Evidence for heteromeric gap junction channels formed from rat
connexin43 and human connexin37
P. R.
Brink1,
K.
Cronin1,
K.
Banach1,
E.
Peterson1,
E. M.
Westphale2,
K. H.
Seul2,
S. V.
Ramanan1, and
E. C.
Beyer2
1 Department of Physiology and
Biophysics, State University of New York at Stony Brook, Stony
Brook, New York 11974; and
2 Department of Pediatrics,
Washington University, St. Louis, Missouri 63110
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ABSTRACT |
Homomeric gap junction channels are composed solely of one
connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from
each other. A heteromeric gap junction channel is one that contains
different connexins within either or both hemichannels. The existence
of heteromeric forms has been suggested, and many cell types are known
to coexpress connexins. To determine if coexpressed connexins would
form heteromers, we cotransfected rat connexin43 (rCx43) and human
connexin37 (hCx37) into a cell line normally devoid of any connexin
expression and used dual whole cell patch clamp to compare the observed
gap junction channel activity with that seen in cells transfected only
with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or
rCx43, in which one population was tagged with a fluorescent marker to
monitor heterotypic channel activity. The cotransfected cells possessed
channel types unlike the homotypic forms of rCx43 or hCx37 or the
heterotypic forms. In addition, the noninstantaneous transjunctional
conductance-transjunctional voltage
(Gj/Vj)
relationship for cotransfected cell pairs showed a large range of
variability that was unlike that of the homotypic or heterotypic form.
The heterotypic cell pairs displayed asymmetric voltage dependence. The
results from the heteromeric cell pairs are inconsistent with summed
behavior of two independent homotypic populations or mixed populations
of homotypic and heterotypic channels types. The
Gj/Vj
data imply that the connexin-to-connexin interactions are significantly
altered in cotransfected cell pairs relative to the homotypic and
heterotypic forms. Heteromeric channels are a population of channels
whose characteristics could well impact differently from their
homotypic counterparts with regard to multicellular coordinated
responses.
homotypic channels; heteromeric channels; heterotypic channels; voltage gating
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INTRODUCTION |
GAP JUNCTION CHANNELS provide an intercellular pathway
between adjacent cells for ions and small solutes such as second
messenger molecules. Unlike other ion channels, the gap junction
channel consists of two hemichannels or connexons, each contributed by one of two adjacent cells. Hemichannels are oligomers, formed by six
protein subunits called connexins. Connexins belong to a gene family
with 13 identified members (3). On the basis of hydropathic studies,
the connexins are predicted to have four transmembrane domains, with
the amino and the carboxy termini located on the cytoplasmic side.
Adjacent cells can synthesize the same or different connexins, and in
any one cell two or more connexins can potentially be coexpressed.
Therefore, three different generic types of channels are possible:
1) homomeric/homotypic channels, in
which both interacting hemichannels are composed of the same connexin,
2) heterotypic channels, in which
the gap junction channel is formed by two hemichannels each composed of a different connexin, and 3) the
heteromeric channel, in which each hemichannel contains at least two
different connexins.
Macroscopic junctional currents for homotypic and heterotypic channels
have been studied using the Xenopus
laevis oocyte expression system. In general, homotypic
channels show symmetric voltage dependence, whereas heterotypic forms
generate asymmetric junctional currents in response to symmetric
transjunctional voltage
(Vj) steps (1,
2, 22, 29, 31-34). Heteromeric forms have been implicated in two
studies using the oocyte system (1, 28) and in a study of connexin43
(Cx43) containing osteoblasts transfected with connexin45
(11). Biochemical analysis of coexpressing cell systems
has also indicated the presence of heteromeric forms (9, 21).
A few predictions are possible for the heteromeric case when both the
unitary currents and macroscopic currents can be monitored. The number
of heteromeric channel types possible with only two coexpressing
connexins is large. If human connexin37 (hCx37) and rat connexin43
(rCx43) are coexpressed and are freely capable of mixing and forming
heteromeric hemichannels, then in any individual cell there are
26 or 64 possible forms and 4,096 heteromeric gap junction channel types could, in theory, exist. The
chances of forming a homotypic gap junction channel are then 1 in 4,096 (0.0002).
