Alteration of Cx43:Cx40 expression ratio in A7r5 cells
Janis M.
Burt,
Anna M.
Fletcher,
Timothy D.
Steele,
Yan
Wu,
G. Trevor
Cottrell, and
David T.
Kurjiaka
Department of Physiology, University of Arizona, Tucson, Arizona
85724
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ABSTRACT |
Connexins (Cx) 40 and 43 are coexpressed by several cell types at
ratios that vary as a function of development, aging, and disease.
Because these connexins form heteromeric channels, changes in
expression ratio might be expected to significantly alter the connexin
composition of the gap junction channel population and, therefore, gap
junction function. To examine this possibility, we stably transfected
A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector
encoding antisense Cx43. Cx43 mRNA continued to be expressed in the
antisense transfected clones, although levels were inversely related to
the number of copies of antisense DNA incorporated into the genome.
Protein levels, quantified in the clones with the highest and lowest
Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels,
although the trends predicted by the Cx43:Cx40 mRNA ratio were
preserved. Electrical coupling did not differ significantly between
clones, but the clone with elevated Cx43:Cx40 protein expression ratio
and unchanged Cx43 banding pattern was significantly better dye coupled
than the parental A7r5 cells. These results suggest that as the
Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.
gap junctions; connexin40; connexin43; heteromeric channel; antisense
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INTRODUCTION |
GAP JUNCTIONS are
clusters of channels that connect cytosols of neighboring cells such
that small molecules can readily diffuse between cells (for review, see
Ref. 6). Two hemichannels (connexons) comprise a
functional channel. Six subunits, called connexins, comprise each
connexon. To date, 15 connexins have been identified in the mammalian
genome. They are distinguished from one another by their predicted
molecular masses in kilodaltons, with connexin43 (Cx43) and 40 (Cx40)
representing connexins of 43 and 40 kDa, respectively. When all 12 connexins comprising the functional channel are identical, the channel
is referred to as homomeric/homotypic. In their homomeric/homotypic
forms, the various connexins exhibit unique functional properties such
as phosphorylation- and voltage-dependent gating and permselectivity.
Many cells express more than one connexin, some of which can combine to
form functional heteromeric and heterotypic channels. When the
connexins comprising a connexon are not identical, the connexon is
heteromeric. When the connexin composition of the two connexons
comprising a channel differ, the channel is heterotypic. Despite the
failure of Cx40 and Cx43 to form functional homomeric/heterotypic channels in the Xenopus oocyte expression system
(36), Cx40 and Cx43 form such channels in mammalian cells
(Cottrell GT and Burt JM, unpublished observations) (31).
In addition, these connexins form heteromeric channels
(17), and recent evidence suggests that they form
functional heteromeric/heterotypic channels (Cottrell GT and Burt JM,
unpublished observations) (16). Available data indicate
that these mixed channels display unique conductance and gating properties.
Vascular smooth muscle cells express Cx43 and Cx40 to varying degrees
(2, 3, 8, 9, 22, 25, 29). The homomeric/homotypic channels
formed by Cx43 and Cx40 exhibit unique 1) unitary
conductances (1, 7, 26, 34, 35), 2)
voltage-dependent gating properties (4, 5, 7), and
3) permeability characteristics (1, 7, 12, 34,
35). Focusing on the latter, Cx43 channels permit free exchange
between cells of both anionic and cationic molecules of significant
size. In contrast, Cx40 channels appear to mediate the exchange of
cations far more readily than anions (see, however, Ref.
12) and of small molecules far more readily than large
molecules. As a result, the functional ramifications of heteromeric
Cx43-40 channel formation could be profound. For example,
heteromeric channels of Cx40 and Cx43 exhibit a broad array of unitary
conductances defined at either end by the conductances of the
homomeric/homotypic channels (17). It is likely that this
array of conductances is indicative of a broad array of permeability properties. Cx43-40 heteromeric channels also exhibit
voltage-dependent gating properties distinct from either the Cx43 or
Cx40 homomeric/homotypic channels. Given the differences in
phosphorylation-dependent gating behavior of homomeric/homotypic Cx43
vs. Cx40 channels, it is likely that heteromeric channels will display
a range of sensitivity to the pathways involved in acute regulation of
cell processes. Thus cells that form heteromeric channels may be able
to adjust their communication properties over a broad physiological
range acutely through phosphorylation-dependent mechanisms and over a
longer time frame by regulating the ratio of expressed connexins.
