© 2000 American Heart Association, Inc.
UltraRapid Communications |
Formation of Heterotypic Gap Junction Channels by Connexins 40 and 43
From the Department of Physiology and Biophysics (V.V., P.R.B.), State University of New York at Stony Brook, NY; Department of Physiology (R.W.), University of Bern, Switzerland.
Correspondence to Peter R. Brink, Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794. E-mail peter{at}patch.pnb.sunysb.edu
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Abstract—Gap junctions formed between transfected cells expressing connexin (Cx) 40 and Cx43 (Cx43-RIN, Cx40-HeLa, and Cx43-HeLa) revealed a relationship, gj=f(Vj), at steady state, that is typified by a nonsymmetrical behavior similar to that previously reported for other heterotypic channels (gap junction conductance [gj]; transjunctional voltage [Vj]). The unitary conductance of the channels was sensitive to the polarity of Vj. A main state conductance of 61 pS was found when the Cx43 cell was stepped positively or the Cx40 cell negatively (Vj=70 mV); the reverse polarities yielded a conductance of 100 pS. These heterotypic channels were permeable to carboxyfluorescein. In addition, two other heterotypic forms are illustrated to demonstrate that endogenous Cx45 expression cannot explain the results. The demonstration of heterotypic Cx40–Cx43 channels may have implications for the propagation of the electrical impulse in heart. For example, they may contribute to the slowing of the impulse propagation through the junctions between Purkinje fibers and ventricular muscle. The full text of this article is available at http://www.circresaha.org.
Key Words: gap junction • ion channel • electrophysiology
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Introduction |
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Coexpression of connexins (Cx) has been demonstrated in a number of tissues including vascular smooth muscle1 and myocardium.2 For example, Cx43 and Cx40 are present in the sinoatrial (SA)-node of dogs and guinea pigs3 4 5 or Cx43, Cx40, and Cx45 in the atrioventricular (AV)-node of humans, rats, and guinea pigs.6 7 8 This finding immediately raises the question about the types of gap junction channels present. In principle, three types of channels can be constituted, ie, homotypic, heterotypic, and heteromeric channels.
Homotypic gap junction channels are defined to consist of two identical hemichannels made from one type of connexin. Such channels have been studied extensively under in vitro conditions (eg, see References 99 –11). Heteromeric channels are defined to consist of two hemichannels composed of more than one type of connexin. However, the existence of such channels in situ is still controversial. Some authors concluded from in vitro and in vivo studies that heteromeric channels exist, exhibiting properties not profoundly different from those of their homotypic counterparts12 13 14 (but see also References 1515 ). Other authors concluded from in vitro studies that there is no unambiguous functional evidence for the presence of heteromeric channels.16 Heterotypic channels consist of two different hemichannels, each of which is made of a different type of connexin. Such channels were demonstrated unequivocally in vitro.12 16 17 18 19 20 However, not every homomeric hemichannel will form a functional gap junction channel with another homomeric hemichannel derived from a different connexin.21
A number of investigators have reported that Cx40 and Cx43 are unable to form functional heterotypic channels between injected oocytes20 22 and transfected HeLa cells.23 This finding was explained as being the result of low levels of connexin expression and/or structural incompatibility between the extracellular loops E1 and E2 of Cx43 and Cx40.23 There are several reasons that heterotypic Cx40–Cx43 channels may exist. One argument relies on functional evidence. Examining isolated myocytes from adult rat hearts, two authors of the present study have recently obtained electrophysiological data indicative of heterotypic Cx40–Cx43 channels (L. Polonchuk, V.V., J.A. Haefliger, H. Eppenberger, R.W., unpublished data, 1998). Another argument is based on structural similarities. A comparison of the amino acid residues of Cx40 and Cx43 indicates that 80% of E1 and 53% of E2 are conserved. A similar degree of homology exists between Cx37 and Cx43, ie, 83% and 55% for E1 and E2, respectively, two candidates that have been shown to establish heterotypic channels between oocytes21 22 and transfected N2a cells.12
The aim of the present study has been to investigate the possible formation of heterotypic gap junction channels between Cx40 and Cx43. For this purpose, we have used HeLa cells transfected with either Cx40 or Cx43 and RIN cells transfected with Cx43. The transfectants were cocultured (Cx43-RIN cells and Cx40-HeLa cells; Cx40-HeLa cells and Cx43-HeLa cells), and the putative formation of heterotypic channels was assessed using a dual-voltage clamp method. In contrast to previous studies, the analysis of multichannel currents as well as single-channel currents revealed properties consistent with the presence of heterotypic Cx40–Cx43 channels.
