Gating Connexin 43 Channels Reconstituted in Lipid Vesicles by
Mitogen-activated Protein Kinase Phosphorylation*
Doo Yeon
Kim
,
Yoonseok
Kam
,
Soo Kyung
Koo§, and
Cheol O.
Joe
¶
From the
Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, Taejon 305-701 and the
§ Laboratory of Genetic Disease, Department of Biomedical
Science, National Institute of Health, Seoul 122-020, Republic of Korea
 |
ABSTRACT |
The regulation of gap junctional permeability by
phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were
reconstituted into unilamellar phospholipid liposomes. The activities
of the reconstituted channels were measured by monitoring liposome
permeability. Liposomes containing the Cx43 protein were fractionated
on the basis of permeability to sucrose using sedimentation in an
iso-osmolar density gradient. The gradient allowed separation of the
sucrose-permeable and -impermeable liposomes. Liposomes that were
permeable to sucrose were also permeable to the communicating dye
molecule lucifer yellow. Permeability, and therefore activity of the
reconstituted Cx43 channels, were directly dependent on the state of
Cx43 phosphorylation. The permeability of liposomes containing Cx43
channels was increased by treatment of liposomes with calf intestinal
phosphatase. Moreover, liposomes formed with Cx43 that had been
dephosphorylated by calf intestinal phosphatase treatment showed
increased permeability to sucrose. The role of phosphorylation in the
gating mechanism of Cx43 channels was supported further by the
observation that phosphorylation of Cx43 by mitogen-activated protein
kinase reversibly reduced the permeability of liposomes containing
dephosphorylated Cx43. Our results show a direct correlation between
gap junctional permeability and the phosphorylation state of Cx43.
 |
INTRODUCTION |
Intercellular communication is necessary in multicellular
organisms to maintain tissue homeostasis and is involved in diverse cellular activities including metabolic cooperation and the exchange of
signaling information for harmonized control of cell growth and
differentiation (1-4). Gap junction channels play an important role in
intercellular communication by providing a direct pathway for the
movement of molecular information among cells (4, 5). Gap junction
channels mediate the communication between adjacent cells by the
open-closed gating of an aqueous pore permeable to ions and small
molecules. Thus, gap junction channels are regarded as cytoplasmic
bridges that allow cell-to-cell passage of ions, nutrients, and second
messenger regulatory molecules (6-9).
Intercellular communication through the gap junction is believed to be
modulated during cellular processes such as cell cycle progression
(10-12), embryogenesis, and development (13, 14). A correlation exists
between decreases in gap junctional communication (GJC)1 and the cellular
events that lead to cell cycle progression; for example, GJC is reduced
during the G1 to S phase transition of the cell cycle (11,
12). In addition, factors that affect cell proliferation were found to
inhibit GJC, including tumor promoters (15, 16), growth factors (10,
17), carcinogens (18, 19), and oncogene products (20-22). The temporal
loss of GJC in proliferating cells was further supported by the
observed growth retardation in cells transfected with overexpressing
connexin genes (23-25).
It has been proposed that GJC is modulated by a mechanism that involves
the posttranslational phosphorylation of gap junction proteins.
Connexin 43 (Cx43) is a major gap junction protein found in animal
heart and brain (26, 27). Studies have implicated phosphorylation of
Cx43 on tyrosine and/or serine residues as a regulatory mechanism for
channel gating. Two types of Cx43 phosphorylation events have been
described. One type is the tyrosine phosphorylation of Cx43 by
oncogene-induced tyrosine kinases (21, 22, 28). The other type involves
the rapid phosphorylation of Cx43 on serine residues in cells
stimulated to grow (10-12, 29, 30). However, the biochemical mechanism
by which posttranslational modification is associated with the
modulation of gap junction channels remains obscure.
It has been suggested that mitogen-activated protein (MAP) kinase plays
an important role in the loss of GJC during mitotic division (31).
Therefore, we sought to determine whether MAP kinase is directly
involved in the gating mechanism of gap junction channels. Detailed
studies of GJC have been severely constrained by the fact that gap
junction channels are not exposed to the extracellular space, and
access to the channels is only available via the cytoplasm or dialyzed
cytoplasm in a whole-cell patch clamp configuration. Thus, it is
difficult to correlate unequivocally the effects of agents that alter
channel phosphorylation with channel permeability, as any of these
agents might elicit secondary metabolic effects. One approach is to
make gap junction channels accessible to study by splitting the
isolated gap junctions and reconstituting them in an artificial lipid
bilayer. Several studies report the induction of channel activities in
a lipid bilayer by incorporating gap junction proteins into lipid
vesicles (32-36).
