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Biochem. J. (2007) 408, 375–385 (Printed in Great Britain) doi:10.1042/BJ20070550 375
The C-terminus of connexin43 adopts different conformations in the Golgi
and gap junction as detected with structure-specific antibodies
Gina E. SOSINSKY*†1, Joell L. SOLAN‡, Guido M. GAIETTA*, Lucy NGAN*, Grace J. LEE*, Mason R. MACKEY*
and Paul D. LAMPE‡
*National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA 92093-0608, U.S.A., †Dept. of
Neurosciences, University of California, San Diego, La Jolla, CA 92093-0608, U.S.A., and ‡Molecular Diagnostics Programme, Division of Public Health Sciences, Fred Hutchinson
Cancer Research Center, Seattle, WA 98109, U.S.A.
The C-terminus of the most abundant and best-studied gap-
junction protein, connexin43, contains multiple phosphorylation
sites and protein-binding domains that are involved in regulation
of connexin trafficking and channel gating. It is well-documented
that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals
at least three connexin43-specific bands (P0, P1 and P2). P1 and
P2 are phosphorylated on multiple, unidentified serine residues
and are found primarily in gap-junction plaques. In the present
study we prepared monoclonal antibodies against a peptide
representing the last 23 residues at the C-terminus of connexin43.
Immunofluorescence studies showed that one antibody (design-
ated CT1) bound primarily to connexin43 present in the
Golgi apparatus, whereas the other antibody (designated IF1)
labelled predominately connexin43 present in gap junctions. CT1
immunoprecipitates predominantly the P0 form whereas IF1
recognized all three bands. Peptide mapping, mutational
analysis and protein–protein interaction experiments revealed that
unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding.
The IF1 paratope binds to residues Pro375–Asp379 and requires
Pro375 and Pro377. These proline residues are also necessary for
ZO-1 interaction. These studies indicate that the conformation of
Ser364/Ser365 is important for intracellular localization, whereas
the tertiary structure of Pro375–Asp379 is essential in targeting and
regulation of gap junctional connexin43.
Key words: confocal microscopy, connexin, electron microscopy,
gap junction, membrane protein structure, phosphorylation,
trafficking.
INTRODUCTION
Gap junction assembly and degradation is a rapid and highly
regulated process and one that is critical to the functionality of
a cell [1]. Gap junctions play a dynamic role in developmental
regulation, ionic transmission, signal transduction pathways and
metabolic co-operation. Specifically, gap junction structures are
critical in co-ordinating responses, regulation of cell ensembles,
helping to synchronize the action of neurons, transport of second
messenger molecules and removal of secreted ions. Many distinct
human diseases result from connexin mutations including hearing
loss, cataracts, impaired nerve conduction and skin abnormalities
(see current reviews [2–4]). The majority of these disease-
related defects are site-specific mutations that result in misfolding
or mistrafficking of connexins. For example, nine different
Cx32 (connexin32) mutations associated with CMTX (X-linked
Charcot-Marie-Tooth disease) cause either no protein expression
or aberrant cellular localization [5].
Cx43 (connexin43) is the most ubiquitous connexin with
widespread tissue expression, and many of the major steps in
the Cx43 life cycle have been characterized. Connexins typically
have a relatively short half-life (∼1.5–5 h, with specific times
depending on the connexin and cell type; [6]). The short connexin
half-life is a key element of coupling regulation as it allows very
dynamic and acute changes in gap junction regulation and GJC
(gap junctional communication) [1,7]. For Cx43, oligomerization
of the monomer into a hexamer (connexons) occurs in the
TGN (trans-Golgi network) [8] and connexons are then moved
by vesicles for insertion into the plasma membrane [9–11].
Connexons are hypothesized to migrate to cell–cell contact areas
where they dock with a partner connexon in an adjoining cell and
add to the edges of a plaque [9,10] or create a new plaque [12].
However, another mechanism for connexon delivery directly to
junctional areas has recently been proposed [13]. The gap-junction
plaques are then internalized as either whole plaques (annular
junctions) or as vesicles for lysosomal and/or proteosomal
degradation. What is not known is how the movement of each
particular Cx43 species (monomer, hexamer, dodecamer and
degradation product) is controlled during its life cycle, although
unspecified phosphorylation events have been implicated [14,15].
Phosphorylation of Cx43 can regulate the kinetics of Cx43
trafficking, assembly, gating and turnover in a cell-cycle-stage-
specific manner. In unstimulated NRK (normal rat kidney) cells,
Cx43 isolated from immunoprecipitated cell lysates shows three
bands on Western blots [15] with approximate molecular masses
of 42, 44 and 46 kDa often referred to as the P0, P1 and P2 forms of
Cx43. Treatment of cell lysates with alkaline phosphatase causes
loss of the 44 and 46 kDa bands with a commensurate increase in
the 42 kDa species, indicating that phosphorylation is responsible
for the migration shift. However, this phosphorylation-dependent
shift in migration is not simply due to the addition of the
mass of phosphate (80 Da). Instead, there are probably specific
phosphorylation events that can induce a conformational change
in Cx43 that is detectable in SDS/PAGE. For example, it has been
shown that phosphorylation on some combination of Ser325,Ser
328
or Ser330 is necessary to form the P2 isoform [16]. Since the P2
isoform has been shown to be present in gap-junction plaques [15],
phosphorylation at these sites appears to induce a conformational
Abbreviations used: BFA, brefeldin A; Cx, connexin; Cy5, indodicarbocyanine; DAPI, 4,6-diamidino-2-phenylindole; EM, electron microscopy; GST,
glutathione transferase; MDCK, Madin–Darby canine kidney; NRK, normal rat kidney; PKC, protein kinase C; TGN,
trans
-Golgi network.
1To whom correspondence should be addressed (email gsosinsky@ucsd.edu).