If a sixfold symmetry axis is assumed and, furthermore, if interaction
energies are rotationally symmetric, but not chiral, and if the
energies of interaction for one hemichannel are not influenced by the
configuration of another, then far fewer heteromeric forms are
predicted. In this case, there are 12 possible distinct heteromeric
hemichannel forms and 1 of each homomeric hemichannel form, 14 forms
total within a cell. For any two coupled coexpressing cells, there are
14 × 14 possible combinations or 196 types of gap junction
channel types possible, including the formation of a heterotypic type.
For all 196 forms, only one would be a homotypic hCx37 and one a rCx43
and two configurations of a single heterotypic form would exist
(hCx37-rCx43 vs. rCx43-hCx37). In addition, a homomeric hemichannel
linked to any heteromeric hemichannel in an adjacent cell is considered
to be a heteromeric gap junction channel. It is impossible to know
whether there are significant differences in gating or conductance for
so many potential forms. However, this scenario predicts the
probability of observing a homotypic hCx37 channel or rCx43 channel to
be <0.0052 (1/196). The probability of observing heterotypic channel
types would be only slightly better (0.0104). For the cases given, the
observation of channel conductances unlike homotypic hCx37 or rCx43
provides strong evidence for the presence of heteromeric and/or
heterotypic forms. Figure 1
shows a schematic for the 14 total forms possible in any one cell
coexpressing 2 mixable connexins. There are 12 mixed hemichannels and 2 homomeric hemichannels. If the ratio of Cx43 to Cx37 is large, then the
predicted forms would tend to arise from the heteromeres shown on the
left of Fig. 1. The inverse would
result in forms arising from the heteromeres shown on the
right of Fig. 1.

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Fig. 1.
Schematic representation of the types of hemichannels possible with two
equally expressed connexins capable of mixing. It is assumed that there
is 6-fold symmetry. There are 12 heteromeric hemichannel types and 2 homotypic types. Cx43, connexin43; Cx37, connexin37.
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Our aim was to investigate the effect of cotransfection of two
connexins via dual whole cell patch clamp. We examined mouse neuroblastoma cells [Neuro-2a (N2a)] cotransfected with hCx37 and
rCx43 and compared the data to those obtained from cells with homotypic
channels or heterotypic channels.
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MATERIALS AND METHODS |
Cell culture.
Experiments were performed with mouse N2a neuroblastoma cells (American
Type Culture Collection CCL-131), since these cells are normally devoid
of any connexin expression and contain no endogenous gap junction
channels that we have detected (19, 27). N2a cells were individually
transfected with the hCx37 cDNA or with the rCx43 cDNA in the vector
pSFFV-neo (7). This vector drives eukaryotic expression with the
splenic focus-forming virus LTR, incorporates a splice donor and
acceptor (from the SV40 early gene) in the 3' untranslated region
followed by a polyadenylation signal, and allows selection of
neomycin-resistant N2a clones in a 0.25 mg/ml active concentration of
G418 (Geneticin, Life Technologies). These stable transfectants have
been extensively characterized previously (19, 24, 25, 27). To generate coexpressing clones, the coding region and 5' untranslated
sequence from rCx43 (clone G2 from Ref. 4) was subcloned into pZeoSV (Invitrogen) between the Spe I and
Kpn I sites. This vector drives eukaryotic expression with the SV40 early gene promoter/enhancer, causes no splicing of the expressed mRNA, uses the SV40 polyadenylation signal, and allows selection with the non-cross-resistant antibiotic Zeocin (Invitrogen). N2a/hCx37 cells were transfected with linearized pZeoSV-rCx43 using lipofectin reagent (Life Technologies), and individual, single clones were selected with 0.25 mg/ml Zeocin. RNA was
prepared from these cells and from cultured bovine aortic endothelial
cells (BAECs) as a positive control and analyzed by RNA blotting with
an equal mixture of 32P-labeled
probes for rCx43 and hCx37 (19). Immunoblots of whole cell lysates (50 µg total protein/lane) were reacted with mouse monoclonal antibodies
to a synthetic peptide representing amino acids 252-270 of rCx43
(Chemicon) or affinity-purified rabbit polyclonal antibodies directed
against a bacterial fusion protein representing the carboxy-terminal
tail of hCx37 (8), followed by incubation with peroxidase-conjugated
secondary antibodies and detection by enhanced chemiluminescence
(Amersham) as described in Ref. 13. Immunofluorescence microscopy was
performed with these same primary antibodies according to Ref. 13.
Electrophysiology.
