From the Cardiovascular Research Laboratory, Departments of Medicine (Cardiology) and Physiology, University of California at Los Angeles School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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The cause of altered ionic homeostasis leading to
cell death during ischemia and metabolic inhibition is unclear.
Hemichannels, which are precursors to gap junctions, are nonselective
ion channels that are permeable to molecules of less than
Mr 1000. We show that hemichannels open
upon exposure to calcium-free solutions when they are either
heterologously overexpressed in HEK293 cells or endogenously
expressed in cardiac ventricular myocytes. In the presence of normal
extracellular calcium, hemichannels open during metabolic inhibition.
During ischemia and other forms of metabolic inhibition, activation of
relatively few hemichannels will seriously compromise the cell's
ability to maintain ionic homeostasis, which is an essential step
promoting cell death.
Ischemia, hypoxia, and other forms of metabolic inhibition cause
rapid disturbances in ionic homeostasis including intracellular Na and
Ca gain and K loss that contribute to cellular injury and death (1);
however, the mechanism is uncertain. Recent studies using dye uptake
assays in immortalized cells have shown that some types of hemichannels
open in low extracellular Ca (2). Hemichannels are the precursors to
gap junctions (for a review, see Ref. 3); two hemichannels aligned end
to end form an intercellular communicating channel. If they are
activated in nonjunctional plasma membrane, hemichannels are
nonselective and exchange intracellular K for extracellular Na or Ca
due to the asymmetrical intracellular/extracellular distribution of
these cations.
We have previously described a nonselective current that is activated
by metabolic inhibition in isolated ventricular myocytes (4). The
purpose of this study was to investigate whether activated nonjunctional hemichannels might be responsible for this current. The
findings are positive, indicating that nonjunctional hemichannels may
be involved in the pathogenesis of ionic disturbances during myocardial
ischemia and hypoxia.
Molecular Biology--
Plasmid encoding
GFP1
(CLONTECH, Palo Alto, CA) was fused to the C
terminus of Cx43 using the polymerase chain reaction overlap extension
method (4, 5). The chimeric product was subcloned and sequenced. Cx43
provided by Dr. Bruce Nicholson (State University of New York, Buffalo,
NY) was subcloned into pCDNA3. Both native and chimeric constructs
used the cytomegalovirus promoter. Transfection was carried out using
the calcium phospate method (6).
Microscopy--
The expression of Cx43-GFP was determined by
examining cell monolayers grown on glass coverslips using a Nikon
microscope fitted with a Xenon lamp and the appropriate filters
(excitation bandpass, 450-480 nm; emission cutoff, <515 nm). Cardiac
myocytes were examined for calcein and dextran-fluorescein loading
using the same filters. Magnification was ×400 in the original slides, which were then scanned and assembled using an Adobe
Photoshop/Macintosh G3 combination.
Electrophysiology--
Whole cell and single channel currents
were recorded with an Axopatch 200A clamp amplifier and a Digidata 1200 data acquisition system using pClamp software (Axon Instruments, Foster
City, CA). For the whole cell clamp experiments, the patch pipette
contained 140 mM KCl, 1 mM MgCl2, 5 mM NaCl, and 10 mM HEPES, pH 7.4, for HEK293
cells or 115 mM cesium glutamate, 30 mM TEA-OH,
10 mM HEPES, 3 mM NaCl, 1 mM
MgCl2, 1 mM NaH2PO4, 5 mM sodium pyruvate, and 1 mMNa-ADP for isolated
ventricular myocytes. The bath solution contained 140 mM
NaCl, 1 mM MgCl2, 5.4 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.2, with or without 10 mM dextrose for HEK293 cells; CsCl was
substituted for KCl for isolated ventricular myocytes. For the 0 Ca
solution, CaCl2 was removed, and 2 mM EGTA was
added (estimated free Ca, 1 nM). Metabolic inhibitors
(which were dissolved in dimethyl sulfoxide as appropriate) or
LaCl3 was added directly to the bath solution. For
halothane, the appropriate bath solution was mixed with halothane and
vigorously shaken. Excess halothane was suctioned off, and the
halothane-saturated solution was covered with mineral oil throughout
its use. Bath solutions were exchanged using a rapid solution exchanger
with a 90% exchanger time of <500 ms (7). For single channel
recordings, the patch pipette contained the appropriate bath solution
described above.
Dye Uptake--
HEK293 monolayers were washed twice with Ca-free
Tyrodes containing 2 mM EGTA (EGTA-Tyrodes) and then
incubated in EGTA-Tyrodes containing 1% Lucifer Yellow for 30 min at
room temperature. Cells were extensively washed with normal Tyrodes
containing 1.8 mM Ca before imaging. Isolated adult cardiac
myocytes (8) were washed by resuspension and centrifugation in
EGTA-Tyrodes and then incubated in normal Tyrodes or EGTA-Tyrodes
containing 150 µM calcein or 1% dextran-fluorescein for
30 min at room temperature. They were washed in normal Tyrodes three
times before imaging.
