© 2000 American Heart Association, Inc.
UltraRapid Communications |
A Novel Anionic Inward Rectifier in Native Cardiac Myocytes
From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nev.
Correspondence to Dayue Duan, MD, PhD, Department of Physiology and Cell Biology/351, University of Nevada School of Medicine, Reno, NV 89557-0046. E-mail dduan{at}med.unr.edu
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Abstract |
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Abstract—Although the cationic inward rectifiers (Kir and hyperpolarization-activated If channels) have been well characterized in cardiac myocytes, the expression and physiological role of anionic inward rectifiers in heart are unknown. In the present study, we report the functional and molecular identification of a novel chloride (Cl-) inward rectifier (Cl.ir) in mammalian heart. Under conditions in which cationic inward rectifier channels were blocked, membrane hyperpolarization (-40 to -140 mV) activated an inwardly rectifying whole-cell current in mouse atrial and ventricular myocytes. Under isotonic conditions, the current activated slowly with a biexponential time course (time constants averaging 179.7±23.4 [mean±SEM] and 2073.6±287.6 ms at -120 mV). Hypotonic cell swelling accelerated the activation and increased the current amplitude whereas hypertonic cell shrinkage inhibited the current. The inwardly rectifying current was carried by Cl- (ICl.ir) and had an anion permeability sequence of Cl->I->>aspartate. ICl.ir was blocked by 9-anthracene-carboxylic acid and cadmium but not by stilbene disulfonates and tamoxifen. A similar ICl.ir was also observed in guinea pig cardiac myocytes. The properties of ICl.ir are consistent with currents generated by expression of ClC-2 Cl- channels. Reverse transcription polymerase chain reaction and Northern blot analysis confirmed transcriptional expression of ClC-2 in both atrial and ventricular tissues and isolated myocytes of mouse and guinea pig hearts. These results indicate that a novel ICl.ir is present in mammalian heart and support a potentially important role of ClC-2 channels in the regulation of cardiac electrical activity and cell volume under physiological and pathological conditions. The full text of this article is available at http://www.circresaha.org.
Key Words: channel, Cl- • action potential • cell volume
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Introduction |
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At least six distinct types of sarcolemmal Cl- currents have been functionally identified in cardiac myocytes.1 2 3 4 5 These include Cl- currents activated by intracellular cAMP-PKA (ICl.PKA), PKC (ICl.PKC), intracellular Ca2+ (ICl.Ca or Ito2), extracellular ATP (ICl.ATP), cell volume (ICl.vol), and a basally active Cl- current (ICl.b). It has been well established that ICl.PKA is mediated by cardiac expression of an isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel,6 7 8 and recent data suggest that ICl.vol and ICl.b may be attributed to cardiac expression of a member of the ClC Cl- channel superfamily,9 ClC-3.10 11 Functional data suggest that ICl.PKC and ICl.ATP may also be attributed to activation of CFTR Cl- channels.12 13 14 15 16 Although the molecular entity responsible for ICl.Ca remains to be determined, it may be encoded by a member of the new CLCA gene family.17 With physiological Cl- gradients, all of these Cl- currents activate predominantly at depolarized voltages, exhibit outward rectification, and thus contribute significantly to shortening of action potential duration; although activation of ICl.Ca under conditions of intracellular Ca2+ overload may, in addition, contribute to the development of oscillatory delayed afterdepolarizations.3 5
Recently, another member of the ClC Cl- channel
family, ClC-2, has been cloned originally from rat heart and
brain18 and then from rabbit heart.19
Although it has been shown that functional expression of rat ClC-2
(rClC-2) and rabbit cardiac ClC-2 (ClC-2
) mRNA in Xenopus
oocytes gives rise to an inwardly rectifying,
hyperpolarization-activated
Cl- conductance that is modulated by changes in
cell volume and extracellular pH,18 19 20 inwardly
rectifying hyperpolarization-activated
Cl- currents with properties resembling ClC-2
have yet to be identified in native cardiac myocytes. However,
hyperpolarization-activated ClC-2 channels
expressed in heterologous expression systems bear a striking
resemblance to the well-characterized
hyperpolarization-activated cationic
pacemaker current, If (or
Ih), found in many mammalian cardiac cell
types21 22 and are now known to belong to the HCN
