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INTRODUCTION |
Gap junctions provide a direct intercellular route allowing
adjacent cells in tissues and organs to communicate signaling and
regulatory molecules (1, 2). Gap junctions discriminate between
molecules mainly on the basis of size (<1 kDa) and charge, and the
channels are gated by transmembrane voltage (3) and chemically,
involving cytoplasmic concentrations of hydrogen and calcium ions (4,
5). Accessory proteins (6, 7) and putative soluble factors (8) are also
candidates in the regulation of the gating of gap junctions. Mutations
detected in the cDNA encoding connexin
(Cx)1 protein subunits of gap
junctions have shown that Cx32 and Cx26 are implicated in
Charcot-Marie-Tooth X-linked peripheral neuropathy and sensorineural
deafness, respectively (9, 10).
A key event in the formation of gap junction channels is the
oligomerization of connexins into hexameric hemichannels (connexons). These align and interact with connexons in the plasma membrane of
adjacent cells to form an electrically sealed intercellular channel
(11). Oligomerization of connexins was reported to occur after exit
from the endoplasmic reticulum (ER) (12), and oligomeric assembly is
promoted in the Golgi apparatus (13). These properties suggest that
connexins are unusual membrane proteins since protein folding and
oligomerization in the membrane are usually requirements for exit from
the ER (14, 15).
Several studies have shown that connexins accumulate in intracellular
stores that correspond to the ERGIC/Golgi in cultured cells (16-18)
and tissues (19), and growth factors appear to regulate connexin
transport between these stores and the gap junction (20). Transport of
membrane proteins in general from such intracellular stores to the
plasma membrane is likely to involve tubulovesicular networks (21, 22).
Gap junctions are dynamic structures, with their constituent connexin
proteins having high turnover rates (16, 23). Since the extent of
intercellular communication is a function of the number of gap junction
channels participating in coupling (24) and because gap junctions are
rapidly removed from the plasma membrane (16, 25, 26), the
translocation of connexins from intracellular sites to the plasma
membrane compensates for this high turnover to maintain and modulate
coupling between cells.
We have used connexin-26, -32, and -43 fused to the calcium reporter
aequorin to study the trafficking pathways that lead to the generation
of functional gap junction channels (18, 27, 28). Gradations in the
cytoplasmic calcium levels surrounding various stations as they
trafficked along the secretory pathway and at the gap junction were
determined (18). To overcome the nonfunctionality of the Cx26-Aeq
chimera, the 16-amino acid carboxyl-terminal tail of Cx26 was replaced
with the 156-amino acid carboxyl-terminal tail of Cx43, producing a
construct (Cx26/43T-Aeq) whose behavior could not be distinguished from
that of Cx26 (18). In this work, the inherent chemiluminescent activity
of a series of Cx-Aeq chimeras was used to quantify levels of connexins
and their oligomeric status in intracellular stores and at the plasma
membrane, and a method was devised to measure the kinetics of
translocation between these stores and the plasma membrane.
Applications of drugs that disrupt protein trafficking and the
cytoskeleton (brefeldin A, nocodazole, and monensin) pointed to
differences in the properties of trafficking of the Cx26, Cx32, and
Cx43 chimeras to plasma membranes and gap junctions.
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EXPERIMENTAL PROCEDURES |
Materials--
All cell culture reagents and plasticware were
from Life Technologies, Inc.; other reagents were obtained from Sigma
unless specified.
Measurement of Delivery of Connexins to the Plasma Membrane from
Intracellular Stores--
COS-7 cells were transfected with plasmid
cDNA encoding Cx32-Aeq, Cx43-Aeq, Cx26/43T-Aeq, and Cx32/43T-Aeq
(18); trypsinized; seeded onto glass coverslips (8 × 104/100 µl); and processed for photon counting as
described (27). Four h prior to commencement of experiments, cells were
incubated in tissue culture medium containing 3 mM EGTA to
give nominal free calcium and a 5 µM final concentration
of coelenterazine (Molecular Probes, Inc.). Coverslips were fixed onto
a perfusion chamber and brought into contact with a fiber-optic bundle
attached to a photon-counting camera (Photek 216) (29). Cells were
perfused with Krebs-Ringer-Henseleit solution containing nominal free
Ca2+ (KRH
Ca2+; 120 mM NaCl, 25 mM Hepes, 4.8 mM KCl, 1.2 mM
KH2PO4, 1.2 mM MgSO4,
and 1 mM EGTA, pH 7.4) for 10 min to remove excess
coelenterazine. Plasma membrane-associated recombinant protein was
selectively consumed by perfusing the cells with KRH+Ca2+
(120 mM NaCl, 25 mM Hepes, 4.8 mM
KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 1.3 mM
CaCl2, pH 7.4). Recombinant protein that has been
previously exposed to Ca2+ (causing the loss of bound
coelenterazine) cannot contribute to further chemiluminescence
measurements. This procedure is shown in Fig. 1.