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376 G. E. Sosinsky and others
change that allows inclusion in the gap-junction plaque and is
detectable by the migration shift. Conversely, phosphorylation
on Ser368 can be detected on the P0 isoform [17], thus gel
electrophoresis cannot discern this event, though it has dramatic
effects on channel selectivity [18].
Clearly the conformation of proteins can be regulated via
phosphorylation to affect subcellular localization and protein–
protein interactions. In addition, antibodies can be specific for
distinct antigen conformations [19]. In the present study, we
examined the binding properties of two anti-Cx43 monoclonal
antibodies, CT1 and IF1, which were made to the same
23-amino-acid C-terminal peptide immunogen, but discerned
distinct tertiary Cx43 structures found in different subcellular
locales. We used a combination of epitope mapping experiments,
immunolabelling and correlative light and electron microscopy to
show that these antibodies are detecting distinct conformations
of the C-terminus in the Golgi apparatus (CT1) and the plasma
membrane (IF1). We show that the CT1 antibody detects a
conformation which is influenced by phosphorylation on specific
serine residues, whereas the IF1 antibody detects a phosphoryl-
ation-independent conformation important for protein–protein
interactions.
MATERIALS AND METHODS
Antibodies
The anti-GST (glutathione transferase), Cx43CT1 (where CT
is C-terminus), Cx43IF1, Cx43NT1 (where NT is N-terminus)
and mouse monoclonal antibodies were made at the Hutchinson
Center’s Antibody Development Shared Resource and are
available on a cost recovery basis from the Fred Hutchinson
Cancer Research Center (http://www.fhcrc.org/science/
shared_resources/antibody_development/products/connexins/).
Cx43IF1 was named as such because it performs well in immuno-
fluorescence and can immunoprecipitate Cx43 effectively but
does not perform well in a Western immunoblot. To avoid con-
fusion of the Cx43CT1 antibody with GST–Cx43CT constructs,
we refer to these antibodies as CT1, IF1 and NT1. GST protein
or peptides corresponding to rat Cx43 residues 1–20 (NT1)
and residues 360–382 (CT1 and IF1) synthesized with a C-
terminal or N-terminal cysteine respectively, for linkage to KLH
(Keyhole Limpet haemocyanin) were used for immmunization
as previously described [17]. Residues 360–382 are identical
for rodent and human Cx43. The anti-Cx43 polyclonal antibody
C6219, purchased from Sigma–Aldrich, was generated using a
synthetic peptide corresponding to the C-terminal segment of the
cytoplasmic domain (amino acids 363–382 with N-terminally
added lysine). The anti-ZO-1 and Cx43 (13–8300) monoclonal
antibodies were purchased from Zymed/Invitrogen. The latter
antibody primarily recognizes the P0 band and was raised against
a C-terminal peptide (amino acids 360–376). The anti-giantin
polyclonal antibody was purchased from Covance Research
Products.
Cell lines and culture conditions
All culture reagents were obtained from Gibco BRL, Life
Technologies. MDCK (Madin–Darby canine kidney), NRK cells
or Rat1 fibroblasts were grown at 37 ◦C in DMEM (Dulbecco’s
modified Eagle’s medium) supplemented with 10%FBS (fetal
bovine serum), 100 units/ml penicillin, 100 mg/ml streptomycin
and 2 mM glutamine and aerated with 5 %CO2. MDCK cells
were used to create stable lines expressing WTCx43 (where WT
is wild-type) or Cx43 with serine to alanine mutations at Ser364
or Ser365. Site-directed mutagenesis was performed using the
GeneTailor site-directed mutagenesis kit (Invitrogen) on Cx43
ligated into pIREShyg (Clontech). Cells were transfected using
an Amaxa Nucleoporator, selected with 200 µg/ml hygromycin
and colonies were isolated using cloning rings. HeLa cells stably
expressing a serine to alanine mutation at Ser368 have been
described previously [17].
Peptide constructs
The Cx43 sequence encoding for the C-terminal amino acids 236–
382 of Cx43 was cloned into a pGEX-2TK vector to generate
GST–Cx43CT. C-terminus deletion/truncation constructs 6S
(missing amino acids 364–373), T374 (missing amino acids 375–
382) and T379 (missing amino acids 380–382) were all cloned
into the pGEX-2T vector to generate the respective GST deletion
constructs [20]. In addition, a C-terminal construct with Pro375 and
Pro377 mutated to phenylalanine residues (denoted as P375/7F)
was also cloned in the same manner. GST constructs were
transformed into DH5αEscherichia coli, and GST fusion proteins
were expressed and purified on glutathione–agarose. For CT1
epitope mapping, fusion proteins were run on SDS/PAGE (10%
polyacrylamide gel), blotted on to nitrocellulose and probed
with a monoclonal anti-GST antibody (IgG2b) and the CT1
antibody (IgG2a), then visualized with a fluorescent dye-labelled
isotype-specific secondary antibody [Alexa Fluor®680 goat anti-
mouse IgG2b (Molecular Probes) and IRDye800 donkey
anti-mouse IgG2a (Rockland Immunochemicals), both extens-
ively cross-reacted against other species] and directly quantified
using the Li-Cor Biosciences Odyssey infrared imaging system
and associated software. All quantifications of gel densitometry
are based on three to five independent measurements. For IF1
epitope mapping, a pulldown approach was utilized. IF1 antibody
(IgG2a) and GST fusion proteins bound to glutathione–agarose
were incubated in 1 %(v/v) Triton X-100 and 1%(w/v) BSA in
PBS at 4 ◦C for 1 h. Beads were washed three times in the same
buffer and run on SDS/PAGE for Western blot analysis. Blots
were incubated with an anti-GST antibody, then visualized with
Alexa Fluor®680 goat anti-mouse IgG2b to detect the deletion
constructs and IRDye800 donkey anti-mouse IgG2a to detect IF1
antibody that bound to the deletion constructs.