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Materials and Methods |
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Cells and Culture Conditions
Experiments were carried out on human HeLa cells transfected with cDNA coding for mouse Cx40, Cx43, and Cx45 and rat islet tumor (RIN) cells transfected with cDNA coding for rat Cx43 and chicken Cx45 DNA. HeLa cells and RIN cells were grown in DMEM and RPMI 1640 medium, respectively, supplemented with 10% FCS, 100 µg/mL streptomycin, and 100 U/mL penicillin. The media also contained 0.4 mg/mL G418 (Geneticin, Life Technologies). The cells were passaged weekly, diluted 1:10, and kept at 37°C in a CO2 incubator (5% CO2/95% ambient air). For later identification, some cultures were previously tagged with cell tracker green (5-chloromethyl-fluorescein diacetate; Molecular Probes).12 Tagged cells expressing one type of connexin and nontagged cells expressing the other type of connexin were mixed and seeded onto sterile glass coverslips placed in multiwell culture dishes (
104 cells/cm2).
Electrophysiological measurements were carried out
on cells cultured for 1 to 3 days.
Solutions
During experiments, the cells were superfused with bath solution
containing (in mmol/L) CsCl 110, KCl 5,
CaCl2 2, MgCl2 1, and HEPES
10 (pH 7.4). The patch pipettes were filled with saline containing
(in mmol/L) CsCl 110, MgCl2 0.1,
CaCl2 0.1, EGTA 3, and HEPES 10 (pH
7.2).
Electrical Measurements
Glass coverslips with adherent cells were transferred to
an experimental chamber perfused with bath solution at room temperature
(21°C to 23°C). The chamber was mounted on the stage of an inverted
microscope (Olympus IMT2). Patch pipettes were pulled from glass
capillaries (code 7052; A-M Systems) with a horizontal puller (Sutter
lnstruments). When filled, the resistance of the pipettes measured 1 to
3 M
. Experiments were carried out on mixed cell pairs. A
dual-voltage clamp method and whole-cell recording were used to
control the membrane potential of both cells and to measure
currents.9 10 Each cell was attached to a patch pipette
connected to a separate micromanipulator (WR-88; Narishige Scientific
Instrument) and amplifier (Axopatch 200). Initially, the membrane
potential of cell 1 and cell 2 was clamped to the same value,
V1=V2.
V2 was then changed to establish a
transjunctional voltage,
Vj=V2-V1.
Currents recorded from cell 2 represent the sum of two
components, the junctional current, Ij, and
the membrane current of cell 2, Im, 2; the
current obtained from cell 1 corresponds to
Ij. To measure
Ij, both cells were held at the same
holding potential, ie, Vh=0 mV. The voltage
of one of the cells was then stepped to different levels.
Signal Recording and Analysis
Voltage and current signals were recorded on chart
paper (Gould RS 2400; Gould Instruments) and videotape (Neurocorder
DR-384; Neuro Data Instruments). For offline analysis, the
current signals were filtered at 1 kHz (low-pass filter), digitized
with a 12-bit A/D converter (DT21EZ, Data Translation), and stored with
a personal computer. Data acquisition and analysis were
performed with custom-made software.10 24
Curve-fitting and statistical analysis were done with
SigmaPlot and SigmaStat, respectively (Jandel Scientific). The data are
presented as mean values ±1 SEM.
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Results |
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The formation of homotypic channels consisting of Cx40 or Cx43 has been demonstrated in several studies examining cells in primary culture11 and transfected cells.9 10 25 26 For illustration, Figures 1A
and 1B show records from a pair of
transfected Cx40-HeLa cells and a pair of transfected Cx43-RIN cells,
respectively. Starting from a Vh of 0 mV,
bipolar pulses of 800 ms in duration were administered to establish
Vj gradients of identical amplitude and
either polarity. Vj was then altered from
±10 to ±110 mV using increments of 20 mV. The associated currents
Ij increased proportional with
Vj and showed a voltage- and time-dependent
inactivation. The amplitudes of Ij were
determined at the beginning (Ij, inst;inst: instantaneous) and end of each pulse
(Ij, ss; ss: steady state) to
estimate the conductances gj, inst and
gj, ss, respectively. The values of
gj, ss were normalized with respect to
gj, inst and plotted versus
Vj. As shown in Figure 1C, this
resulted in a symmetrical relationship for both types of cell pairs
(•: Cx40-HeLa cells; 
: Cx43-RIN cells). The analysis
revealed the following Boltzmann parameters: Cx40
(continuous curve): Vj,0=56 mV,
gj, min=0.18, z=2.1; Cx43:
Vj,0=66 mV,
gj, min=0.23, z=2.3 (dashed
curve). These findings are consistent with previous reports on
Cx409 26 and Cx43 gap
junctions.10 11 25
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In the case of pairs of Cx40-HeLa cells, the single-channel
conductances of the main state and residual state,
j, main and
j, residual, determined in preparations with
one operational channel, turned out to be 135 and 26 pS, respectively
(data not shown). In the case of pairs of Cx43-RIN cells, these
conductances averaged 76 and 18 pS (data not shown). These values are
consistent with previous reports on Cx409 26 and
Cx43 channels.10 11 25
To examine whether heterotypic Cx40–Cx43 channels are formed, Cx40-HeLa cells were cocultured with Cx43-RIN or Cx43-HeLa cells (see Materials and Methods). Dual whole-cell voltage-clamp experiments were performed on cell pairs with one cell stained with cell tracker and the other devoid of it. Of 23 cell pairs examined, 20 exhibited functional coupling at the macroscopic or microscopic current level, whereas 3 showed no coupling. In the case of Cx40-HeLa–Cx43-RIN preparations, gj averaged 3.8±1.6 nS (n=12); in the case of Cx40-HeLa–Cx43-HeLa preparations, gj averaged 6.8±2.9 nS (n=8).