Translocation of Connexins to the Plasma Membrane--
Cell
culture, transfection, and processing for photon counting were carried
out as described above. Coelenterazine (5 µM final concentration) in Ca2+-free Dulbecco's modified Eagle's
medium was added 4 h prior to photon counting. The following
inhibitors were added 1 h prior to start of experiments: brefeldin
A (5 µg/ml), nocodazole (20 µg/ml; Calbiochem), and monensin (20 µM). Cells exposed to nocodazole were processed prior to
experiments as described (31). During the experiments, all perfusion
media (KRH
Ca2+ and KRH+Ca2+) contained the
appropriate drug at the above concentrations. The effect of inhibitors
on the recovery of chemiluminescent activity at the plasma membrane was
measured at time intervals between 5 and 25 min using the above protocol.
Oligomeric Status of Recombinant Connexins in Intracellular
Stores--
COS-7 cells (1 × 107) transfected with
cDNA encoding each recombinant connexin and propagated for 48 h were scraped into phosphate-buffered saline, pH 7.4; pelleted by
centrifugation (1500 × g, 10 min); and resuspended in
1 ml of homogenization buffer (10 mM triethanolamine, 1 mM EDTA, 10 mM sodium acetate, 1 mM
dithiothreitol, and 250 mM sucrose, pH 7.4) supplemented
with protease inhibitor mixture (0.5 µg/ml pepstatin A, 5 µg/ml
leupeptin, 5 µg/ml chymostatin, 5 µg/ml antipain, 5 µg/ml
aprotinin, and 10 µg/ml phenylmethylsulfonyl fluoride). Cells were
dispersed by passage through a fine gauge needle (25 gauge, 10 times)
and sonicated (10 × 20 s; Decon Laboratories, Ltd.). The
resultant suspension was centrifuged (500 × g, 15 min) to obtain a post-nuclear supernatant. The post-nuclear supernatant was
separated on 15-ml linear Nycodenz gradients as described by Hammond
and Helenius (32) into 16 (1 ml) fractions, and their densities were
calculated from refractive index measurements. The subcellular
compartments corresponding to the ER (17-22% (w/v) Nycodenz,
fractions 1-5), ERGIC (13-16% (w/v) Nycodenz, fractions 6-11), and
the Golgi (9-12% (w/v) Nycodenz, fractions 12-16) were verified by
assay across the gradients of mannosyltransferase, Golgi 58-kDa
protein, and galactosyltransferase, respectively (18). Fractions
corresponding to each subcellular compartment were pooled. To determine
the distribution of Cx-Aeq chimeras across the gradient, 500 µl from
each pool was incubated with an equal volume of buffer (1 mM EDTA, 500 mM NaCl, 5 mM
-mercaptoethanol, and 10 mM Tris-HCl, pH 7.4) and 5 µM coelenterazine for 2 h in the dark at 4 °C.
Chemiluminescent activity was then measured by luminometry (Berthold)
after injection of 50 mM CaCl2 into the sample.
The remainder of the pooled ER, ERGIC, and Golgi samples from the
Nycodenz gradients (see above) were dialyzed against distilled
H2O (removal of Nycodenz was monitored by refractometry), concentrated under vacuum to ~50 µl, and mixed with an equal volume of 2× solubilization buffer (20 mM triethanolamine, 20 mM EDTA, and 20 mM dithiothreitol, pH 9.2).
Dodecyl maltoside (0.2%, w/v) was added to solubilize membranes, but
to keep oligomeric forms of connexins intact (13), and each pooled
sample was mixed by orbital rotation for 1 h at 4 °C. Pooled
fractions corresponding to the ER, ERGIC, and Golgi were added to
separate preformed sucrose gradients (5 ml, 10-40% (w/v) sucrose)
containing dodecyl maltoside (0.2%, w/v) and centrifuged for 22 h
at 150,000 × g to determine the oligomeric status of
connexins within the ER, ERGIC, and Golgi according to sedimentation
rates (12). Fractions (10 × 0.5 ml) were collected from the
bottom of the tube, and following the removal of 20 µl from each for
refractive index measurements, the chemiluminescent activity of each
fraction was measured. Under the above conditions (10-40% sucrose,
22 h, 150,000 × g), the resultant sucrose
gradient was categorized as follows: <15% (w/v) sucrose, monomer
(connexin); 15-33% (w/v) sucrose, oligomeric intermediates; and
>33% (w/v) sucrose, hexamer (connexon) (according to the migration of
known molecular mass proteins) (13).
