© 1997 American Heart Association, Inc.
Articles |
Conductances and Selective Permeability of Connexin43 Gap Junction Channels Examined in Neonatal Rat Heart Cells
From the Department of Physiology (V.V., R.W.), University of Bern (Switzerland) and the Department of Physiology (F.F.B.), University of Rochester (NY).
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Abstract |
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Abstract Myocytes from neonatal rat hearts were used to assess the conductive properties of gap junction channels by means of the dual voltage-clamp method. The experiments were carried out on three types (groups) of preparations: (1) induced cell pairs, (2) preformed cell pairs with few gap junction channels (1 to 3 channels), and (3) preformed cell pairs with many channels (100 to 200 channels) after treatment with uncoupling agents such as SKF-525A (75 µmol/L), heptanol (3 mmol/L), and arachidonic acid (100 µmol/L). In group 1, the first opening of a newly formed channel was slow (20 to 65 ms) and occurred 7 to 25 minutes after physical cell contact. The rate of channel insertion was 1.3 channels/min. Associated with a junctional voltage gradient (Vj), the channels revealed multiple conductances, a main open state [
j(main state)], several substates
[j(substates)], and a residual state
[j(residual state)]. On rare occasions, the channels
closed completely. The same phenomena were observed in groups 2 and 3.
The existence of j(residual state) provides an
explanation for the incomplete inactivation of the junctional current
(Ij) at large values of Vj in cell pairs with
many gap junction channels. The values of j(main state)
and j(residual state) gained from groups 1, 2, and 3
turned out to be comparable and hence were pooled. The fit of the data
to a Gaussian distribution revealed a narrow single peak for both
conductances. The values of j were dependent on the
composition of the pipette solution. Solutions were as follows: (1) KCl
solution, j(main state)=96 pS and
j(residual state)=23 pS; (2) Cs+
aspartate- solution, j(main state)=61 pS
and j(residual state)=12 pS; and (3)
tetraethylammonium+
aspartate- solution, j(main state)=19 pS
and j(residual state)=3 pS. The respective
j(main state)-to-j(residual state) ratios
were 4.2, 5.1, and 6.3. This indicates that the residual state
restricts ion permeation more efficiently than does the main state.
Transitions of Ij between open states (main open state,
substates, and residual state) were fast (<2 ms), and transitions
involving the closed state and an open state were slow (15 to 65 ms).
This implies the existence of two gating mechanisms. The residual state
may be regarded as the ground state of electrical gating controlled by
Vj; the closed state, as the ground state of chemical
gating.
Key Words: neonatal rat heart • single-channel conductance • gap junction • connexin43 • selective permeability
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Electrical coupling between cardiac cells enables impulse propagation and hence is essential for a coordinated contraction of the heart. As in other syncytia, in cardiac tissues the functional cell-to-cell communication is mediated by gap junctions.1 It is well established that gap junctions of vertebrate tissues differ in molecular structure, physiological properties, such as sensitivity to voltage, second messengers, intracellular Ca2+, and H+, and pharmacological responses (for review, see Sáez et al2 ). Early studies performed on pairs of adult ventricular heart cells suggested that cardiac gap junctions are insensitive to Vj.3 4 5 6 However, more recent experiments with pairs of embryonic and neonatal heart cells have indicated that cardiac gap junctions are sensitive to large Vj gradients.7 8 9 10 At steady state, gj decreases with increasing Vj in a sigmoidal fashion without, however, declining to zero. Conceivably, in early experiments the Vj sensitivity was masked by series resistances located in the recording path and marginal Vj gradients.6 10
Gap junctions constitute assemblies of intercellular channels. Each channel consists of two hemichannels (connexons) composed of six transmembrane proteins (connexins). So far, six different connexins have been identified in vertebrate heart tissues encoded by a multigene family, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50.11 12 This opens the possibility that cardiac cells express different connexins and thereby form homotypic, heterotypic, and heteromeric channels.13 Hence, examination of primary heart cells renders it difficult to assess the biophysical properties of connexin-specific gap junctions and gap junction channels. The wide spectrum of published data on single-channel properties and gap junction properties may originate partially from the diversity of the channel structure.
Over the last decade, pairs of cells dispersed from hearts of different animal species and different developmental stages have been used to examine the properties of gap junction channels. In these preparations, gap junctions typically consisted of hundreds of channels. This situation often caused methodological problems and experimental limitations for electrophysiologists. For example, in order to resolve single-channel currents, the activity of most gap junction channels had to be suppressed by exposure to uncoupling agents, such as lipophilic substances.14 However, this approach may be challenged for several reasons: (1) The uncoupling agents might interfere with the processes under investigation. (2) The discrete current steps observed under these conditions are not easily ascribable to the functional states of a channel. A problem is that the basal current level and, hence, the resting state of a channel are not known. (3) During washout of lipophilic agents, the number of operational channels is variable. (4) Every other steplike increase in Ij may result from a different channel. This renders it difficult to examine the kinetic properties of single gap junction channels.
The aim of the present investigation has been to circumvent some of these difficulties. Adopting an alternative approach, we have reinvestigated the electrical properties of gap junction channels of neonatal rat heart cells, whose major gap junction protein is Cx43.15 The experiments were performed on induced cell pairs16 17 and preformed cell pairs using the dual voltage-clamp method. This approach permitted the following tasks: (1) examination of conductive properties of single channels in the absence of uncoupling agents, (2) comparison of single-channel data gained in the absence and presence of uncoupling agents, and (3) comparison of single-channel data with multichannel data. Preliminary results have been published previously.18
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of Cardiac Cells
Myocytes were isolated from neonatal rat hearts (2-day-old Wistar rats) by means of an enzymatic procedure.19 In brief, pieces of ventricular tissue were dissociated with trypsin (code 210234, Boehringer-Mannheim). The dispersed cells were suspended in culture medium (M199, GIBCO) containing 20 U/mL penicillin, 20 µg/mL streptomycin (code 2212, Seromed), 2 µg/mL vitamin B12 (code V-2876, Sigma Chemical Co), and 10% neonatal calf serum (Boehringer-Mannheim). The cell suspension was diluted to a density of 1x105 to 2x105 cells/mL. Aliquots of 1.5 mL were plated into multiwell culture dishes (22-mm diameter) containing glass coverslips. The cells were incubated at 37°C in a CO2 incubator (2% CO2/98% ambient air; the pH of the medium was 7.4). After 24 hours, the culture medium was replaced by another medium containing 5% serum. The cells were ready for use 6 hours after plating.
Solutions and Chemicals
Experiments were carried out in modified Krebs-Ringer solution
(mmol/L): NaCl 150, KCl 4, CaCl2 2, MgCl2 1,
CsCl 2, BaCl2 1, glucose 5, pyruvate 2, and HEPES 5 (pH
7.4). This solution was designed to minimize K+ currents
across the cell membrane.19 Patch pipettes were filled
with saline containing (mmol/L) Cs+ aspartate-
110, NaCl 10, TEA+ Cl- 10, MgCl2
1, CaCl2 1, HEPES 5 (pH 7.2), EGTA 10 (pCa 
8), and MgATP
3 filtered through 0.22-µm pores. In some experiments,
Cs+ aspartate- was replaced by an equimolar
amount of KCl or TEA+ aspartate-. The 
value was determined at 22°C using a laboratory conductivity meter
(PW 9505, Philips).
