Multiple Connexins Form Gap Junction Channels in Rat
Basilar Artery Smooth Muscle Cells
Xing Li, J. Marc Simard
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Abstract—Three connexins, Cx43, Cx40, and Cx37, have been found by protein or mRNA analysis to be prominent in
mammalian blood vessels, but electrophysiological characterization of gap junction channels in freshly isolated vascular
smooth muscle cells (SMCs) has not previously been reported. We used a dual-perforated patch-clamp technique to study gap
junction conductances in SMC pairs from rat basilar arteries. Macroscopic junctional conductance (Gj) measured in 98 cell
pairs with either Cs1 or K1 ranged between 0.68 and 24.8 nS. In weakly coupled cells (Gj,5 nS), single-channel currents were
readily resolved without pharmacological uncoupling agents, allowing identification of 4 major unitary conductances. Two of
these conductances, 80 to 120 pS and 150 to 200 pS, corresponded to the major conductance states for homotypic channels
formed from Cx43 or Cx40, which we confirmed were present in smooth muscle by immunofluorescence analysis. Two other
conductances, 220 to 280 pS and .300 pS, were identified that have not been previously reported in vascular SMCs.
Macroscopic recordings revealed currents that deactivated incompletely over a broad range of transjunctional potentials. In
about half of the pairs, we identified macroscopic as well as single-channel currents that exhibited marked voltage asymmetry,
consistent with nonhomotypic, ie, either heterotypic or heteromeric channels. Our data indicate that basilar artery SMCs are
coupled in vivo in a richly complex manner, involving Cx43, Cx40, and other large-conductance channels, and that a
significant number of couplings involve putative nonhomotypic channels. (Circ Res. 1999;84:1277-1284.)
Key Words: gap junction n connexin 43 n connexin 40 n vascular smooth muscle n patch clamp
G
ap junction channels are formed from connexins, the
products of a large multigene family with 13 members
in mammals.1 Despite the large number of molecularly
distinct connexins known, only 3, Cx43, Cx40, and Cx37,
have been found by protein or mRNA analysis to be prominent in mammalian blood vessels.2 Although reliably identified, functional characterization of gap junction channels
formed by these connexins in vascular tissue has been
limited. There are several reports on electrophysiological
characterization of gap junction channels formed by these 3
connexins in expression systems3– 8; in the A7r5 cell line9 –13;
or in serially passaged explant cultures from arterial,9 corpus
cavernosum,14,15 or umbilical cord16 tissue. To date, however,
there are no reports on electrophysiological study of freshly
isolated native vascular preparations.
Electrophysiological study of gap junction channels in
freshly isolated SMCs is important, because SMCs undergo
modulation from a contractile to a synthetic phenotype in
culture, with phenotypic modulation being associated with
alterations in expression of Cx43 and Cx40.16 –18 Also, in a
tissue such as the vessel wall, in which only 3 connexins are
expected, electrophysiological methods may be used to identify them with high sensitivity (single channels) and high
spatial resolution (isolated cell pairs), thereby complementing
immunochemical methods. Moreover, electrophysiological
methods are best for assessing involvement of nonhomotypic
(heterotypic or heteromeric) channels. Homotypic channels
are composed solely of 1 connexin type and exhibit characteristic conductance and voltage dependence. Conversely,
both heterotypic channels, which contain 2 homomeric
hemichannels each made from different connexins, and heteromeric channels, which contain different connexins within
either or both hemichannels, exhibit distinguishable conductance and voltage dependence.6,7,19
In this report, we used a dual-perforated patch-clamp
technique to study gap junction conductances in freshly
isolated SMC pairs from rat basilar arteries. We present
immunofluorescence and electrophysiological evidence that
basilar artery SMCs are coupled in a complex manner by
channels formed from Cx43, Cx40, and other highconductance channels and that a significant number of couplings involve putative nonhomotypic channels.
Materials and Methods
Cell Preparation
SMCs were isolated from rat (Wistar, 180 to 250 g) basilar arteries
as previously described.20 The identity of the cells was assured by
their elongated, phase-bright appearance with phase-contrast microscopy21 and by immunostaining with a-actin monoclonal antibody.
Received September 8, 1998; accepted March 30, 1999.
From the Departments of Neurosurgery (X.L., M.S.) and Physiology (M.S.), University of Maryland School of Medicine, Baltimore, Md.