Effect of Inhibitors on Plasma Membrane-associated
Connexins--
COS-7 cells transiently expressing various Cx-Aeq
chimeras were incubated in Ca2+-free Dulbecco's modified
Eagle's medium containing an inhibitor of protein trafficking (5 µg/ml brefeldin A, 20 µg/ml nocodazole, or 20 µM
monensin) for 1-6 h prior to start of measurement of chemiluminescence. The amount of plasma membrane-associated recombinant connexin at each time interval was quantified for each protein as
described above.
To investigate the effects of inhibitors on total recombinant protein
in cells (at the plasma membrane and in intracellular stores), COS-7
cells (1 × 107) expressing Cx-Aeq chimeras were
exposed to brefeldin A, nocodazole, or monensin for 2 or 6 h.
Cells were subsequently harvested in hyposmotic buffer (1 mM EDTA, 5 mM
-mercaptoethanol, and 20 mM Tris, pH 7.4) and freeze-thawed (five cycles). Nuclei
were removed from the suspension by centrifugation (500 × g, 15 min), and the protein concentration of the supernatant
was determined (Pierce). Reconstitution of the extracted recombinant
protein with coelenterazine and measurement of chemiluminescent
activity were carried out as described above.
Intercellular Transfer of Lucifer Yellow--
HeLa cells were
microinjected with plasmid cDNA encoding wild-type or chimeric
connexins (28). Transient expression of recombinant protein in >95%
cells was observed 24 h later, and cells were then incubated in
brefeldin A (5 µg/ml) or nocodazole (20 µg/ml) for 2 or 6 h.
Lucifer yellow (5% (w/v) in 0.3 M LiCl) was injected into
the cytoplasm, and 10 min later, cells were fixed in paraformaldehyde (4% (v/v) in phosphate-buffered saline). Dye transfer was quantified by viewing cells on a fluorescence microscope (Zeiss) fitted with appropriate filters.
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RESULTS |
Delivery of Cx-Aeq Chimeras to the Plasma Membrane Occurs at
Different Rates--
The addition of Ca2+ to cells
previously maintained in Ca2+-free media has been shown to
selectively trigger plasma membrane-associated aequorin (18, 30). The
remaining chemiluminescence in a population of cells expressing
chimeric connexins (i.e. intracellular stores that are
unaffected by the addition of KRH+Ca2+ to the cells
formerly maintained in KRH
Ca2+) was calculated at the end
of each experiment by exposing cells to 5 mM
Ca2+ in H2O; this allowed the relative amounts
of Cx-Aeq at the plasma membrane and sequestered in intracellular
stores to be determined. Following consumption of all plasma
membrane-associated recombinant protein, cells were perfused for
variable times in KRH
Ca2+. By measuring the recovery of
chemiluminescent activity at the plasma membrane at different time
points (Fig. 1), it became evident that
connexins were delivered to the plasma membrane at different rates
(Fig. 2). After consumption of all Cx-Aeq
chimeras initially present at the plasma membrane, the rate of
appearance of chimeras at the plasma membrane was Cx26/43T-Aeq (5 min) > Cx43-Aeq (10 min) > Cx32-Aeq
Cx32/43T-Aeq (15 min) (Fig.
2, C (panel i), B (panel
ii), A (panel iii), and D
(panel iii), respectively).

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Fig. 1.
Schematic diagram showing how the recovery of
chemiluminescent activity of Cx-Aeq chimeras at the plasma membrane was
determined. Reconstituted Cx-Aeq chimeras at the plasma membrane
were maintained by incubation of cells in
Ca2+-free Dulbecco's modified Eagle's
medium/KRH Ca2+ (A). Selective consumption of
plasma membrane-associated Cx-Aeq chimeras by addition of 1.3 mM Ca2+ to the perfusion medium does not
trigger intracellular stores of recombinant protein (B).
Coelenterazine is unavailable to bind to the Cx-Aeq chimeras once it
has been consumed at the plasma membrane. Subsequent incubation of
cells in EGTA (KRH Ca2+) permits trafficking of
reconstituted Cx-Aeq (from pre-existing intracellular locations) to the
plasma membrane (C). Newly delivered Cx-Aeq at the plasma
membrane can be detected by readdition of Ca2+ to the
perfusion medium (KRH+Ca2+) as described above
(D).
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Fig. 2.