To further explore the properties of Ij in
mixed cell pairs, the bipolar pulse protocol (see Figures 1A
and 1B) was used to alter Vj from ±10 to ±150
mV and generate a family of currents. Figure 2A illustrates an experiment with a
Cx40-HeLa–Cx43-RIN preparation. Inspection of the current signals
indicated that the size and time course of
Ij are not only sensitive to the amplitude
of Vj but also to its polarity. This means
Ij was asymmetrical at each
Vj. Specifically, stepping
Vj to make the inside of the Cx43-RIN cell
negative or the inside of the Cx40-HeLa cell positive resulted in a
large Ij, inst with pronounced
inactivation. Conversely, stepping Vj to
make the inside of the Cx43-RIN cell positive or the inside of the
Cx40-HeLa cell negative led to a small
Ij, inst with marginal or no inactivation.
Consistent with these findings, inversion of the
Vj polarity during bipolar pulses revealed
that Ij recovery is faster in the former
case. Experiments performed on Cx40-HeLa–Cx43-HeLa preparations (data
not shown) yielded Ij patterns comparable
to those seen in Cx40-HeLa–Cx43-RIN cell pairs.
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Figures 2C and 2D summarize the data gathered from
Cx40-HeLa–Cx43-RIN (n=10) and Cx40-HeLa–Cx43-HeLa preparations (n=6).
They show the normalized relationships
gj, inst versus
f(Vj) () and
gj, ss versus
f(Vj) (•). Both plots were
strongly asymmetrical. The instantaneous gj
increased when the cell expressing Cx43 was made negative inside and
decreased when it was made positive. The increase in
gj, inst peaked at 1.15 between
Vj=-100 and -150 mV. The decrease in
gj, inst reached a value of 0.7 at
Vj=150 mV. In contrast, the steady-state
gj declined when the Cx43 cell was made
negative or positive. In the former case, it decreased in a sigmoidal
fashion to a quasi stable level at a Vj of
150 mV. In the latter case, it decreased gradually without reaching
a plateau, ie, it followed nearly Ij,
inst. The continuous curves at negative values of
Vj represent the best fit of data
to the Boltzmann equation using the following parameters:
Figure 2C: Vj,0=-82 mV,
gj, min=0.31, gj,
max=1.03, z=1.86; Figure 2D:
Vj,0=-89 mV, gj,
min=0.33, gj, max=0.98,
z=1.67. When compared with the respective homotypic channels
(see Figure 1C), the heterotypic channels exhibit a more
negative Vj,0 and a broader voltage
sensitivity. This is consistent with previous reports on
heterotypic Cx26–Cx3216 17 18 and Cx37–Cx43
channels.12
Diffusion experiments on Cx40-HeLa–Cx43-RIN cell pairs indicated that
Cx40–Cx43 channels are permeable to 5- (and 6-)
carboxyfluorescein (Molecular Probes), the
fluorescent anionic dyes (Figure 2B).
Figure 3 illustrates experiments designed
to study the Vj sensitivity of single
heterotypic channels. Figure 3A shows records of a
Cx40-HeLa–Cx43-RIN cell pair whose gap junction contained a single
operational channel. Biphasic pulses were applied to cell 1
(V1; Cx43-RIN cell) while gap junction
currents were recorded from cell 2 (I2;
Cx40-HeLa cell). As illustrated for
Vj=±70, ±90, and ±110 mV,
I2 showed three discrete levels
corresponding to the closed state (continuous lines), main state, and
residual state (dashed lines). The analysis indicated that the
unitary currents are smaller when cell 1 is depolarized (positive
Vj) than when it is hyperpolarized
(negative Vj). Moreover, residual currents
were preferentially seen in the latter case, ie, voltage gating was
virtually absent at positive Vj. The
observed single channel conductances were j,
main=60/100 pS (positive/negative
Vj); j,
residual=14 to 20 pS (negative
Vj).