Experiments were carried out on transfected N2a cell pairs with the
dual whole cell voltage clamp method (5, 16, 25, 27). During the
experiments, the cells were bathed in a solution containing (in mM) 180 CsCl or KCl, 1 CaCl2, 1.8 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES) (pH 7.1-7.3). For the whole cell recording, the
pipette solution contained (in mM) 180 CsCl, 1 ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 0.1 CaCl2, 1.8 MgCl2, and 10 HEPES (pH 7.0). In
some experiments, the pipette and external bathing media contained 110 mM salt rather than 180 mM and the salts used were NaCl, LiCl, KCl,
RbCl, or tetramethylammonium chloride. Flow of the intercellular
junctional current
(Ij) was
induced by different voltage protocols. For the recording of the
macroscopic current-voltage relationship, both cells were held at a
holding potential of 0 mV. From this holding potential, the voltage of
one cell was stepped to varying voltages (Vj of
10-150 mV or
120 mV, in 20-mV increments). For
all experiments shown, the first voltage step was negative. After
holding the potential for 400 ms or 4 s, respectively, the voltage was
flipped to the equal but opposite polarity for the same time. For
single-channel or multichannel recordings, one cell of the pair was
stepped to different voltages for many seconds to minutes to observe a
number of channel events. For current recording data and analyses, see Brink et al. (5). All macroscopic records were filtered at 1 kHz, and
all channel recordings were filtered at 0.5 kHz (see Figs. 4 and 5) or
0.2 kHz (see Fig. 6). All records shown are currents observed in the
nonstepped cell held at membrane voltage (Vm) of 0 mV.
These recordings then represent
Ij only. In the coexpressing cells, steady state was not assured even after 4-s step
durations. Thus, for all transjunctional conductance
(Gj)/Vj graphs, the averages of the last five data points at 400 ms or 4 s were
used to compute the junctional conductance. The term "noninstantaneous" is used to indicate the
Gj/Vj
relationships that do not represent the instantaneous
Gj but do not
necessarily represent the steady-state either.
Cell tagging methods.
Cells were grown to confluency. Medium containing 10 µM cell tracker
green (5-chloromethylfluorescein diacetate, Molecular Probes) was
applied to the cells, and cells were incubated in the dark at 37°C
for 30 min. The medium was aspirated off, fresh medium with no dye was
applied, and the cells were again incubated in the dark at 37°C for
30 min. Both the tagged cells and cells containing no tag but
transfected with another connexin were trypsinized, mixed, and plated
out. After 12-24 h, cell pairs in which one cell of a pair
fluoresced were patched.
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RESULTS |
Coexpression in N2a cells.
Expression of connexins in cells derived from clones of transfected N2a
cells was initially determined by RNA blotting (Fig. 2). RNA derived from cultured BAECs was
used as a positive control, since these cells express both Cx37 and
Cx43 mRNAs (14) and showed hybridization to the expected endogenous
Cx37 mRNA [1.7 kilobase (kb)] and Cx43 mRNA (3.3 kb) (Fig.
2, lane 1). Neither the Cx37 nor the
Cx43 probe hybridized to the RNA derived from untransfected N2a cells
(lane 5). Cells transfected solely
with hCx37 showed hybridization to an mRNA of ~2.7 kb, as expected for the recombinant-derived mRNA (lane
4). This 2.7-kb hCx37 mRNA was also detected in total
RNA derived from individual cotransfected clones
(lanes 2 and
3), which also showed hybridization
to a band of ~2 kb (the size expected for the mRNA derived from
pZeoSV-rCx43). The identity of the rCx43 and hCx37 bands was confirmed
by blots hydridized with each probe alone (data not shown). The
intensity of hybridization of hCx37 and rCx43 probes was approximately
equivalent; because this pointed to expression of a similar magnitude,
these clones were chosen for further experiments. Production of
connexin proteins in the serially transfected N2a cells was examined
immunochemically. Immunoblots of whole cell lysates of the coexpressing
N2a cells showed a major immunoreactive Cx37 band of ~37 kDa and an
immunoreactive Cx43 band of ~45 kDa, which were not detectable in
untransfected N2a cells or N2a cells transfected with the plasmid
vectors alone (Fig. 3). There was
relatively little Cx43 produced by these cells, as indicated by
comparison with blots of heart homogenates (not shown), requiring
substantial exposure of the N2a blots and an increase in the background
of nonspecific bands (compare lanes 1 and 2 in Fig. 3). We attempted to
visualize the connexin proteins produced in these cells by
immunofluorescence. Staining with either anti-Cx37 or anti-Cx43
antibodies (which will intensely stain other Cx37- and Cx43-expressing
cells such as aortic endothelial cells) yielded only occasional small
spots of fluorescence between cells, suggesting that there were only
very small gap junctions between these cells (data not shown).