Cx-Aeq chimeras are trafficked to the plasma
membrane at different rates. Following initial consumption of
aequorin (see Fig. 1B), the appearance of Cx32-Aeq
(A), Cx43-Aeq (B), Cx26/43T-Aeq (C),
and Cx32/43T-Aeq (D) at the plasma membrane was quantified
after perfusion of cells in KRH Ca2+ for 5 (panels
i), 10 (panels ii), and 15 min (panels iii)
and subsequent addition of KRH+Ca2+. The total amount of
unconsumed Cx-Aeq (i.e. located intracellular stores) was
determined following cell lysis in 5 mM Ca2+ as
described under "Experimental Procedures." Data are plotted as
means (n = 4).
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Effects of Inhibitors on the Translocation of Connexins to the
Plasma Membrane--
The amount of Cx32-Aeq (Fig.
3A), Cx43-Aeq (Fig.
3B), Cx26/43T-Aeq (Fig. 3C), and Cx32/43T-Aeq
(Fig. 3D) detected at the plasma membrane increased with
time. The translocation of various connexins to the plasma membrane was
influenced by various inhibitors of protein trafficking and depended on
the connexin isoform to which the aequorin reporter was attached.
Delivery of Cx32-Aeq, Cx43-Aeq, and Cx32/43T-Aeq from intracellular
stores to the plasma membrane was inhibited by 83 ± 4, 87 ± 4, and 81 ± 4%, respectively, in cells treated with brefeldin A
(Fig. 3, A, B, and D, respectively) compared with untreated cells. Treatment of cells expressing these chimeric proteins with nocodazole had a much lower relative effect on
their movement to the plasma membrane; nocodazole reduced the amount of
Cx32-Aeq, Cx43-Aeq, and Cx32/43T-Aeq at the plasma membrane by 29 ± 16, 4 ± 7, and 7 ± 7%, respectively (Fig. 3,
A, B, and D respectively). In
contrast, delivery of Cx26/43T-Aeq to the plasma membrane was almost
completely inhibited by nocodazole treatment (89 ± 5% inhibition
of translocation to the plasma membrane). However, translocation of
Cx26/43T-Aeq was affected to a lesser extent by exposure to brefeldin A
(16 ± 11% inhibition of movement to the plasma membrane) (Fig.
3C). The movement of all connexins to the plasma membrane
was inhibited by monensin treatment (Fig. 3, A-D). A
combination of brefeldin A and nocodazole completely inhibited
trafficking of all Cx-Aeq chimeras (data not shown).

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Fig. 3.
Effect of inhibitors of protein trafficking
on the delivery of Cx-Aeq chimeras from internal stores to the plasma
membrane. Trafficking of Cx32-Aeq (A), Cx43-Aeq
(B), and Cx32/43T-Aeq (D) to the plasma membrane
(PM) was inhibited by treatment of cells with brefeldin A
( ), but was only slightly affected by nocodazole treatment ( )
compared with untreated cells ( ). In contrast, movement of
Cx26/43T-Aeq to the plasma membrane (C) was inhibited by
incubation of cells in nocodazole and was relatively unaffected by
brefeldin A treatment. Trafficking of all Cx-Aeq chimeras was blocked
by monensin ( , A-D). All data are plotted as the
mean ± S.E. (n = 4).
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Intracellular Stores of Connexins: Location and Oligomeric
Status--
The intracellular location of the Cx-Aeq chimeras was
analyzed using a subcellular fractionation approach. Cx32-Aeq,
Cx43-Aeq, and Cx32/43T-Aeq accumulated in the Golgi region of COS-7
cells (Table I) as determined by their
comigration with galactosyltransferase (18). Cx26/43T-Aeq was found
predominantly in the ERGIC (revealed by its comigration with Golgi
58-kDa protein), with only 21 ± 5% of the total chimera being
transferred to the Golgi apparatus (Table I). Analysis of connexin
assembly demonstrated that the oligomerization process commenced in the
ER, and the formation of connexons, i.e. protein migrating
at the 9 S position on sucrose gradients (13, 36), was promoted as the
connexin trafficked along the secretory pathway (Fig.
4); the extent of oligomerization (determined on the basis of the amount of connexon formed) of each
connexin was Cx43-Aeq
Cx26/43T-Aeq > Cx32/43T-Aeq > Cx32-Aeq (Fig. 4, B, C, D, and
A, respectively). However, the fusion of aequorin to the
carboxyl-terminal tail of each connexin appeared to hinder the
oligomerization of Cx-Aeq chimeras relative to that observed with
wild-type connexins (28). The results show that Cx26/43T-Aeq was
assembled into connexons at an earlier stage than observed with the
Cx32-Aeq, Cx43-Aeq, or Cx32/43T-Aeq chimera, and its maximal
oligomerization into connexons occurred, importantly, in the ERGIC
environs of the cell (Fig. 4C). However, it was noted that
the Cx-Aeq chimeras were not fully oligomerized in these intracellular
stores, and they existed as a mixture of hexameric connexons and
non-hexameric oligomers (Fig. 4).