In the present study, we reconstituted Cx43 gap junction channels in
liposomes and measured the permeability with the use of iso-osmolar
sucrose/urea density gradient sedimentation (34). Our results indicate
that MAP kinase phosphorylation of Cx43 directly modulates channel gating.
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EXPERIMENTAL PROCEDURES |
Reagents--
Lissamine rhodamine B-labeled
phosphatidylethanolamine (PE) was purchased from Avanti Polar
Lipids, Inc. (Birmingham, AL). Bovine phosphatidylcholine, egg
phosphatidylserine,
N-octyl-
-D-glucopyranoside (octylglucoside),
leupeptin, phenylmethylsulfonyl fluoride, calf intestinal phosphatase
(CIP) (conjugated to beaded agarose), and cyanogen bromide
(CNBr)-activated Sepharose 4B beads were obtained from Sigma. CHAPS and
dimethyl suberimidate·2HCl (DMS) were purchased from Pierce. MAP
kinase (Erk2) was purchased from New England Biolabs (London, United
Kingdom). [
-32P]ATP (5000 Ci/mmol) was from Amersham
Pharmacia Biotech.
Preparation of anti-Cx43 Antibody--
The sequence of synthetic
peptide SSRASSRPRPDDLEI corresponded to amino acid residues 368 to 382 of the C-terminal region of Cx43 (26). The peptide was conjugated to
keyhole limpet hemocyanin, and antisera were then prepared by the
method of Beyer et al. (38).
Immunoaffinity Purification of Cx43--
Cx43 was
immunoaffinity-purified by the modified method of Rhee et
al. (36). Brain tissue from Sprague-Dawley rats (~1.3 g) was
minced in 25 ml of 5 mM NaHCO3 buffer (pH 8.2)
containing 5 mM EDTA and 1 mM
phenylmethylsulfonyl fluoride, and the homogenate was centrifuged at
48,000 × g for 10 min. The crude membrane fraction pellet was solubilized in buffer A (50 mM sodium phosphate
(pH 7.4), 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, and 0.75% CHAPS). After centrifugation at
100,000 × g for 1 h, the supernatant was loaded
onto an immunoaffinity column in which anti-Cx43 antibody was
cross-linked to CNBr-activated Sepharose 4B. After successive washes
with buffer A and buffer A plus 0.5 M NaCl, the column was
equilibrated with buffer A in which CHAPS was replaced with 80 mM octylglucoside. Bound Cx43 was eluted from the column by
brief exposure to 50 mM sodium acetate buffer (pH 2.4)
containing 10 mM KCl, 459 mM urea, 0.1 mM EDTA, 80 mM octylglucoside, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM leupeptin. The
affinity column eluent was rapidly neutralized by adding it directly to
0.3 volumes of neutralization buffer (1 M HEPES (pH 7.4),
10 mM KCl, 459 mM urea, 0.1 mM
EDTA, and 80 mM octylglucoside). To prepare a denatured
form of Cx43, a portion of the affinity column eluent was incubated at
30 °C for 3 h before it was mixed with the neutralization
buffer. All the procedures were carried out at 4 °C.
Gel Electrophoresis and Immunoblot
Analysis--
Immunoaffinity-purified Cx43 protein was analyzed by an
8% SDS-polyacrylamide gel. For staining the gel-bound protein with silver nitrate, the gel was preincubated in a mixture of ethanol, acetic acid, and water (4:1:5, v/v/v) for 1 h followed by an
additional incubation in ethanol, acetic acid, and water (5:5:90,
v/v/v) for 2 h. Protein bands were cross-linked by soaking the gel
in a solution containing 1% glutaraldehyde and 0.5 M
sodium acetate for 30 min. The gel was rinsed extensively with
deionized water, and the protein bands were stained in a freshly made
0.8% ammoniacal silver nitrate solution containing 0.02 N
NaOH. After washing, the images were developed by incubating the gel in
a solution containing 0.05% citric acid and 0.1% formaldehyde for 5 to 10 min. The reaction was stopped by transferring the gel into 5% Tris buffer containing 2% acetic acid. For immunoblot analysis, the
gel-bound protein bands were electrotransferred to a nitrocellulose membrane, and the membrane was blocked by incubation for 1 h at 37 °C in 3% gelatin in Tris-buffered saline (25 mM
Tris-HCl (pH 8.3), 50 mM NaCl). The blocked membrane was
then incubated in Tris-buffered saline containing 0.05% (v/v) Tween 20 and 10 µg/ml anti-Cx43 antibody with gentle shaking for 1 h at
37 °C. The membrane was transferred to Tris-buffered saline
containing 0.05% (v/v) Tween 20 and incubated for 30 min with buffer
changes every 10 min. The washed membrane was then incubated for 1 h at 37 °C with 0.5 µg/ml alkaline phosphatase-conjugated
anti-rabbit IgG. The Cx43 bands were developed in 0.1 mg/ml nitro blue
tetrazolium and 0.05 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 10 mM Tris buffer (pH 9.5) containing 10 mM
MgCl2 and 5 mM NaCl.