HEK293 cells were transfected with wild-type Cx43 or Cx43 linked
in frame at the C terminus to the green fluorescent protein (Cx43-GFP).
In monolayers transfected with Cx43-GFP, the pattern of expression
showed long lines of fluorescence that were consistent with the
formation of gap junctions in regions of contact between adjacent
transfected cells, in addition to some perinuclear and plasma membrane
fluorescence (Fig. 1, D and
F). These long fluorescent lines were not observed between
adjacent nontransfected and transfected cells (Fig. 1D),
suggesting that the endogenous connexin-43 in the nontransfected HEK293
cells (9) either did not pair with the Cx43-GFP or was present at too
low a level (9) to form detectable fluorescent structures with the
exogenous Cx43-GFP. Cells transfected with GFP alone showed a
homogenous pattern of fluorescence (Fig. 1B) that did not
transfer to adjacent nontransfected cells, which is consistent with the
size permeation characteristics of gap junctions (10).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
Cellular distribution of GFP and Cx43-GFP in
transfected HEK293 cells. A, bright-field image of confluent
HEK293 cells, one of which is transfected with GFP. B,
fluorescence image of A illustrating GFP fluorescence
filling the cytoplasm of the transfected cell (middle), with
no detectable endogenous fluorescence in the adjacent nontransfected
cells. C-F, bright-field (C and E)
and fluorescence images (D and F) of HEK293 cells
transfected with Cx43-GFP. Fluorescent lines consistent with gap
junctions are present at the contact regions between transfected cells
but not between transfected and nontransfected cells.
To examine whether Cx43-GFP formed functional plasmalemmal
hemichannels, individual HEK293 cells from nonconfluent monolayers were
patch-clamped in the whole cell mode (Fig.
2, A and B).
Consistent with previous studies (2, 11-16), the removal of
extracellular Ca rapidly induced a large current, with a linear
current-voltage relationship that reversed at 5 ± 1 mV
(n = 4). Current amplitude at
80 mV averaged 838 ± 173 pA. No voltage- or time-dependent inactivation was
evident when voltage clamps were used in place of the voltage ramp
(data not shown). Replacement of extracellular Na and K with
N-methyl-D-glucamine (Mr
195) shifted the reversal potential in the negative direction by only
7 ± 1 mV (data not shown), indicating a relatively nonselective
current with significant permeability to large cations. The current was
reversibly blocked by 68 ± 6% using the gap junction blocker
halothane (Refs. 2 and 17; Fig. 2, A and B). In
nontransfected cells, removing extracellular Ca had a much smaller
effect on the current-voltage relationship (Fig. 2D).
Current amplitude at
80 mV increased from 26 ± 6 to 179 ± 48 pA, which was presumably attributable to either the activation of
endogenous hemichannels or the nonspecific effects of Ca removal on
membrane conductance.
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To determine whether linking GFP to the C terminus of Cx43 artificially
promoted hemichannel opening at low extracellular Ca, we co-transfected
HEK293 cells with wild-type Cx43 and GFP fused to the subunit of
Na,K-ATPase (
NaKP-GFP) as a marker for identifying transfected
cells. Individual fluorescent cells examined in the whole cell patch
clamp mode produced similar linear nonselective currents in response to
the removal of extracellular Ca, as with Cx43-GFP. Currents were
blocked by La and, to a lesser extent, by halothane (Fig.
2D), possibly indicating that GFP linkage may modify
halothane sensitivity.
Single channel currents were recorded from HEK293 cells expressing Cx43-GFP (Fig. 2C). With 1.8 mM Ca in the patch pipette, no single channel currents were observed (five cells). However, with low free Ca (1 nM) in the patch pipette, single channel currents were detected in six of nine cells transfected with Cx43-GFP, but in none of the nontransfected cells. Furthermore, halothane inhibited the channels, with the NPo decreasing from 0.28 ± 0.02 to 0.13 ± 0.03 (n = 3). The single channel conductance of the fully opened channel averaged 120 ± 25 pS. Several substate conductance levels were also observed, although their dwell times were too inconsistent to analyze quantitatively.