The goal of the current study was to develop smooth muscle cell lines
that would facilitate exploration of the ramifications of coexpression
of Cx40 and Cx43 at varying ratios. Toward this end, we stably
transfected A7r5 cells, from a vascular smooth muscle cell line in
which Cx40 and Cx43 are naturally coexpressed (23, 24),
with a vector encoding antisense Cx43 (Cx43AS). Clones of these cells
as well as cells of the parental population were isolated, and mRNA and
protein levels were determined. The communication properties of those
clones with the most extreme Cx43:Cx40 protein ratios were determined.
A clone with elevated Cx43:Cx40 protein ratio wherein Cx43
phosphorylation was unchanged displayed significantly enhanced dye
coupling despite comparable levels of electrical coupling, suggesting
that by controlling the expression ratio of these proteins, the cells
directly regulate the types of communicated molecules. A clone with
elevated Cx43:Cx40 protein ratio with significant amounts of Cx43
phosphorylation failed to show increased communication levels,
suggesting that cells can regulate the communication pathway through
signaling cascades as well. These cell lines should prove useful in
understanding the rules of heteromeric channel assembly and the
physiological significance of heteromerization to intercellular
communication and cell function.
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MATERIALS AND METHODS |
Vectors.
A7r5 cells were transfected with pRSV-Cx43AS, pCMV-Cx43AS [obtained
from Dr. Alan Lau (15)], or pRc/CMV (Invitrogen, San Diego, CA). The pRSV vector was made by excising full-length Cx43 from
clone G2B (courtesy of Dr. Eric Beyer) with the restriction enzyme
EcoRI and then subcloning the excised fragment into the unique SalI cloning site of pRXneo (obtained from Dr. Roger
Miesfeld) by using a blunt-end ligation strategy.
Transfection and cell culture.
A7r5 cells were transfected after subculture and growth to 50-75%
confluence level in 60-mm plates. Cells were exposed to pRSV-Cx43AS,
pCMV-Cx43AS, or pRc/CMV vector DNA as a calcium phosphate/DNA precipitate (Profection; Promega) according to the manufacturer's instructions. Neomycin-resistant cells were selected at 250 µg/ml G418 (GIBCO BRL). At confluence, they were dilution cloned. Cells were
maintained, after their expansion from single-cell clones, in DMEM
(GIBCO) supplemented with 10% fetal bovine serum (FBS) and antibiotics
(3% pennicillin, 5% streptomycin) at 37°C and 5% CO2.
For transfectants, culture medium also contained 250 µg/ml G418.
RNA isolation, detection, and quantification.
Preliminary screening of total RNA by standard Northern techniques
suggested that Cx43 was elevated in cells that had been transfected
with the Cx43 antisense vector. Because only mRNA that successfully
exits the nucleus can be translated, we determined the Cx43 and Cx40
mRNA content of the cytoplasmic fraction of total RNA. Cytoplasmic RNA
was isolated as follows. Confluent cells (maintained in 10% FBS) were
washed with PBS, aspirated "dry," and lysed (0.65% Nonidet P-40,
0.15 M NaCl, 0.01 M Tris · HCl, and 0.0015 M MgCl2,
pH 7.4) such that nuclei remained intact and stuck to the dish.
The lysate was collected and centrifuged to remove cellular debris.
Urea buffer (7 M urea, 0.35 M NaCl, 0.01 M Tris · HCl, 0.01 M
EDTA, and 1% SDS, pH 7.4) was added to the lysate, and the mixture was
phenol extracted twice and chloroform extracted once. RNA was then
precipitated in ethanol, pelleted, and dried.