ZO-1 pulldown assays were performed by incubating fusion
proteins bound to glutathione–agarose with NRK cell lysates
in the presence of 1 %(w/v) BSA. Cells were lysed in PBS
containing 0.5 %Triton X-100, 0.5 %deoxycholate, 50 mM
NaF, 500 µMNa
3VO4, 2 mM PMSF and 1×complete protease
inhibitors (Roche Diagnostics), followed by centrifugation at
13 000 gat 4 ◦C for 10 min. Beads were washed three times in
PBS containing 0.5 %Triton X-100 and 0.5 %deoxycholate, run
on SDS/PAGE and blotted on to nitrocellulose. Blots were cut
in half to separate GST fusion proteins from the ZO-1 migratory
position. The lower half of the blot was probed with a monoclonal
anti-GST antibody and the upper half of the blot was probed with
a monoclonal anti-ZO-1 antibody. Blots were then incubated with
IRDye800 donkey anti-mouse secondary antibody and directly
quantified using the Li-Cor system.
Immunodetection of Cx43 from heart
Mouse studies were conducted under FHCRC Institutional
Animal Care and Use Committee approval. Inbred mice (4 months
of age in a FVB/N:C57BL6 background) were anaesthetized
(avertin; 0.1 ml/3 g of body weight) and killed using cervical
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Cellular localization of Cx43 species 377
Figure 1 Different and specific subcellular localizations occur in unstimulated NRK cells with CT1 and IF1
NRK cells immunolabelled with (A) IF1 or (B) CT1 show punctate membrane labelling characteristic of gap junctional staining or intracellular labelling respectively. Cx43 was immunoprecipitated
from NRK lysates with either a polyclonal antibody anti-Cx43 (Sigma), IF1 or CT1, separated by SDS/PAGE, Western blotted and probed with the NT1 antibody (C). CT1 recognizes only the lowest
band, P0, whereas IF1 binds to all three bands, P0, P1, P2.
dislocation. Hearts were excised and placed either in ice-cold PBS
for 30 s (control group) or incubated without coronary perfusion in
non-oxygenated glucose-free PBS with 1.8 mM calcium at 37 ◦C.
After 5, 15 or 30 min of incubation, hearts were longitudinally
bisected and sonicated in Laemmli sample buffer with 50 mM
NaF, 500 µMNa
3VO4, 2 mM PMSF and 1×complete
protease inhibitors for Western blot analysis [SDS/PAGE; (10%
gels)].
Peptide competition assays
Replicate lanes of recombinant GST–Cx43CT were blotted on
to nitrocellulose, then probed with CT1 antibody alone or CT1
antibody plus 10 µg/ml of a peptide containing Cx43 residues
360–382 (pep360) or a peptide containing Cx43 residues 368–
382 (pep368), then visualized using fluorescent-dye-labelled
secondary antibody [IRDye800-conjugated donkey anti-mouse
IgG (Rockland Immunochemicals)] and directly quantified using
the Li-Cor system.
Alkaline phosphatase treatments
Cells were lysed with 0.2 %SDS in PBS, clarified by microcen-
trifugation and treated with 100 units/ml alkaline phosphatase at
37 ◦C for 30 min. Lysates were run on SDS/PAGE (10%gel),
blotted and co-incubated with CT1 (IgG2a) and NT1 (IgG1),
then visualized with isotype-specific secondary antibodies [Alexa
Fluor®680 goat anti-mouse IgG2a (Molecular Probes) and
IRDye800 donkey anti-mouse IgG1 (Rockland Immunochemi-
cals)] and directly quantified using the Li-Cor system.
Immunofluorescence and confocal microscopy
Immunolabelling procedures followed those previously described
in [21]. Fluorescence microscopy was performed using a BioRad
MRC-1024 or an Olympus FluoView confocal microscope (Bio-
Rad). Samples were labelled with secondary antibodies linked
to FITC, Cy5 (indodicarbocyanine) or rhodamine (Jackson
ImmunoResearch Laboratories). CT1, IF1, anti-calnexin and anti-
giantin antibodies were used at 1:250 dilutions. Nuclei were
labelled with DAPI (4,6-diamidino-2-phenylindole) according
to the manufacturer’s instructions (Molecular Probes). All fluo-
rescent secondary antibodies were used at a 1:100 dilution. It
should be noted that we have observed microtubule-like staining
in MDCK cells that do not express Cx43 using the CT1 antibody.
This staining was not observed in NRK, HeLa or the same MDCK
cells if they had been transfected with Cx43.
Immunoperoxidase staining and preparation of samples for EM
(electron microscopy)
NRK cells were plated on MatTek dishes as described previously
[21]. CT1 and IF1 labelling for EM was performed with a
1:250 dilution, whereas the goat anti-mouse HRP (horseradish
peroxidase) conjugate was used at a dilution of 1:200. Cells were
immunolabelled following a previously described protocol [22],
fixed with 4 %(w/v) paraformaldehyde, 0.1 %glutaraldehyde in
1×PBS and reacted for 5–8 min in 0.05 mg/ml DAB (diamino-
benzidine) with 0.01 %H2O2.Afterwashingin1×PBS, the
labelled cells were fixed with 2%glutaraldehyde in 1×PBS
buffer for 20 min, washed five times with 1×PBS, post-fixed
with 0.5 %osmium tetroxide in 1×PBS for 30 min and washed
five times in double-distilled water. Cells were then dehydrated
in an ethanol series and embedded in Durcupan ACM epoxy
resin. Ultramicrotomy was performed using a Reichert Ultracut
E ultramicrotome (Leica) and a diamond knife (Diatome U.S.)
to produce 80–140 µm thick sections. Sections were collected on
50 mesh gilder copper grids. CT1 samples were stained en bloc
with 2 %uranyl acetate overnight whereas IF1 sections were post-
stained for 15 min in 2 %aqueous uranyl acetate. Transmission
EM images were obtained with a JEOL JEM-1200EX electron
microscope (JEOL U.S.A.) operating at 60 kV.