Sucrose Gradient Analysis of Cx43 Assembly--
The oligomeric
state of immunoaffinity-purified Cx43 was examined by sucrose density
sedimentation as described by Falk et al. (39).
Immunoaffinity-purified Cx43 protein was loaded onto a 5-ml linear
gradient of 5 to 20% (w/v) sucrose containing 10 mM Hepes
(pH 7.4), 459 mM urea, 0.1 mM EDTA, and 80 mM octylglucoside. After centrifugation for 16 h at
200,000 × g, the gradients were fractionated, and 0.1 ml fractions were collected. Aliquots (50 µl) from the gradient
fractions were transferred to microwell plates, and the amount of
Cx43 in each fraction was quantified by enzyme-linked immunosorbent
assay using anti-Cx43 antibody. Standard proteins with known
sedimentation coefficients (ovalbumin, 3.5 S; bovine serum albumin, 4.3 S) were subjected to sedimentation on a separate gradient and used to
determine the S value of Cx43 oligomers.
Cross-linking Analysis of Cx43 Assembly--
The quaternary
structure of immunoaffinity-purified Cx43 protein was examined by
chemical cross-linking using DMS as a coupling agent (40, 41). Aliquots
of immunoaffinity-purified Cx43 protein (~10 µg in 180 µl) in 10 mM triethanolamine buffer (pH 8.3) containing 80 mM octylglucoside, 10 mM KCl, and 0.1 mM EDTA were mixed with 20 µl of 10 mM
triethanolamine buffer (pH 8.3) containing 100 mM DMS.
After incubation for 8 h at 4 °C, the cross-linking reaction was stopped by adding 1 µl of 1 M glycine buffer (pH 7.4)
to each sample and incubating the reaction mixtures at room temperature for 30 min. The cross-linked protein samples were then denatured by
adding an equal volume of SDS sample buffer containing 125 mM Tris-Cl (pH 6.8), 20% SDS, 10%
-mercaptoethanol,
and 20% glycerol. The cross-linked complexes were analyzed by
electrophoresis on a 4 to 15% SDS-polyacrylamide gradient gel followed
by immunoblot analysis using anti-Cx43 antibody as described above.
Reconstitution of Cx43 Channels into Liposomes--
Cx43
channels were reconstituted in unilamellar phospholipid vesicles. A
procedure for the incorporation of gap junction protein in unilamellar
liposomes has been described (36). The lipids phosphatidylcholine,
phosphatidylserine, and lissamine rhodamine B-labeled PE were dissolved
in chloroform at a molar ratio of 2:1:0.05. Fluorescently labeled PE
was used as a marker for liposomes in the gradient fractions. The lipid
mixture was dried to a thin film under a stream of nitrogen gas, and
the lipid film was suspended in 10 mM HEPES buffer (pH 7.4)
containing 459 mM urea, 10 mM KCl, 0.1 mM EDTA, and 80 mM octylglucoside.
Immunoaffinity-purified Cx43 was then added to the mixture, and the
protein:lipid ratio was adjusted to 1:45 (w/w) for most procedures
employed in this study. The protein-lipid mixture was applied to a
Sephadex G-50 (Amersham Pharmacia Biotech) column (2 × 100 cm),
and the column was eluted with a buffer containing 10 mM
HEPES (pH 7.4), 459 mM urea, 10 mM KCl, and 0.1 mM EDTA at the flow rate of 0.1 ml/min. The liposomes were
collected in the void volume of the column, whereas octylglucoside was
retained by the column. The liposomes were concentrated by
centrifugation at 240,000 × g for 18 h at 4 °C.