Cytoplasmic RNA was denatured and slotted onto ZetaProbe GT
membrane (Bio-Rad), in duplicate, at 10 µg/sample. The duplicate blots were probed for connexin mRNAs with the use of end-labeled (30) 48-mer oligomers or random primer-labeled cDNAs. The
Cx40 48-mer was complementary to bases 876-923 in the coding
sequence of rCx40; the Cx43 48-mer was complementary to bases
714-762 in the coding sequence of rCx43. Random primer-labeled
cDNA probes (Prime-a-gene Kit from Promega, used according to the
manufacturer's instructions) were made by using a 591-bp fragment of
the rCx40 sequence or a 1393-bp fragment of rCx43. Sample comparisons
were made after the connexin content of a sample was normalized to its
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content, which was
detected by using random primer-labeled GAPDH made from a 770-bp
fragment of the hGAPDH sequence. For both types of probes, hybridizations and washes were done in standard sodium phosphate solutions at 65°C overnight (ZetaProbe GT manual) or in Quickhyb (Stratagene) at 68°C for 1-2 h with comparable results.
Radioactivity was visualized via PhosphorImager (Molecular Dynamics),
and sample intensity was assessed using the software provided by the manufacturer.
Sample intensity was converted to micrograms of bound cDNA probe or
micromoles of oligomer probe as follows. The specific activity
(cpm/µg or cpm/µmol) of each probe was calculated after trichloroacetic acid (TCA) precipitation. A membrane strip spotted with
105, 104, 103, 102, and
101 cpm (determined by scintillation counting) of the probe
used to hybridize each blot was imaged simultaneously with each blot. The screen intensity of each spot was plotted as a function of the
spotted counts per minute to verify the linear range of detection and
to establish the relationship between image intensity and probe counts
per minute. Counts per minute hybridized to each sample could then be
calculated, and the micrograms (cDNA probe) or micromoles (oligo probe)
of connexin could be calculated from the specific activity of the
probe. With the cDNA probes, Cx43 was 2.357 times more likely
to be detected than Cx40 because of the differing lengths of the
template DNA (1393/591). Consequently, comparisons of Cx40 and Cx43
contents from cDNA probes were made after the Cx40 results were
multiplied by 2.357. This correction was not used for the data derived
from end-labeled oligomer probe, wherein each copy of mRNA should
hybridize with only one copy of probe.
Modified Southern analysis.
To estimate the number of copies of the antisense Cx43 sequence stably
incorporated into the genome, we screened our transfectants for the
amount of RSV (Rous sarcoma virus) promoter sequence in the DNA as
follows. Cells were pelleted and subsequently lysed by heating at
100°C for 10 min in a solution containing 0.4 M NaOH and 10 mM EDTA,
and cells were then slotted onto nylon membrane (Zeta-Probe GT). The
blot was hybridized with an RSV cDNA random primer-labeled probe, which
was made by using an ~320-bp fragment of RSV that was released from
pRXneo after digestion with restriction enzymes HinfI and
HindIII and then gel purified. RSV content was visualized
and quantified as described in RNA isolation, detection, and
quantification. After image analysis was completed, the
blot was stripped and probed with random primer-labeled GAPDH cDNA. The relative RSV content of each clone was determined after the RSV
data were normalized to the GAPDH data.
Detection of Cx43 antisense mRNA.
The antisense mRNA could not be detected in any of the
antisense-transfected clones by using the procedures described for detection of mRNA. To increase our chances of detecting the antisense mRNA, we developed a ribonuclease protection assay (RPA). Total RNA (10 µg) and riboprobe were hybrized overnight at 42°C and treated with
RNase A/RNase T1 (Ambion RPA kit), and probe/RNA complexes were
visualized on an Instant Imager (Packard Instruments) after
electrophoretic separation on 8 M urea, 4% polyacrylamide gels.