RESULTS
In the present study, we report on the binding properties of two
monoclonal antibodies, CT1 and IF1, that were made to the same
peptide representing the last 23 amino acids (360–382) of Cx43
but preferentially labelled different subcellular fractions of
Cx43. In NRK cells, IF1 stained only gap-junction plaques
whereas CT1 labelled mainly perinuclear Golgi-like structures
(Figures 1A and 1B respectively). This perinuclear CT1 staining
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The Authors Journal compilation c
2007 Biochemical Society
378 G. E. Sosinsky and others
Figure 2 CT1 staining co-localizes with giantin, a Golgi marker, whereas IF1 staining is localized to gap-junction plaques
(A,B,D,E,G,H,Jand K) Single-channel fluorescence images are shown in black and white (left-hand and middle columns) whereas the right-hand column contains the colourized overlay of
the left-hand and middle panel images [FITC, green; Cy5, red; merge between the two colours will appear in yellow] along with DAPI counterstaining (blue) to mark cell nuclei (C,F,Iand L).
Strong co-localization of staining is shown between CT1 and anti-giantin (D–F), and IF1 and anti-Cx43 (G–I). CT1 co-stains poorly with junctional anti-Cx43 (A–C) and IF1 does not co-stain with
anti-giantin (J–L).
was also seen in rat fibroblasts and MDCK cells expressing
Cx43 (results not shown). We hypothesized that these antibodies
bound to distinct Cx43 populations due to their recognition of
conformationally distinct epitopes. In order to determine their
specificity, we also tested the ability of these antibodies to
immunoprecipitate Cx43 as compared with a polyclonal Cx43-
specific antibody sold by Sigma that was generated against a
similar peptide (amino acids 363–382). SDS/PAGE typically
separates Cx43 into three macroscopic bands on a Western blot
that correspond to different phosphorylated forms of Cx43 (called
P0 or NP, P1 and P2). The Sigma antibody performs well in im-
munoblotting (recognizing all three isoforms) and immunofluo-
rescence assays yielding apparent gap junctional and perinuclear
localization [23].
Immunoprecipitations of Cx43 from NRK whole-cell lysates
using IF1, CT1 or the Sigma antibody were detected using
a Cx43 antibody to the N-terminus that detects all three
phospho-isoforms (designated NT1). CT1 immunoprecipitated
primarily the P0 form, whereas IF1 and the Sigma polyclonal
antibody immunoprecipitated the P0, P1 and P2 forms (Fig-
ure 1C).
Co-localization studies with CT1 and IF1
In order to further address specific organellar localization, we
performed double immunofluorescence labellings with either CT1
or IF1 and the Sigma polyclonal antibody anti-Cx43 (Figures 2A–
2C) or a polyclonal antibody against giantin (Figures 2D–2F).
Giantin is a membrane-bound component of the cis and medial
Golgi [24] that has been shown to play a role in transport of
vesicle-associated tethering factor [25]. We found that CT1 and
Sigma polyclonal antibodies co-labelled intracellular structures
in a perinuclear location (yellow in the merged image, Figure 2C;
the CT1 staining is more intense than that of the Sigma antibody)
but to a far less extent the punctate, gap junctional Cx43 (shown
in red) present in the plasma membrane. This was also reflected in
the Western blot in Figure 1(C) where the predominant species
recognized by CT1 is P0, though very faint bands possibly co-
migrating with the phosphorylated species were present. The
CT1 labelling mainly co-localized with giantin (Figures 2D–2F;
yellow in the merged image, Figure 2F). In the merged images
(Figures 2C, 2F, 2I and 2L), DAPI staining (shown in blue) was
included as a nuclear marker to help distinguish different cells.
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Cellular localization of Cx43 species 379
Figure 3 The CT1 epitope is found in the Golgi apparatus but is not required for Golgi entry
Immunoperoxidase labelling with CT1 in NRK cells shows an enhancement of staining in the Golgi apparatus region. (A) A low magnification image of the perinuclear region. The two arrows
point to two regions of Golgi stacks that are shown at higher magnification in (B). The magnification in (B)is∼3.7 times that of (A). Note the overall staining of the Golgi stacks (arrows) as
well as membrane staining in some vesicles (arrowhead). (C) Conversely, immunoperoxidase labelling with IF1 heavily stains gap junctions (arrows) without any significant intracellular labelling.
(D) The 2×enlargement of the centre gap junction reveals small vesicles lying underneath the gap junction. (E) For comparison, NRK cells that have been CT1-immunoperoxidase-labelled reveal
no significant gap junction staining (e.g. arrow).
Similar CT1 labelling patterns were found in Rat1 fibroblasts
and Cx43-transfected MDCK cells indicating that localization in
the Golgi apparatus is not cell-type-specific (results not shown).
Conversely, IF1 staining overlapped significantly with that of the
Sigma polyclonal antibody at apparent gap junctional structures
(Figures 2G–2I; yellow in the merged image, Figure 2C) but
did not overlap with that of the anti-giantin antibody (Fig-
ures 2J–2L).
Higher resolution subcellular localizations of CT1 to the Golgi
apparatus and IF1 to gap-junctional plaques by EM
Localization at EM resolution allows us to look at the distribution
of CT1 staining in the region of the cell containing the Golgi
apparatus. When using CT1 with stronger fixation conditions
than those used for light microscopy, immunoperoxidase methods
provided the best sensitivity with minimum background [26].
Light microscopy of immunoperoxidase-stained samples showed
similar staining patterns to the fluorescence images (results
not shown). Figure 3(A) shows a low magnification electron
microscopic view of the perinuclear area of the cell. The specimen
has been counter-stained with uranyl acetate in order to visualize
the cell components and this counter-staining accounts for the
dark colour of the nucleus. This sample has good ultrastruc-
tural preservation particularly in the Golgi membrane stacks and
small vesicles surrounding the Golgi apparatus. A higher (∼3.7×)
magnification view (Figure 3B) revealed uniform staining across
the Golgi stacks (arrows) consistent with the confocal images
in Figures 2(A) and 2(D). In addition some, but not all, of the
trafficking vesicles in the Golgi region appear to be highlighted
by the peroxidase reaction product (see example denoted by the
arrowhead).