Channel Activity of Cx43 in Liposomes--
The permeability of
the Cx43 channels reconstituted in liposomes was examined by the
technique of transport-specific density shift as described (34). The
liposomes were layered onto linear, 0 to 400 mM,
iso-osmolar sucrose density gradients in which a reverse linear
gradient of 459 to 0 mM urea in the same buffer compensated
the sucrose gradient osmotically. The gradient was then centrifuged at
250,000 × g for 12 h at 4 °C. The distribution of fluorescently labeled liposomes in the gradient was photographed under UV illumination in the dark. The gradient was eluted through a
hole formed at the bottom of a centrifuge tube, and the distribution of
the lipid vesicles was determined by measuring the intensity of
rhodamine fluorescence in each fraction with a spectrofluorometer (LS-3B, Perkin-Elmer) at 590 nm excited at 560 nm. To measure channel
activity of Cx43 in the liposomes, the loss of lucifer yellow
fluorescent communication dye from the liposomes was monitored. Liposomes formed with Cx43 in the presence of 3 mM lucifer
yellow were layered on a gradient. After centrifugation at 250,000 × g for 12 h, gradients were eluted from the bottom.
Each fraction was monitored for the fluorescence of lissamine rhodamine
B-labeled PE in the liposome membrane. The amount of lucifer yellow
retained in the liposomes was quantified by measuring its fluorescence intensity at 530 nm excited at 428 nm.
Dephosphorylation of Cx43--
Cx43 (~30 µg) which was
either solubilized in detergent or incorporated in liposomes was mixed
with 0.1 volume of 50 mM Tris buffer (pH 8.0) containing 1 mM EDTA, 80 mM octylglucoside, and 50 units/ml
agarose-conjugated CIP. The reaction mixtures were incubated for 3 h at 30 °C with gentle agitation. In the control experiment, an
equal amount of agarose beads was added to the mixture to replace the
agarose-conjugated CIP. The mixtures were then centrifuged for 5 min at
1,000 × g to remove the agarose beads and conjugated
enzyme. Dephosphorylated protein samples were resolved by
SDS-polyacrylamide (8%) gel electrophoresis (PAGE) followed by
immunoblot analysis with anti-Cx43 antibody. The permeability of
liposomes containing dephosphorylated Cx43 was examined by sedimentation in an iso-osmolar sucrose density gradient.
Phosphorylation of Cx43 by MAP Kinase
Treatment--
Dephosphorylated Cx43 (~30 µg) was mixed with 0.1 volume of a buffer containing 100 mM Tris-Cl (pH 7.4), 10 mM MgCl2, 1 mM ATP, and 50 units/ml
MAP kinase. The reaction mixture was incubated for 3 h at 30 °C
in the presence or absence of [
-32P]ATP (0. 1 mCi/ml).
Protein samples phosphorylated in the presence of
[
-32P]ATP were resolved by 8% SDS-PAGE followed by
autoradiography. The permeability of liposomes formed with the MAP
kinase-treated Cx43 was compared with that of untreated control Cx43 by
sedimentation analysis in an iso-osmolar density gradient.
 |
RESULTS |
Purification of Cx43--
Cx43, immunoaffinity-purified from rat
brain using anti-Cx43 antibody, was resolved on an SDS-polyacrylamide
(8%) gel, and a protein band with a molecular size of ~41 kDa was
detected by silver staining. Immunoblot analysis identified the band as
Cx43 (Fig. 1). The anti-Cx43 antibody
used in this study was directed against a nonconserved cytoplasmic
epitope and may not cross-react with other types of connexins (27, 38).
Other connexin family proteins expressed in rat brain (27) were also
probed using antibodies against connexin 32 (Cx32) and connexin 26 (Cx26), but no corresponding protein bands were detected (data not
shown).

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Fig. 1.
SDS-PAGE of Cx43 and the corresponding
immunoblot. Solubilized membrane proteins from rat brain
(lane 1) and Cx43 immunoaffinity-purified with an antibody
specific to the C-terminal region of Cx43 (amino acids 368 to 382)
(lane 3) were subjected to electrophoresis on an
SDS-polyacrylamide (8%) gel and visualized by silver staining.
Immunoblot analysis was carried out using the same antibody (lane
2).