Riboprobes were transcribed (using Promega's Riboprobe transcription systems according to manufacturer's recommendations) from linearized vector from the SP6 or T7 promoters. The Cx43AS probe was 541 nucleotides in length, 483 of which were complementary to bases 1-483 of the antisense sequence. Cx43 sense mRNA, Cx40, and GAPDH were also detected. The Cx43 probe was 315 nucleotides in length, 233 of which were complementary to bases 915-1148 of the Cx43 sequence. The Cx40 probe was 497 nucleotides in length, 447 of which
were complementary to bases 597-1039 of the Cx40 sequence. The
GAPDH probe (transcribed from pTRI-GAPDH-Rat from Ambion) was 383 nucleotides in length, 316 of which complemented the 3' terminus of the
coding sequence. Specific activity for all probes was determined after
TCA precipitation.
Western analysis.
Total protein was isolated from 100-mm dishes of confluent cells by
using standard procedures and was then quantified (Pierce BCA Protein
Assay Kit). Proteins were electrophoretically separated on 10% SDS
gels, electrophoretically transferred to nitrocellulose (Hybond ECL,
Amersham Pharmacia Biotech), and blocked with 5% nonfat dry milk
(NFDM) in Tris-buffered saline supplemented with 0.5% Tween 20 (TTBS)
(30). Blots were incubated overnight at 4°C with primary
antibody, polyclonal affinity-purified Cx43 (Sigma), or Cx40 (Dr. Alex
Simon) diluted 1:4,000 in 1% NFDM-TTBS. After several washes, blots
were incubated with either horseradish peroxidase (HRP)-conjugated
anti- rabbit Ig (diluted 1:5,000; Amersham Pharmacia Biotech) or
35S-labeled anti-rabbit Ig (0.4 µCi/ml; Amersham
Pharmacia Biotech). HRP signal was detected with the use of enhanced
chemiluminescence (ECL; Pierce SuperSignal) and X-ray film. Connexin
expression levels were quantified from blots probed with
35S-labeled secondary antibody, which was visualized in a
position-specific manner with the use of an Instant Imager (Packard
Instruments). For quantification, each gel was loaded with several
samples, protein ladder, and either Cx40 or Cx43 standards. Standards
were prepared by using glutathione S-transferase
(GST)-fusion protein constructs of the COOH termini of Cx43 and Cx40
(provided by Drs. Alan Lau and David Paul, respectively). GST-fusion
proteins were purified directly from bacterial (BL21) lysates with the
affinity matrix Glutathione Sepharose 4B. Fusion proteins were eluted
under mild conditions and gel purified. Cx43 COOH terminus was released from the GST and gel purified again; Cx40-GST was used as a standard directly. The protein content of the gel-purified proteins was determined, and amounts corresponding to 0.5-8 pmol of the Cx protein were loaded either in separate lanes or in a staggered fashion
in the same lane. The counts from these standards were used to
construct a standard curve against which the connexin content of the
samples could be compared.
Dye injections and junctional conductance measurements.
The extent of dye coupling was determined as follows. Cells were plated
at confluent density (15,000 cells/cm2) onto glass
coverslips. Twenty-four hours after cells were plated, the glass
coverslips were mounted in a perfusion chamber and visualized on a
microscope equipped with differential interference contrast and
fluorescence optics. A microelectrode containing 0.5% Lucifer yellow
was lowered onto the surface of a cell, and the cell was impaled by
overcompensation of the capacitance compensation feature of the
amplifier (WPI 700). The number of cells containing dye was determined
5 min after injection.
Junctional conductance (gj) was determined by
using dual-whole cell voltage-clamp strategies as described previously
(20). To minimize the effects of series resistance, we
restricted our analysis of gj to pairs wherein
the sum of pipette series resistances was between 20 and 35 M
. Under
these recording conditions, gj is
underestimated. However, when the series resistances of compared groups
do not differ, gj is underestimated to the same
extent in those groups. Any differences in gj
between groups with equal series resistances would remain significant
after correction for series resistance. Cells were bathed in a solution
containing (in mM) 142.5 NaCl, 4 KCl, 1 MgCl2, 5 glucose, 2 sodium pyruvate, 10 HEPES, 15 CsCl, 10 tetraethylammonium chloride
(TEACl), 1 BaCl2, and 1 CaCl2. The patch
pipette contained (in mM) 124 KCl, 14 CsCl, 9 HEPES, 9 EGTA, 0.5 CaCl2, 5 glucose, 9 TEACl, 3 MgCl2, and 5 Na2ATP. The pH and osmolarity of both solutions were
adjusted to 7.2 and 322 mosM, respectively. Electrodes were fabricated from 1.2-mm glass (Corning 6020, AM Systems) on a Flaming/Brown Micropipette Puller (model P87; Sutter Instruments).