Arachidonic acid (code A-9673, Sigma) was dissolved in hexane (10 mmol/L), aliquoted, and stored at -20°C. Before use, the solvent was removed under a stream of N2, and the dry substance was dispersed in Krebs-Ringer solution. Formation of fatty acid micelles was achieved by sonification at 35 kHz for 90 s. Heptanol (Fluka) and SKF-525A (code EI-105, Smith Kline & French; now commercially available as Proadifen, Biomed Research Laboratories) was directly added to the saline.
Electrical Measurements
Glass coverslips with adherent cells were transferred to the
experimental chamber and superfused with modified Krebs-Ringer solution
at room temperature (20°C to 24°C). The experimental chamber was
mounted on the stage of an inverted microscope equipped with
phase-contrast optics (Diaphot-TMD Nikon, Nippon Kogaku). Patch
pipettes were pulled from glass capillaries (code GC150TF-10, Clark
Electromedical Instruments) with a horizontal puller (DMZ-Universal,
Zeitz-Instrumente). When filled with solution, the pipettes had direct
current resistances of 3 to 6 M
(tip diameter, 1 to 2 µm). A
video system (CCD camera, Panasonic WV-CD51/6, Matsushita Electric
Industrial; monitor, JVC TM-916EM, Victor Co) allowed the optical
supervision of the cells and pipettes during an experiment.
Two types of experiments have been performed. In one case, two single
cells were brought into contact with two patch pipettes attached to
separate amplifiers and pushed against each other to induce the
formation of gap junction channels ("induced cell pair"
approach16 ). In the other case, two pipettes were
connected to the cells of a cell pair spontaneously formed in culture
("preformed cell pair" approach). In both configurations, the
dual voltage-clamp method in conjunction with tight-seal whole-cell
recording was used to control individually the membrane
potential of the two cells and to measure separately the current flow
through both pipettes.6 Initially, the membrane potential
of both cells (cell 1 and cell 2) was clamped to the same voltage,
usually -45 mV (V1=V2). Thereafter, the
membrane potential of one of the cells was changed to establish a
junctional voltage gradient,
Vj=V2-V1. In the case of two
separate cells, the currents measured with pipettes 1 and 2 correspond
to current flow through the membrane of cell 1 and cell 2
(I1 and I2), respectively. In case of a cell
pair, a coupling current, Ij, of identical amplitude but
opposite polarity is superimposed on I1 and
I2.
Voltage and current signals were recorded on videotape after pulse code modulation (Neurocorder DR-886, Neuro Data Instruments) and on a chart recorder (Gould RS 3400, Gould Instruments). For later analysis, the signals were filtered at 1 kHz (eight-pole Bessel, -3 dB), digitized at 3.33 kHz with a 12-bit A/D converter (IDA 12120, Indec Systems), and transferred to a personal computer. Data acquisition and signal analysis were performed using the software C-Lab (Indec Systems). The results are presented as mean±1 SEM. Curve fitting and statistical analysis was done with the software SigmaPlot and SigmaStat, respectively (Jandel Scientific).
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Results |
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Formation of Gap Junctions
In order to examine the de novo formation of gap junction channels, two separate myocytes in proximity were selected visually. Each cell was connected to a patch pipette to form a gigaohm seal. After disruption of the membrane patches, the whole-cell recording mode was established. The membrane potential of cell 1 (V1) and cell 2 (V2) was clamped to different levels to create a sustained voltage gradient (Vj=V2-V1) across the presumptive junctional membrane area. Thereafter, the cells were maneuvered against each other to establish a physical membrane-to-membrane contact. This was achieved by gentle displacement of the patch pipettes via micromanipulators. Fig 1A
illustrates a successful
example of gap junction formation. It shows the time course of channel
insertion plotting gj (Ij/Vj)
versus time. Early during the experiment, a sustained Vj
was present. Later on, Vj gradients were induced by
repetitive application of voltage pulses to cell 1 (amplitude, 50 mV;
duration, 200 ms; and frequency, 1 Hz). Physical contact between the
cells was established at t=0 minutes. The first opening of the first
channel inserted occurred at t=21 minutes. Thereafter, channel
formation continued. At t=33 minutes, this process seemingly came to a
halt.
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Using this procedure, we found successful formation of gap junction
channels in 6 of 108 preparations (success rate, <6%). In 54
experiments, the cells remained in physical contact for >20 minutes;
in 17 experiments, for >40 minutes. Yet, no sign of Ij was
detectable. This is consistent with the low incidence of
electrical coupling in spontaneously formed cell pairs in fresh
cultures. For example, 6 hours after plating, 50% of the cell pairs
examined showed no electrical coupling. In the experiments with
successful gap junction formation, the time to first channel opening
ranged from 7 to 22 minutes (average delay, 18.4±2.6 minutes). The
speed of subsequent channel insertion was also variable. For
example, 12 minutes after the first channel insertion, 11 to 14
channels were operational in the most advanced case, but only one
channel was operational in the least developed case.
Fig 1B
summarizes the results from four prominent coupling experiments.
Two preparations with only one or two channels inserted have not been
included in this analysis. Values of gj were
determined every minute, averaged, and plotted versus time after first
channel opening (t=0 minutes). Hence, the resulting plot emphasizes the
process of gap junction formation. It shows that gj
increased with time in a sigmoidal manner, suggesting that channel
insertion is a cooperative process. After 11 minutes, gj
reached an apparent plateau at 0.73±0.07 nS. The maximal rate of
channel insertion was determined as 1.3 channels/min16
(assumption: j(main state)=61 pS; see "Comparison of
Single-Channel Conductances").
Two kinds of factors may be responsible for the low rate of success in detecting de novo formation of gap junctions (<6%; see above): (1) methodological difficulties and (2) biological constraints. With respect to factor 1, random drift of the micromanipulators can lead to the loss of a gigaohm seal and hence to an early end of an experiment. Also, mechanical instability can alter continuously the contact area between the cells. Conceivably, this renders it difficult for connexons to dock with each other. With respect to factor 2, formation of gap junctions could depend on specific cellular requirements. However, none of the parameters considered turned out to be severely critical: (1) temperature (20°C to 35°C), (2) [Ca2+]o (0 to 5 mmol/L), (3) [Ca2+]i (18 to 240 nmol/L), (4) cAMP (0 to 6 mmol/L), (5) enzymes used to isolate the myocytes (trypsin, collagenase), (6) cell substratum (glass, collagen), and (7) time interval between cell dispersion and experiments (6 to 48 hours). This suggests that the density of connexons in the cell membrane is scarce and/or the fluidity of the membrane lipids is low.