Immunoperoxidase methods using the IF1 antibody heavily
stained gap-junction plaques with almost no intracellular staining
(Figure 3C). Higher magnification of the centre gap junction (Fig-
ure 3D) revealed staining of small vesicles, which were
presumably part of the degradation component of the connexin life
cycle [9]. The immunoreactivity for IF1-labelled gap junctions is
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380 G. E. Sosinsky and others
Figure 4 An increase in the amount of available epitope for CT1 binding
occurs after BFA or alkaline phosphatase treatment and during hypoxia in
heart
(A) NRK cells were treated with BFA or not (CON). Western blot analysis of cell lysates were
performed using NT1 to detect total Cx43 (NT1 panel) and CT1-reactive Cx43. (B) Immunoblot
analysis of lysates from mouse heart exposed up to 30 min of hypoxia and detected with an
antibody to total Cx43 (NT1; top panel) and CT1 (bottom panel). (C) NRK cell lysates treated with
alkaline phosphatase (AP) or not (CON). Blots were probed with both NT1 (left-hand panel) and
CT1 (right-hand panel) to detect total Cx43. Signals from (C) were directly quantified and the
ratio of CT1 to NT1 binding determined for three experiments (D). Protein loads were equalized
in all lanes.
significantly heavier when compared with gap junction labelled
with CT1, even with the more contrasting en bloc uranyl acetate
counter-stain (Figure 3E, arrows). The contrast in the CT1-
labelled gap junctions is primarily due to mass density and also
to some slight specific staining that is also seen in the light
microscopy images.
The CT1 epitope is present before Cx43 enters the
Golgi apparatus
Since the CT1 epitope appeared to be enriched in the Golgi
apparatus we examined whether this epitope was required for
Golgi entry. To address this question, we treated NRK cells with
BFA (brefeldin A), which results in Golgi disassembly. NRK cells
were incubated with BFA for 5 h and whole cell lysates were run
on SDS/PAGE, blotted and co-incubated with NT1 (IgG1)and
CT1 (IgG2a) antibodies. Consistent with previous results from
Laird et al. [12], the NT1 signal showed that BFA treatment
resulted in enrichment of the 42 kDa or P0 isoform at the
expense of the P1 and P2 forms and accumulation of a transiently
phosphorylated 43 kDa isoform (Figures 4A and 4B, NT1; BFA
lane labelled P1/2). Probing of the same blot with the CT1 antibody
showed that this antibody recognized only the 42 kDa and 43 kDa
isoforms of Cx43 (Figure 4A, CT1) and that this signal was
enriched upon BFA treatment (Figure 4A, CT1). Immunofluo-
rescence of CT1- and IF1-stained BFA-treated samples was
performed with a Cx43 polyclonal antibody or giantin antibody
co-localizations (results not shown). These images demonstrated
that the CT1 staining remained intracellular, with some peri-
nuclear concentrations, but the fluorescence was more dispersed
throughout the cytoplasm and did not entirely co-localize with
giantin. Most plaques that had been stained with IF1 and the Cx43
polyclonal antibody appeared to have been internalized during
the 5 h BFA protocol as previously reported [12]. Therefore these
results indicate that the CT1 epitope occurs on Cx43 prior to its
entry into the Golgi apparatus.
CT1 epitope increases in ischaemic heart
We and others have shown that in hypoxic heart, the SDS/PAGE
migration of Cx43 shifts from slower-migrating isoforms to the P0
isoform and that this corresponds to a loss of gap-junction plaques
at the intercalated disc [16]. We used the CT1 antibody to examine
whether its binding would increase during mouse heart hypoxia.
Hearts were incubated in non-oxygenated PBS for 0–30 min at
37 ◦C, lysed in SDS sample buffer, run on SDS/PAGE, blotted and
probed with the CT1 and NT1 antibodies. The CT1 signal was
increased by over 50 %(56 %+
−30.5 %) in as early as 5 min and
increased to 6-fold (6.31 +
−2.47) over control levels by 30 min of
ischaemia (Figure 4B). At the later time points, the CT1 signal
increase clearly coincided with an increase in the P0 form of
Cx43, consistent with the idea the CT1 senses a conformation
of Cx43 that is not typically found in the slower-migrating
isoforms of Cx43.
CT1 recognizes primarily the P0 form of Cx43
The Cx43 migration shift during hypoxia is a result of dephos-
phorylation on specific serine residues [16]. A similar migration
shift was first shown by Musil et al. [14], by in vitro treatment
of cell lysates with alkaline phosphatase, where the P1 and P2
bands of Cx43 are lost with a commensurate increase in the
faster migrating P0 form (Figure 4C, AP lane). In order to further
characterize how the phosphorylation status of Cx43 influences
the binding of CT1, NRK whole cell lysates, treated with alkaline
phosphatase, were run on SDS/PAGE, blotted and incubated with
CT1 and NT1. In Figure 4(C), a Western blot of cell lysate (left-
hand panel, CON lane) blotted with the NT1 antibody and detected
with an IgG1-specific secondary shows the three Cx43 bands
(P0, P1 and P2) as expected. Probing of the same blot with CT1
recognized only the P0 form (right-hand panel, CON lane). When
NRK cell lysates were treated with alkaline phosphatase prior to
SDS/PAGE, the NT1 signal showed Cx43 running exclusively as
P0 (left-hand panel, AP lane) and the CT1 signal showed increased
binding to P0 (right-hand panel, AP lane). Quantification of this
blot (Figure 4D) indicated that the amount of available epitope that
was recognized by CT1 increased more than 2-fold upon alkaline
phosphatase treatment. This combined with the hypoxic heart
results indicate that CT1 binds to a non-phosphorylated epitope
that is biologically relevant during the heart tissue response to
insult.