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RESULTS |
Initially, 17 A7r5 clones and 7 pRSV-Cx43AS clones were isolated,
and the mRNA contents of each were quantified by using oligomer probes.
The Cx43:Cx40 mRNA ratios for these clones varied as a function of Cx43
content (Fig. 1), suggesting that the
level of Cx43 expression largely determined the ratio. The data
revealed a fairly broad range of Cx43 content as well as Cx43:Cx40
ratios. The Cx43:Cx40 ratio of the pRSV-Cx43AS clones increased
linearly (r2 = 0.81) with Cx43 content. The
clone with the highest Cx43:Cx40 ratio, 6B5N, and that with the lowest
ratio, 2A6, as well as several isolates of the parental population,
were evaluated for expression levels by using both the oligomer and
cDNA probe strategies. Although the absolute values of the results from
the two strategies differed (Table 1),
both strategies indicated that the Cx43:Cx40 ratio was elevated in the
6B5N vs. A7r5 cells and reduced in the 2A6 vs. A7r5 cells.

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Fig. 1.
Cx43:Cx40 mRNA ratio vs. Cx43 mRNA content of A7r5 cells
and clones. Data were determined from cytoplasmic RNA probed with
end-labeled oligomer. Note the broad range of values for Cx43 and the
Cx43:Cx40 ratio. The ratio was linearly related to Cx43 content
(r2 = 0.82, P < 0.05). Cx,
connexin; RSV, Rous sarcoma virus; Cx43AS, antisense Cx43 clone.
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Table 1.
Cx43 and Cx40 mRNA and protein contents and Cx43:Cx40 ratios of A7r5
parental cells and several A7r5 cell clones
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Because Cx43 mRNA levels were elevated (relative to the parental A7r5
cells) in several of the antisense Cx43-transfected clones (Fig. 1), we
determined whether the Cx43 mRNA content of the antisense-transfected
clones was related to the number of copies of the antisense gene stably
incorporated into the DNA of the clones. This was accomplished by
determining the RSV DNA levels by using a modified Southern analysis as
described in MATERIALS AND METHODS. RSV promoter sequence
was detected in all of the pRSV-Cx43AS clones but not in nontransfected
A7r5 cells (Fig. 2). Cx43 mRNA content of
the pRSV-Cx43AS clones was inversely related to their RSV content (Fig.
3). Cx40 mRNA levels of the pRSV-Cx43AS
clones were unrelated to their RSV content. These results link low RSV
content (Cx43AS content) to enhanced Cx43 mRNA content in most of the
pRSV-Cx43AS clones but also indicate that as Cx43AS mRNA levels
increased, Cx43 mRNA levels decreased. Furthermore, these results
suggest that the 6B5N cells might have significantly less Cx43AS mRNA
than the 2A6 cells. To demonstrate that these clones actually express
the antisense mRNA and to evaluate relative expression levels, we used
RPA to detect the Cx43AS as well as Cx43 and GAPDH sense mRNAs. Figure
4 demonstrates that both 2A6 and 6B5N
cells actively express the antisense Cx43 mRNA.

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Fig. 2.
Slot blot shows RSV (left lane, short
exposure; middle lane, longer exposure) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; right lane)
content of pRSV-Cx43AS clones as well as A7r5 controls. Note the low
RSV content of the 6B5N clone and the high content of the 2A6 clones.
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Fig. 3.
Cx43 and Cx40 mRNA levels (relative to levels observed in
control A7r5 cells) vs. RSV levels in the pRSV-Cx43AS transfectants.