Correspondence to J. Marc Simard, Department of Neurosurgery, University of Maryland School of Medicine, 22 South Greene St, Baltimore, MD
21201. E-mail [email protected]
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
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1278
Multiple Connexins Form Gap Junction Channels
Immunofluorescence Microscopy
Sections (12 to 16 mm) were prepared from freshly frozen rat basilar
arteries. Sections were exposed to Cx40 antibody (affinity-purified
rabbit anti-Cx40, Chemicon International Inc; dilution 1:200) at
room temperature for 1 hour and then at 4°C overnight and then were
treated with affinity-purified goat anti-rabbit FITC-conjugated secondary antibody (Chemicon; dilution 1:400) at room temperature for
1 hour. The protocol for Cx43 (mouse monoclonal anti-Cx43,
Chemicon; dilution 1:200) was similar, except that sections were
maintained with primary antibody at 4°C for 36 hours, and a rabbit
anti-mouse secondary antibody (Chemicon; dilution 1:300) was
used. Sections processed without primary antibody served as negative controls. Sections were mounted using ProLong antifade mounting medium (Molecular Probes). Immunolabeled sections were
examined on a Nikon Eclipse E600 microscope equipped with a
1003 objective. Images were captured and processed using a Sony
DKC-5000 digital camera and a personal computer with Adobe
Photoshop 5.0.
Patch-Clamp Experiments
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Patch-clamp experiments were performed at room temperature using
extracellular solution containing (in mmol/L) NaCl 145, KCl 5,
MgSO4 2, HEPES 10, and glucose 12.5, pH 7.4. Pairs of pipettes
pulled from the same capillary tube (0.8 to 1.1 mm3100 mm;
Kimax-51, Kimble) were used to reduce differences in tip resistance
(1.5 to 3 MV). For some experiments, both cells were studied using
a dual nystatin-perforated patch technique (DNPPT),22 with pipettes
containing (in mmol/L) CsCl 130, MgSO4 z 6H2O 8, and HEPES 10,
and nystatin 165 mg/mL, pH 7.2. For other experiments, 1 cell was
studied with a nystatin-perforated whole-cell technique and the other
with a conventional whole-cell technique, with one pipette containing (in mmol/L) KCl 55, K2SO4 75, MgCl2 z 6H2O 8, HEPES 10, and
tetraethylammonium z Cl 5, and nystatin 165 mg/mL, pH 7.2, and the
other containing (in mmol/L) KCl 145, MgCl2 z 6H2O 2, CaCl2 z
2H2O 4.2, EGTA 5, tetraethylammonium z Cl 5, HEPES 10, glucose
10, and ATP z 2Na 3, pH 7.2.
Cells with seal resistances .2 GV were recorded using patchclamp amplifiers (Axopatch 200A, Axon Instruments, Inc). Series
resistance of each pipette was 7 to 25 MV, thus '15 to 50 MV total.
Total measured access resistance was 36.960.7 MV (n53). Singlecell input resistance was 23.469.9 GV (n59; 130 mmol/L CsCl).
Both cells of a pair were voltage clamped at the same holding
potential (HP-40 or –10 mV). Transjunctional voltage (Vj) was
generated by applying 6-second step pulses from –100 to 1100 mV
to 1 cell. The change in current in response to Vj recorded from the
other cell held at a constant HP was considered junctional current
(Ij). Junctional conductance (Gj) was calculated as Ij/Vj. A 10-mV,
100-ms pulse was applied to the pulsed cell 500 ms before each
6-second test pulse to assess stationarity of Gj during the experiment.
After strong test pulses, Gj recovered as a first-order exponential
(t53.1 seconds), showing '70% recovery 500 ms after 60-mV
pulses (n53). To ensure full recovery, a 9-second interval was
always used between test pulses. Records of voltages and currents
filtered at 1 kHz were recorded on a digital tape recorder (DTR1200, Biologic, Echirolles, France).
Data Analysis
Current signals were played back offline, filtered at 50 to 500 Hz,
and sampled at 500 to 2000 Hz. Single-channel analysis was
performed with the CED Patch and Voltage Clamp Program, version
6.34 (CED, Cambridge, UK), or by hand. When single-channel
activity and noise were low, all-points amplitude histograms were
constructed to determine single-channel conductance. Otherwise,
single-channel transitions were measured by hand. All macroscopic
current records were recorded in the nonpulsed cell of each pair.