Recruitment of Newly Formed Gap Junction Channels
Fig 2 illustrates the formation and subsequent
operation of a new gap junction channel. The signals were recorded
from an induced cell pair during insertion of the first channel. Traces
I1 and I2 represent the currents
recorded from cells 1 and 2, respectively, driven by a sustained
voltage gradient, Vj=-70 mV (V1=-2 mV,
V2=-72 mV). Synchronous deflections in I1 and
I2, comparable in amplitude and of opposite polarity,
indicate the operation of a gap junction channel. The first transition
(upward deflection in I1, downward deflection in
I2) was slow (65 ms) and reflects the first opening of
the newly formed channel. I1 and I2 eventually
settled for a stable level corresponding to current flow through the
fully open channel. The subsequent current transitions were fast (<2
ms) and resulted from Vj-dependent channel gating. During
this mode of activity, I1 and I2 did not return
to the reference level (solid lines). Instead, the currents fluctuated
between two discrete levels corresponding to the fully open state and
the residual state (dashed lines). The latter is attributable to
incomplete channel closure. An analysis of the discrete current
levels revealed the following j values:
j(main state)=67 pS and j(residual
state)=13 pS. Insertion of additional channels in this preparation
yielded the same phenomena (data not shown). The same behavior of
Ij was observed in all preparations with induced channel
formation. Therefore, the sequence of events ("slow channel opening,
followed by fast channel flickering") provides a phenomenological
pattern to identify the recruitment of new channels.
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Single-Channel Events Examined in Preformed Cell Pairs
The results presented so far demonstrate that induced cell
pairs are useful to elucidate the current flow through single Cx43
channels. This approach enabled us to identify unambiguously the
existence of two discrete conductance states, j(main
state) and j(residual state). However, the low success
rate (see "Formation of Gap Junctions") renders this method
impractical for routine examinations of gap junction channels in
neonatal rat heart cells. For example, not enough data were available
to carry out quantitative analyses of single-channel
conductances. Hence, we resorted to experiments on preformed cell
pairs. The rationale was as follows: The studies with induced cell
pairs led to further insight into the operation of gap junction
channels, with respect to channel transitions and conductance levels in
particular.16 17 With this knowledge at hand, we set out
to analyze Ij signals from preformed cell pairs in
a more objective way.
In two cases of preformed cell pairs, the gap junctions consisted of
few channels, ie, one to three. Hence, they allowed the identification
of single-channel events without pharmacological interventions. In
these cases, the following pulse protocol was adopted. Starting from a
common holding potential (V1=V2=-45 mV), a
voltage pulse was administered repetitively (25 to 100 mV, 200 ms, 1
Hz) to one of the cells to establish a Vj gradient. The
associated Ij signals invariably exhibited fast transitions
between two discrete levels attributable to j(main
state) and j(residual state) (data not shown).
In the other group of preformed cell pairs (n=7), the gap junctions consisted of many channels, ie, 100 to 200. To visualize single-channel events, the preparations were superfused with an uncoupling agent to fully block the intercellular current flow. For this purpose, we used lipophilic substances, such as heptanol, a long-chain alkanol,5 20 arachidonic acid, an unsaturated fatty acid,19 or SKF-525A, a blocker of cytochrome P-450 enzymes.21 Thereafter, the uncoupling agent was washed out slowly. During the resulting recovery, previously blocked channels became active one after another. The pattern "slow opening, followed by fast flickering" (see "Recruitment of Newly Formed Gap Junction Channels") was used to monitor the reactivation of channels and to identify discrete current levels. As soon as more than two channels were involved, the uncoupling agent was administered again.
Fig 3 illustrates the procedure of transient chemical
uncoupling. The holding potential of cell 1 and cell 2 was set to -45
mV, and depolarizing test pulses were administered to cell 2 (25 mV,
200 ms, 1 Hz; traces V1 and V2) to evoke
Ij flow (trace I1). Application of 75
µmol/L SKF-525A led to a gradual decrease of Ij. During
this process, the gain of I1 was increased 10-fold to
resolve single-channel events (dashed vertical line). After complete
disappearance of Ij signals, the SKF-525A was slowly washed
out (see arrows). At the same time, the test pulses were ceased, and
V2 was depolarized to 0 mV to establish a sustained
Vj gradient of 45 mV. After a short delay (dashed
horizontal lines), single-channel events became apparent (trace
I1).
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Fig 4 shows current records obtained early during
washout of SKF-525A. In this case, Vj was maintained at
-60 mV (V1=12 mV and V2=-48 mV). The paired
records in the upper panel document the first channel reopening
(left side) and reclosure (right side); ie, I1 and
I2 started from the zero current level (solid lines) and
ended at this level after 3 s. In between these events,
I1 and I2 were jumping between two discrete
levels corresponding to the fully open state of the channel and the
residual state (dashed lines). The current records in the lower
panel repeat the early and late episodes of I1 and
I2 at an expanded time scale. They demonstrate that the
channel reopening and reclosure involved slow transitions (25 ms)
between the completely closed state and the fully open state, whereas
regular flickering involved fast transitions (<2 ms) between the fully
open state and the residual state. Therefore, reopening of a channel
after chemical uncoupling resembled the first opening of a newly formed
channel (see Fig 2). The analysis of the current records in
Fig 4 yielded conductances of 68 and 13 pS for j(main
state) and j(residual state), respectively.
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After recovery of the first channel, in some experiments the protocol
with a maintained Vj gradient was replaced by
Vj pulses. Fig 5 illustrates records
obtained in this way. V1 and V2 were clamped to
the same level, ie, -45 mV. Thereafter, a hyperpolarizing pulse was
administered periodically to cell 2 (100 mV, 200 ms, 1 Hz) to establish
a Vj gradient (trace V2). The voltage pulse was
accompanied by regular channel gating with fast transitions (trace
I1). The current fluctuated between two levels
corresponding to the fully open state, j(main state),
and the partially closed state, j(residual state)
(dashed line). During the voltage pulse, I1 did not return
to the zero current level (solid line) but promptly did so when the
driving force was reduced to zero at the end of the pulse.
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Fig 6 documents the sequential activation of gap
junction channels in another preparation observed during washout of
SKF-525A. Vj was maintained at -60 mV (V1=12
mV and V2=-48 mV). Reopening of a first channel provoked
two discrete current levels in I1 and I2,
reflecting the fully open state and the residual state. Reopening of a
second channel (see arrows) gave rise to three additional current
levels corresponding to 2·j(main state),
j(main state)+j(residual state), and
2·j(residual state). Hence, in the presence of two
operational channels (n=2), the total residual conductance
of the preparation was N·j(residual state), ie,
2·12 pS=24 pS (see dashed lines).
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Ij records collected late during washin of SKF-525A
revealed a sequence of events mirroring those depicted in Figs 3 and 4.
Specifically, the closure of the last channel involved a slow
transition starting at the main open state and ending at the closed
state (data not shown). However, for practical purposes, the results
presented in the present study were gained from washout
periods.
Comparison of Single-Channel Conductances
The single-channel records gained from (1) induced cell
pairs, (2) preformed cell pairs with weak coupling, and (3) preformed
cell pairs with strong coupling (after treatment with SKF-525A) were
analyzed separately. The following values were obtained: (1)
j(main state)=62.3±0.6 pS (n=45, three cell pairs) and
j(residual state)=12.6±0.4 pS (n=19), (2)
j(main state)=61.0±0.7 pS (n=31, two cell pairs) and
j(residual state)=11.7±0.3 pS (n=21), and (3)
j(main state)=60.5±0.5 pS (n=181, seven cell pairs) and
j(residual state)=11.9±0.2 pS (n=103). Statistical
analysis revealed no significant differences for either
j(main state) or j(residual state)
between group 1 and group 2 (by Student's t test,
P<.17 and .07) or groups 1+3 and group 3 (P<.09
and .43), respectively. This indicates that SKF-525A does not affect
the single-channel conductances. Hence, the data from the three
experimental groups were combined and sampled in 2-pS bins for further
analysis (see "Selective Permeability of Single
Channels").