Note the inverse relationship between Cx43 mRNA content and RSV content
(r = 0.698, P < 0.05). Cx40 mRNA
content was not related to RSV content (r = 0.28, P > >0.05).
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Fig. 4.
Cx43AS mRNA was detected by ribonuclease protection assay
in 2A6 (lane 2) and 6B5N (lane 3) cells but not
in A7r5 cells (lane 4). Interestingly, the 6B5N cells would
appear to have more Cx43AS than the 2A6 cells. GAPDH and Cx43 were
detected in all samples. Lane 1 shows nondigested probes.
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Because complete knockdown of Cx43 was not observed in any of the
pRSV-Cx43AS clones, we stably transfected A7r5 cells with the
pRcCMV-Cx43AS vector used by Goldberg et al. (15) to
significantly reduce Cx43 expression in Rat 1 cells. Using RPA
techniques, we found that Cx43 mRNA levels were reduced by 24-43%
in three CMV-Cx43AS clones. RPA was also used to verify the elevation
of Cx43 mRNA observed by Northern techniques in the 6B5N cells.
Consistent with the data presented in Table 1, Cx43 mRNA was elevated
4.8-fold above that in A7r5 controls in the 6B5N cells (Table
2).
Despite the failure of the antisense Cx43 mRNA in these clones to
produce substantial knockdown of sense Cx43 mRNA, the data described
indicated that we had successfully isolated stable cell lines with
expression ratios differing by approximately fivefold. To determine
whether protein levels were well predicted by the mRNA levels, we used
Western blots to assay Cx43 and Cx40 levels in total protein isolated
from each of these cell lines. Initially, bound antibody was visualized
by using HRP-conjugated secondary antibody and enhanced
chemiluminescent techniques. A typical blot is shown in Fig.
5. Note that the banding pattern of Cx43
in the 6B5N and 2A6 cells is comparable to that of the parental A7r5 cells, suggesting that phosphorylation of Cx43 was comparable in these
cell types. In contrast, the CMV23 cells displayed high levels of the
more slowly migrating forms of Cx43, which have been demonstrated by
others (18, 19, 21) to be indicative of phosphorylation of
the protein and loss of communication.

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Fig. 5.
Western blots of total protein isolated from the
indicated cell lines and probed for Cx40 or Cx43 content, as indicated.
Protein loading (in µg) is indicated below each lane.
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Quantification of expression ratios using ECL proved difficult because
of variability in signal from blot to blot. Therefore, we evaluated
Cx40 and Cx43 levels in total protein from 6B5N, 2A6, CMV23, and
parental A7r5 cells by using a radioactive secondary antibody and
protein standards against which samples could be compared (see
MATERIALS AND METHODS). Typical blots for both connexins are shown in Fig. 6. The blots reveal
that both the Cx40 and Cx43 contents of the clones differed from that
of the parental cells. Relative to the A7r5 parental cells, the 6B5N
cells contained less Cx40 and comparable amounts of Cx43, the 2A6 cells
contained more Cx40 and less Cx43, and the CMV23 cells contained more
of both proteins. The results from multiple samples are summarized in
Fig. 7. These connexin contents predict
ratios (see Table 1) that differ by more than threefold between the 2A6
and 6B5N cells.

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Fig. 6.
Western blots of total protein (70 µg/lane) from CMV23
(lane A), 6B5N (lane B), 2A6 (lane C),
and A7r5 (lane D) cells probed with Cx43 (A) or
Cx40 antibody (B) and visualized with
35S-labeled antirabbit Ig. Cx43- and Cx40-specific bands
are labeled; n.s. band represents nonspecific binding of the secondary
antibody. Protein standards (A: 0.5, 1, 2, and 4 pmol;
B: 1, 2, 4, and 8 pmol) were loaded in a staggered fashion
at ~15-min intervals (left lane) in each blot.
C: counts associated with each of the protein standard bands
increase linearly as a function of their protein contents.
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Fig. 7.
Cx43 and Cx40 contents of A7r5, 6B5N, 2A6, and CMV23
clones. Cx43 data were derived from 4 sample sets; Cx40 data were
derived from 3 sample sets.