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Table I
Subcellular distribution of Cx-Aeq chimeras in COS-7 cells
The amount of Cx-Aeq chimeras in the ER, ERGIC, and Golgi compartments
was determined by chemiluminescence measurement following Nycodenz
fractionation of COS-7 cells. Results are given as the percentage of
total cellular Cx-Aeq in each compartment and are expressed as the
means ± S.E. (n = 3).
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Fig. 4.
Oligomeric status of intracellularly located
Cx-Aeq chimeras. Following subcellular fractionation of COS-7
cells expressing Cx-Aeq chimeras on Nycodenz gradients, the extent of
oligomerization of Cx32-Aeq (A), Cx43-Aeq (B),
Cx26/43T-Aeq (C), and Cx32/43T-Aeq (D) was
determined as described under "Experimental Procedures. Monomeric
connexins (white bars, M), oligomeric connexins
(gray bars, O), and hexameric connexons
(black bars, H) were categorized on the basis of
their velocity sedimentation on sucrose gradients (13). Subcellular
fractions were first isolated by centrifugation on Nycodenz gradients,
and Nycodenz and sucrose gradient profiles are shown in E
and F, respectively. Error bars represent S.E.
(n = 3).
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Removal of Connexins from the Plasma Membrane Occurs at Different
Rates--
Cx-Aeq chimeras specifically associated with the plasma
membrane accounted for 2-5% of the total recombinant protein
expressed by COS-7 cells, with the majority of Cx-Aeq chimeras being
located in intracellular stores (Fig. 4). However, Cx32-Aeq (3 ± 0.2%), Cx43-Aeq (4 ± 0.5%), Cx26/43T-Aeq (5 ± 0.3%), and
Cx32/43T-Aeq (4 ± 0.4%) (Fig. 5,
A-D at t = 0, respectively) were detected at the plasma membrane in different amounts. Trafficking of Cx32-Aeq (Fig. 5A), Cx43-Aeq (Fig. 5B), or Cx32/43T-Aeq
(Fig. 5D) to the plasma membrane was inhibited by incubating
COS-7 cells expressing each of these chimeric connexins in brefeldin A. Under these conditions, the time required for the removal of 50% of
Cx32-Aeq, Cx43-Aeq, and Cx32/43T-Aeq from the plasma membrane
(t1/2) was 198 ± 24, 168 ± 19, and
165 ± 27 min, respectively. The majority of Cx32-Aeq (85 ± 5%), Cx43-Aeq (76 ± 4%), and Cx32/43T-Aeq (74 ± 6%) was
removed from the plasma membrane following exposure to brefeldin A for
6 h. Treatment of COS-7 cells expressing Cx32-Aeq, Cx43-Aeq, or
Cx32/43T-Aeq with nocodazole did not greatly affect the amount of
recombinant protein associated with the plasma membrane even after
6 h of incubation (13 ± 7, 13 ± 6, and 21 ± 8%
decreases, respectively) compared with untreated cells (Fig. 5,
A, B, and D, respectively). In
contrast, removal of Cx26/43T-Aeq from the plasma membrane took place
more rapidly (t1/2 = 54 ± 10 min) when its
trafficking to the plasma membrane was inhibited by nocodazole (Fig.
5C). Brefeldin A treatment of cells expressing Cx26/43T-Aeq
resulted in a longer residence time of this chimera at the plasma
membrane (13 ± 5% decrease) compared with other Cx-Aeq chimeras
(Fig. 5C). Incubation of cells with monensin prevented
trafficking of all Cx-Aeq chimeras to the plasma membrane (Fig. 3) and
inhibited their subsequent removal from the cell periphery (Fig. 5,
A-D). In complementary experiments using HeLa cells
expressing various Cx-Aeq chimeras, both the amount of Cx-Aeq chimeras
present at the plasma membrane and their loss from the cell surface
induced by various inhibitors of protein trafficking were no different
from what was observed in COS-7 cells (data not shown).

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Fig. 5.
Removal of Cx-Aeq chimeras from the plasma
membrane occurs at different rates in COS-7 cells. The amount of
Cx32-Aeq (A), Cx43-Aeq (B), Cx26/43T-Aeq
(C), and Cx32/43T-Aeq (D) present at the plasma
membrane (PM) was determined following incubation of cells
in brefeldin A ( ), nocodazole ( ), or monensin ( ) for 0-6 h.
Quantification was by chemiluminescence measurements, and data are
given as the mean ± S.E. (n = 4).