Selective Permeability of Single Channels
The experiments presented so far were carried out with a
pipette solution containing Cs+ aspartate- as
a major charge carrier (see "Solutions and Chemicals"). In the
experiments to be described next, we used pipette solutions containing
KCl or TEA+ aspartate- instead. Fig 7 illustrates appropriate records gained from
preformed cell pairs during washout of SKF-525A. Application of voltage
pulses to cell 2 (traces V2) led to current signals with
two distinct levels attributable to the fully open state and the
residual state (traces I1). In the presence of KCl
(pipette) solution, j(main state) and
j(residual state) turned out to be 96 pS and 25 pS,
respectively (top current record, Vj=-90 mV). In the
presence of TEA+ aspartate- (pipette)
solution, the respective conductances were 20 and 3 pS (bottom current
record, Vj=-100 mV). Hence, both j
values decreased drastically with increasing size of the major charge
carriers. For comparison, in the case of Cs+
aspartate- (pipette) solution, the conductances were 61
and 12 pS, respectively (middle current record, Vj=-75
mV). Ijs recorded in the presence of TEA+
aspartate- (pipette) solution often showed a critical
signal-to-noise ratio. In these cases, segments of current traces were
analyzed using an all-point procedure. Application of this
method to the I1 signal in Fig 7, bottom trace, revealed
that the residual current level is significantly different from the
reference current level (P<.001).
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The single-channel currents determined in the presence of KCl,
Cs+ aspartate-, and TEA+
aspartate- (pipette) solution were used to calculate
j. The values of j(main state) and
j(residual state) collected were sampled in 2- and 1-pS
bins, respectively. Fig 8 summarizes the resulting
frequency histograms. The j(main state) and
j(residual state) data gave rise to the distributions on
the right side and left side, respectively. Each data group described a
binomial distribution and thus was fitted with a Gaussian distribution.
The smooth curves correspond to the best fit of the data using a
nonlinear least-squares algorithm. For the KCl (pipette) solution (Fig 8A), the mean values of j(main state) and
j(residual state) were 95.6±0.5 pS (n=273, 10
preparations) and 22.7±0.2 pS (n=161, 10 preparations), respectively.
For the Cs+ aspartate- (pipette) solution (Fig 8B), j(main state) and j(residual state)
were 60.8±0.4 pS (n=257, 12 preparations) and 12.0±0.1 pS (n=143),
respectively. For the TEA+ aspartate-
(pipette) solution (Fig 8C), j(main state) and
j(residual state) were 18.7±0.2 pS (n=169, three
preparations) and 2.9±0.1 pS (n=42, three preparations),
respectively.
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A comparison of the j(main state) data and
j(residual state) data allows some insight into the
process of ion permeation through Cx43 channels. For example, the
values of both j(main state) and
j(residual state) decrease with increasing molecular
mass of the monovalent ionic charge carriers involved. This may reflect
a decrease of the ionic mobility in aqueous solution with increasing
ionic size or a discrimination brought about by the structure of the
channel pore. In addition, considering the mean conductance values, it
turns out that the ratio of j(main state) to
j(residual state) is not constant. It decreases from 4.2
to 5.1 and 6.3 for the pipette solutions containing KCl,
Cs+ aspartate-, and TEA+
aspartate-, respectively. This suggests that the residual
state exerts a more prominent restriction to ion permeation than does
the fully open state.
To test this hypothesis, we performed statistical analyses with
limited data. The global data sets were reduced to cases where
j(main state) and j(residual state) were
identifiable as consecutive channel events. Numeric analyses
yielded the following values for the j(main
state)-to-j(residual state) ratio: KCl solution (n=75),
4.14±0.04; Cs+ aspartate- (n=38), 5.12±0.03;
and TEA+ aspartate- (n=25), 6.51±0.11. Test
analysis revealed that these ratios are significantly different
from each other (by Kruskal-Wallis ANOVA, P<.0001). This
allows the conclusion that the residual state restricts ion permeation
more efficiently than does the main state.
Fig 9 shows a plot of the single-channel conductance
(j) versus conductivity of the pipette solution ().
The values of j(main state) and
j(residual state) were taken from Fig 8; the values of
for the different pipette solutions have been determined with a
conductivity meter and were as follows (22°C): KCl, 12.3 mS/cm;
Cs+ aspartate-, 8.3 mS/cm; and
TEA+ aspartate-, 4.7 mS/cm. In order to
construct the graph, the values of j and were
normalized relative to those of the KCl (pipette) solution. The filled
circles and open circles correspond to the j(main state)
and j(residual state) data, respectively. The dashed
line represents the function =m·j, where
m indicates slope (m=1). It assumes that the ionic mobilities in the
channel pore and in aqueous solution are the same. However, the graph
shows that this is not the case. The relationships
=f[j(main state)] and
=f[j(residual state)] both exhibit a slope >1.
This implies that the ions experience a restriction during the passage
through the channels in both the Cs+
aspartate- and TEA+ aspartate-
solutions. The graph also shows that the restriction is more pronounced
for j(residual state) than for j(main
state). This suggests that the transition from the fully open state to
the residual state is accompanied by an increase of the ability of the
channel to discriminate between ions.
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indicates mean values of
, mean values of
Single Channels Exhibit Multiple Conductance States
Some single-channel records revealed additional conductance
states between j(main state) and
j(residual state). To examine these events, we used the
KCl (pipette) solution because it promises the largest j
readings (see "Selective Permeability of Single Channels"). Fig 10 shows two representative examples
obtained from preformed cell pairs early during washout of SKF-525A,
ie, when a single channel was operational (see "Single-Channel Events
Examined in Preformed Cell Pairs"). In Fig 10A, Vj was
maintained at 50 mV (V1=-45 mV and V2=5 mV).
The associated records I1 and I2 revealed
three discrete levels. Since the signals were symmetrical, the
transitions reflect gap junctional events (transition times, <2 ms).
Hence, the three current levels reflect the main open state (dotted
lines), a substate (short dashes), and the residual state (long
dashes). The analysis revealed the following conductances:
j(main state)=93 pS, j(substate)=36 pS,
and j(residual state)=22 pS. Thus, the step between
j(substate) and j(residual state) was 14
pS. Note that I1 and I2 did not return to the
reference level (solid lines); ie, the channel failed to close
completely. In Fig 10B, a hyperpolarizing pulse was applied to cell 2
to set Vj to -75 mV (V1=-45 mV and
V2=-120 mV). As a consequence, the channel exhibited fast
transitions (<2 ms) involving three discrete current levels (see trace
I1). The levels correspond to j(main state)
of 100 pS (dotted line), j(substate) of 33 pS (short
dashes), and j(residual state) of 22 pS (long dashes).
Hence, the difference between j(substate) and
j(residual state) was 11 pS. Again, I1 (and
I2, not shown) failed to return to the reference current
level during the Vj pulse (solid line).
|
When compared with the main open state and residual state, substates
were observed less frequently. They were seen preferentially in
conjunction with short Vj pulses of large amplitude (200
ms, >75 mV) and with long Vj pulses of intermediate
amplitude (>1 s, 50 mV). Occasionally, as many as three different
substates were discernible in a given record. As a rule, the
current steps involving substates corresponded to conductances
equivalent to 1/9 to 1/7 of j(main state).