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Intercellular communication was characterized in the 6B5N, 2A6, and
CMV23 clones and was compared with that in A7r5 parental cells to
evaluate whether the change in Cx43:Cx40 ratio and the apparent
phosphorylation state of Cx43 influenced communication. gj was not different among the four cell types
(P > 0.06, ANOVA), although with a larger sample size
it is likely that a significant difference would be observed for the
CMV23 cells (Table 3). Although gj is underestimated in these experiments
because of the series resistances of the recording electrodes (Table
3), since series resistances were comparable across cell types,
gj is underestimated to the same degree across
cell types. Therefore the absence of differences in
gj among cell types is significant.
On the basis of reported differences in Cx40 vs. Cx43 channel
selectivity (1, 35), we hypothesized that the permeability of the junctions to large dye molecules would, despite the absence of
differences in gj, be significantly greater in
6B5N than in A7r5 or 2A6 cells. Furthermore, because the appearance of
Cx43 in bands that migrate more slowly on SDS-polyacrylamide gels is associated with increased phosphorylation and reduced coupling, we
hypothesized that the CMV23 cells, despite their higher Cx43:Cx40 ratio, would also be poorly coupled. To test these hypotheses, we
determined the extent of Lucifer yellow dye coupling in these cell
lines. Representative dye injections for the four cell types are shown
in Fig. 8, and data from multiple
injections are summarized in Fig. 9. The
6B5N cells were significantly better dye coupled (Lucifer Yellow) than
the other cell types. These data indicate that gap junction
permeability was elevated when the Cx43:Cx40 ratio was elevated (6B5N
cells); but this enhanced permeability can be compromised by
posttranslational modification (e.g., phosphorylation) of the Cx43
protein (CMV23 cells).

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Fig. 8.
Lucifer yellow dye coupling was extensive in 6B5N cells
(D and E) compared with that in A7r5
(A), CMV23 (B), and 2A6 cells (C).
A: 2 neighboring cells contain small amounts of dye.
B and C: low levels of dye were observed in only
1 neighboring cell (electrode observed in bottom of C).
D: the brightest cell was injected; dye was observed in many
neighboring cells very quickly after injection (picture at 5 min).
E: differential interference contrast image of cells in
D with injected cell marked by
asterisk.
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Fig. 9.
Dye coupling is significantly enhanced in 6B5N cells
(asterisk) compared with A7r5, 2A6, and CMV23 cells. The number of
injected cells was as follows: A7r5, 61; 6B5N, 59; 2A6, 20; and CMV23,
21.
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DISCUSSION |
Recent data indicate that Cx40 and Cx43 form functional
heteromeric (17), homomeric/heterotypic (31),
and heteromeric/heterotypic channels (Cottrell GT and Burt JM,
unpublished observations). Thus as many as 196 channel types (4,
17) are theoretically possible in cells that coexpress these
proteins. To understand what the functional consequences of this
diversity of channel types might be, clones of a particular cell line
that express different ratios of the connexins of interest are needed.
The goal of the present study was to generate A7r5 cell clones that display different Cx43:Cx40 expression ratios and to begin evaluation of whether they display different junctional properties.
Multiple clones of Cx43AS-transfected, vector-only-transfected, and
nontransfected A7r5 cells were isolated. These clones displayed a range
of Cx43:Cx40 mRNA ratios. In some clones, Cx40 mRNA levels were altered
to a far greater extent than Cx43 mRNA levels. However, the changes in
Cx40 mRNA levels were unrelated to the efficiency of transfection,
which suggests that the variability in Cx40 levels represents
spontaneous clonal variation. In general, clones with elevated
Cx43:Cx40 mRNA ratio also had elevated Cx43:Cx40 protein ratio;
however, the mRNA levels for each connexin were not strictly predictive
of the protein levels for the connexins. The failure of mRNA levels to
accurately predict protein levels may indicate that connexin gene
expression is regulated at multiple levels, transcriptionally as well
as posttranscriptionally. The finding that changes in the level of one
connexin did not consistently alter expression of the other indicates
that expression of these two connexins must be independently regulated.