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Fig. 2.
RNA blot demonstrating the expression of both human Cx37 (hCx37) and
rat Cx43 (rCx43) in cotransfected clones. Total RNA derived from bovine
aortic endothelial cells (lane 1), 2 independent cotransfected clones of Neuro-2a (N2a) cells
(lanes 2 and
3), N2a cells solely transfected
with Cx37 (lane 4), and
untransfected N2a cells (lane 5) was
hybridized with a mixture of probes for Cx37 and Cx43. Arrowheads,
migration of the 18S and 28S rRNAs. Cx43 probe hybridizes to an
endogenous mRNA of 3.3 kilobases (kb) and a transfection-derived mRNA
of 2 kb. Cx37 probe hybridizes to an endogenous mRNA of 1.7 kb and a
transfection-derived mRNA of 2.7 kb. Clones
9 and 10 (the 2 cotransfected clones) are shown and represent those used for dual whole
cell patch clamp.
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Fig. 3.
Immunoblot demonstrating the presence of Cx37 and Cx43 polypeptides in
cotransfected N2a cells. Blots of lysates of N2a cells transfected with
pSFFV-neo vector alone (lane 1,
left and
right) or clone
9 transfected with hCx37 and rCx43
(lane 2,
left and right, respectively) were reacted with
anti-Cx37 or anti-Cx43 antibodies as indicated.
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Single-channel conductance in homomeric/homotypic rCx43 and hCx37
cell pairs.
To examine the effect of the coexpression of two different connexins on
junctional coupling, experiments were performed on cells expressing
solely hCx37 or rCx43 and then compared with cells coexpressing hCx37
and rCx43. Weakly coupled N2a cell pairs allowed the observation of
single-channel events. The hCx37 channel was previously described as a
330- to 400-pS channel with a 63-pS substate (27). rCx43 has also been
characterized and has a unitary conductance of 80-90 pS in 150 mM
salt (25) and 96 pS in 120 mM KCl (24). Figure
4 illustrates the homomeric forms of hCx37 and rCx43 transfected into N2a cells. In both cases, the pipette solution contained 180 mM CsCl, as described in
MATERIALS AND METHODS. In this study,
the unitary conductance of hCx37 was 360 pS and for rCx43 it was 115 pS. Both of these values are in the same range as previous reports for
hCx37 (27) and rCx43 (Refs. 24, 25, 30; see Ref. 5 for hCx43). Both
records illustrate the typical gating found for these two homomeric
connexin types. A substate is present in the recording showing the
hCx37 homotypic channel. Its conductance was 66 pS [1/(25 mV/1.65
pA)]. rCx43 shows few substates when
Vj values of 70 mV or less are used.

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Fig. 4.
A: recording from a cell pair
transfected solely with hCx37. Transjunctional voltage
(Vj) = 25 mV. B: recording from a cell pair transfected with rCx43 only.
Vj = 25 mV. In both cases, the pipette solution contained 180 mM CsCl
as the major solute (see MATERIALS AND
METHODS). Bathing solution also contained 180 mM CsCl
(see MATERIALS AND METHODS). Typical
gating behaviors for these two homomeric connexins are shown. Both
records were taken 10 s after the onset of the step. Five experiments
were monitored with the same results for both hCx37 and rCx43.
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Single-channel conductance in heterotypic cell pairs of rCx43 and
hCx37.
Heterotypic cell pairs also revealed unitary activity, as shown in Fig.
5. At the single-channel level, the gating
of the heterotypic channel is asymmetric. For Fig. 5, a step from
Vj of 0 to
60 mV was applied and subsequently stepped briefly back to
Vj = 0 mV and
then to +60 mV. The recordings are from the Cx43 cell that was held at
a Vm = 0 mV. The
stepping cell was the Cx37 cell. The pipette solution was the 180 mM
CsCl solution. Stepping the potential of the Cx37 cell to positive
values resulted in a gating behavior for the heterotypic channel that
was similar to homotypic Cx37. The opposite polarity
revealed gating behavior that appears almost voltage independent. The
unitary conductance for the positive step shown in Fig. 5,
bottom, revealed a transition of 175 pS and a substate of 55 pS. We never observed a complete closure from
the 175-pS state to zero conductance. Instead, the closure to the 55-pS
substate and a subsequent opening back to the 175-pS level were common.