Fast Versus Slow Channel Transitions
As already mentioned (see "Recruitment of Newly Formed Gap
Junction Channels"), Cx43 channels operate in two different modes.
The modes can be distinguished based on the speed of Ij
transitions. Fast transitions were complete within the response time of
the experimental setup, ie, <2 ms, whereas slow transitions lasted 15
to 65 ms. For example, associated with the first opening of a newly
formed channel, Ij underwent a slow transition from the
closed state to the main open state (see Fig 2). The slow transition
was followed by episodes of fast transitions between the main open
state, the substates, and the residual state. Fast transitions did not
involve the closed state. Likewise, slow transitions were seen in
conjunction with the reopening of a channel previously blocked by
lipophilic agents, such as heptanol, arachidonic acid,
and SKF-525A (see Fig 4). Again, the slow transition was followed by
trains of fast transitions involving the main open state, the
substates, and the residual state. The inverse sequence of events was
observed during pharmacological uncoupling. A manifestation of this
phenomenon is also visible in Fig 4. After a series of fast
transitions, Ij exhibited a slow transition from the
residual state to the closed state.
Fig 11 illustrates that slow transitions occurred not
only between the closed state and the main open state but between other
states as well. It shows records gained from a preformed cell pair
during removal of SKF-525A. V1 and V2 were held
at 5 and -70 mV, respectively, to establish a sustained Vj
of -75 mV (traces not shown). The signals I1 and
I2 show an episode starting with a first channel reopening;
ie, both current traces underwent a slow transition (15 ms) between
the zero current level (solid lines) and a level corresponding to the
main open state. After a few milliseconds, I1 and
I2 exhibited a fast transition (<2 ms) to the residual
state (dashed lines). About 2.5 s later, I1 and
I2 slowly declined to the zero level (transition time,
35 ms). Shortly thereafter, the currents slowly returned to the
residual level (transition time, 55 ms). Slow transitions have also
been seen between the main open state and closed state.
|
These results indicate that transitions between
j(residual) and j(main state) usually are
fast (<2 ms) and transitions involving the closed state are slow (15
to 65 ms). Conceivably, fast events reflect biophysical channel gating
governed by Vj, whereas the slow transitions reflect
chemical gating controlled by uncoupling agents or
insertion/degradation of channels.
Relationship Between gj and Vj
Using preformed cell pairs, we have also examined the dependency
of gj on Vj in the presence of different
pipette solutions. To carry out these experiments, Vj
gradients of long duration (5 to 40 s), variable amplitude (up
to 130 mV), and either polarity were administered to one of the cells,
and Ij values were recorded from the other cell. Fig 12A shows original records (V2,
I1) obtained with the KCl (pipette) solution at a
Vj of -110 mV (V1=-40 mV and
V2=-150 mV). Typical for this condition, Ij
showed a time-dependent decay. For analysis, the amplitude of
Ij was determined at the beginning [Ij(inst)]
and end [Ij(ss)] of each Vj pulse to
calculate the conductances
gj(inst)=Ij(inst)/Vj and
gj(ss)=Ij(ss)/Vj. The values of
gj(ss) were then normalized with respect to
gj(inst) and plotted versus Vj. The graph in
Fig 12B summarizes the results obtained for pipette solution containing
KCl (filled circles) and TEA+ aspartate- (open
circles). For clarity, only half the data sets have been depicted
(negative Vj values, KCl solution; positive Vj
values, TEA+ aspartate- solution). The smooth
curves represent the best fits of the data to the Boltzmann
equation, assuming that each channel contains two symmetrical gates in
series:
gj([1-gj(min)]/{1+exp[A(Vj–Vj,(0))]}+gj(min)),
where gj(min) is the normalized conductance at large
Vj, and Vj,(0) corresponds to Vj at
which gj is half-maximally inactivated. A is
the maximal steepness of the relationship and expresses the gating
charge, zq(kT)-1, where z is the
equivalent number of unitary positive charges (q) moving through the
entire electric field applied, and k and T represent
Boltzmann's constant and temperature in degrees kelvin, respectively.
The analysis yielded the following parameters: for
the KCl solution, Vj,(0)=-51.5 mV/51.2 mV,
gj(min)=0.28/0.25, and z=3.1/2.9; for the TEA+
aspartate- solution, Vj,0=-59.5 mV/59.0 mV,
gj(min)=0.15/0.16, and z=2.0/2.3 (for negative/positive
values of Vj). Therefore, in the case of both pipette
solutions examined, the data yielded a bell-shaped relationship that
was nearly symmetrical. Interestingly, gj(min) and z were
smaller with TEA+ aspartate- solution. In the
two groups of cell pairs, the average gj(inst) was
1.96±0.7 nS (four cell preparations) and 1.78±0.7 nS (seven
preparations), respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study presents evidence that Cx43 gap junction channels possess various conductance states: a main open state, several substates, a residual state, and a closed state. The data offer an explanation for the incomplete decline of gj at large Vj values. In this respect, Cx43 channels resemble the channels of other vertebrate or invertebrate gap junctions.16 17 22 The underlying experiments involved application of Vj gradients, use of lipophilic uncoupling agents, and substitution of pipette solutions.
De Novo Formation of Gap Junction Channels
It has been suggested that precursors of gap junction channels are
present as hemichannels in cell membranes. Evidence supporting this
view emerged from electrophysiological
studies. For example, expression of bovine Cx44, rat Cx46, or chicken
Cx56 in Xenopus oocytes induced voltage-gated currents
attributable to connexons.23 24 25 Pharmacological
interventions imposed on differentiated cells led to similar
results.26 27 Hence, the formation of gap junction
channels is expected to occur by docking of hemichannels of adjacent
cells. Our data on induced pairs of neonatal rat heart cells are
consistent with this view.
Two single myocytes, when brought into physical contact, showed first signs of channel formation within 7 to 25 minutes. For the same type of cells, Rook et al7 previously reported a delay of 5 to 15 minutes. For comparison, pairing transfected HeLa cells expressing Cx4017 and parental insect cells (for references, see Bukauskas and Weingart16 ) revealed a delay of 28 to 45 minutes and <5 minutes, respectively. Various factors can be responsible for the diversity in delay, eg, differences in the number of hemichannels per unit membrane area or differences in the fluidity of cell membranes. However, the involvement of cell adhesion molecules or cellular signal transduction cannot be excluded.
In neonatal rat heart cells, the maximal rate of channel insertion was
1.3 channels/min. This value sets an upper limit for the turnover rate
of Cx43 channels. Pairs of these cells contain 460 gap junction
channels [gj=28 nS, Reference 1010 ; j(main
state)=61 pS, the present study]. Considering a half-life for Cx43
channels of 3 hours (assessment of gj by L. Firek and R.
Weingart, unpublished data, 1992), this amounts to a turnover rate of
1.3 channels/min. For comparison, studies with insect cells revealed
maximal insertion rates of 2.5 channels/min (for C6/3616 )
and 60 channels/min (for hemocytes28 ). Interestingly,
pulse-chase labeling yielded a half-life of 1.9 hours for Cx43 in
neonatal rat myocytes.15
Resolving Single-Channel Data From Preformed Cell Pairs
The method of induced pairs was useful to elucidate the properties
of Cx43 channels in neonatal rat heart cells. However, the experiments
with successful channel formation were rather scarce (<6%).