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Incubation of cells with nocodazole or monensin for up to 6 h had
little effect on the total levels of Cx-Aeq chimeras expressed (Table
II). Prolonged exposure to brefeldin A (6 h) decreased the amount of all Cx-Aeq chimeras in cells (82 ± 9%). This probably represents removal from intracellular stores owing
to degradation (16) and therefore did not affect the quantification of
brefeldin A-induced loss of connexin from the plasma membrane.
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Table II
Effect of protein trafficking inhibitors on the amount of Cx-Aeq
chimeras in COS-7 cells
The Cx43-Aeq content of cells was determined following treatment of
cells with inhibitors of protein trafficking for 2 or 6 h. The
results are expressed as the means ± S.E. (n = 3)
and are representative of the effects of protein trafficking inhibitors
on all Cx-Aeq chimeras.
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Intercellular Dye Transfer Depends on a Threshold Amount of
Connexin at the Plasma Membrane--
Intercellular transfer of Lucifer
yellow was determined following treatment of HeLa cells expressing
Cx-Aeq chimeras and wild-type connexins with brefeldin A or nocodazole
(Table III). After a 2-h incubation in
brefeldin A (when 2-3% the total recombinant connexin was present at
the plasma membrane), dye transfer between cells expressing Cx32 and
Cx43 (as Cx-Aeq chimeras or wild-type connexins) was not affected
(Table III). However, after a 6-h exposure to brefeldin A, conditions
that lowered the amount of connexin at the plasma membrane to <1%,
the transfer of Lucifer yellow between cells expressing these connexins
was abolished (Table III). Despite its minor effect on the amount of
Cx32-Aeq, Cx43-Aeq, and Cx32/43T-Aeq (13 ± 7, 13 ± 6, and
21 ± 8% decreases, respectively) at the plasma membrane,
nocodazole treatment of HeLa cells expressing these chimeric connexins
for 6 h resulted in a markedly lower efficiency of dye
coupling.
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Table III
Dye transfer between HeLa cells transfected with wild-type or chimeric
connexins
The intercellular transfer of Lucifer yellow was assessed following
treatment of HeLa cells expressing wild-type connexins or various
Cx-Aeq chimeras with brefeldin A or nocodazole for 2 or 6 h (see
"Experimental Procedures"). Enhanced green fluorescent protein was
injected as a control. Data are given as the percentage of cells
transferring Lucifer yellow to two or more neighbors (mean ± S.E.), and >20 colonies were injected in each experiment.
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Intercellular dye transfer between cells expressing Cx26/43T-Aeq or
wild-type Cx26 was reduced by incubation in nocodazole for 2 h
(~1.5% of total Cx26/43T-Aeq at the plasma membrane) or 6 h
(<1% of total Cx26/43T-Aeq at the plasma membrane) (Fig.
5C). However, despite the loss of coupling observed
following nocodazole treatment, a detectable level of dye transfer
remained (Table III). Brefeldin A treatment of cells expressing
Cx26/43T-Aeq or wild-type Cx26 did not significantly alter levels of
cell-to-cell coupling (Table III).
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DISCUSSION |
The inherent chemiluminescent properties of connexins fused to the
calcium-dependent photoprotein aequorin have allowed
trafficking pathways to be studied. We showed that the time of transfer
of various Cx-Aeq chimeras from intracellular stores to the plasma membrane depended on the connexin isoform. Stores of chimeric protein
were detected in different intracellular locations. Cx43-Aeq resided in
a different environs of the Golgi compared with Cx32-Aeq and
Cx32/43T-Aeq, whereas Cx26/43T-Aeq was found predominantly in the ERGIC
in COS-7 cells (18). The calcium environments surrounding these
intracellular connexin stores were different (18). The present results
do not allow us to distinguish between chimeras translocating at the
same speed but from different intracellular locations to the plasma
membrane or whether they trafficked at different rates from spatially
separate stores (33). Since Cx32-Aeq and Cx32/43T-Aeq are in similar
intracellular locations and take a similar time to reach the plasma
membrane, both taking longer than Cx43-Aeq, it is concluded that the
cytoplasmic carboxyl-terminal domain has little influence on the
trafficking properties of these two chimeras. The results with
Cx26/43T-Aeq as well as other constructs in which the carboxyl-terminal
tail of Cx43 was removed or truncated (28, 34) support the conclusion
that the trafficking of connexins to gap junctions is not dependent on
the integrity of the carboxyl-terminal tail.