The conductance of the intermediate state was 120 pS. The opposite
polarity generated a unitary conductance of 98 pS. Some substate
activity is also apparent. This result is the same as that found for
three other experiments of paired heterotypic channels that were weakly
coupled. These conductances and the asymmetric behavior are very
different from the homotypic forms shown in Fig. 4 and the heteromeric
forms shown in Fig. 6.

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Fig. 5.
Representative recording of heterotypic channels. Data shown in
middle panel were taken from cell of a
heterotypic pair that was transfected with rCx43. It was held at
membrane voltage
(Vm) = 0 mV,
while the other cell was stepped from 0 to 60 mV and then
momentarily stepped to 0 mV again and finally to +60 mV. Stepping the
hCx37-transfected cell in the positive direction resulted in a unitary
channel conductance of 175 pS with a 120-pS main state and a 50-pS
subconductance. Stepping in the opposite polarity revealed a 98-pS
conductance. Bathing solution was CsCl saline, and the pipette solution
was CsCl as well (see MATERIALS AND
METHODS). Four other experiments with heterotypics
yielded the same results. O, open; C, closed;
Ij, junctional
current.
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Fig. 6.
Representative multichannel recordings of hCx37- and
rCx43-cotransfected cells. Multichannel activity in a cell pair
recorded during
Vj = ±50 mV
(A) or
Vj= +40 mV
(B). Different conductance levels
are indicated as dotted lines. Amplitude histograms for every data
segment are shown on right. Vertical
scales are representative for the current set and the amplitude
histogram. More detailed representations of the unitary activity are
shown below both A and
B, with a greater time scale and with
heights of the current levels indicated by arrows. Total number of
experiments = 5.
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Single-channel conductance in cell pairs cotransfected with rCx43
and hCx37.
In weakly coupled pairs of cotransfected cells, unitary channel events
could be observed. Figure 6 shows representative current traces
recorded from a multichannel preparation. The pipette solution was 180 mM CsCl. Six different conductance levels can be observed in Fig.
6A: 280, 220, 200, 150, 70, and 35 pS.
Again, the channel activity was monitored from the cell held at
Vm = 0 mV. Three of those conductance states were also observed in the current traces
presented in Fig. 6B (opposite
polarity). The majority of these conductance states are unlike the
unitary conductance levels of the homomeric hCx37 or rCx43 channels
(24, 25, 27). Another feature of the cotransfected cell pairs is
illustrated in Fig. 6, A and
B. During the application of voltage
steps, no transitions to
Ij = 0 pA could
be observed. This indicates that one or more channels were open at all
times. Assuming there is only one constantly open channel that means a
unitary conductance of 130 pS (50 mV/6.4 pA) is found for that
channel. The conductance states recorded from other
cotransfected cells yielded similar observations
(n = 5). The multichannel recordings
are inconsistent with a homomeric/homotypic population of hCx37 and
rCx43 channels. They are also inconsistent with heterotypic behavior.
The data provide strong evidence for the presence of heteromeric
and/or heterotypic forms. The 280-, 200-, and 150-pS channels
have no identifiable counterparts for any of the conductive states of the homotypic forms or the heterotypic forms. The 70- and 35-pS conductances cannot unequivocally be attributable to substates of Cx37
or Cx43 or as substates or main states of heteromeric forms.
Macroscopic transjunctional current in homomeric, heterotypic, and
heteromeric gap junctions.
Many of the cell pairs examined generated macroscopic
Ij in which
unitary activity was not observable. These data provided a monitor of
the voltage-dependent behavior for the homotypic, heterotypic, and
heteromeric forms.
The range of junctional conductance was 1.0-10 nS for hCx37,
0.3-11 nS for rCx43, 0.1-9 nS for the heterotypic forms, and 0.25-11 nS for the cotransfected cells. In cell pairs that
produced macroscopic
Ij, the
voltage-dependent inactivation of the current was examined. Figure
7 shows original macroscopic current traces recorded from the homotypic hCx37 and rCx43 cell lines. The hCx37 recording (Fig. 7A) exhibits strong
voltage dependence. Only at a
Vj of 20 mV or
less is the Ij
constant over the duration of the voltage pulse. The data shown for
hCx37 demonstrate the strong voltage-dependent inactivation of
Ij. Neither a
variation of the main cation in the pipette solution
(Cs+,
Na+,
K+,
Li+, tetramethylammonium cation,
Rb+) nor a prolongation of the
voltage pulse influenced the shape of the curve. Weaker voltage
dependence is observed for rCx43 (Fig.