Therefore, this approach is not suited as a routine method for these
cells. Conceivably, this may be true for primary dispersed cells in
general. The use of weakly coupled pairs spontaneously formed in
culture offered an alternative. Screening cell cultures at different
times after plating (up to 8 hours), we found abundant pairs with no
channels or many channels but only two pairs with one or two channels.
Hence, from a practical point of view, this method was also tedious.
Another possibility examined involved the use of lipophilic agents, eg,
SKF-525A, to uncouple pairs with many channels. These substances
provoke a dose-dependent impairment of gj.21
To visualize single-channel events arising from identified channels,
the engram "slow opening, followed by fast flickering" (see
"Recruitment of Newly Formed Gap Junction Channels") was most
useful. It allowed us to observe the reactivation of previously blocked
channels and to detect single-channel events, analyze them, and
interpret them within the framework of the knowledge gained from
induced cell pair experiments. The data gained in this way were
appropriate to assess j, but not channel kinetics.
Single-Channel Conductances
We found that Cx43 channels exhibit several prominent conductance
states, a main open state, several substates, a residual state, and a
closed state. Statistical analysis revealed that the
experimental methods adopted (see "Unraveling of Single-Channel Data
From Preformed Cell Pairs") has no influence on the values of
j(main state) and j(residual state). This
suggests that (1) the channels formed spontaneously, and those forced
to form with the induced cell pair approach are identical, and (2) the
uncoupling agents used do not affect j. Subconductance
states were interposed between the residual state and the main open
state. They were mainly associated with Vj pulses of short
duration and large amplitude (200 ms and >75 mV, respectively) or long
duration and moderate amplitude (>1 s and 50 mV, respectively).
Ij transitions attributable to substates corresponded to
conductances of 1/9 to 1/7 of j(main state). Because of
their rare incidence, no attempt was made to quantify the
substates.
Single-channel experiments were performed in the presence of different
pipette solutions. We assumed that the pipette solution was in
equilibrium with the cytosol before current measurements (a few minutes
versus tens of minutes). It turned out that j values
depend on the ions available as charge carriers. Under the condition
most closely matching the physiological situation,
ie, in the presence of Cs+ aspartate- (see Fig 7B), j(residual state) and j(main state)
were 12 and 61 pS, respectively. In the presence of KCl
(KCl>Cs+aspartate) and
TEA+ aspartate-
(TEA+aspartate<Cs+aspartate),
the values of both j(residual state) and
j(main state) grew larger (23 and 96 pS) and smaller (3
and 19 pS), respectively. Moreover, j(residual state)
was more responsive to a change in than was j(main
state). This becomes evident from the following comparisons: (1) The
j(main state)-to-j(residual state) ratios
for KCl, Cs+ aspartate-, and TEA+
aspartate- solution were 4.2, 5.1, and 6.3, respectively
(see Fig 9); ie, they did not remain constant. (2) The
j(KCl)-to-j(Cs+
aspartate-) ratios for the main state and residual state
were 1.6 and 2.1, respectively; the corresponding values for the
j(KCl)-to-j(TEA+
aspartate-) ratio were 4.8 and 8.3. These comparisons
suggest that the residual state represents a more prominent
barrier for ion permeation than the main open state. This conclusion
supports the idea that the main state–residual state transitions
provide a mechanism for the modulation of intercellular signaling based
on solute size.16
Recently, it has been reported that neonatal rat myocytes express not
only Cx43 but also Cx45.15 This finding is intriguing,
since in our experiments we did not see currents attributable to such
channels. However, Cx45 channels appear to exhibit properties
distinctly different from those of Cx43 channels both at the
microscopic and macroscopic current level. For example, SKHep1 cells
endogenously expressing Cx45 were reported to have a
j of 32 pS and a Vj of 13.4
mV.29 Therefore, based on
electrophysiological criteria, homotypic
channels (Cx43-Cx43, Cx45-Cx45) and heterotypic channels (Cx43-Cx45)
should be readily discernible in neonatal rat heart cells.
In the past, several investigators have attempted to assess the
conductance of gap junction channels in neonatal rat heart cells. Some
found histograms with a single peak: 50 to 60 pS (pipette solution,
K+ glutamate-, temperature not
stated),30 33 pS (Cs+ aspartate-,
22°C),19 and 45 pS (Cs+
aspartate-, 22°C).10 Others reported
histograms with two peaks: 20/45 pS (K+
gluconate-, 21°C)7 and 21/43 pS
(K+ gluconate-, 20°C).31 For
comparison, transfection of rat Cx43 into N2A and SKHep1 cells yielded
histograms with one peak (KCl, 80 pS; K+
glutamate-, 57 pS; 20°C to 22°C)32 and
several peaks sensitive to Vj33 (for an
explanation, see Reference 1616 ), respectively. However, these studies
did not allow for distinguishing between the residual state and the
closed state; ie, their j values are likely to
correspond to differences between conductance states. Hence, direct
comparisons between our data and those of these groups pose
problems.
Cardiac myocytes have also been used before to study selective
permeabilities of gap junction channels. Examining neonatal rat heart
cells, Rook et al7 reported
j(KCl)-to-j(K+
gluconate-) ratios of 1.5 and 1.3 for their two
conductance peaks. In heart cells of adult guinea pigs, Weingart and
Rüdisüli34 found a
j(KCl)-to-j(K+
aspartate-) ratio of 1.4. For comparison, Veenstra et
al32 reported the following values for the
j(KCl)-to-j(K+
glutamate-) ratio: 1.4 for rat Cx43, 1.35/1.2 for chicken
Cx43, and 1.2 for chicken Cx45. However, these conductance ratios
cannot be compared directly with ours because no distinction was made
between j(main state) and j(main
state)-j(residual state) (see also above).
Transitions Between Channel States
Our single-channel records revealed two kinds of current
transitions, fast ones (<2 ms) and slow ones (15 to 65 ms). Fast
transitions occurred between open-channel states, ie,
j(main state), j(substates), and
j(residual state). They resemble the transitions of
voltage-gated ion channels and thus may reflect rapid conformation
changes of connexins brought about by movements of polar groups. The
existence of substates suggests that each connexin provides a subgate.
Slow transitions occurred between j(closed state) and an
open-channel state. They were evident in conjunction with washin and
washout of lipophilic uncoupling agents (see Fig 4) and thus may result
from chemical channel gating. Conceivably, slow transitions may involve
interactions between connexins and membrane lipids, thus giving rise to
slow modifications of the channel structure. Occasionally, slow
transitions were also seen in the absence of uncoupling agents.