Dye transfer experiments confirmed that the current observed in low extracellular Ca was due to functional hemichannels (Fig. 3). HEK293 cell monolayers containing either all nontransfected cells (Fig. 3, A and B) or a mixture of transfected and nontransfected cells (Fig. 3, C-F) were exposed to nominally Ca-free media containing 1% Lucifer Yellow (Mr 522). After 30 min, Lucifer Yellow was washed out with Ca-containing media, and the cells were imaged. Cells expressing Cx43-GFP showed the brightest dye uptake (Fig. 3, C and D). Contacting nontransfected cells also showed a dimmer fluorescence, which is consistent with dye transfer from the adjacent transfected cells via gap junctions. No dye uptake occurred in nontransfected cells that were not close to transfected cells, suggesting that they did not take up Lucifer Yellow. The entry of Lucifer Yellow and the subsequent transfer to contacting cells indicate that the putative Cx43 hemichannels opened by low extracellular Ca are nonselective and are large enough to allow molecules of at least Mr 522 to enter, which is consistent with the known properties of gap junctions (18). Comparable results were obtained when wild-type Cx43 was transfected alone (Fig. 3, E and F), although the successfully transfected cells could not be unequivocally identified.
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These findings establish that Cx43 forms functional hemichannels with
typical properties of gap junctions. Linkage to GFP did not
fundamentally alter hemichannel properties. We therefore proceeded to
test the effects of metabolic inhibition. HEK293 cells expressing
Cx43-GFP were patch-clamped (whole cell mode) with 1.8 mM
[Ca]o and exposed to the metabolic inhibitors carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 10 µM) and iodoacetate (IAA; 1 mM) to inhibit
oxidative and glycolytic metabolism, respectively (Fig. 2, E
and F). After ~15 min, a large linear current developed
that was similar to that induced by low [Ca]o
but ~2-fold larger (1720 ± 282 pA at 80 mV; reversal potential,
7 ± 1.5 mV; n = 5). This current was
reversibly inhibited by 73 ± 8% by halothane and irreversibly
inhibited by 97 ± 1% by 1 mM
[La]o (Fig. 2, E and F).
In contrast, in nontransfected cells, only a small increase in current
from 39 ± 15 to 132 ± 49 pA was observed during metabolic
inhibition (Fig. 2H).
To rule out a nonspecific effect of the metabolic inhibitors, we tested
alternative combinations of metabolic inhibitors: 5 µM
rotenone (a mitochondrial inhibitor rather than an uncoupler like FCCP) + 1 mM IAA or 10 µM FCCP + 10 mM
2-deoxyglucose (a glycolytic inhibitor). Although the activation time
course differed, a similar current developed in transfected cells (Fig.
2H). The combination of FCCP + IAA also activated the
hemichannel current in cells co-transfected with wild-type Cx43 and
NaKP-GFP (Fig. 2H). Halothane and La inhibited the
putative hemichannel currents (Fig. 2H), although to a
slightly lesser degree.
We also recorded single channel currents from cell-attached patches on
Cx43-GFP-transfected cells during metabolic inhibition with FCCP + IAA
(Fig. 2G) with normal extracellular Ca (1.8 mM) in the patch pipette and bath. After a delay of 8-20 min,
noisy-appearing currents were observed. Single channel amplitude was
similar to that induced by low [Ca]o (5 ± 1.2 versus 4.5 ± 0.9 pA at 40 mV). However, the
kinetics differed, due to a greater substate occupancy during metabolic
inhibition. These are likely to be Cx43-GFP hemichannels, because the
NPo decreased significantly from 0.33 ± 0.13 to 0.21 ± 0.11 (n = 3; Fig. 2G)
upon superfusion with halothane, and channel activity was never
observed during metabolic inhibition in nontransfected cells.
We have previously described a nonselective La-sensitive current activated by metabolic inhibition in isolated cardiac ventricular myocytes (4, 8). Because Cx43 is the most abundant gap junctional protein in these cells (19), we investigated whether this current could be explained by the activation of Cx43 hemichannels. To detect whether functional hemichannels were present, isolated myocytes were incubated for 30 min at room temperature (22 °C) in the absence of extracellular Ca with 150 µM calcein (Mr 623) present. After washing out calcein with Ca-containing solution, ~50% of the rod-shaped cells were fluorescent, indicating calcein uptake (Fig. 4A, bottom panel), as compared with only ~3% of rod-shaped myocytes after incubation with calcein in media containing 1.8 mM Ca (Fig. 4A, top panel). Dead myocytes took up dye under both conditions. No rod-shaped cells became fluorescent after exposure to dextran-linked fluorescein (Mr 1500-3000) in either the absence or presence of Ca (Fig. 4B), which is consistent with the known Cx43 dye transfer characteristics (Mr cutoff, ~1000). Rounded dead myocytes, however, readily took up this dye.