gene family.23 24 25 Because of similarities in inward
rectification and relatively slow activation during membrane
hyperpolarization, it is possible that coexpression
of ClC-2 Cl- channels in some native cardiac
myocytes, which also express If (HCN), may
in part explain earlier observations that suggested some anion
sensitivity of the
hyperpolarization-activated
If.26 27 28
In the present study, using both electrophysiological and molecular biological techniques, we tested the hypothesis that endogenous expression of ClC-2 may be responsible for a novel, inwardly rectifying hyperpolarization-activated anion conductance in native atrial and ventricular myocytes isolated from mouse and guinea pig hearts. The data demonstrate that, under conditions during which cationic inwardly rectifying channels are blocked or eliminated, inwardly rectifying currents with an anion permeability of Cl->I->>aspartate (Asp-) can be detected in a percentage of mouse and guinea pig atrial and ventricular myocytes. The biophysical and pharmacological properties of this inwardly rectifying Cl- current (ICl.ir) are nearly identical to the known properties of cloned ClC-2 Cl- channels expressed in heterologous expression systems. Finally, we present evidence using the reverse transcription–polymerase chain reaction (RT-PCR) and Northern blot analysis that confirms transcriptional expression of a ClC-2 homologue in both atrial and ventricular tissue and cells from mouse and guinea pig heart. These results provide the first evidence that a novel ICl.ir, which may be encoded by ClC-2, is functionally expressed in mammalian heart. ICl.ir, like cationic inward rectifiers, may play an important role in the regulation of action potential duration, resting membrane potential, and pacemaker activity under both physiological and pathophysiological conditions. A preliminary report describing these results has been published.29
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Materials and Methods |
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Electrophysiological Measurements
Whole-cell currents were measured from single atrial and ventricular myocytes enzymatically isolated from mouse and guinea pig hearts using the tight-seal, whole-cell, voltage-clamp technique,30 as previously described.16 31 To prevent contamination from Ca2+ and K+ currents, and the cationic inward rectifiers IK1 and If, nisoldipine (1 µmol/L), 4-aminopyridine (4-AP, 2 mmol/L), Ba2+ (2 mmol/L), and Cs+ (10 mmol/L) were present continuously in the extracellular bath solutions and cations in the intracellular pipette solution and, in some experiments, in the bath solution were replaced by the large impermeant cation, N-methyl-D-glucamine (NMDG). Hypotonic (230 mOsm/kg H2O, measured by freezing-point depression) bath solutions contained (mmol/L) NaCl 100, MgCl2 1, CaCl2 1, BaCl2 2, NaH2PO4 0.33, CsCl 10, HEPES 10, glucose 5.5, [Cl-]o 118; pH 7.4. In some experiments, NaCl was replaced with equimolar concentrations (100 mmol/L) of NaI or Na-aspartate. The isotonic and hypertonic bath solutions were the same as the hypotonic solution except the osmolarity was adjusted to 290 mOsm/kg H2O and 360 mOsm/kg H2O by adding mannitol. The pipette solution contained (in mmol/L) NMDG-Cl 118, MgATP 5, NaGTP 0.1, ethylene-glyco-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) 5, HEPES 5, [Cl-]i 118; pH 7.4, 290 mOsm/kg H2O using mannitol. In low [Cl-]i (20 mmol/L) pipette solution, 98 mmol/L NMDG-Cl was replaced by NMDG-aspartate. A bridge (3 mol/L KCl in agar salt) between the bath and a Ag/AgCl reference electrode immersed in pipette solution was used to minimize changes in liquid junction potential, and junction potentials were zeroed before formation of the membrane-pipette seal. Cell dimensions (length and width) were roughly estimated with a calibrated graticule in the microscope eyepiece. Experiments were conducted at room temperature (22°C to 24°C).
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
RT-PCR of total RNA prepared from cardiac tissues using the
Trizol reagent (Life Technologies) or from enzymatically dispersed
individual cardiac myocytes using SNAP total RNA isolation kit
(Invitrogen) was performed as previously described.10 The
primer region (forward: 5'-TGGGAGGAGCAGCAGCTGAA-3', reverse:
5'-CAGAGTGCATGCACCTCT-GTGGT-3') is specific for rClC-2 (GenBank
accession No. X64139)20 and corresponds to
nucleotides 2515 to 2822, generating a
307-nucleotide amplification product. Automatic
nucleotide sequencing was performed on both strands using
the dideoxy nucleotide chain termination method (Genetic
Analyzer, Model 310, Perkin Elmer).