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Figure 3B shows an experiment carried out on a
Cx40-HeLa–Cx43-RIN cell pair whose gap junction consisted of several
operational channels. This time a bipolar voltage-ramp protocol was
used to alter Vj from -50 to 50 mV and
back to -50 mV again. The ramps evolved at a rate of 100 mV/200 ms
(see V1 and
V2). The associated junctional current
changed linearly with time with a distinct break
Vj=0 mV
(I2). The dashed lines were aligned to the
shallower segments and correspond to a slope conductance of 0.64 nS,
indicating the involvement of at least 10 channels. The steeper segment
yielded a slope conductance of 1 nS. Conceivably, the break in slope
reflects the Vj sensitivity of the
channels. An involvement of channel inactivation seems unlikely because
of the short duration of the ramp and the small amplitude of
Vj.
Figure 3C summarizes the combined results from the
Cx40-HeLa–Cx43-RIN cell pairs (n=7). The plot j,
main=f(Vj) (•)
demonstrates that over the voltage range examined, ie, ±150 mV, the
channels exhibit a strong dependence on Vj
polarity and a weak dependence on Vj
amplitude. Because of limitations of resolution, it was not possible to
gain reliable data for Vj<||30||mV. The
plot j,
residual=f(Vj) (
)
indicates that the channels also exhibit a weak dependence on
||Vj||.
Figure 3D compares the I-V relationships of the
single-channel data documented in Figure 3C and the multichannel
data presented in Figure 2C. The currents
Ij, inst (•) and
Ij, main (•) were normalized with
respect to the values at Vj=-150 mV,
averaged, and plotted versus Vj,
respectively. The currents Ij, residual
() were scaled to account for the normalization of the
Ij, main values, averaged, and plotted
versus Vj. The analysis yielded the
following slopes for the relationships
Ij(normalized)=f(Vj):
multichannel currents:
-3.93x10-3/-6.85x10-3
mV-1
(r2=0.98/0.99) for positive/negative
Vj; single-channel currents, main state:
-3.90x10-3/-6.74x10-3
mV-1
(r2=0.99/0.99); residual state:
-8.99x10-4/-7.23x10-4
mV-1
(r2=0.83/0.98).
Involvement of Intrinsic Connexins. The possible
contribution of endogenous coupling in the mixed-cell pairs
was explored by studying a number of relevant combinations of HeLa and
RIN cells. For this purpose, small test pulses (10 mV, 70 ms) were
administered to one cell of a pair to determine
gj without interference from voltage and
time dependence. The Table summarizes the results
gathered from the different types of cell pairs. A comparison of the
gj data indicates that both HeLa cells and
RIN cells express a marginal level of intrinsic connexins. Pairs
consisting of identical parental cells (HeLa–HeLa and RIN–RIN)
yielded a weak coupling (0.04 and 0.13 nS) or no coupling at all (90%
and 83%). Similarly, mixed pairs containing one transfectant
(Cx43-RIN–HeLa, Cx43-HeLa–HeLa, and Cx40-HeLa–HeLa) also showed weak
coupling (not detectable, not detectable, and 0.04 nS, respectively) or
no coupling (100%, 100%, and 87%, respectively). In contrast, mixed
pairs consisting of two transfectants (Cx40HeLa–Cx43-RIN,
Cx40-HeLa–Cx43-HeLa) yielded significant coupling (3.8 and 6.8 nS,
respectively) and few coupling failures (0% and 20%, respectively).
Hence, these pairs exhibit a 170- and 190-fold or larger
gj than the respective control pairs
(Cx40HeLa–Cx43-RIN versus Cx40-HeLa–RIN and Cx43-RIN–HeLa;
Cx40-HeLa–Cx43-HeLa versus Cx40-HeLa–HeLa and Cx43-HeLa–HeLa). This
suggests that coupling in pairs of transfected cells occurs by
exogenous rather than endogenous connexins. The
gj data for homotypic pairs of Cx40-HeLa,
Cx43-HeLa, and Cx43-RIN cells in the Table are comparable to
those previously reported for these cell
lines.9 23 27 Transfection increased the
intercellular coupling at least 100-fold.27
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The Table also summarizes the unitary conductances determined
from these experiments. Pairs of parental cells showed a value of 40 to
50 and 30 pS (HeLa–HeLa and RIN–RIN), respectively. This is in
agreement with Cx45–Cx45 channels in HeLa cells.28 29
Pairs of Cx43-RIN cells and Cx43-HeLa cells yielded a value of 76 and
72 pS, respectively, indicative of Cx43–Cx43
channels,10 11 25 whereas pairs of Cx40-HeLa gave a value
of 135 pS, comparable with Cx40–Cx40 channels.9 26 Pairs
of Cx40-HeLa–HeLa or Cx40-HeLa–RIN might be expected to form
Cx40–Cx45 channels. A few unitary conductance values yielded 30 to 40
and 20 pS, which are not consistent with the Cx40–Cx43
heterotypic channel in the present study. The unitary conductances
quoted for the pairs Cx40-HeLa–Cx43-RIN and Cx40-HeLa–Cx43-HeLa refer
to the values prevailing at Vj=±70 mV.