The scatter plot in Figure 3C was fit to a linear regression equation
(Origin 5.0, Microcal). The multiple gaussian distributions in Figure
4 were fit using the nonlinear, least-squares method of MarquardtLevenberg (Origin 5.0). When symmetrical about the 0-mV axis, the
voltage dependence of the steady-state conductance was analyzed by
Figure 1. Cx40 and Cx43 immunofluorescence in rat basilar
artery. L indicates vessel lumen; E, endothelial layer; and SM,
smooth muscle layer. Bar510 mm.
measuring currents at the end of 6-second test pulses and fitting
nominal (Figure 5C, 5F, and 5I) or normalized (Figure 6) data to the
Boltzmann equation,23 as follows:
Gj-ss5(Gmax2Gmin)z{11exp[(V1/22Vj)/k]}21z{11exp[(V1/21Vj)/k]}211Gmin
where Gj-ss is the steady-state conductance; Gmax and Gmin are the
maximum and minimum steady-state conductances, respectively; Vj
is transjunctional voltage; V1/2 is the midpoint potential for the
negative portion of the curve (the midpoint potential for the positive
portion is given by –V1/2); and k is the “steepness” of voltage
dependence. Data were fit to the Equation using the nonlinear,
least-squares method of Marquardt-Levenberg (Origin 5.0). All data
are given as mean6SD. When appropriate, statistical significance
was assessed using the Student t test.
Results
Immunofluorescence Microscopy
Study of basilar artery sections disclosed the presence of both
Cx40 and Cx43 immunostaining in the smooth muscle cell
layers of the arterial wall (Figure 1), similarly to previous
reports on cerebral arterioles.24 Cx40 was readily detected in
the endothelial layer as well, but Cx43 was not. In smooth
muscle, Cx43 staining appeared as sparse punctate immunofluorescent signal, whereas Cx40 staining appeared as elongated clusters of signals around 1 or more cells.
Electrical Coupling Between Cell Pairs
Seventy pairs tested with CsCl in both pipettes showed
junctional conductance (Gj) ranging from 0.68 to 24.8 nS
(8.765.1 nS). Twenty-eight pairs tested with K1 in both
pipettes showed Gj59.666.5 nS, which was not different
from the value with Cs1 (by t test, P.0.05). Among the 70
pairs studied with CsCl, 52 measurements were performed
within 5 hours after cell dissociation, and 18 were obtained at
18 to 24 hours after dissociation. Values of Gj in these 2
subgroups, 8.365.6 and 9.663.2 nS, were not different (by t
test, P.0.05), which suggests that there was no important
change in cell coupling with time during the period of study.
Li and Simard
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Figure 2. Recordings of the gap junction channels of 80 to 120
pS, 150 to 180 pS, and 220 to 280 pS. A, Single-channel recordings from a pair of cells (101696) held at 0 mV (upper trace)
and – 40 mV (lower trace); Vj540 mV; dual voltage clamp with 1
conventional and 1 perforated whole-cell configuration; KCl.
Gaussian fit-of-amplitude histogram revealed openings of 90,
103, 94, and 75 pS. B, Single-channel recordings from a pair of
cells (092396) held at –20 mV (upper trace) and – 40 mV (lower
trace); Vj520 mV; DNPPT; CsCl. Gaussian fit-of-amplitude histogram revealed openings of 176 and 35 pS. C, Single-channel
recordings from a pair of cells (970507); both cells held at
HP-10 mV, whereas 1 cell was pulsed to generate Vj520 mV;
DNPPT; CsCl. Gaussian fit-of-amplitude histogram revealed
openings of 212 and 267 pS.
Single-Channel Junctional Currents
When cell pairs were weakly coupled (Gj,5 nS), high-gain
recordings revealed single-channel junctional events, which
were identified by the characteristic feature that, in recordings
from the 2 cells, currents were of equal size and opposite
polarity.
Junctional channel events were quantified in 21 weakly
coupled pairs. Events of 2 different conductances were
frequently identified. These were conductances of 80 to 120
pS, identified in almost all pairs (Figure 2A), and of 150 to
200 pS, identified in 16 of 21 pairs (Figure 2B). As found in
systems expressing only a single connexin, conductances of
80 to 120 pS and of 150 to 200 pS are characteristic for the
open mainstates of homotypic Cx4325–27 and Cx405,28 channels, respectively.