Northern Blot Analysis
To confirm the expression of ClC-2 in cardiac tissues, Northern
blot analysis was performed as described
previously.10 16 A 420-bp rClC-2 cDNA probe was
radiolabeled with 32P by random
priming.32 Hybridization was performed under the same
conditions overnight. The filters were washed at high stringency (3
times in 2x SSC at room temperature for 5 minutes then twice in 0.2x
SSC/0.1% SDS at 65°C for 30 minutes) to ensure specificity of
labeling. Filters were exposed to film, and
autoradiography was performed using a BioRad
phosphoimager (Hercules).
Data Analysis
Data are presented as mean±SEM. Student’s t
test or ANOVA with Scheffé contrasts was used to determine
statistical significance. A two-tailed probability (P) of
5% was considered significant.
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Results |
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Novel Volume-Regulated Inward Rectifier Cl- Current (ICl.ir) in Mouse Heart
With the cationic inward rectifier blockers (Cs+ 10 mmol/L and Ba2+ 2 mmol/L) in the extracellular bath solution and a large impermeable cation NMDG in the intracellular pipette solution to replace all permeable cations, hyperpolarization of the cell membrane activated a time-dependent inward current in both atrial and ventricular myocytes isolated from mouse heart. Figure 1A
shows an example of the currents
recorded from ventricular myocytes under isotonic,
hypotonic, and hypertonic conditions. The current activated on
hyperpolarization from -40 to -140 mV under
isotonic (290 mOsm/kg H2O) conditions. Subsequent
exposure of the same cells to hypotonic (230 mOsm/kg
H2O, 21% hypotonic) bath solutions caused
significant cell swelling and caused a further increase in current
amplitude (Figure 1). Overall, cell areas during hypotonic cell
swelling were estimated to increase from 1633±165 to 2234±176
µm2 (n=5, P<0.05). Hypotonic cell
swelling not only increased current amplitudes but also accelerated the
kinetics of current activation. As shown in Figure 1B, the
time-dependent activation of the current at hyperpolarized potentials
was relatively slow in onset and could be best fit by a biexponential
function. Under isotonic conditions (left panel of Figure 1B),
the mean activation time constants at -120 mV were
1=179.7±23.4 ms and
2=2073.6±287.6 ms (n=5). Under hypotonic
conditions (right panel of Figure 1B), both the fast activation
time constant (1=97.5±8.5 ms at -120 mV,
n=5, P=0.011 versus isotonic condition) and the slow
activation time constant (2=656.4±113.6 ms at
-120 mV, P=0.002 versus isotonic condition) were
significantly reduced. Hypertonic cell shrinkage caused inhibition of
the current (Figures 1A and 1C). Under isotonic conditions, we
observed similar
hyperpolarization-activated currents in 5
of 52 (9.6%) ventricular myocytes, which was further
regulated by hypotonic cell swelling and hypertonic cell shrinkage.
Figure 1C shows the mean current-voltage (I-V)
relationship of the
hyperpolarization-activated inward currents
under isotonic (
), hypotonic (
), and hypertonic (
) conditions.
These currents had a strong inwardly rectifying I-V
relationship with mean reversal potentials (Erev)
of 2.0±3.8 mV, 3.3±3.4 mV, and -1.7±3.3 mV (n=5, P=NS),
under isotonic, hypotonic, and hypertonic conditions, respectively,
which were very close to the predicted equilibrium potential of
Cl- (ECl=0 mV) with a
symmetrical Cl- gradient
([Cl-]o/[Cl-]i=118/118
mmol/L).
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As shown in Figure 2A, the time-dependent
inwardly rectifying currents in mouse ventricular myocytes
remained unchanged when all cations in the extracellular solution were
also replaced by NMDG. These currents, however, were significantly
reduced by extracellular cadmium (0.3 mmol/L), which has been
shown to block inwardly rectifying Cl- current
in noncardiac cells.33 34 Furthermore, as shown in Figure 2B, when [Cl-]i
was partially substituted with equimolar Asp-
(98 mmol/L; [Cl-]i
20 mmol/L), the hypotonic cell swelling–activated inward
current was significantly smaller than the current recorded with
high [Cl-]i (see Figures 1
and 2A), and the reversal potential of the current
shifted to more negative potential with a mean value of -43.8±1.6 mV
(n=4), which is very close to the predicted ECl
(-45.5 mV). Again, these currents were significantly blocked by
extracellular cadmium. These results strongly indicate that the current
in mouse ventricular myocytes is neither
If nor a swelling-induced nonspecific
cationic inward rectifier current.35 36 It may
represent a novel volume-regulated Cl-
inward rectifier current, ICl.ir.