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Oligomeric State of Immunoaffinity-purified Cx43--
The assembly
of immunoaffinity-purified Cx43 was examined by velocity sedimentation
in a sucrose density gradient, and the composition of the purified Cx43
complex was deciphered by chemical cross-linking with DMS. In previous
studies, connexins were isolated from insoluble lipid membranes under
denaturing conditions with the use of ionic detergents. It is possible
that these isolation conditions alter the secondary structure of
connexin proteins, influencing the ability of connexin subunits to
assemble into gap junctional connexons, which are composed of six
connexin subunits. Therefore we sought to determine whether Cx43
solubilized with a nonionic detergent (octylglucoside) under
nondenaturing conditions maintained its oligomeric state after
purification. To accomplish this, we subjected immunoaffinity-purified
Cx43 to sucrose density gradient sedimentation. The gradient was
fractionated, and the amount of Cx43 in each fraction was determined by
enzyme-linked immunosorbent assay using an anti-Cx43 antibody. Two
distinct Cx43 protein peaks corresponding to 5 S and 9 S particles were detected. Previous studies (39) have demonstrated that the 5 S Cx43
population contributes the monomeric form of the protein, whereas the 9 S species represents the hexameric form. Our data showed that only
one-half of the purified Cx43 protein was assembled into connexons.
When the Cx43 samples were denatured with a low pH treatment (pH 2.2),
the 9 S Cx43 population was dissociated to yield the 5 S form (Fig.
2A).

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Fig. 2.
Analysis of connexon assembly.
A, sucrose density gradient analysis of Cx43 assembly. The
denatured ( ) or nondenatured Cx43 protein ( ) was loaded onto a
linear gradient of 5 to 20% (w/v) sucrose (total 5 ml) containing 10 mM Hepes (pH 7.4), 459 mM urea, and 80 mM octylglucoside. After centrifugation for 16 h at
200,000 × g, gradients were fractionated, and the
amount of Cx43 in each fraction was quantified by enzyme-linked
immunosorbent assay using anti-Cx43 antibody. The Cx43 protein peaks
corresponding to 5 S (monomeric connexins) and 9 S particles (assembled
gap junction connexons) are marked. B, chemical
cross-linking analysis of Cx43 assembly. The quaternary structure of
immunoaffinity-purified Cx43 was examined by chemical cross-linking
procedures using DMS as a coupling agent. Cx43 was incubated either in
the presence or absence of 10 mM DMS for 8 h at
4 °C, and the treated proteins were resolved on a 4 to 15%
SDS-polyacrylamide gradient gel followed by immunoblot analysis using
anti-Cx43 antibody. DMS cross-linked the ~41 kDa Cx43 monomers
(lane 1) to form complexes that migrated at ~210 kDa
(lane 2).
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The quaternary structure of immunoaffinity-purified Cx43 was also
examined by chemical cross-linking with DMS. DMS cross-linking of Cx43
monomers (~41 kDa; Fig. 2B, lane 1) produced a
protein complex that migrated at ~210 kDa (Fig. 2B,
lane 2). This 210-kDa estimate may not represent an accurate
measurement of the molecular size of a connexon complex, as chemically
cross-linked proteins often migrate farther than expected on
SDS-polyacrylamide gels (42).
Permeability of Cx43 Channels Formed in Unilamellar
Liposomes--
We next incorporated immunoaffinity-purified Cx43
protein into unilamellar phospholipid liposomes. The size of the
liposomes is strongly influenced by the lipid:detergent ratio and by
the chain length of the detergent molecules. In Rhee et al.
(36), phospholipid unilamellar vesicles were constructed under the same experimental conditions as ours. The size distribution of the vesicles
in that study was examined by filtration over a size exclusion high
performance liquid chromatography column, and the average vesicle
diameter was estimated to be 45 nm. Assuming a bilayer thickness of 4.0 nm and an average area of 0.7 nm2/phospholipid molecule
(43), the protein:lipid ratio used in this study should give rise to
contain an average of one gap junction channel per vesicle (36). The
Poisson distribution predicts that 67% of the vesicles will contain at
least one connexon.
The permeability of reconstituted Cx43 channels was examined by
sedimentation velocity analysis of liposomes in an iso-osmolar sucrose
density gradient (34). Liposomes that were formed in the absence of
Cx43 or with denatured Cx43 were sucrose-impermeable; thus they
migrated only a short distance into the gradient and banded near the
top of the gradient (Fig. 3A,
tubes 1 and 3). In contrast, liposomes formed
with intact Cx43 were fractionated into two populations within the
gradient, a sucrose-permeable fraction that migrated near the bottom of
the gradient and a sucrose-impermeable fraction that migrated near the
top (Fig. 3A, tube 2). It has been suggested that
sucrose-permeable liposomes that contain open Cx43 channels
continuously equilibrate their internal solution with the surrounding
external solution and thus migrate to a more dense position in the
gradient. Liposomes that are impermeable to sucrose, either because
they lack Cx43 channels or contain a closed form of the channels, are
buoyed by the internal urea buffer, which is lighter than the external
sucrose buffer. Impermeable liposomes are therefore retained at the top
of the gradient (36).