They were taken from the experiments already described in Figure 3. Finally, when examining Cx40 and Cx43 transfectants, we
rarely noticed signs of endogenous channels (see also
Reference 99 ). Conceivably, they may even be suppressed after
transfection from a functional point of view.
The bipolar pulse protocol was then used to study multichannel currents
from a Cx43-HeLa–Cx45-HeLa cell pair (Figure 4A) and a Cx40-HeLa–Cx45-HeLa cell pair
(Figure 4B). In the former case, the stepping
Vj to make the inside of the Cx45-HeLa cell
negative or the inside of the Cx43-HeLa cell positive resulted in an
Ij, inst with pronounced inactivation.
Conversely, stepping Vj to make the inside
of the Cx45-HeLa cell positive and the inside of the Cx43-RIN cell
negative led to an Ij, inst with
distinct activation. In the latter case, the current signals revealed a
similar picture, ie, Ij
inactivated when the inside of the Cx45-HeLa cell was made
negative or the inside of the Cx40-HeLa was made positive.
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Figures 4C and 4D show the analysis of the current
records depicted in Figures 4A and 4B. They
represent the normalized relationships gj,
inst versus Vj () and
gj, ss versus
Vj (•). Both plots were strongly
asymmetrical. In the case of Cx43-HeLa–Cx45-HeLa (Figure 4C),
the instantaneous gj decrease was more
pronounced when the cells expressing Cx45 were made negative. In the
case of Cx40-HeLa–Cx45-HeLa (Figure 4D), the instantaneous
gj decreased when Cx45 cells were made
positive. In contrast, the steady-state gj
increased when the cell expressing Cx45 was made positive inside and
decreased when it was made negative. Note that
gj was far from steady state at the end of
the 400-ms pulse applied (see Figures 4A and 4B).
When compared with Cx40-HeLa–Cx43-RIN (see Figures 2A and 2C) and Cx40-HeLa–Cx43-HeLa cell pairs (see Figure 2D), the
currents and conductances of Cx43-HeLa–Cx45-HeLa (Figures 4A and 4C) and Cx40-HeLa–Cx45-HeLa cell pairs (see Figures 4B and 4D) behave differently. They deviate with regard to
Vj dependence, gating polarity, current
patterns, kinetics of inactivation, and the presence or absence of
activation of Ij. This suggests that
Cx43–Cx45 and Cx40–Cx45 channels are likely not to be involved in the
Cx40-HeLa–Cx43-RIN and Cx40-HeLa–Cx43-HeLa cell pairs.
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Our experiments indicate that the incidence of electrical coupling in parental HeLa cells and RIN cells is low. After transfection with Cx40 or Cx43, coupling was enhanced
200-fold in Cx40-HeLa–Cx43-HeLa
and Cx40-HeLa–Cx43-RIN cell pairs. This suggests formation of
heterotypic Cx40–Cx43 channels.
The multichannel and single-channel data presented indicate
that Cx40–Cx43 channels are present in Cx40-HeLa–Cx43-HeLa and
Cx40-HeLa–Cx43-RIN cell pairs. With regard to multichannel data, the
relationships gj,
inst=f(Vj) and
gj,
ss=f(Vj) were
asymmetrical, a property seen in heterotypic gap junctions whose
connexons exhibit widely different properties (eg,
Cx26–Cx3216 17 18 or Cx37–Cx43.12 21 In
the case of Cx40–Cx43, rectification was prominent for both
relationships, ie, gj,
ss=f(Vj) and
gj,
inst=f(Vj) (see
Figures 2C and 2D). Voltage gating was almost exclusively
observed at negative Vj. This is
consistent with the notion that Cx40 is gating with positive
polarity (V.V., R.W., P.R.B., unpublished observations, 1999)
and Cx43 with negative polarity.30 With regard to
single-channel data, the function j,
main=f(Vj) was
characterized by two phenomena, a strong dependence on
Vj polarity and a weak dependence on
Vj amplitude (see Figure 3C).
Although the latter has been anticipated on theoretical grounds, the
former is not.31 Because of limited resolution,
values of gj, main (and
gj, residual) could not be determined
accurately for Vj<||30||mV. Hence, the
function gj,
main=f(Vj)
remains undefined over this voltage range. It may undergo an
abrupt change at Vj=0 mV or a gradual
change over a narrow range of Vj values,
ie, from -30 to 30 mV. The function j,
residual=f(Vj)
showed a distinct voltage dependence for either
Vj polarity.