Presumably, slow events starting from j(closed state)
(see Fig 4) or ending at j(closed state)
represent recruitment of a new channel or removal of an
existing channel, respectively. Coexistence of fast and slow
Ij transitions has been observed previously in neonatal rat
heart cells,20 transfected HeLa cells expressing
Cx40,17 and insect cells.16
Comparison of Microscopic and Macroscopic Currents
Experiments on preformed cell pairs revealed that the relationship
gj(ss)=f(Vj) is bell-shaped and nearly
symmetrical (see Fig 12). The data were best described by Boltzmann's
equation. It assumes that gj depends on a single population
of channels with two states, that the energy difference between the
states is a linear function of Vj, and that the transitions
between states are reversible and obey first-order kinetics. A similar
relationship between gj(ss) and Vj has been
found previously in neonatal rat heart cells,10
Xenopus oocytes injected with mouse Cx43
mRNA,35 SKHep1 cells expressing human Cx43,36
and RIN cells expressing rat Cx43.37
Our single-channel data suggest that the incomplete decay of
gj(ss) at large Vj is due to partial closure of
individual channels; ie, gj(min) reflects
j(residual state). Initially, this concept has been
proposed for insect cells16 and HeLa cells expressing
various mouse connexins, such as Cx4017 (see also
Bukauskas and Weingart,22 1995). This conclusion is
further supported by the close match of the values of
gj(min) and j(residual
state)/j(main state) for pipette solution containing KCl
(0.28/0.25 for negative/positive Vjs versus 0.24) and
TEA+ aspartate- (0.15/0.16 versus 0.16). It is
also compatible with the finding that gj(min) and
j(residual state)/j(main state) were
larger in the former case. According to Fig 12, the ionic composition
of the pipette solution also affected the other Boltzmann
parameters, ie, Vj(0) and z, not only
gj(min). Vj(0) was smaller in the presence of
KCl (51 versus 59 mV), whereas z was larger (3.1/2.9 versus 2/2.3). To
explain these findings, additional experiments are needed.
The Boltzmann parameters gained from our experiments [KCl
(pipette) solution: Vj(0)=-51 mV/51 mV,
gj(min)=0.28/0.25, and z=3.1/2.9] are comparable to those
previously reported for ventricular cells of neo-natal
hamsters38 [Vj(0)=-69 mV/61 mV,
gj(min)=0.34/0.32, and z=2.8/2.4; K+
glutamate- (pipette solution), room temperature], atrial
cells of adult rats39 [Vj(0)=42.5 mV,
gj(min)=0.22, and z=1.1; CsCl (pipette) solution, room
temperature], and SKHep1 cells transfected with human
Cx4333 [Vj(0)=-58 mV/64 mV,
gj(min)=0.38/0.36, and z=3; CsCl (pipette) solution,
temperature not stated). Conceivably, the larger gj(min)
values reported by the latter group may have resulted from the use of
strongly coupled cell pairs (see Reference 3838 ).
Gating of Cx43 Gap Junction Channels
The data presented suggest the following model for the
operation of a Cx43 channel. Each channel possesses two different
gating mechanisms, one controlled by an electrical signal and the other
controlled by a chemical signal. Hence, it is responsive to two kinds
of stimuli. On the one hand, the residual state may be regarded as
ground state of electrical gating. In this mode, the channel undergoes
fast transitions (<2 ms, corresponding to the frequency response of
the recording system; conceivably, the true channel transitions
are faster) between the residual state and a substate or the main open
state. The probability of a channel to be in the main open state is
controlled by Vj. At small values of Vj, open
probability is close to 1, at large values of Vj, it
approaches 0. The decline of Ij with Vj is
determined primarily by the open probability and, to a lesser degree,
by occasional substates. On the other hand, the closed state may be
regarded as ground state of chemical gating. In this mode, a channel
exhibits slow transitions (tens of milliseconds) between the closed
state and any one open state. A similar model has been proposed
previously for the murine Cx40 channel17 and for an insect
channel of unidentified gap junction protein.16
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Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported by the Swiss National Science Foundation (31-36046.92 and 31-45554.95), the Schweizerische Herzstiftung, and the Swiss Department of Foreign Affairs. We are grateful to Marlis Herrenschwand for her expert technical assistance and to Prof Silvio Weidmann for his critical comments on the manuscript.
|
Footnotes |
|---|
Reprint requests to R. Weingart, Department of Physiology, Bühlplatz 5, CH-3012 Bern, Switzerland.
Previously published as preliminary results in abstract form (Experientia. 1995;51:A68).
Received July 8, 1996; accepted January 21, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J. Kang, N. Kang, D. Lovatt, A. Torres, Z. Zhao, J. Lin, and M. Nedergaard Connexin 43 Hemichannels Are Permeable to ATP J. Neurosci., April 30, 2008; 28(18): 4702 - 4711. [Abstract] [Full Text] [PDF]
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 G. Kanaporis, G. Mese, L. Valiuniene, T. W. White, P. R. Brink, and V. Valiunas Gap Junction Channels Exhibit Connexin-specific Permeability to Cyclic Nucleotides J. Gen. Physiol., March 31, 2008; 131(4): 293 - 305. [Abstract] [Full Text] [PDF]
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S. M. Kelly, J. K. VanSlyke, and L. S. Musil Regulation of Ubiquitin-Proteasome System mediated Degradation by Cytosolic Stress Mol. Biol. Cell, November 1, 2007; 18(11): 4279 - 4291. [Abstract] [Full Text] [PDF]
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 E. Louis, C. D. E. Boschi, G. J. Ortega, and E. Fernandez Role of transport performance for neuron cell morphology FASEB J, March 1, 2007; 21(3): 866 - 871. [Abstract] [Full Text] [PDF]
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 H.-Z. Wang, P. R. Brink, and G. J. Christ Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1366 - C1376. [Abstract] [Full Text] [PDF]
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J. K. VanSlyke and L. S. Musil Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function Mol. Biol. Cell, November 1, 2005; 16(11): 5247 - 5257. [Abstract] [Full Text] [PDF]
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 F. Vogalis, C. C. Hegg, and M. T. Lucero Electrical Coupling in Sustentacular Cells of the Mouse Olfactory Epithelium J Neurophysiol, August 1, 2005; 94(2): 1001 - 1012. [Abstract] [Full Text] [PDF]
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 J. Shibayama, W. Paznekas, A. Seki, S. Taffet, E. W. Jabs, M. Delmar, and H. Musa Functional Characterization of Connexin43 Mutations Found in Patients With Oculodentodigital Dysplasia Circ. Res., May 27, 2005; 96(10): e83 - e91. [Abstract] [Full Text] [PDF]
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P. Beauchamp, C. Choby, T. Desplantez, K. de Peyer, K. Green, K. A. Yamada, R. Weingart, J. E. Saffitz, and A. G. Kleber Electrical Propagation in Synthetic Ventricular Myocyte Strands From Germline Connexin43 Knockout Mice Circ. Res., July 23, 2004; 95(2): 170 - 178. [Abstract] [Full Text] [PDF]
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 V. Valiunas, S. Doronin, L. Valiuniene, I. Potapova, J. Zuckerman, B. Walcott, R. B. Robinson, M. R. Rosen, P. R. Brink, and I. S. Cohen Human mesenchymal stem cells make cardiac connexins and form functional gap junctions J. Physiol., March 15, 2004; 555(3): 617 - 626. [Abstract] [Full Text] [PDF]