In some pairs, we observed junctional channels of greater
conductances than expected in SMCs. One that was frequently identified had a value of 220 to 280 pS (Figure 2C),
greater than any reported for homotypic channels due to Cx43
or Cx40. Another large conductance, identified in 2 of 21
pairs, had a conductance .300 pS and very fast open-close
transitions (Figure 3). Macroscopic recordings showed strong
June 11, 1999
1279
voltage-dependent reduction of Ij, with sporadic and distorted
large-conductance openings at high Vj (.u640 mVu) that
were not present at low Vj (Figure 3B). The probabilities
of opening of this channel were 0.80, 0.35, and 0.087
at Vj 220, –30, and – 40 mV, respectively. Also, the relationship between single-channel current amplitude and Vj revealed voltage-dependent unitary conductance saturation
(Figure 3C).
We measured 3118 single-channel events (21 pairs) elicited at – 60#Vj#160 mV. Compilation of these data into an
event amplitude histogram (Figure 4A) revealed a broad
spectrum of conductances ranging from 10 to 362 pS with 4
dominant conductances, based on fitting to a multiple gaussian function. Events obtained at low and at high Vj were
analyzed separately. Figure 4B shows a histogram for channel
events recorded at Vj5610, 620, and 630 mV, and Figure
4C is the comparable histogram for events obtained at
Vj5640, 650, and 660 mV. The subset of data obtained at
low Vj was more homogeneous than the combined data set,
with the 4 dominant conductances being more easily distinguishable in the histogram (Figure 4B versus 4A). By
contrast, the subset of data obtained at higher Vj showed a
broad grouping of conductances at .125 pS and fewer
openings at .300 pS. The 4 dominant conductances from
data at low Vj had fitted values of 48, 96, 182, and 245 pS
(Figure 4B). Values at 96 and 182 pS were attributed to the
open mainstates of Cx43 and Cx40. The smallest peak at 48
pS, which contributed significantly to the histogram, is
similar in value to that reported for the subconductance states
of rat homotypic Cx43 and Cx40 gap junction channels found
in expression systems.5,25–28 The fourth peak at 245 pS,
corresponding to events of 220 to 280 pS, is currently not
attributed.
Macroscopic Junctional Currents
Application of test pulses to various values of VjÞ0 mV
revealed currents that usually deactivated (decayed) over the
course of several seconds (Figure 5A, 5D, and 5G). The
current-voltage relationship for instantaneous junctional currents was usually linear with reversal at 0 mV (Figure 5B, 5E,
and 5H; E). The instantaneous conductance-voltage relationship typically exhibited only weak, if any, voltage dependence (Figure 5C, 5F, and 5I, E).
The time course of decay of currents was usually similar
for test pulses of equal magnitude and opposite polarity (ie,
junctional currents exhibited largely symmetric time dependence of deactivation; Figure 5A, 5D, and 5G). In some pairs,
deactivation was slow and incomplete (Figure 5A), whereas
in others, deactivation was more rapid (Figure 5G). When
deactivation was slow, the time course was usually complex
and nonexponential (Figure 5A and 5D), whereas when
faster, the time course was usually exponential (Figure 5G).
Invariably, deactivation was incomplete during 6-second test
pulses (Figure 5A, 5D, and 5G).
The transjunctional potential at which deactivation occurred correlated with the rate of deactivation. When currents
deactivated slowly, they generally did so only during larger
test pulses. In some pairs, deactivation was observed only at
Vj.u640 mVu (Figure 5C, F). Conversely, when currents
1280
Multiple Connexins Form Gap Junction Channels
Figure 3. Recordings of the .300-pS gap junction
channel. A, Single-channel recordings from a pair
of cells (071196p3) held at – 40 mV (lower trace)
and –20 mV (upper trace); Vj-20 mV; DNPPT; CsCl.
Gaussian fit-of-amplitude histogram revealed
openings of 323 and 65 pS. B, Same pair was
given a series of 6-second pulses to yield Vj from
– 60 to 160 mV. Single-channel openings elicited
at Vj-20, –30, and –50 mV are shown. C, Singlechannel Ij-Vj relationship for the same pair. The
amplitude of the single-channel current was linear
at –30 mV#Vj#130 mV (F), with a slope of 332
pS (r50.996, P,0.0001), but saturated at Vj.u640
mVu (E).