CT1 recognizes a sub-region of the C-terminus containing
Ser364/Ser365
In order to determine the specific portion of the C-terminal
peptide that formed the antigenic epitopes for IF1 and CT1,
we performed epitope-mapping experiments using GST fusion
constructs containing Cx43 deletions/truncations/substitutions
and peptide competition. A graphic showing the sequence of the
C-terminal peptide (pep360), a smaller peptide (pep368) and dele-
tion constructs (6S, T374 and T379) is shown in Figure 5(A).
CT1 binding to these deletion constructs was determined by
Western blot (Figure 5B) and then quantified after normalization
to binding of an anti-GST antibody (Figure 5C). CT1 did not re-
cognize a deletion construct missing residues 364–373. However,
c
The Authors Journal compilation c
2007 Biochemical Society
Cellular localization of Cx43 species 381
Figure 5 Epitope mapping of CT1 indicates that its epitope is localized to
residues 364–368
A combination of GST fusion proteins expressing Cx43CT deletion/truncation mutants and
peptide competition were used to determine the epitope recognized by CT1. (A) depicts the
C-terminal amino acid sequence used to generate CT1 and IF1 (bold letters), the deleted
portions of the fusion constructs (6S, T374 and T379) and the amino acids present in the
peptides (pep368 and pep360) used in the present experiments. Western blots were performed
on the deletion/truncation constructs using CT1 and GST antibodies (B). (C) Signals were
directly quantified and the ratio of CT1 to GST binding was determined (
n
=3,
P
<0.001 and
P
<0.05 respectively; Student’s
t
test values). Western blots were performed on GST–Cx43CT
using CT1 alone, or in the presence of the immunizing peptide, pep360, or a peptide comprising
amino acids 368–382, pep368 (D). (E) CT1 signal from experiments as shown in (D)were
quantified and values in the presence of pep360 and pep368 were different from controls (
n
=3,
P
<0.001 and
P
<0.05 respectively; Student’s
t
test values). Taken together, the graphs in
(C)and(E) indicate the primary epitope determinant is between amino acids 364–368.
it recognized truncation constructs at amino acid residues 374
and 379 (Figure 5B). The 364–373 region contains three di-
serine repeats, Ser364/Ser365 ,Ser
368/Ser369 and Ser372 /Ser373.To
further define the epitope, peptide competition experiments were
performed, using the immunizing peptide, pep360, and a peptide
containing residues 368–382 (Figure 5D). We found that pep360
was much more effective at inhibiting CT1 binding (10-fold
inhibition) than pep368 (40 %inhibition), indicating that Ser364–
Ala367 is the primary epitope for the CT1 antibody (Figure 5E).
Given the fact that CT1 primarily recognized the dephosphoryl-
ated form of Cx43, we hypothesize that non-phosphorylated Ser364
and/or Ser365 are critical features of the CT1 epitope.
To further confirm that Ser364 and Ser365 are a requirement
for CT1 binding, full-length Cx43 was transfected into MDCK
cells that do not natively express any Cx43 [27]. Cells containing
unmodified Cx43 (WTCx43), Cx43 containing a serine to alanine
substitution at Ser364 (S364A) and Cx43 with a serine to alan-
ine mutant at Ser365 (S365A) were prepared. CT1 did not bind
either of these mutants efficiently (Figure 6), indicating that
serine residues at both of these positions were required for
CT1 recognition. Even when there was faint recognition (as in
the S364A lane) CT1 reacts with the P0 form only. To further
determine that Ser368 was not influencing CT1 binding, HeLa
cells stably expressing a serine to alanine mutation at Ser368
Figure 6 Mutation of either Ser364 or Ser365 affects CT1 antibody binding
WTCx43 or Cx43 containing serine to alanine mutations at residues Ser364,Ser
365 or Ser368
were transfected into either MDCK cells or HeLa cells. Western blot analysis was performed on
lysates from cells expressing these constructs or the parental, Cx43-negative cells. Blots were
incubated with NT1, to detect total Cx43, and CT1 (top panels). Signals were directly quantified
and the ratio of CT1 to NT1 determined (bottom panel). Ser364 and Ser365 values were different
from WTCx43 (
n
=5and
P
<0.001, Student’s
t
test values).
were also analysed (Figure 6). CT1 bound as well to S368A
as to WTCx43. It should be noted that all of these mutants
make predominantly the P0 form of Cx43. Figure 6 also shows
that CT1 is specific for Cx43, as it did not react with HeLa or
MDCK cell lysates that do not endogenously express Cx43. Taken
together, these results indicate that CT1 recognizes the Cx43
fraction that has not been post-translationally modified at Ser364
and/or Ser365.
IF1 recognizes a sub-region of Cx43 close to the end of the
C-terminus
Epitope mapping for IF1 was performed slightly differently as
it performs poorly in a Western blot. However, since IF1 is
able to recognize Cx43 by immunoprecipitation, a pulldown ap-
proach was utilized. Glutathione-bead-immobilized GST fusion
constructs were incubated with IF1 for 1 h, washed and run on
SDS/PAGE. Western blots were performed using an anti-mouse
antibody to detect IF1 (Figures 7A and 8A, top panels) and anti-
GST to detect the GST proteins (bottom panel). Full-length GST–
Cx43CT was the most efficient, followed by 6S and T379; T374
was particularly inefficient (Figure 7), pulling down 20-fold less
IF1, whereas T379 pulled down 5-fold less (both significantly
different from CT1, P<0.001, Student’s ttest). Since deletion
c
The Authors Journal compilation c
2007 Biochemical Society
382 G. E. Sosinsky and others
Figure 7 IF1 binding to deletion/truncation constructs indicates that the
epitope is near the C-terminal end of the sequence of Cx43
GST fusion proteins expressing Cx43CT deletion/truncation mutants were incubated with IF1
and assessed for their ability to bind or pulldown the antibody. Fusion proteins immobilized on
glutathione–agarose were either incubated with IF1 (+IF1 Ab) or without (No Ab). Western blot
analysis on these samples were performed using a monoclonal anti-GST antibody to detect the
fusion proteins (bottom panel). IF1 was detected with the anti-mouse secondary antibody (top
panel). Signals were directly quantified and the ratio of IF1 to GST signal determined (graph;
n
=5and
P
<0.001, Student’s
t
test values). *
P
<0.001 compared with the GST43 control.