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A. G. Kleber Cell-to-Cell Coupling Between Host and Donor Cells in the In Situ Myocardium Circ. Res., June 13, 2003; 92(11): 1176 - 1178. [Full Text] [PDF]
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V. Valiunas, E. C. Beyer, and P. R. Brink Cardiac Gap Junction Channels Show Quantitative Differences in Selectivity Circ. Res., July 26, 2002; 91(2): 104 - 111. [Abstract] [Full Text] [PDF]
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A. P. Moreno, M. Chanson, J. Anumonwo, I. Scerri, H. Gu, S. M. Taffet, and M. Delmar Role of the Carboxyl Terminal of Connexin43 in Transjunctional Fast Voltage Gating Circ. Res., March 8, 2002; 90(4): 450 - 457. [Abstract] [Full Text] [PDF]
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 Y. Qu and G. Dahl Function of the voltage gate of gap junction channels: Selective exclusion of molecules PNAS, January 22, 2002; 99(2): 697 - 702. [Abstract] [Full Text] [PDF]
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G. T. Cottrell and J. M. Burt Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1559 - C1567. [Abstract] [Full Text] [PDF]
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 V. Valiunas, J. Gemel, P. R. Brink, and E. C. Beyer Gap junction channels formed by coexpressed connexin40 and connexin43 Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1675 - H1689. [Abstract] [Full Text] [PDF]
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 L. Polontchouk, J.-A. Haefliger, B. Ebelt, T. Schaefer, D. Stuhlmann, U. Mehlhorn, F. Kuhn-Regnier, E. R. De Vivie, and S. Dhein Effects of chronic atrial fibrillation on gap junction distribution in human and rat atria J. Am. Coll. Cardiol., September 1, 2001; 38(3): 883 - 891. [Abstract] [Full Text] [PDF]
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A. O. Martin, M.-N. Mathieu, C. Chevillard, and N. C. Guerineau Gap Junctions Mediate Electrical Signaling and Ensuing Cytosolic Ca2+ Increases between Chromaffin Cells in Adrenal Slices: A Role in Catecholamine Release J. Neurosci., August 1, 2001; 21(15): 5397 - 5405. [Abstract] [Full Text] [PDF]
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 T. A.B. van Veen, H. V.M. van Rijen, and T. Opthof Cardiac gap junction channels: modulation of expression and channel properties Cardiovasc Res, August 1, 2001; 51(2): 217 - 229. [Abstract] [Full Text] [PDF]
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H.-Z. Wang, N. Day, M. Valcic, K. Hsieh, S. Serels, P. R. Brink, and G. J. Christ Intercellular communication in cultured human vascular smooth muscle cells Am J Physiol Cell Physiol, July 1, 2001; 281(1): C75 - C88. [Abstract] [Full Text] [PDF]
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H. S. Tamaddon, D. Vaidya, A. M. Simon, D. L. Paul, J. Jalife, and G. E. Morley High-Resolution Optical Mapping of the Right Bundle Branch in Connexin40 Knockout Mice Reveals Slow Conduction in the Specialized Conduction System Circ. Res., November 10, 2000; 87(10): 929 - 936. [Abstract] [Full Text] [PDF]
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D. S. He and J. M. Burt Mechanism and Selectivity of the Effects of Halothane on Gap Junction Channel Function Circ. Res., June 9, 2000; 86 (11): e104 - e109. [Abstract] [Full Text] [PDF]
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V. Valiunas, R. Weingart, and P. R. Brink Formation of Heterotypic Gap Junction Channels by Connexins 40 and 43 Circ. Res., February 4, 2000; 86 (2): e42 - e49. [Abstract] [Full Text] [PDF]
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M. Srinivas, R. Rozental, T. Kojima, R. Dermietzel, M. Mehler, D. F. Condorelli, J. A. Kessler, and D. C. Spray Functional Properties of Channels Formed by the Neuronal Gap Junction Protein Connexin36 J. Neurosci., November 15, 1999; 19(22): 9848 - 9855. [Abstract] [Full Text] [PDF]
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V. Valiunas, D. Manthey, R. Vogel, K. Willecke, and R. Weingart Biophysical properties of mouse connexin30 gap junction channels studied in transfected human HeLa cells J. Physiol., September 15, 1999; 519(3): 631 - 644. [Abstract] [Full Text] [PDF]
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G. J. Christ, M. Spektor, P. R. Brink, and L. Barr Further evidence for the selective disruption of intercellular communication by heptanol Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H1911 - H1917. [Abstract] [Full Text] [PDF]
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D. S. He, J. X. Jiang, S. M. Taffet, and J. M. Burt Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells PNAS, May 25, 1999; 96(11): 6495 - 6500. [Abstract] [Full Text] [PDF]
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C. Castro, J. M. Gomez-Hernandez, K. Silander, and L. C. Barrio Altered Formation of Hemichannels and Gap Junction Channels Caused by C-Terminal Connexin-32 Mutations J. Neurosci., May 15, 1999; 19(10): 3752 - 3760. [Abstract] [Full Text] [PDF]
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G. J. Christ and P. R. Brink Analysis of the Presence and Physiological Relevance of Subconducting States of Connexin43-Derived Gap Junction Channels in Cultured Human Corporal Vascular Smooth Muscle Cells Circ. Res., April 16, 1999; 84(7): 797 - 803. [Abstract] [Full Text] [PDF]
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A. Muller, M. Lauven, R. Berkels, S. Dhein, H.-R. Polder, and W. Klaus Switched single-electrode voltage-clamp amplifiers allow precise measurement of gap junction conductance Am J Physiol Cell Physiol, April 1, 1999; 276(4): C980 - C987. [Abstract] [Full Text] [PDF]
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D. T. Kurjiaka, T. D. Steele, M. V. Olsen, and J. M. Burt Gap junction permeability is diminished in proliferating vascular smooth muscle cells Am J Physiol Cell Physiol, December 1, 1998; 275(6): C1674 - C1682. [Abstract] [Full Text] [PDF]
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R. Vogel and R. Weingart Mathematical model of vertebrate gap junctions derived from electrical measurements on homotypic and heterotypic channels J. Physiol., July 1, 1998; 510(1): 177 - 189. [Abstract] [Full Text] [PDF]
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G. Calero, M. Kanemitsu, S. M. Taffet, A. F. Lau, and M. Delmar A 17mer Peptide Interferes With Acidification-Induced Uncoupling of Connexin43 Circ. Res., May 19, 1998; 82(9): 929 - 935. [Abstract] [Full Text] [PDF]
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P. R. Brink, K. Cronin, K. Banach, E. Peterson, E. M. Westphale, K. H. Seul, S. V. Ramanan, and E. C. Beyer Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37 Am J Physiol Cell Physiol, October 1, 1997; 273(4): C1386 - C1396. [Abstract] [Full Text] [PDF]
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A. P. Moreno, M. Chanson, J. Anumonwo, I. Scerri, H. Gu, S. M. Taffet, and M. Delmar Role of the Carboxyl Terminal of Connexin43 in Transjunctional Fast Voltage Gating Circ. Res., March 8, 2002; 90(4): 450 - 457. [Abstract] [Full Text] [PDF]
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J. M. B. Anumonwo, S. M. Taffet, H. Gu, M. Chanson, A. P. Moreno, and M. Delmar The Carboxyl Terminal Domain Regulates the Unitary Conductance and Voltage Dependence of Connexin40 Gap Junction Channels Circ. Res., April 13, 2001; 88(7): 666 - 673. [Abstract] [Full Text] [PDF]
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