7B). Here, a time- and
voltage-dependent decay of the current can first be observed at a
Vj of 50 mV.
Figure 7, C
(n = 12) and D (n = 5), shows the noninstantaneous conductance (400 ms after the
initiation of the step) plotted against
Vj. The data from each experiment are displayed for both cell types. For
both homotypic forms, the
Gj vs.
Vj relationships
are the same as reported previously (25, 30, 33, 34).

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Fig. 7.
Voltage-dependent kinetics of homotypic junctional membrane currents.
Representative original traces of
Ij recorded from
hCx37 (A), rCx43
(B). Voltage protocol is described
in MATERIALS AND METHODS.
A and
B: short-voltage protocol with a pulse
duration of 400 ms before the flipping of the voltage. In
A, pipette salt used was NaCl; for
B, KCl was used.
C and
D: normalized conductance voltage
plots for hCx37 (from
A; n = 12) and rCx43 (from B;
n = 5). Data obtained with the short
protocol are represented by open symbols, data obtained with the long
protocol (4-s voltage pulse) are represented by filled symbols and
connected by a line. For C, currents
registered with pipette solutions containing varying main cationic
charge carriers were used (Cs+,
Na+,
K+,
Li+, tetramethylammonium cation,
Rb+). For
D, cation was either
Cs+ or
K+.
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Heterotypic hCx37 and rCx43 junctional conductance shows
voltage-dependent asymmetry that is consistent with previous
observations in oocyte pairs (32-34). The data shown in Fig.
8, A and
B, show the two recording modes.
Either the rCx43-transfected cell was stepped (Fig.
8A) or the hCx37-transfected cell
was stepped using the short protocol (0.4 s) described in
MATERIALS AND METHODS. The records
shown in Fig. 8A are measurements of
Ij recorded in the hCx37 cell held at
Vm = 0 mV,
whereas Fig. 8B shows currents recorded in the rCx43 cell held at
Vm = 0 mV. The
data are from two different experiments. Figure
8C summarizes the data from six
experiments, which are all plotted relative to the rCx43 cell being
stepped and the hCx37 cell being held at
Vm = 0 mV. The Vo (which is the
Vj where
Gj is one-half
the maximal measured conductance for
Gj) was
~70-80 mV for the negatively gated rCx43-positively gated hCx37
heterotypic case. Pipette solutions of 110 mM KCl and 180 mM CsCl were
used in experiments for Fig.
8C. The data in Fig. 8
are consistent with previous studies that indicate that Cx37 gates
positively and Cx43 gates negatively (15, 18).

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Fig. 8.
Voltage-dependent kinetics of junctional membrane currents and
noninstantaneous junctional conductance for heterotypic hCx37-rCx43 are
shown. A:
Ij tracing for
the short (400 ms) 150-mV pulse protocol described in
MATERIALS AND METHODS, in which the
stepped cell was transfected with rCx43 and the cell transfected with hCx37 was held at
Vm = 0 mV.
Recording in the latter cell (with hCx37) is shown.
B:
Ij tracing for
another cell pair, in which cell transfected with hCx37 was stepped and
rCx43 cell was held at
Vm = 0 mV. These
records were made with 110 mM NaCl saline bathing the cells and a
pipette solution of 110 mM KCl. C:
noninstantaneous normalized transjunctional
conductance-Vj
relationship. Filled symbols represent experiments done with 110 mM
NaCl bath and 110 mM KCl pipette solution, and open symbols represent
experiments done with cell pairs bathed in 180 mM CsCl and with a
pipette solution of 180 CsCl. Total number of experiments = 6.
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Data obtained from cell pairs transfected with both hCx37 and rCx43 are
much more scattered than those obtained from homotypic or heterotypic
cell pairs, especially when the short protocol (400-ms step duration)
is employed (Fig. 9). The voltage-dependent decrease of the normalized conductance is shifted to higher voltages than those observed for homotypic or heterotypic when a short (400 ms)
protocol is used. The longer protocol reveals a
Gj/Vj relationship that approximates that obtained for rCx43.