The significance of altered protein ratios to junctional function
appears profound. Although junctional conductance was comparable across
cell types, the 6B5N cells were >25 times better dye coupled than the
A7r5 cells. Given the similarity of series resistances across cell
types, it is highly unlikely that complications arising from series
resistance would cause a 25-fold underestimate of gj in the 6B5N vs. A7r5 cells. Thus the data
suggest that the nearly 2-fold difference in Cx43:Cx40 expression ratio
in 6B5N vs. A7r5 cells (0.67 vs. 0.37) caused a >25-fold difference in the extent of dye coupling with no change in electrical coupling.
If heteromeric channel assembly is a random process, then the relative
contribution of each of the 196 possible channel types to the total
pool of channels should reflect the expression ratio of the two
connexins. Assuming random assembly, the probability of forming
a specific connexon can be calculated as
where %Cx1 is the percentage of connexin1 in the
total pool of connexins, n = 6 (connexins in a
connexon), and r is the number of subunits of
Cx1 in the heteromer of interest. The probability of
forming a specific channel can be calculated as the product of the two
connexon probabilities. Assuming random assembly, probability theory
predicts that an increase in Cx43:Cx40 expression ratio from 0.37 to
0.67 (A7r5 vs. 6B5N) would result in an ~118-fold increase in
homomeric/homotypic Cx43 channels and a 14-fold increase in the channel
population wherein both connexons contain four or more Cx43 subunits
(see Table 4). We do not know the
connexin composition of the channel types that mediate the bulk of the dye permeation observed in our studies. Homomeric/homotypic Cx40 channels appear to be highly cation selective, whereas Cx43 channels are reasonably well permeated by ions of both charges as well as by
large fluorescent dyes. The permeation properties of Cx40/43 heterotypic and heteromeric channels are unknown. However, published data indicate that the behavior of such channels is not readily predicted by the behavior of the homomeric/homotypic channels. He et
al. (17), Valiunas et al. (31), and Cottrell
and Burt (unpublished work) have demonstrated that single-channel
amplitudes and voltage-dependent gating were significantly different in
the heterotypic and heteromeric settings relative to the
homomeric/homotypic setting, suggesting that heteromeric channels may
exhibit different selectivities and gating properties. Probability
theory predicts that small changes in expression ratio could
significantly alter the connexin composition of the channel population.
Our data suggest that changes in expression ratio profoundly influence
gap junction selectivity.
If such changes in expression ratio were to occur in vivo during
development (11, 33) or as a consequence of disease, injury, or disturbed flow (13, 14, 27, 28, 32), then our
data suggest that electrical signaling could be preserved while
intercellular exchange of metabolites and large second messenger signaling molecules would be greatly altered. In cells that rely on
electrical signals to coordinate their activity, such as the heart and
blood vessels, the capacity to maintain electrical signaling while
dramatically altering the exchange of other types of signaling molecules could be of considerable importance to organ function and
animal survival. Kurjiaka et al. (20) demonstrated that A7r5 cells maintained in 1% vs. 10% serum were significantly better dye coupled despite comparable levels of electrical coupling. The
present data suggest that this enhanced dye coupling could result from
an increase in the Cx43:Cx40 expression ratio in the growth-arrested cells.
In summary, we have generated several cell lines that express a range
of Cx43:Cx40 protein ratios. The data from these cell lines suggest
that junctional permeability can be regulated not only through
phosphorylation of channel proteins but also through alteration of the
connexin composition of heteromeric channels. The latter strategy would
likely occur over a longer time course, comparable to protein turnover
rates, and is thus complementary to the phosphorylation strategy.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood
Institute Grants HL-58732 and HL-07249, Arizona Disease Control Research Commission Award 1-217, and American Heart Association, Arizona Affiliate Award AZGS-35-97.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. M. Burt, Dept. of Physiology, Univ. of Arizona, AHSC, Rm 4103, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail:
jburt{at}u.arizona.edu).
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.
Received 1 June 2000; accepted in final form 15 September 2000.
 |
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