content was determined with
Roti®-Quant according to the manufacturer’s instructions (Roth, Karlsruhe,
Germany). An aliquot of the supernatant corresponding to 500 ␮g of protein was
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