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
deactivated rapidly, deactivation was observed during smaller
test pulses. In some pairs, deactivation was strongly voltage
dependent and was observed at Vj.u610 mVu (Figure 5I, F).
When the time dependence of deactivation was similar for
positive and negative pulses of equal magnitude, the voltage
dependence of Gj-ss was symmetric about the 0-mV axis.
Normalized values of Gj-ss (G9j-ss) for 17 pairs (130 mmol/L
CsCl) showing symmetric voltage dependence were fit to the
Equation (Figure 6). Several features were notable: (1) the
voltage dependence of deactivation (k in the Equation) was
relatively steep, with values ranging from 1.7 to 4.0 mV,
reflecting the observation that Gj-ss frequently transitioned
from near maximum to near minimum in a single 10-mV
step; (2) the midpoint potential (V1/2 in the Equation) varied
broadly, from –13 mV to – 46 mV; and (3) deactivation was
never complete during 6-second test pulses, as indicated by
values of Gmin ranging from 0.16 to 0.34.
Putative Nonhomotypic Conductance
Figure 4. Event amplitude histogram of single-channel gap
junction conductances. A, Event amplitude histogram (bin width,
6 pS) comprising 3118 events elicited at – 60 mV#Vj#160 mV
pooled from 21 pairs. The histogram was fit to a multiple gaussian distribution with mean values indicated. B, Event amplitude
histogram (bin width, 8 pS) constructed from 1532 events from
panel A elicited at –30 mV#Vj#130 mV. The histogram was fit
to a multiple gaussian distribution with mean values of 48, 96,
182, and 245 pS. C, Event amplitude histogram (bin width, 8
pS) constructed from 1586 events from panel A elicited at
Vj5640, 650, and660 mV.
When identical connexons are coupled, the voltage dependence of Gj-ss tested from Vj50 mV should be symmetrical
about the 0-mV axis, because the kinetics of deactivation of
the 2 connexons should be identical. Conversely, if different
connexons with different deactivation kinetics are coupled,
the voltage dependence of the Gj-ss will be asymmetrical about
the 0-mV axis. These observations form the basis for the
electrophysiological test for nonhomotypic conductance, ie,
asymmetry of voltage dependence Gj-ss.
In addition to recordings presented above showing symmetric voltage dependence of Gj-ss, many of our recordings
exhibited considerable voltage asymmetry. Figure 7A presents an example in which currents only deactivated during
strong positive pulses, with no deactivation during comparable negative pulses. Note also in these records that at strongly
positive Vj, Ij activated appreciably during the first few
seconds before gradually deactivating to steady-state values,
whereas at strongly negative Vj, Ij activated slowly and only
by a small amount. Voltage asymmetry such as this suggests
the presence of nonhomotypic channels. We calculated the
ratio of Gj-ss obtained at 160 mV and – 60 mV (smaller
value/larger value) for 64 pairs (130 mmol/L CsCl). This ratio
was ,0.67 in 32 of 64 pairs, a value consistent with “marked
asymmetric voltage dependence.”19 Thirteen pairs were more
sensitive to positive Vj, and 19 pairs were more sensitive to
negative Vj, suggesting no systematic error. Figure 7B illustrates the G9j-ss-Vj relationship for 7 pairs that were more
sensitive to negative Vj.
Li and Simard
June 11, 1999
1281
Figure 5. Symmetric macroscopic junctional currents. A, D, and G, Original traces of Ij in 3 different cells recorded in response to Vj generated by
step pulsing the other cell of the pair from –100 to
1100 mV in 20-mV steps (A, 122397p1) or from
– 60 to 160 mV in 10-mV steps (D, 120396p1; G,
021297p2). B, E, and H, Ij-Vj relationships for the
recordings in panels A, D, and G, respectively,
with values of instantaneous Ij (Ij-ins, E) and steadystate Ij (Ij-ss, F) indicated. C, F, and I, Gj-Vj relationships from the recordings displayed in panels A,
D, and G, respectively, with values of instantaneous Gj (Gj-ins, E) and steady-state Gj (Gj-ss, F)
indicated. For the 3 panels, nonlinear leastsquares fit of Gj-ss to the Equation gave values of
Gmin/Gmax50.20, 0.18, and 0.12; V1/2-60, –25, and
–16 mV; and k514, 4.0, and 3.3 mV, for panels C,
F, and I, respectively. All data obtained with
DNPPT and CsCl.