Significant interference from endogenous connexins is
unlikely because of the low level of expression. This conclusion is
based on several observations. For example, coupling between parental
cells and between Cx40 or Cx43 transfectants and parental cells is
rudimentary or absent (see Table). Moreover, the incidence of
single-channel currents attributable to intrinsic connexins is
extremely low in pairs of transfectants. Furthermore, pairs of
Cx45-HeLa cells with Cx40-HeLa or Cx43-HeLa (Cx43-RIN) cells yielded
asymmetrical relationships
gj=f(Vj)
distinctly different from those seen in Cx40-HeLa–Cx43-HeLa or
Cx40-HeLa–Cx43-RIN cell pairs (compare Figures 2 and 4).
Most striking is the change in Vj polarity
responsible for channel gating. Such properties were not detectable in
our data from Cx40-HeLa–Cx43-HeLa or Cx40-HeLa–Cx43-RIN cell pairs
(compare Figures 2 and 4). This suggests that intrinsic
connexins are expressed at a low level of expression. Finally, the
properties of single channels seen between parental cells and between
Cx40 or Cx43 transfectants and parental cells are different from those
observed in Cx40-HeLa–Cx43-HeLa or Cx40-HeLa–Cx43-RIN cell pairs (see
Table).
What are the mechanisms underlying rectification of
gj, inst and gj,
ss observed for Cx40–Cx43 gap junctions and gap junction
channels? On the one hand, gj, ss
reflects the properties of voltage-sensitive gating of the channels.
Hence, rectification of gj, ss can
be explained by the opposite gating polarity of Cx40 and Cx43 (see
above). On the other hand, gj, inst
reflects the properties of j, main.
Hence, rectification of gj, inst can
be explained by the properties of hemichannels arranged in series. It
has been shown that the symmetrical functions
gj=f(Vj)
of a homotypic gap junction require two hemichannels with an identical
nonlinear I/V relationship.31 Compared
with this, two hemichannels with different properties led to the
asymmetrical functions characteristic of heterotypic gap junctions.
This suggests that the hemichannel functions hc,
main=f(Vm) of Cx40
and Cx43 arranged in series determine the properties of
j,
main=f(Vj).
Rectification of j, main (and
j, residual) may be caused by
interactions between ionic charge carriers and channel structures.
Alternatively, it may reflect alterations of the effective channel
geometry caused by the electric field. Another possibility is that the
channels exhibit rapid gating beyond the time resolution of the data
recording (1 to 2 ms). Yet another possibility is partial
channel block. However, the data in Figure 3C argue against this
because the block did not increase with increasing
Vj.
The heart is a prominent organ whose tissues coexpress Cx40, Cx43, and Cx45.32 33 During maturation, mouse hearts show overlapping (left ventricle) and complementary (atrium) expression of Cx40, Cx43, and Cx45.32 33 34 In adult hearts, Cx40 and Cx43 are distributed unequally: SA-node: Cx40>>Cx43; atrium: Cx40>Cx43; AV-node: Cx43>Cx40 (however, Cx45 is the major connexin); Purkinje fiber: Cx40>>Cx43; and ventricle: Cx43>>Cx40. Cx45 distribution is controversial. Coppen et al32 have demonstrated prevalent Cx45 expression in conduction tissues whereas Alcolea et al33 have presented data that indicate a downregulation of Cx45 throughout the pacemaker-conducting system such that the distribution appears to be slight in adult heart. Hence, prominent regions with coexpression of Cx40 and Cx43, and possibly Cx45, include junctional interphases in tissues such as Purkinje fibers and ventricular muscle or atrial muscle and AV-node as well as specialized tissues (ie, atrium, AV-node). Thus the properties of Cx40–Cx43, Cx45–Cx40, and/or Cx45–Cx43 heterotypic channels may lead to the following phenomena. At junctional interphases between specialized tissues, rectification of Cx40–Cx43 channels is expected to facilitate intercellular current flow when the Cx40 cell is depolarized and to impair it when the Cx43 cell is depolarized. Similarly, for the Cx40–Cx45 channel, depolarization of the Cx40 cell will facilitate current flow whereas depolarization of the Cx45 cell will impair current flow. Hence, for these two cases, propagation of action potentials will be accelerated orthodromically and decelerated antidromically. For the Cx43–Cx45 case when the Cx45 cell is depolarized, intercellular current flow is facilitated whereas depolarization of the Cx43 cell will impair current flow. Any one of these combinations could result in enhanced or impaired action potential propagation from conducting to working myocardium, depending on the connexin distribution and amount within the individual cells. In specialized tissues, the intercellular resistance is expected to be reduced when compared with the presence of homotypic channels alone. The decrease depends on the fraction of heterotypic channels. This follows from the serial arrangement of Cx40–Cx43 hemichannels versus Cx40–Cx40 and Cx43–Cx43 hemichannels and the possible involvement of some fraction of homotypic and heterotypic channels including Cx45 hemichannels.16
Currents carried by heterotypic Cx40–Cx43 channels represent a novel finding. The induced cell pair approach may have prevented their detection in transfected HeLa cells.23 This would be the case provided Cx40–Cx43 channels form more slowly than others. Presumably, such channels were not seen in paired Xenopus oocytes because of the low expression level of exogenous connexins or the suboptimal temperature for processing vertebrate proteins.20 22 It is also possible that the formation of Cx40–Cx43 channels relies critically on the presence of cell adhesion molecules such as L-CAM or cadherins,35 36 a requirement ideally met in RIN and HeLa cells. Interestingly, Cx26–Cx37, Cx32–Cx37, and Cx32–Cx43 establish no heterotypic channels in injected oocytes37 but do so in transfected HeLa cells (F.F. Bukauskas, R.W., unpublished data, 1995).