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Fig. 9.
Voltage-dependent kinetics of junctional membrane currents and
noninstantaneous junctional conductance for heteromeric hCx37-rCx43 are
shown. A: record using the short
150-mV protocol. B: record using
the longer protocol. C: data from 14 experiments [10 short (open symbols) and 4 long (filled symbols)
protocol]. Data show greater scatter than either homotypic form
or the heterotypic form. Total number of channels present between N2a
cell pairs is small, often less than the total theoretical heteromeric
forms. Variation is consistent with notion that in any particular
experiment one is only viewing a fraction (subsets) of the total number
of channel types possible.
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DISCUSSION |
Morphological studies have shown that different connexins are not only
coexpressed in the same tissue but that they also may occur in
identical gap junctional plaques (10, 17, 18). Stauffer (21)
demonstrated, after coexpression of Cx32 and Cx26 in an insect cell
line, the occurrence of both proteins in the same gap junctions.
Although this evidence is compelling, mass measurements have not been
able to confirm Cx32-Cx26 heteromeric forms (20). Our approach was to
examine if coexpression of hCx37 and rCx43 in the same cell line
results in conductance and gating properties atypical of homotypic and
heterotypic gap junction channels. We performed these studies by stable
transfection of N2a cells with individual connexin sequences and
cotransfection with two connexins, since the parental N2a cells are
devoid of connexin expression and detectable gap junction channels.
Previous studies of gap junction channels in N2a cells transfected with hCx37 and rCx43 (19, 25, 27), in which macroscopic and microscopic records allowed determination of voltage-dependent behavior and illustration of unitary activity, are consistent with our present findings. N2a transfectants produce relatively small amounts of gap
junction proteins; therefore, detection of single-channel events are
facilitated. We typically detected <100 channels in a cell pair. We
were able to confirm expression of Cx37 and Cx43 in the cotransfected
N2a clones by RNA blotting and immunoblotting. We could not compare
localization of these connexins by immunofluorescence, possibly because
the amounts of connexin produced were below the detection limits of
this system. Estimates by others of the sensitivity of immunostaining
(23) suggest that connexin immunoreactivity would only be visualized if
all of the connexin channels were concentrated in one or two plaques
and, furthermore, each channel needed to be labeled. Unfortunately, the
ratios of the two connexin types can neither be modulated nor
rigorously determined. The biochemical data illustrate that both
connexins are synthesized. The electrophysiological data indicate that
the two connexins in question are capable of mixing, based on channel
conductances and different voltage dependence relative to homotypic and
heterotypic forms.
Cx37 and Cx43 were chosen because they are known to coexpress in vivo
in endothelial cells (19). Cx37 and Cx43 are known to form heterotypic
channels as well (33, 34), a finding confirmed in this study. On the
experimental level, hCx37 and rCx43 offer the possibility that they
exhibit distinctive electrophysiological properties. Cx37 is the most
voltage-sensitive connexin, with a half-maximal inactivation at ±25
mV and a single-channel conductance of 350-400 pS (25, 27). On the
other hand, Cx43 exhibits a weak voltage dependence, with a
half-maximal inactivation at
Vj values of
±60-70 mV (30). Figure 4 shows examples of hCx37 and rCx43
under identical
Vj and ionic
conditions. The unitary conductance of hCx37 is in the range previously
reported, as is that of rCx43. The records also illustrate the gating
behavior of the two homotypic gap junction channels. The heterotypic
channels of hCx37 and rCx43 represent a novel observation. Our data
indicate that the heterotypic channel has configuration-dependent
conductance.
The coexpression of hCx37 and rCx43 resulted in a channel population in
which gating behavior could not be predicted by the two connexins
alone. In the absence of any heteromeric gap junction channel, only
three types of gap junction channels may occur. Two cases have both
hemichannels of a gap junction channel formed of either rCx43 or hCx37
(homotypic), and the other type has two heterotypic forms (mirror
images). The
Vo for these
cases are 70, 25, and 80 mV for 400-ms protocol, respectively. Although one would expect a variability depending on the expression level of the
different channels, the resulting
Gj/Vj
relationship should display a
Vo between 25 and
80 mV. This was not the case. The N2a cell line cotransfected with
hCx37 and rCx43, therefore, did not exhibit the voltage-dependent
characteristics of either homotypic or heterotypic forms. This result
implies that the homomeric voltage gate requires some form of
interaction between the individual connexins, which might not be
implemented or is impaired in the heteromeric channels.