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Apart from nonhomotypic channels, voltage asymmetry
might also be caused by asymmetric run-up or run-down, but
this was not observed with our perforated patch method. To
ensure that asymmetry did not result from sensitivity to
different membrane potentials, both cells of a pair were tested
using the same pulse protocol. Figure 8A shows Ij recorded
from cell b of a pair, held at –10 mV, while cell a was pulsed
to yield Vj. Figure 8B shows Ij recorded from cell a of the pair,
held at –10 mV, while cell b was pulsed to yield Vj. The
“mirror image” of voltage dependence of G9j-ss (Figure 8D)
indicated that the gap junction channels were more sensitive
to Vj driving Ij in the direction from cell a to cell b.
Polarization of Vm could also play a role in asymmetric
voltage dependence, and so we examined the effect of HP.
Figure 8C shows Ij recorded from the same pair with the same
protocol as in Figure 8B, except that both cells were held at
– 40 mV instead of –10 mV. The identical G9j-ss-Vj curve
(Figure 8E) suggested that changing Vm within the physiological range of – 40 to –10 mV did not change sensitivity to
Vj. Similar observations were made in 5 other pairs.
Additional evidence for nonhomotypic conductances was
also obtained from junctional channel recordings in weakly
coupled cell pairs. In some pairs, the single-channel unitary
conductance exhibited various degrees of heterogeneity. Figure 9 shows an example. At positive Vj, the major conductance of single-channel events was 150 pS, whereas at
negative Vj, the major single-channel conductance was 215 to
257 pS and was more strongly voltage dependent.
Discussion
This is the first report to systematically examine gap junction
conductances in freshly isolated vascular SMCs. Previous
reports on gap junction channels formed by Cx43, Cx40, and
Cx37 found in vascular tissue have characterized these
channels in expression systems,3– 8 in the A7r5 cell line,9 –13 or
in serially passaged cultured cells derived from explanted
vascular tissue.9,14 –16 As noted in the introduction, study of
ion channels in freshly isolated SMCs is important, because
channel expression in these cells is altered by in vitro
culture.22
Figure 6. Symmetric macroscopic steady-state
junctional conductances. Steady-state conductance, measured at the end of 6-second test
pulses, was obtained at 10-mV intervals in 17
pairs showing symmetric voltage dependence.
Normalized values (G9j-ss) were fit to the Equation
using an iterative nonlinear least-squares method.
Values from the fit were –13 mV#V1/2#– 46 mV,
1.7 mV#k#4.0 mV, and 0.16#Gmin#0.35. Values
from individual cells are plotted (symbols), as are
the resulting fits (lines). All data obtained with
DNPPT and CsCl.
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Multiple Connexins Form Gap Junction Channels
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Figure 7. Asymmetric macroscopic steady-state junctional conductances. A, Original records of Ij elicited during test pulses to
Vj of –100 to 1100 mV in 20-mV steps (122397p2). B, Normalized steady-state values of Gj (G9j-ss) from 7 pairs exhibiting
“marked asymmetric voltage dependence,” with more deactivation at negative Vj than at positive Vj. All data obtained with
DNPPT and CsCl.
We confirmed that Cx43 and Cx40, which have been
previously identified immunochemically in cerebral and other
blood vessels,24 can be identified electrophysiologically and
by immunofluorescence in basilar artery SMC pairs. Gap
junction channels with unitary conductances of 80 to 120 pS
and 150 to 200 pS, consistent with Cx43 channels25–27 and
Cx40,5,28 were observed in most cell pairs. Our findings
regarding conductances ,200 pS in basilar artery cells were
comparable with those reported in A7r5 cells9 –13 and in other
cultured cells of vascular origin.9,14,15
A major finding of this study was that there were 2 groups
of channel events with conductances .200 pS not previously
reported in SMCs. One group of high conductance channels,
observed in 10% of weakly coupled pairs, had a slope
conductance of 332 pS (–30 mV#Vj#30 mV), with both
single-channel conductance and probability of opening showing strong voltage sensitivity. Because of the sensitivity to
voltage, the large conductance openings tended to saturate
and inactivate at Vj.u630 mVu, resulting in disappearance of
channel openings .300 pS at high transjunctional voltages
(Figure 3C). Although these features essentially duplicate
observations made in transfected cells expressing Cx37,8,29
additional work involving protein or RNA identification
would be required for confirmation. Notably, Cx37 is regarded as an endothelial connexin2,3 and has not been
reported in vascular SMCs.30
The other group of high conductance channels exhibited a
conductance of 220 to 280 pS, with a mean of 245 pS.