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Similar ICl.ir was also observed in mouse
atrial myocytes (16 of 113, 14.2%). The relative anion selectivity of
ICl.ir was further examined by replacing
[Cl-]o with an equimolar
concentration (100 mmol/L) of iodide
(I-) or aspartate
(Asp-) (ECl48.3 mV)
under hypotonic conditions. Cells were held at -40 mV, and test
potentials were applied at an interval of 10 seconds from -140 mV to
+80 mV for 2 seconds in +20-mV increments and then to +40 mV for 400 ms
before return to the holding potential (see Figure 3 and inset). In four different mouse
atrial cells, reduction of
[Cl-]o caused a shift of
Erev of the current from +3.5±0.7 mV
(Cl-) to +33.4±4.6 mV
(I-) and +47.2±1.2 mV
(Asp-), respectively. The permeability ratios
(permeability ratio of anion X with respect to
Cl-,
Px/PCl) were then
calculated from the shifts of Erev using the
modified Goldman-Hodgkin-Katz equation.37 The cell
swelling–activated inward rectifier channel had an estimated
PI/PCl of 0.22±0.09 (n=4)
and PAsp/PCl of 0.04±0.01
(n=4), suggesting that the current is conducted through an anion
selective channel with a relative anion permeability of
Cl->I->>Asp-.
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Effects of Cl- Channel Blockers on
ICl.ir
Several compounds including arylaminoalkyl benzoates derivatives
such as 9-anthracene-carboxylic acid (9-AC) and disulfonic stilbene
derivatives such as
4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) have
been identified as effective blockers of a variety of
cardiac5 38 39 and noncardiac Cl-
channels.40 Therefore, we assessed the effects of 9-AC and
SITS on the swelling-induced ICl.ir in
mouse atrial myocytes. As shown in Figure 4A, 1 mmol/L of 9-AC significantly
(P<0.001 versus control at all test potentials) blocked
ICl.ir (Figure 4A-a) in a
voltage-independent manner (Figure 4A-b). The inhibition of the
current amplitude at test potentials of -140, -120, -100, -80,
-60, and -40 mV was 47.1±1.9%, 50.6±1.7%, 48.8±2.2%,
46.6±2.7%, 46.3±2.9%, and 45.0±3.8%, respectively (n=4,
P=NS for voltage dependence). In contrast,
ICl.ir was not sensitive to disulfonic
stilbene derivatives. As shown in Figure 4B, 1 mmol/L SITS
failed to affect the current in mouse atrial cells. The changes in
current densities after SITS were -3.0±1.7%, -1.3±2.6%,
-6.2±4.4%, -2.3±3.5%, -4.2±1.6%, and -5.4±4.0% at test
potentials of -140, -120, -100, -80, -60, and -40 mV,
respectively (n=5, P=NS versus control at all test
potentials).