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Acknowledgments |
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This work was supported by a National Institutes of Health grant (GM55263, to P.R.B.) and a Swiss National Science Foundation grant (31-55297.98, to R.W.). The authors would like to thank Dr T. Steinberg for parental RIN cells and Cx43-transfected cells, Dr K. Willecke for Cx45-HeLa cells, and Dr E. Beyer for advice and support. The authors acknowledge the expert technical assistance of Laima Valiuniene.
Received December 3, 1999; accepted January 3, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Christ GJ, Spray DC, el-Sabban M, Moore LK, Brink PR. Gap junctions in vascular tissues. Evaluating the role of intercellular communication in the modulation of vasomotor tone. Circ Res. 1996;79:631–646.
2. Beyer EC. Gap junctions. Int Rev Cytol. 1993;137C:1–37.
3.
Kanter HL, Laing JG, Beau SL, Beyer EC, Saffitz JE.
Distinct patterns of connexin expression in canine Purkinje fibers and
ventricular muscle. Circ Res. 1993;72:1124–1131.
4.
Kanter HL, Laing JG, Beyer EC, Green KG, Saffitz JE.
Multiple connexins colocalize in canine ventricular myocyte
gap junctions. Circ Res. 1993;73:344–350.
5.
ten Velde I, de Jonge B, Verheijck EE, van Kempen MJ,
Analbers L, Gros D, Jongsma HJ. Spatial distribution of connexin43, the
major cardiac gap junction protein, visualizes the cellular network for
impulse propagation from sinoatrial node to atrium. Circ
Res. 1995;76:802–811.
6. Gourdie RG, Green CR, Severs NJ, Thompson RP. Immunolabelling patterns of gap junction connexins in the developing and mature rat heart. Anat Embryol (Berl). 1992;185:363–378.[Medline] [Order article via Infotrieve]
7. Davis LM, Rodefeld ME, Green K, Beyer EC, Saffitz JE. Gap junction protein phenotypes of the human heart and conduction system. J Cardiovasc Electrophysiol. 1995;6:813–822.[Medline] [Order article via Infotrieve]
8.
van Kempen MJ, Fromaget C, Gros D, Moorman AF, Lamers
WH. Spatial distribution of connexin43, the major cardiac gap junction
protein, in the developing and adult rat heart. Circ Res. 1991;68:1638–1651.
9. Bukauskas FF, Elfgang C, Willecke K, Weingart R. Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells. Biophys J. 1995;68:2289–2298.[Medline] [Order article via Infotrieve]
10.
Brink PR, Ramanan SV, Christ GJ. Human connexin 43 gap
junction channel gating: evidence for mode shifts and/or
heterogeneity. Am J Physiol. 1996;271:C321–C331.
11.
Valiunas V, Bukauskas FF, Weingart R. Conductances and
selective permeability of connexin43 gap junction channels examined in
neonatal rat heart cells. Circ Res. 1997;80:708–719.
12. Brink PR, Cronin K, Banach K, Peterson E, Westphale EM, Seul KH, Ramanan SV, Beyer EC. Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37. Am J Physiol. 1997;273:C1386–C1396.
13. Brink PR, Valiunas V, Christ GJ. Homotypic, heterotypic and heteromeric gap junction channels. In: Peracchia C, ed. Gap Junctions. New York, NY: Academic Press. In press.
14.
He DS, Jiang JX, Taffet SM, Burt JM. Formation of
heteromeric gap junction channels by connexins 40 and 43 in vascular
smooth muscle cells. Proc Natl Acad Sci U S A. 1999;96:6495–6500.
15.
Bevans CG, Harris AL. Direct high affinity modulation
of connexin channel activity by cyclic nucleotides.
J Biol Chem. 1999;274:3720–3725.