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Fig. 3.
Permeability of reconstituted Cx43 channels
to sucrose and lucifer yellow. Unilamellar liposomes composed of
phosphatidylcholine, phosphatidylserine, and lissamine rhodamine
B-labeled PE at a molar ratio of 2:1:0.05 were formed with purified
Cx43. The protein:lipid ratio was typically 1: 45 (w/w). A,
the liposomes were fractionated in an iso-osmolar sucrose density
gradient on the basis of sucrose permeability. Liposomes marked with
the fluorescently labeled lipid were illuminated with UV light in the
dark. Shown are liposomes formed without Cx43 (tube 1),
liposomes formed with Cx43 (tube 2), liposomes formed with
denatured Cx43 (tube 3). B, the distribution of
liposomes formed with Cx43 in the presence of 3 mM lucifer
yellow was also examined after sedimentation. The fractions were eluted
from the bottom of the gradient tube, and the amount of liposomes in
each fraction was quantified by measuring the fluorescent intensity of
lissamine rhodamine B-labeled PE in the liposome membrane (panel
1). The distribution of lucifer yellow within the gradient was
examined by measuring the fluorescence intensity at 530 nm excited at
428 nm (panel 2).
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We next monitored the transfer of lucifer yellow, a well known
communicating dye molecule, through reconstituted Cx43 channels. Liposomes were formed with or without Cx43 in the presence of 3 mM lucifer yellow, and the loss of the entrapped lucifer
yellow from the liposomes was monitored. After velocity sedimentation, the liposomes in the iso-osmolar density gradient were eluted from the
bottom of the tube. The amount of lucifer yellow retained in liposomes
was quantified by measuring its fluorescence intensity at 530 nm
excited at 428 nm. Liposomes formed with Cx43 fractionated into two
populations within the gradient, but lucifer yellow was detectable only
in the sucrose-impermeable fraction. Sucrose-permeable liposomes, which
contained open Cx43 channels, specifically released lucifer yellow,
whereas sucrose-impermeable liposomes retained the dye. These results
suggest that sucrose-permeable channels formed with Cx43 are also
permeable to lucifer yellow (Fig. 3B). The respective
permeability of liposomes formed with varying protein:lipid ratios is
shown in Fig. 4. The data indicate that
the fraction of sucrose-permeable liposomes increases with increasing
concentrations of Cx43 incorporated.

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Fig. 4.
Changes in the permeability of liposomes by
the amount of Cx43 incorporation. A, liposomes were
formed with varying amounts of Cx43. The lipid:protein (w/w) ratios
were 100:1 (tube 1), 50:1 (tube 2), 25:1
(tube 3), and 5:1 (tube 4). Liposomes were then
separated in an iso-osmolar sucrose density gradient on the basis of
sucrose permeability. B, the gradients were then
fractionated from the bottom, and the amount of liposomes in each
fraction was quantified by measuring the fluorescence intensity of
lissamine rhodamine B-labeled PE in the liposome membrane.
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Increased Channel Permeability by Dephosphorylation of
Cx43--
The effect of Cx43 phosphorylation on gap junctional
permeability was presented (Fig. 5). Cx43
preparations from rat brain are known to contain both the
phosphorylated and nonphosphorylated forms of the protein (27). Our
immunoaffinity-purified Cx43 was treated with CIP either before or
after incorporation into liposomes. Treatment of Cx43 with CIP
converted the 43-kDa phosphorylated form (Fig. 5, lanes 1 and 3) into the dephosphorylated 41-kDa form (Fig. 5,
lanes 2 and 4). The fraction of permeable liposomes containing Cx43 was increased by treating the liposome with CIP (Fig.
5, tube 2). The distribution analysis of liposomes after sedimentation revealed that 15% of the total liposome population was
sucrose-permeable. However, 27% of the total liposome population became sucrose-permeable if the liposomes were treated with CIP (5 units for 3 h) before sedimentation. The hypothesis that
dephosphorylation of Cx43 increases gap junctional permeability is
further supported by our subsequent experiment, which showed an
increase in the permeability of liposomes formed with CIP-treated Cx43
(Fig. 5, tube 4). The fraction of permeable liposomes formed
with dephosphorylated Cx43 was about 2-fold greater than that observed
with control liposomes incorporated with intact Cx43 (Fig. 5,
tubes 3 and 4).