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Electrophysiological evidence also supported the existence of
functional hemichannels in isolated myocytes. After the calcein dye
uptake protocol, brightly fluorescent rod-shaped myocytes were
patch-clamped (whole cell mode). The removal of extracellular Ca (with
or without EGTA present) rapidly induced a current with a linear
voltage relationship that reversed near 0 (n = 6; Fig. 5, A-C). Current amplitude at
80 mV averaged 7 ± 1.8 pA/pF (n = 6). The
current was reversibly blocked by 1.8 mM Ca or 2 mM La.
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To determine whether the endogenous hemichannels in myocytes could be
activated by metabolic inhibition with normal extracellular Ca,
calcein-labeled myocytes were exposed to rotenone (5 µM)
in dextrose-free Tyrode's solution containing 1.8 mM Ca.
After 8-10 min, a linear nonrectifying current developed that was
fully blocked by 2 mM La (Fig. 5, D-F). The
reversal potential of the La-sensitive component was 5.6 ± 5.2 mV
compared with 3.9 ± 2.5 mV for the current induced by Ca removal.
Myocytes underwent a rapid and progressive contracture as the current
activated, indicating a severe disturbance of ionic homeostasis and
intracellular Ca overload. We have previously shown (4) that this
current is nonselective to molecules as large as
N-methyl-D-glucamine (Mr
195), which is consistent with hemichannel properties in HEK293 cells
(Fig. 2).
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DISCUSSION |
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The first major finding of this study is that both wild-type Cx43 and the Cx43-GFP assemble into functional hemichannels when heterologously overexpressed in HEK293 cells. The hemichannels open upon the removal of extracellular Ca, are nonselective and permeable to large molecules such as Lucifer Yellow (Mr 450), and are reversibly blocked by halothane and irreversibly blocked by La. To our knowledge, this study is the first detailed electrophysiological characterization of hemichannels formed from heterologously expressed Cx43 and defines a convenient, useful assay system for future structure-function studies. The findings are generally consistent with previously described studies of heterologously expressed Cx46 (13, 15, 16) and Cx56 (12) and studies of native hemichannels in skate (20) and catfish retina (11). Whether other connexins are activated by metabolic inhibition, however, is unknown. Our study also documents the presence of functional [Ca]o-sensitive hemichannels in normal ventricular myocytes, suggesting a novel potential explanation for the "Ca paradox" (21).
The most significant finding is the demonstration that endogenous and
heterologously expressed Cx43 or Cx43-GFP hemichannels open when
subjected to metabolic inhibition, even when extracellular Ca remains
normal. In a beating cardiac myocyte, 10 open hemichannels producing a
100 pA (0.7 pA/pF) current at 80 mV would increase the Na influx by
75% (22), and the fully developed current (5 pA/pF or about 70 open
hemichannels) would increase the Na influx 5-fold. This represents a
tiny fraction of the estimated 2.6 × 106 junctional
connexins in a typical cardiac myocyte (23). To the extent that the
Na-K pump is unable to compensate for this additional Na load,
intracellular Na accumulation and K loss result (22), causing membrane
depolarization, intracellular Ca overload by reverse Na-Ca exchange,
and possibly direct Ca entry through hemichannels. We cannot absolutely
exclude that hemichannel density in isolated myocytes is artifactually
increased by the isolation procedure. However, existing evidence
suggests that gap junction plaques are pulled off intact with either
one myocyte or its partner, rather than separating into plaques of
hemichannels (24).
The gap junction blocker halothane has been shown to have
cardioprotective effects during myocardial ischemia (25), which could
be mediated in part by inhibition of the Cx43 hemichannel. Also,
activators of protein kinase C have been shown to prevent Cx43
hemichannels from opening during the removal of extracellular Ca (5),
raising the possibility that the role of protein kinase C in ischemic
preconditioning may be linked in part to the suppression of hemichannel activation.
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ACKNOWLEDGEMENT |
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We thank Dr. Yujuan Lu for technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Specialized Center of Research in Sudden Cardiac Death P01 HL52319, National Institutes of Health Grants RO1 HL36729, RO1 HL44880, and R29 HL51129, by a grant-in-aid (to S. A. J.) and research fellowship (to R. K.) from the American Heart Association, Greater Los Angeles Affiliate, and by the Laubisch Fund, the Chizuko Kawata Endowment, and the Maude Cody Guthman Endowment.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cardiovascular
Research Laboratory, Depts. of Medicine (Cardiology) and Physiology, University of California at Los Angeles School of Medicine, 675 Circle
Dr. South, Los Angeles, CA 90095. Tel.: 310-825-9029; Fax: 310-206-5777; E-mail: jweiss{at}mednet.ucla.edu.
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ABBREVIATIONS |
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The abbreviations used are: GFP, green fluorescent protein; Cx, connexin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IAA, iodoacetate; MI, metabolic inhibition..
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REFERENCES |
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