In those experiments for which unitary activity was observed for
cotransfected cell pairs, no unitary event like hCx37 or rCx43 could be
observed. A number of other channel conductances were observed (Fig.
4). Comparison with Fig. 6 indicates that there are channel types with
conductances intermediate between the two homotypic forms and smaller
conductance states as well. Two general questions arise. How many
different types of channels can be predicted to occur when hCx37 and
rCx43 are freely capable of mixing and forming heteromeric hemichannels
and what is the probability of occurrence for homotypic channels under
such conditions? With the assumption of sixfold symmetry, 196 types of
gap junction channel are then possible between two coexpressing cells.
If only a limited number of heteromeric hemichannel types were allowed, 3 vs. 12 for example, then the total number of combinations is 5 × 5 or 25 (3 heteromeric forms and 1 each of the homomeric
hemichannels equals 5). The total number of gap junction channel types
is 25 vs. 196. With this scheme, the probability of observing a
homotypic channel of either type is 0.04 and the probability of
observing heterotypic channels becomes 0.08. For the case of only two
heteromeric forms, the probabilities of observing homotypic or
heterotypic channels are 0.063 and 0.13, respectively. For the case of
one heteromeric form with one each of the homomeric hemichannel forms (3 × 3 = 9 total combinations), the probabilities of
observing a homotypic channel are 0.1 and 0.2 for the heterotypic
mirror image forms.
In our experiments, homotypic rCx43 exhibited a single-channel
conductance of 115 pS in 180 mM CsCl, which is in good accordance with
previous results (5, 25, 27). For homotypic hCx37, the single-channel
conductance was 360 pS in 180 mM CsCl, which is similar to previous
reports (27). Our experimental data for the cotransfected cells show
channel conductance states of 280, 220, 200, 150, 70, and 35 pS, with
180 mM CsCl as the major solute in the pipette. The majority of
single-channel conductances cannot be described by a
homotypic/heterotypic population of hCx37 and rCx43 channels. Instead,
it points to a free combination of connexins in one hemichannel. We
observed multichannel activity in a number of cotransfected cell pairs
and, as Fig. 5 illustrates, could identify a number of conductive
states. Even with the assumption that each conductive state arises from
a distinct heteromere, this does not represent an upper limit for the
number of heteromeric forms but does equate to a lower boundary. It is
entirely possible that many heteromeric forms have similar or identical
conductances and/or gating properties and would thus be
indistinguishable from one another using dual whole cell patch clamp
methods.
Could the unitary conductances seen in the coexpressing cell pairs be
substates of homotypic hCx37 or rCx43? If this were the case, then the
macroscopic records would reflect
Gj/Vj
relationships intermediate between homotypic hCx37 and rCx43.
Furthermore, the multitude of states seen in the weakly coupled pairs
is not consistent with the magnitude or frequency of subconducting
states for either homotypic form. hCx37 has a pronounced 63-pS substate
(25, 27) that persists for seconds, whereas rCx43 has two easily
observed substates of ~60 and ~30 pS that are usually infrequent
unless large Vj
steps are employed (26, 30).
The macroscopic
Ij and unitary
current data illustrated here are consistent with the formation of
functional heteromeric gap junction channels by two members of group II
or
-type connexins (3, 12). The single-channel data shown here
provide evidence for a number of conductive states that cannot be
identified as either homotypic main states or substates. The same is
true for the heterotypic channels. The existence of heteromeric forms
implies that cells coexpressing connexins can produce a spectrum of
channel types, each form potentially having unique specific
permselective and gating characteristics. This potential plethora of
channel types could well be critical to multicellular processes such as differentiation or coordinated contraction in both excitable and nonexcitable cells. For example, alterations in the ratios of two
connexins, leading to possible changes in distribution of heteromeric
channels, may have functional consequences for intercellular communication in endothelial cells. An example is reported in Larson et
al. (14), which recently demonstrated that abundances of Cx37 and Cx43
are differentially affected by cellular growth, density, and tumor
growth factor-
.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-31299 (to P. R. Brink) and HL-45466 (to E. C. Beyer).
 |
FOOTNOTES |
Address for reprint requests: P. R. Brink, Dept. of Physiology and
Biophysics, SUNY at Stony Brook, Stony Brook, NY 11794.
Received 15 November 1996; accepted in final form 26 June 1997.
 |
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