Although this channel was prominent in the event histogram
Figure 8. Asymmetric macroscopic junctional currents are not
due to polarity of testing configuration or membrane potential.
A, Original traces of Ij recorded from cell b of a pair (121597p2)
during step pulses from – 60 to 160 mV applied to cell a; both
cells held at –10 mV. B, Ij recorded from cell a, elicited by the
same pulse protocol applied to cell b of the same pair as shown
in panel A. C, Ij recorded from cell a of the same pair, using the
same pulse protocol as in panel B, but with both cells held at
– 40 mV. D, Normalized Gj-ss (G9j-ss)-vs-Vj relationship for the recordings in panels A (F) and B (Œ). E, G9j-ss-vs-Vj curve for the
recordings in B (Œ) and C (‚).
of Figure 4B, it is difficult to attribute this conductance to any
specific connexin. In the SMC line A7r5, in which Cx43 and
Cx40 are coexpressed, single-channel conductances .200 pS
have not been reported,9 –13 which makes it difficult to
attribute a conductance of 245 pS to any nonhomotypic
combination of these connexins. It is possible that the 245-pS
channel represents an alternate state of Cx373 or a nonhomotypic channel of 220 to 280 pS conductance, as found in cells
cotransfected with Cx43 and Cx37,7 but the presence of an
as-yet-unidentified connexin cannot be excluded. Additional
work will be required to identify this important channel in
basilar artery cells.
The second major finding of this study concerns identification of putative nonhomotypic channels in freshly
isolated SMC pairs. The following two observations
strongly implicate involvement of nonhomotypic, presumably heterotypic/heteromeric channels: (1) some macroscopic recordings demonstrated marked asymmetric voltage dependence for the steady-state current, and (2) some
junctional channel recordings showed events with marked
asymmetric voltage dependence. Also, we observed activation (turning on) during step pulses away from Vj50
mV, but only in pairs showing asymmetry of voltage
dependence. Although voltage asymmetries can be due to
Li and Simard
June 11, 1999
1283
Heart Association, Maryland affiliate. We thank Lioudmila
Melnitchenko for her expert technical assistance.
References
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Figure 9. Asymmetry single-channel events. Asymmetric
voltage-dependent single-channel conductances were recorded
from a pair (012297) using DNPPT and CsCl; both cells held at
–10 mV; Vj was generated by applying 6-second step pulses to
1 cell. Single-channel events in response to Vj5620, 630, and
640 mV showed asymmetric macroscopic as well as unitary
conductances (arrows) at values of Vj of opposite polarity but
identical magnitude.
asymmetric chemical environments, including differences
in pH, [Ca21]i, or phosphorylation status, nonhomotypic
channels account better for activation of current during test
pulses that should deactivate the current.7 Coexpression of
Cx43, Cx40, and possibly other connexins such as Cx37 in
rat basilar artery SMCs could result in heterologous
coupling between adjacent cells. Heterotypic channels can
be formed between Cx43 and Cx37, and between Cx40 and
Cx37, although not between Cx43 and Cx40.6,7,31,32 Heteromeric forms have previously been reported in expression systems7,33,34 and, presumptively, in cultured embryonic cardiac cells.19
In summary, we present electrophysiological evidence that
basilar artery SMC pairs are functionally coupled by Cx43,
Cx40, and other large conductance channels, possibly Cx37.
Many couplings are homotypic, but a substantial number
involve putative nonhomotypic channels, suggesting a high
degree of promiscuity of connexins in native vascular tissue.
The purpose for this richly complex connectivity has yet to be
elucidated.
Acknowledgments
This work was supported by Grant HL42646 from the National
Heart, Lung, and Blood Institute and by a grant from the American
Heart Association, with funds contributed in part by the American
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Multiple Connexins Form Gap Junction Channels in Rat Basilar Artery Smooth Muscle
Cells
Xing Li and J. Marc Simard
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Circ Res. 1999;84:1277-1284
doi: 10.1161/01.RES.84.11.1277
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