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Fig. 5.
Gating mechanism of Cx43 channels by
phosphorylation. Panel 1 (left), liposomes
formed with solubilized Cx43; center, lane 1,
Cx43 in the liposomes was resolved by 8% SDS-PAGE followed by
immunoblot analysis; right, tube 1, the
permeability of liposomes containing Cx43 was analyzed by sedimentation
in an iso-osmolar sucrose density gradient. The relative amount of
sucrose-permeable liposomes was represented by the densitometric
scanning of the gradient after sedimentation using Tina 2.0 software
(FujiFilm). Panel 2 (left), liposomes containing
Cx43 were treated with 5 units of CIP for 3 h at 30 °C;
center, lane 2, the liposome was treated with
CIP, and Cx43 was resolved by 8% SDS-PAGE followed by immunoblot
analysis; right, tube 2, Cx43 in the liposomes
was dephosphorylated, and the liposomes were separated by
sedimentation. Panel 3 (left), solubilized Cx43
was incorporated into liposomes; center, lane 3,
solubilized Cx43 was resolved by 8% SDS-PAGE followed by immunoblot
analysis; right, tube 3, liposomes containing
Cx43 were separated by sedimentation. Panel 4 (left), Cx43 dephosphorylated by CIP treatment was
incorporated into liposomes; center, lane 4,
dephosphorylated Cx43 was resolved by 8% SDS-PAGE followed by
immunoblot analysis; right, tube 4, liposomes
formed with dephosphorylated Cx43 were separated by sedimentation.
Panel 5 (left), a, liposomes were
formed with dephosphorylated Cx43; center, lane
5a, dephosphorylated Cx43 in the liposomes was incubated with 10 µCi of [ -32P]ATP for 3 h and resolved by 8%
SDS-PAGE followed by autoradiography; right, tube
5a, liposomes containing dephosphorylated Cx43 were separated by
sedimentation. Panel 5 (left), b,
dephosphorylated Cx43 was phosphorylated by MAP kinase and incorporated
into liposomes; center, lane 5b, dephosphorylated
Cx43 was incubated with 5 units of MAP kinase and 10 µCi of
[ -32P]ATP for 3 h and resolved by 8% SDS-PAGE
followed by autoradiography; right, tube 5b,
liposomes containing MAP kinase-treated Cx43 were separated by
sedimentation. Panel 5, (left), c, MAP
kinase-treated Cx43 was in turn dephosphorylated by CIP treatment and
incorporated into liposomes; center, lane 5c, MAP
kinase-treated Cx43 was incubated with 10 units of CIP for 5 h and
resolved by 8% SDS-PAGE followed by autoradiography; right,
tube 5c, liposomes containing dephosphorylated Cx43 were
separated by sedimentation.
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Reduced Channel Permeability by MAP Kinase Phosphorylation of
Cx43--
In an effort to further characterize the role of
phosphorylation in the gating of gap junction channels, we used MAP
kinase to phosphorylate solubilized Cx43 (Fig. 5, lane 5b).
The fraction of permeable liposomes containing CIP-treated Cx43 was
reduced by 79% if the dephosphorylated Cx43 became phosphorylated by
MAP kinase (Fig. 5, tubes 5a and 5b). MAP
kinase-dependent gating mechanism of Cx43 channels was
further supported by the observation that MAP kinase-induced inhibition
of channel permeability was reversed by treatment of Cx43 with CIP
(Fig. 5, tube 5c). These data suggest that phosphorylation
of Cx43 by MAP kinase may be directly involved in the channel gating
process. Because protein kinase C has also been implicated in the
regulation of GJC, we tested whether protein kinase C could
phosphorylate the immunoaffinity-purified Cx43. Only a trace amount of
phosphorylated Cx43 was detected by autoradiography, whereas histone
H1, a known substrate protein of protein kinase C, was readily
phosphorylated under the same experimental conditions (data not shown).
 |
DISCUSSION |
Gap junctions are membrane channels that link end-to-end across
the space between juxtaposed cells. In this study, we present evidence that the phosphorylation of connexin is directly involved in
the gating mechanism of gap junction channels. We showed that the
activity of Cx43 channels reconstituted in liposomes was dramatically increased by dephosphorylation of Cx43. The activity of the
reconstitituted Cx43 channels was monitored by measuring the
permeability of the liposomes to sucrose in an iso-osmolar sucrose
density gradient. Several other studies have reported the functional
reconstitution of connexons consisting of Cx26 (35) and Cx32 (36).