Ab, antibody.
of 372–373 or 379–382 did not affect binding (i.e. IF1 bound
to the 6S and T379 deletion constructs; Figure 7A), it appears
that the IF1 paratope binds primarily to the Cx43 region within
residues 375–379 (Pro–Arg–Pro–Asp–Asp). To further define the
role that the proline residues at 375 and 377 play, we performed a
pulldown of IF1 antibody with Cx43 containing Pro375 and Pro377
mutated to phenylalanine residues. Mutation of these proline
residues essentially eliminated IF1 binding to the fusion protein
(Figure 8A) indicating that they are a key feature of the IF1
epitope. Given that IF1 binds to a conformation of Cx43 residing
chiefly in apparent gap-junction plaques, we wondered whether
Pro375 and Pro377 might be part of a conformation important for
protein–protein interactions. We focused on ZO-1, a protein that
requires at least the three C-terminal residues for binding to Cx43
[20,28]. Given the proximity of Pro375 and Pro377 to the C-terminus
and the apparent specificity of IF1 for gap junctional structures, we
hypothesized that these residues would influence ZO-1 binding.
We again utilized a pulldown approach in which we incubated cell
lysates from NRK cells with the GST fusion proteins immobilized
on glutathione beads and ran the bound proteins on SDS/PAGE.
Although GST–CT43 efficiently pulls down ZO-1, neither GST
alone nor the Pro375/Pro377 mutant bound ZO-1 (Figure 8B).
DISCUSSION
The C-terminus of Cx43 is critical for binding of cellular
components such as ZO-1 [20,28–30] and tubulin [31] as well
as binding to the intracellular loop of Cx43 [32]. An illustration
Figure 8 Pro375 and Pro377 are critical for IF1 binding and ZO-1 interaction
with the C-terminus of Cx43
GST fusion proteins containing GST alone, Cx43CT or Cx43CT with Pro375 and Pro377 converted
into phenylalanine residues were bound to glutathione–agarose and incubated with IF1 (A)or
NRK cell lysates (B). After Western blotting of the samples, the membranes were cut to separate
fusion proteins from the IF1 antibody or ZO-1 migratory positions. GST fusion proteins were
probed with a monoclonal anti-GST antibody (Aand B; GST Ab). Bound IF1 was directly detected
using an anti-mouse secondary antibody (A; IF1 Ab). The upper portion of the blot in (B)was
probed with a monoclonal anti-ZO-1 antibody (B; ZO-1Ab). Signals were directly quantified and
the IF1 to GST ratio is presented in (A)(
n
=5and
P
<0.001) and the ZO-1 to GST ratio in (B)
(
n
=4and
P
<0.001, Student’s
t
test value). Ab, antibody.
of the flexible C-terminus as determined by NMR methods [33]
is shown in Figure 9. Using two antibodies generated against a
peptide comprising the C-terminal 23 amino acids of Cx43 (360–
382), we have found that the C-terminus is present in at least two
distinct conformations when present within gap-junction plaques
or in cytoplasmic membranes.
The IF1 antibody epitope mapped to the amino acids
P375RPDD379 andrequiredPro
375 and Pro377 for binding. In addi-
tion, staining with IF1 was found entirely in gap-junction plaques
indicating that the conformation of P375RPDD379 may be different
from intracellular Cx43 species. Since proline residues often
confer rigidity or a kink to tertiary structure, we focused on the role
of proline residues in forming this epitope and hypothesized they
might be able to regulate protein–protein interactions. This kink
is reflected in the NMR structure shown in Figure 9 (region in the
dotted-line box indicated by the arrow). The C-terminus of Cx43
has been shown to bind to the PDZ domain of ZO-1 in a manner
dependent on the last three residues (Leu380–Ile382 ) of Cx43, and
immunofluorescence studies indicate that the ZO-1 and Cx43
interaction occurs primarily at apparent gap junctional structures
[20,28–30]. When we substituted phenylalanine residues for
Pro375 and Pro377, ZO-1 binding was lost. These results indicate
that Cx43 localization to a gap junction implies a specific struc-
tural conformation at residues Arg374–Asp379 that is also important
for ZO-1 binding. Interestingly, another group has shown that this
region of the C-terminus may play a role in regulation at the gap-
junction plaque. Shibayama et al. [34] have identified a peptide
that tightly bound the Cx43CT and induced shifting of residues
376–379 when examined by NMR. This peptide could inhibit gap
junction uncoupling, increase the mean open time of channels
and potentially affect the selectivity of the channel. Taken
altogether, these results show that the conformation of residues
c
The Authors Journal compilation c
2007 Biochemical Society
Cellular localization of Cx43 species 383
Figure 9 Cx43 sequence topology and possible C-terminal phosphorylation sites
Richardson diagram of the secondary structure of the Cx43CT amino acids 250–382 at low pH [49]. The serine residues are coloured white and labelled on the polypeptide chain. The thicker white
segment indicates the region most likely forming the binding pocket for CT1, whereas the thicker grey tube would form the epitope for IF1 as determined by the results presented in the present study.
The dotted-line box highlights the part of the structure that was used as the initial immunogen and the arrow points to the terminal amino acids that are important in ZO-1 binding.
P375RPDD379 is important for regulation of Cx43 in the gap-
junction plaque and may have several functional consequences.
