© 1999 American Heart Association, Inc.
Original Contributions |
Multiple Connexins Form Gap Junction Channels in Rat Basilar Artery Smooth Muscle Cells
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 msimard{at}surgery1.umaryland.edu
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Abstract |
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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 Cs+ or K+ 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.
Key Words: gap junction • connexin 43 • connexin 40 • vascular smooth muscle • patch clamp
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Introduction |
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Gap 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 4 5 6 7 8 ; in the A7r5 cell line9 10 11 12 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 17 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 high-conductance channels and that a significant number of couplings involve putative nonhomotypic channels.
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Materials and Methods |
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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
-actin monoclonal antibody.
Immunofluorescence Microscopy
Sections (12 to 16 µm) 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 100x 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
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
mmx100 mm; Kimax-51, Kimble) were used to reduce differences in
tip resistance (1.5 to 3 M
). 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 · 6H2O 8,
and HEPES 10, and nystatin 165 µg/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 · 6H2O 8,
HEPES 10, and tetraethylammonium ·
Cl 5, and nystatin 165 µg/mL, pH 7.2, and the other containing
(in mmol/L) KCl 145, MgCl2 ·
6H2O 2, CaCl2 ·
2H2O 4.2, EGTA 5,
tetraethylammonium · Cl 5, HEPES 10,
glucose 10, and ATP · 2Na 3, pH 7.2.
Cells with seal resistances >2 G were recorded using patch-
clamp amplifiers (Axopatch 200A, Axon Instruments, Inc). Series
resistance of each pipette was 7 to 25 M, thus 
15 to 50 M
total. Total measured access resistance was 36.9±0.7 M (n=3).
Single-cell input resistance was 23.4±9.9 G (n=9; 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 +100 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 (
=3.1 seconds), showing 70% recovery 500 ms after
60-mV pulses (n=3). 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
(DTR-1200, 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
Marquardt-Levenberg (Origin 5.0). When symmetrical about the 0-mV axis,
the voltage dependence of the steady-state conductance was
analyzed by 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:
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Results |
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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.
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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 (8.7±5.1 nS). Twenty-eight pairs tested with
K+ in both pipettes showed
Gj=9.6±6.5 nS, which was not different
from the value with Cs+ (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, 8.3±5.6 and
9.6±3.2 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.
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 26 27 and Cx405 28 channels,
respectively.
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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 voltage-dependent
reduction of Ij, with sporadic and
distorted large-conductance openings at high
Vj (>||±40 mV||) 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 -20, –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+60 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 Vj=±10, ±20, and ±30 mV,
and Figure 4C is the comparable histogram for events obtained at
Vj=±40, ±50, and ±60 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 26 27 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
Vj
0 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;
). The instantaneous conductance-voltage relationship typically
exhibited only weak, if any, voltage dependence (Figure 5C, 5F, and 5I, ).
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>||±40 mV|| (Figure 5C, •).
Conversely, when currents deactivated rapidly, deactivation was
observed during smaller test pulses. In some pairs, deactivation was
strongly voltage dependent and was observed at
Vj>||±10 mV|| (Figure 5I, •).
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
(G'j-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
When identical connexons are coupled, the voltage dependence of
Gj-ss tested from
Vj=0 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
+60 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
G'j-ss-Vj
relationship for 7 pairs that were more sensitive to negative
Vj.
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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 G'j-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.
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E, G'j-ss-vs-Vj
curve for the recordings in 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
G'j-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.
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Discussion |
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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 4 5 6 7 8 in the A7r5 cell line,9 10 11 12 13 or in serially passaged cultured cells derived from explanted vascular tissue.9 14 15 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
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 26 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 10 11 12 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
mVVj30 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>||±30 mV||, 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 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 10 11 12 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 Vj=0 mV, but only in pairs showing asymmetry of voltage dependence. Although voltage asymmetries can be due to asymmetric chemical environments, including differences in pH, [Ca2+]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.
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Acknowledgments |
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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 Heart Association, Maryland affiliate. We thank Lioudmila Melnitchenko for her expert technical assistance.
Received September 8, 1998; accepted March 30, 1999.
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