Information regarding the gating mechanism of gap junction channels can
be obtained from the assessment of the permeability of hemichannels to
small molecules. Structural, biochemical, and physiological data show
the hemichannels have properties that are similar to those of intact
gap junctions. For example, the activities of hemichannels are, like
those of intact gap junction channels, voltage-dependent.
Trexler et al. (44) demonstrate the
voltage-dependent gating properties of Cx46 hemichannels in excised patches from Xenopus oocytes. Hemichannels have also
been shown to exist in the plasma membrane (45, 46). Several studies have proposed that the electrical coupling between horizontal cells of
the fish retina occurs through hemichannels. Recently, Li et
al. (47) presented data in support of the existence of nonjunctional plasma Cx43 hemichannels in Novicoff hepatoma cells. In
this cell line, they measured properties of fluorescent dye transfer
through nonjunctional hemichannels that were similar to those observed
with gap junction channels. The hemichannels are believed to be
typically in the closed state to prevent the loss of cytoplasmic
solutes and the entry of extracellular ions. But some of the
hemichannels formed in Xenopus oocyte membranes open by
membrane depolarization (48) and induce cation fluxes (44). Open
hemichannels were also shown to form in a system in which Cx32 channels
were reconstituted in lipid vesicles that lacked the cytoplasmic or
membrane factors necessary for maintaining the channels in a closed
state (32, 34, 36). From the result of our study, we propose that Cx43
forms open hemichannels in lipid vesicles. The results herein showed
that liposomes containing open Cx43 channels were permeable to sucrose
and communicating dye (Fig. 3). Indeed we observed an increase in the
fraction of permeable liposomes as more connexon protein was
incorporated (Fig. 4).
Although the gating mechanism of gap junction channels remains obscure,
the rapid and reversible changes in the GJC by protein kinase
activators have led to the hypothesis that gap junction channels
undergo a conformational change by phosphorylation to gate the
channels. Cx43 has been identified as a substrate of MAP kinase (30),
protein kinase C (49), and p34cdc2 kinase (29), and it has been
shown that GJC is rapidly reduced by the increased phosphorylation of
Cx43 on serine residues (11, 12, 29). MAP kinase constitutes one of the
most important protein kinase families for the progression of the cell
cycle. Activated MAP kinase phosphorylates a variety of targets
including other protein kinases and transcription factors (50).
Recently it was shown that Cx43 is a substrate of MAP kinase and that
the phosphorylation of Cx43 is mitosis-specific in many types of cells (11, 12, 29, 30). Comparison of the amino acid sequences of MAP kinase
substrates has identified PX1-2(S/T)P as a consensus phosphorylation site (51-53). Sequence analysis of Cx43 revealed MAP kinase consensus phosphorylation sites at serine 255, 279, and 282. In fact Warn-Cramer et al. (37) confirm that MAP
kinase phosphorylates Cx43 at Ser-255, -279, and -282. Data presented
in this study suggest that phosphorylation of Cx43 by MAP kinase is
directly involved in the gating mechanism of Cx43 channels.
 |
ACKNOWLEDGEMENT |
We thank Dr. S. K. Rhee (Yeungnam
University, Kyungsan, Republic of Korea) for assistance in the
reconstitution of gap junction channels.
 |
FOOTNOTES |
*
This study was supported in part by grants from Korea
Advanced Institute of Science and Technology, Korea Research
Foundation, and Science Research Center for Cell Differentiation (Seoul
National University, Seoul, Republic of Korea).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
82-42-869-2667; Fax: 82-42-869-2610; E-mail:
cojoe{at}sorak.kaist.ac.kr.
 |
ABBREVIATIONS |
The abbreviations used are:
GJC, gap junctional
communication;
Cx43, connexin 43;
PE, phosphatidylethanolamine;
octylglucoside, N-octyl-
-D-glucopyranoside;
CIP, calf intestinal phosphatase;
CNBr, cyanogen bromide;
CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate;
DMS, dimethyl suberimidate;
PAGE, polyacrylamide gel electrophoresis;
Cx32, connexin 32;
Cx26, connexin 26;
MAP, mitogen-activated protein.
 |
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