Conversely, the conformations of Ser364 and Ser365 can appar-
ently regulate the intracellular localization of Cx43 and binding of
the CT1 antibody. The present study indicates that elimination
of serine residues at either or both of these sites can affect the
configuration of the C-terminus and that loss of the CT1 epitope
is correlated with inclusion in a gap junction. Both Ser364 and
Ser365 have been reported to be phosphorylated in response to
increased cAMP levels which leads to increased trafficking of
Cx43 to gap-junction plaques [35–37]. Thus it seems likely that
phosphorylation of Ser364 and/or Ser365 is the modification
that eliminates the CT1 epitope in cells and that this event is correl-
ated with gap-junction formation. Regulation of the CT1 epitope
appears to occur after or upon Golgi exit as treatment of cells
with BFA, which results in the loss of P1and P2 and the appear-
ance of a phosphatase-sensitive band migrating just above the
P0 form [12,38] (Figure 4A, P1/2), showed that both P0 and P1/2
were CT1 reactive, indicating that the CT1 epitope is present
prior to Golgi entry. It is interesting to note that an antibody from
Zymed (13-8300), like the CT1 antibody, has been reported to
bind primarily to non-phosphorylated Cx43 [39]. However, that
antibody did not work well in immunoprecipitation or immuno-
fluorescence experiments [40], possibly indicating distinct
biochemical properties from those of CT1.
These results are consistent with evidence that phosphorylation
could play a role in regulating Cx43 transport from ER to
Golgi, to TGN, to plasma membrane, to gap-junction plaques
and internalization. The C-terminus of Cx43 contains 21 serine
residues, and to date, a total of twelve serine residues and
two tyrosine residues have been reported to be phosphorylated
following stimulation of various kinases (reviewed in [41]).
For example, PKC (protein kinase C) phosphorylates residue
Ser368 whereas MAPK (mitogen-activated protein kinase)
phosphorylation occurs at Ser255,Ser
279 and Ser282 of Cx43 and
causes distinct changes in the gating properties of gap-junctional
channels [42,43]. Phosphorylation events at Ser325,Ser
328 and/or
Ser330 are correlated with the regulation of gap junction assembly
[44] and Ser364 phosphorylation plays a role in enhanced gap
junction assembly [35]. Ser365 has been shown to be phosphoryl-
ated in response to follicle-stimulating hormone partially through
a PKA (protein kinase A)-mediated mechanism [36,37]. Phos-
phorylation of specific serine residues has also been shown to
be regulated in the heart. Notably, Ser364 is the start of a tandem
repeat of Arg–X–Ser–Ser–Arg found in amino acids 362–374 and
a naturally occurring S364P mutation reported in visceroatrial
heterotaxia [45] that in experimental model systems can cause
alteration of gating or expression [46,47]. A hypoxia model of
ischaemia has shown regulation of specific phosphorylation sites
including an increase in PKC-mediated phosphorylation on Ser368
[18] and a decrease in phosphorylation on Ser325/Ser328 /Ser330 [16].
In the present study we show that the CT1 epitope increases
upon hypoxia consistent with increases in Ser368, and is negatively
correlated with Ser325/Ser328 /Ser330 phosphorylation and retention
of Cx43 in gap-junction plaques.
Since the same 23 amino acid peptide was able to generate
two conformationally distinct paratopes, the C-terminus has the
potential to sense and transduce important and specific inform-
ation via conformational changes. NMR analysis of small soluble
domains of connexin peptides in solution has provided insights
into the cytoplasmic structure that in general appears to be highly
disordered (see the recent review in reference [48]). The structure
of a large portion of the Cx43CT domain (amino acids 254–382,
Figure 9) shows one α-helix at segment Ala311–Ser325 and possibly
another at Asp339–Lys345. Even with acidification, there are only
minor changes, resulting in a slight decrease in αhelicity at the N-
terminus, and an increase at the C-terminus of the peptide [33,49]
although there is a dimerization of the C-terminal peptide seen at
low pH. The C-αbackbone of the 254–382 amino acid peptide
as determined by solution structure NMR is shown in Figure 9.
In this Richardson diagram, the positions of all serine residues
are marked in white. With the exception of Ser325, all other serine
residues map on to unordered regions of sequence. Ser325 is found
at the end of one of the two short helices. The flexibility of the C-
terminus may serve a purpose in being able to rapidly adapt locally
new conformations upon phosphorylation, dephosphorylation or
changes in interacting partners. The ability of both CT1 and IF1 to
recognize a conformational change implies that a physical change
occurs to provide a suitable shape with which other connexin-
interacting proteins can perform lock-and-key or induced-fit
mechanisms. Dynamic changes in the interactions of Cx43 with
specific chaperone proteins that occur during the different steps
in trafficking of the protein through its life cycle would fit with
our working hypothesis that the conformation of the C-terminal
c
The Authors Journal compilation c
2007 Biochemical Society
384 G. E. Sosinsky and others
region of Cx43 regulates its exit from the Golgi apparatus and the
stability of a gap junction.
Dr Paul Sorgen (University of Nebraska Medical Center, Omaha, Nebraska, U.S.A.) kindly
provided the NMR co-ordinates for the C-terminus peptide solution structure. Dr Steven
Taffet(Upstate Medical University, SUNY,Syracuse, NY, U.S.A.) generously provided some
of the GST–Cx43CT deletion constructs. We thank Dr Ben Giepmans for advice, Galen
Hand and Lucy Ngan for technical assistance in the early stages of this project and Joshua
Brown for his help with Figure 9. Support was contributed by National Science Foundation
grant MCB0543934 (to G. E.S.), National Institutes of Health grants GM55632 (to P.D. L.),
GM072881 (to G.E.S.) and GM065937 (to G.E. S.). Some of the work included in the
present study was conducted at the National Center for Microscopy and Imaging Research
at San Diego, which is supported by National Institutes of Health Grant RR04050 awarded
to Dr Mark Ellisman.
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Received 23 April 2007/13 August 2007; accepted 23 August 2007
Published as BJ Immediate Publication 23 August 2007, doi:10.1042/BJ20070550
c
The Authors Journal compilation c
2007 Biochemical Society