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ICl.ir in Guinea Pig Cardiac
Myocytes
Hyperpolarization-activated inwardly
rectifying, volume-sensitive, stilbene-insensitive currents with
properties similar to mouse cardiac ICl.ir
were also observed in some guinea pig atrial (6 of 56, 10.7%) and
ventricular (3 of 32, 9.4%) myocytes. Under hypotonic
conditions, it has been reported that an outwardly rectifying
volume-regulated Cl- current
(ICl.vol) is present in 
90% of
atrial myocytes and 30% of ventricular myocytes of
guinea pig heart.31 41 42 43 44 Different from
ICl.ir, however,
ICl.vol is outwardly rectifying,
deactivates at positive potentials, has an anion permeability
sequence of
I->Cl-, and
is sensitive to disulfonic stilbene (such as SITS)
Cl- channel
blockers.31 41 42 44 Figure 5A shows an example of whole-cell
currents recorded from a guinea pig ventricular
myocyte, in which both ICl.vol and
ICl.ir were activated under
hypotonic conditions. In these experiments, currents were recorded
using the same bath and pipette solutions as in Figure 1 and the
same voltage-clamp protocol as in Figure 3. As shown in Figure 5A-a, small
hyperpolarization-activated time-dependent
inward currents could be detected in this cell even under isotonic
conditions. Subsequent hypotonic cell swelling not only increased the
inward currents but also activated large outward currents
(Figure 5A-b). Although the outward currents showed
time-dependent deactivation at positive depolarizing potentials, the
inward currents showed time-dependent activation at negative membrane
potentials. Subsequent exposure of the same cell to 1 mmol/L of
SITS, an effective blocker of cardiac
ICl.vol, caused an inhibition of mainly the
outward current (Figure 5A-d), leaving the time-dependent inward
current largely unaffected (Figure 5A-c). Similar results were
observed in three guinea pig ventricular myocytes, and the
mean I-V curves under isotonic (
), hypotonic(•), and
hypotonic 1 mmol/L SITS (
) conditions are shown in Figures 5B and 5C, respectively. The SITS-sensitive current showed
outward rectification and deactivation at more positive potentials
(Figures 5A-d and 5D), which are typical features of
ICl.vol.10 31 42 The
SITS-insensitive inwardly rectifying current (Figures 5A-c and
5C), however, had properties very similar to those of
ICl.ir observed in mouse cardiac myocytes
described earlier in Figure 1. The estimated current densities
for guinea pig ICl.ir in
ventricular myocytes (-10.4±0.5 pA/pF, at -120 mV, n=3)
and atrial myocytes (-12.8±0.7 pA/pF, at -120 mV, n=3) revealed no
significant differences (P=NS).
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We also examined the Cl- dependence of the
current in guinea pig atrial myocytes using different
[Cl-]i. As shown in
Figure 6A-a, when
[Cl-]i was reduced to
20 mmol/L by replacing NMDG-Cl with equimolar amount (98
mmol/L) of NMDG-aspartate, hypotonic cell swelling induced an
activation of both small slowly activating inward currents and large
outward currents (Figure 6A-a). Subsequent exposure of the same
cell to the ICl.ir blocker
Cd2+ (0.3 mmol/L) caused an inhibition of
mainly the inward currents (Figures 6A-b and 6A-d). Further
exposure of the cell to 10 µmol/L of tamoxifen (TMX), which
blocks ICl.vol in many cardiac and
noncardiac cells10 11 33 41 but has no effect on
ICl.ir in noncardiac
cells,33 caused a further inhibition of the currents
(Figures 6A-c). Similar results were observed in three guinea
pig atrial myocytes, and the mean I-V curves under hypotonic
control (), hypotonic+0.3 mmol/L
Cd2+(•), and hypotonic+0.3 mmol/L
Cd2++10 µmol/L TMX () conditions,
respectively, are shown in Figure 6B. The
Cd2+-sensitive currents showed time-dependent
activation at hyperpolarization potentials (Figure 6A-d) and had an inwardly rectifying I-V relationship
with a mean reversal potential of -42.6±4.4 mV (n=3), which is very
close to the estimated ECl (-45.6 mV). These
properties are very similar to those of
ICl.ir observed in mouse cardiac myocytes
under the same conditions (see Figure 2B). The TMX-sensitive
currents (Figures 6A-e and 6D) were outwardly rectifying and had
typical properties of ICl.vol in the same
tissue described previously.10 31 42 These results
suggest that, in addition to the outwardly rectifying
ICl.vol, hypotonic cell swelling also
activates an inwardly rectifying
ICl.ir in these guinea pig cardiac
myocytes.
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Molecular Expression of ClC-2 in Mouse and Guinea Pig
Heart
The biophysical and pharmacological properties of
ICl.ir in mouse and guinea pig heart
described above, including the anion selectivity, inward rectification,
regulation by cell volume, sensitivity to 9-AC and
Cd2+, and insensitivity to SITS and TMX, are
nearly identical to those known properties of currents generated by
ClC-2 channels expressed in Xenopus
oocytes19 20 45 and mammalian HEK 293
cells,46 suggesting that ClC-2 is a strong molecular
candidate that may be responsible for
ICl.ir. Therefore, we tested for molecular
expression of ClC-2 in mouse and guinea pig heart.