Subcellular fractionation of transfected COS-7 cells demonstrated that
oligomerization of connexins had commenced in the ER and increased as
the proteins trafficked along the secretory pathway, broadly in
agreement with other findings (12). However, the serial process of
oligomerization of Cx-Aeq chimeras into connexons was incomplete, with
up to 65% of chimeric protein in the hexameric form, and we propose
that these intracellular stores exist as a mixture of partially and
fully assembled chimeric connexons. These results raise questions
regarding the oligomerization and assembly of connexons into gap
junctions. First, proteins that oligomerize do so before exiting the ER
(35). The subcellular fractionation and oligomerization approaches
showed that the failure of recombinant connexins to oligomerize fully
did not cause a major retention of chimeric connexins in the ER. The
results suggest that both ERGIC and Golgi stations of the secretory
pathway promote oligomerization and that the partial oligomerization
initiated in the ER is sufficient to permit onward trafficking of
connexins. Second, since the plasma membrane contains mainly hexameric
connexons (13, 38), we propose that the Golgi possesses mechanisms that retain incomplete oligomers but permit the onward trafficking of fully
oligomerized hexameric hemichannels to the plasma membrane. These and
other results (12) reinforce the conclusion that oligomerization is
completed in the Golgi and that oligomerization is a key determinant for the movement of connexons from the Golgi to the plasma membrane. Also, the results show that, despite being present in the Golgi in
similar amounts compared with Cx43, Cx32 oligomerized to a lesser
extent and was also present at the plasma membrane in lower amounts
than Cx43, again highlighting the conclusion that the Golgi is a key
organelle in the oligomerization of these two connexins.
Inhibitors were used to study the routes of gap junction assembly. The
translocation of Cx32-Aeq, Cx43-Aeq, and Cx32/43T-Aeq chimeras to the
plasma membrane was inhibited by brefeldin A, but nocodazole treatment
(39-41) had only minor effects. These results argue strongly that
trafficking of the Cx32 and Cx43 chimeras occurred via the Golgi
apparatus since it was interrupted by disruption of this organelle.
Delivery to the plasma membrane was compromised by nocodazole
treatment, for Lucifer yellow transfer in cells expressing Cx43 and
Cx32 chimeras was reduced by ~30%, but this was probably caused by
nocodazole-induced remodeling of the plasma membrane (42, 43). As
expected, disruption of the Golgi by brefeldin A treatment severely
inhibited dye transfer by cells expressing Cx43 and Cx32 chimeras.
The effects of brefeldin A and nocodazole treatment on cells expressing
Cx26/43T-Aeq were different, with implications for the trafficking
route followed by this chimera. The delivery to the plasma membrane of
the Cx26 chimera, in contrast to that of the Cx32 and Cx43 chimeras,
was largely unaffected by brefeldin A treatment. Also, the cells,
whether expressing Cx26 or its chimera, were functional as demonstrated
by Lucifer yellow transfer. The brefeldin A insensitivity of the
trafficking of the majority of the Cx26 chimera is highly suggestive
that it may have reached the plasma membrane via a route that does not
directly involve the Golgi apparatus. Furthermore, the sensitivity of
the Cx26 chimera to nocodazole treatment suggests that its routing is
crucially dependent on microtubules, in contrast to the minimal
dependence under the same conditions of Cx32 and Cx43 trafficking. The
drug effects on trafficking were mirrored by their influence on Lucifer yellow transfer, for cells expressing the Cx26 chimera treated with
brefeldin A transferred dye almost as efficiently as those expressing
wild-type Cx26. Also, in contrast to its effects on Cx32 and Cx43
chimeras as well as wild-type Cx32 and Cx43, nocodazole inhibited the
assembly of gap junctions constructed of either wild-type Cx26 or the
Cx26 chimera.
Although Cx32 and Cx43 followed the secretory pathway to the plasma
membrane, it should be noted that the role of the Golgi apparatus in
glycosylation, in contrast to oligomerization, is not a concern with
connexins, for they are non-glycosylated proteins. With the Cx26
chimera, its targetting was not dependent on Golgi intactness as
inferred from the lack of brefeldin A sensitivity, and this agreed with
the oligomerization data, showing that it oligomerized in the ERGIC.
The trafficking characteristics of the Cx26 chimeras led to the
conclusion that an alternative pathway accounts for the bulk of the
trafficking of this connexin to the plasma membrane. Intriguingly, the
results suggest that ERGIC may serve as a sorting compartment in the
trafficking of specific connexins. Since COS-7 cells are not polarized,
it is unlikely that these two pathways followed by connexins correspond
to those delivering proteins to the apical or basolateral plasma
membrane domains (44). Results consistent with the existence of an
alternative connexin trafficking route to the plasma membrane have been
obtained by biochemical subcellular fractionation approaches, which
show that the majority of Cx26 appears to bypass the Golgi apparatus in
guinea pig liver (13).