16. Valiunas V, Niessen H, Willecke K, Weingart R. Electrophysiological properties of gap junction channels in hepatocytes isolated from connexin32-deficient and wild-type mice. Pflügers Arch. 1999;437:846–856.
17. Verselis VK, Ginter CS, Bargiello TA. Opposite voltage gating polarities of two closely related connexins. Nature. 1994;368:348–351.[Medline] [Order article via Infotrieve]
18. Bukauskas FF, Elfgang C, Willecke K, Weingart R. Heterotypic gap junction channels (connexin26-connexin32) violate the paradigm of unitary conductance. Pflügers Arch. 1995;429:870–872.
19.
Bukauskas FF, Vogel R, Weingart R. Biophysical
properties of heterotypic gap junctions newly formed between two types
of insect cells. J Physiol (Lond). 1997;499:701 713.
20. White TW, Paul DL, Goodenough DA, Bruzzone R. Functional analysis of selective interactions among rodent connexins. Mol Biol Cell. 1995;6:459–470.[Abstract]
21. White TW, Bruzzone R. Multiple connexin proteins in single intercellular channels: connexin compatibility and functional consequences. J Bioenerg Biomembr. 1996;28:339–350.[Medline] [Order article via Infotrieve]
22. Bruzzone R, Haefliger JA, Gimlich RL, Paul DL. Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell. 1993;4:7–20.[Abstract]
23. Haubrich S, Schwarz HJ, Bukauskas F, Lichtenberg-Frate H, Traub O, Weingart R, Willecke K. Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains. Mol Biol Cell. 1996;7:1995–2006.[Abstract]
24. Krisciukaitis A. Computer programs for investigation of intercellular communication using double whole-cell voltage clamp. Electron Electric Eng. 1997;4:71 83.
25. Banach K, Weingart R. Connexin43 gap junctions exhibit asymmetrical gating properties. Pflügers Arch. 1996;431:775–785.
26.
Beblo DA, Veenstra RD. Monovalent cation permeation
through the connexin40 gap junction channel. Cs, Rb, K, Na, Li, TEA,
TMA, TBA, and effects of anions Br, Cl, F, acetate, aspartate,
glutamate, and NO3. J Gen Physiol. 1997;109:509–522.
27. Traub O, Eckert R, Lichtenberg-Fraté H, Elfgang C, Bastide B, Scheidtmann KH, Hülser DF, Willecke K. Immunohistochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells. Eur J Cell Biol. 1994;64:101–112.[Medline] [Order article via Infotrieve]
28.
Coppen SR, Dupont E, Rothery S, Severs NJ. Connexin45
expression is preferentially associated with the
ventricular conduction system in mouse and rat heart.
Circ Res. 1998;82:232–243.
29. Hülser DF, Rehkopf B, Traub O. Dispersed and aggregated gap junction channels identified by immunogold labeling of freeze-fractured membranes. Exp Cell Res. 1997;233:240–251.[Medline] [Order article via Infotrieve]
30. Steiner E, Ebihara L. Functional characterization of canine connexin45. J Membr Biol. 1996;150:153–161.[Medline] [Order article via Infotrieve]
31.
Vogel R, Weingart R. Mathematical model of vertebrate
gap junctions derived from electrical measurements on homotypic and
heterotypic channels. J Physiol (Lond). 1998;510:177–189.
32.
Coppen SR, Severs NJ, Gourdie RG. Connexin45
(
6) expression delineates an extended conduction system in the
embryonic and mature rodent heart. Dev Genet. 1999;24:82–90.[Medline]
[Order article via Infotrieve]
33.
Alcolea S, Theveniau-Ruissy M, Jarry-Guichard T, Marics
I, Tzouanacou E, Chauvin JP, Briand JP, Moorman AF, Lamers WH, Gros DB.
Downregulation of connexin 45 gene products during mouse heart
development. Circ Res. 1999;84:1365–1379.
34.
Delorme B, Dahl E, Jarry-Guichard T, Briand JP,
Willecke K, Gros D, Theveniau Ruissy M. Expression pattern of connexin
gene products at the early developmental stages of the mouse
cardiovascular system. Circ Res. 1997;81:423–437.
35.
Musil LS, Goodenough DA. Biochemical analysis
of connexin43 intracellular transport, phosphorylation,
and assembly into gap junction plaques. J Cell Biol. 1991;115:1357–1374.
36. Frenzel EM, Johnson RG. Gap junction formation between cultured embryonic lens cells is inhibited by antibody to N-cadherin. Dev Biol. 1996;179:1–16.[Medline] [Order article via Infotrieve]
37.
White TW, Bruzzone R, Wolfram S, Paul DL, Goodenough
DA. Selective interactions among the multiple connexin proteins
expressed in the vertebrate lens: the second extracellular domain is a
determinant of compatibility between connexins. J Cell
Biol. 1994;125:879–892.
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