Cx26/43T-Aeq was internalized from the plasma membrane of COS-7 cells
more rapidly than other connexins. This result and the rapid appearance
of Cx26 at the cell surface via a brefeldin A-insensitive route suggest
that the amount of Cx26 at the plasma membrane can be modulated
independently of other connexin isoforms. Kojima et al. (45)
demonstrated that Cx26 alone was recruited into gap junctions in female
rat liver expressing Cx26 and Cx32 with no associated increase in
protein synthesis or mRNA levels and suggested that intracellular
stores of Cx26 could be selectively mobilized to the gap junction in an
estrogen-dependent manner. Indeed, other examples exist of
proteins that may traffic to the cell surface independently of the
Golgi apparatus (46-49), including major histocompatibility complex
class I molecules (37).
The removal of connexin from the plasma membrane of COS-7 cells treated
with brefeldin A or nocodazole, neither of which inhibits protein
degradation (50, 51), showed that a threshold level of connexin (~1%
of the total cell recombinant protein) was required at the plasma
membrane to ensure functional cell-to-cell dye coupling. Only chimeras
translocated from intracellular stores can be active at the plasma
membrane; once exposed to Ca2+ at the plasma membrane, they
become inactive due to the absence of the cofactor, coelenterazine.
Thus, even in the unlikely event of connexins being recycled back to
the plasma membrane (26), these chimeras can play no further part in
subsequent measurements. These results confirm that we have measured
connexin movement from intracellular stores and not simply monitored
recycling events as observed with other membrane proteins such as the
transferrin receptor (51). The loss of connexin isoforms from the
plasma membrane in cells incubated in brefeldin A or nocodazole was
also abolished by lactacystin (20 µM) and leupeptin (100 µM) (data not shown), indicating that degradation of
Cx-Aeq chimeras was mediated by proteasomal and lysosomal mechanisms,
as also shown for wild-type connexins (25, 26).
No connexin-43 or connexin-32 was detected by immunofluorescence at the
plasma membrane following treatment of cultured cells with brefeldin A
for 6 h (16, 18). A disparity has thus arisen between the extent
of intercellular coupling observed and the amount of protein that can
be detected at gap junctions by immunofluorescence (26). The high
sensitivity of our approach has allowed us to calculate that 0.5-1%
of the total cellular chimeric protein remained at the plasma membrane
even after 6 h incubation of the Cx32-Aeq, Cx43-Aeq, Cx32/43T-Aeq
chimeras with brefeldin A or the Cx26/43T-Aeq chimera with nocodazole.
This low amount of protein was insufficient to allow functional
cell-to-cell coupling as assessed by Lucifer yellow transfer. It was
estimated, based on chemiluminescent data (assuming that one photon
equates to one molecule of recombinant connexin-aequorin (29)), that
1% (no communication) or 2% (detectable cell-to-cell coupling) of
total cellular connexin present at the plasma membrane approximated to
25 and 50 hemichannels, respectively. However, since unpaired
hemichannels are impermeable to Lucifer yellow in the presence of
external calcium (38) and intercellular dye transfer is conditional
upon the successful docking of connexons, cell-to-cell coupling can
probably be achieved by the assembly of relatively few gap junction
intercellular channels. Furthermore, since ~25 hemichannels
calculated at the cell surface of HeLa cells do not permit
intercellular transfer of Lucifer yellow, it is likely that most of
these connexons are unpaired. Other approaches have suggested that
intercellular communication can be mediated by as few as two gap
junction channels (52, 53).
Although attachment of large reporter groups to proteins is unlikely to
be functionally benign, the carboxyl-terminal tail switching studies
with the Cx26 and Cx32 chimeras are strongly indicative that their
trafficking behavior largely mirrors that of wild-type connexins, as
also demonstrated with other reporters used to follow trafficking
characteristics in living cells. For example, extensive studies with
the green fluorescent protein attest to the validity of this general
approach for kinetic analysis of their trafficking along the secretory
pathway (54).
In conclusion, the present approaches have analyzed intercellular
communication by studying connexon assembly and translocation from
intracellular stores to the plasma membrane and the recruitment of
these hemichannels into gap junctions. The results identify an
alternative trafficking route to the gap junction that is followed by
the bulk of Cx26. This routing contrasts with the conventional Golgi
pathway used by Cx32 and Cx43. An alternative trafficking route can
provide an important mechanism for rapidly generating homomeric Cx26
channels independently of channels (homo- or heteromeric) constructed
of other connexins. The identification of routes leading to the
assembly of gap junctions with different permeability properties, constructed of homo- or heteromeric connexins, supports further the
possibility that these intercellular communication channels exhibit
regulatable selectivity to biochemical messengers.