Figure 7A shows an agarose gel depicting
a ClC-2–specific RT-PCR product generated from RNA derived from
mouse atrial and ventricular tissues. The RT-PCR reaction
of total RNA prepared from both atrial and ventricular
tissue with specific primers designed to amplify a
307-nucleotide of rClC-2 (nucleotide positions
2515 to 2822, see Materials and Methods) confirmed the transcriptional
expression of ClC-2 in both atrium and ventricle. The RT-PCR
product was sequenced and determined to be identical to the
previously cloned rClC-2.20 Northern blot
analysis also indicated that ClC-2 mRNA is expressed in both
atrial and ventricular tissue from mouse heart. Figure 7B shows hybridization to a transcript of 3.3 kb, which is
similar in size to ClC-2 mRNA expressed in rat heart.20 We
also amplified a ClC-2–specific RT-PCR product generated from RNA
derived from isolated single atrial and ventricular
myocytes enzymatically dispersed from guinea pig heart (Figure 7C). This RT-PCR reaction used the same specific primers and
amplified a 307-nucleotide product of gpClC-2,
confirming transcriptional expression of ClC-2 in single atrial and
ventricular myocytes.
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Discussion |
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In the present study, we first characterized the biophysical and pharmacological properties of a volume-regulated inwardly rectifying Cl- current, ICl.ir, in mammalian heart. In both atrial and ventricular myocytes of mouse and guinea pig heart, ICl.ir activated slowly on hyperpolarization with a biexponential time course. The current was very sensitive to changes in cell volume. Whereas hypertonic cell shrinkage diminished the current, hypotonic cell swelling increased the current and accelerated the activation kinetics. ICl.ir exhibited strong inward rectification with a reversal potential close to the estimated ECl in symmetrical Cl- and physiological asymmetrical Cl- conditions. The anion selectivity sequence of ICl.ir was Cl->I->>Asp-. Although ICl.ir was strongly blocked by Cd2+ and the carboxylic acid derivative, 9-AC, it was not sensitive to inhibition by effective blockers of ICl.vol such as TMX and the disulfonic stilbene derivative, SITS. All these properties clearly separate ICl.ir from other types of previously described Cl- currents in heart, suggesting that ICl.ir may be mediated by a novel type of Cl- channel.5
ClC-2 belongs to a large ClC gene family of voltage-gated
Cl- channels and is ubiquitously expressed in
many tissues.9 20 Initially, ClC-2 was cloned from rat
heart and brain, but only the brain form was subsequently
sequenced.20 It has 907 amino acids, a molecular mass of
99 kDa, and shares 50% homology with ClC-0 and ClC-1. When
transiently expressed in Xenopus oocytes, ClC-2 channels
activate during hyperpolarization (-90 to
-180 mV) and are further stimulated by cell swelling. ClC-2 channels
exhibit a strong inwardly rectifying instantaneous I-V
relationship and an anion permeability of
Cl-
Br->I-.
The channel is blocked by carboxylic acid derivatives and
Cd2+ but is largely unaffected by disulfonic
stilbene derivatives and TMX.18 19 20 47 When expressed
in mammalian (HEK 293) cells, ClC-2 channels are active under isotonic
conditions but exhibit faster activation kinetics than the channel
expressed in Xenopus oocytes.46 A rabbit
homologue of ClC-2 (ClC-2G) was isolated from a rabbit gastric cDNA
library.45 ClC-2G has also been shown to be ubiquitously
expressed in rabbit19 45 and human48 tissues.
Recently, Furukawa et al49 isolated a ClC-2 clone from
a rabbit heart cDNA library that is identical to ClC-2G. They also
proposed that an alternatively spliced, truncated form of ClC-2,
ClC-2ß, may be specifically expressed in heart. However, in a
subsequent study, they found this truncated ClC-2ß clone is likely to
be an artifact of library construction and not a product of
alternative splicing.19 It has been shown that, when
expressed in Xenopus oocytes, ClC-2G (ClC-2) channels
have similar biophysical and pharmacological characteristics as rClC-2
channels.19 45
Cl- currents with properties similar to those of
heterologously expressed ClC-2 channels have not been specifically
identified in native cardiac myocytes before, despite early reports of
a hyperpolarization-activated
Cl- current in some multicellular cardiac
preparations.26 28 50 These results have generally been
attributed to anion sensitivity of the
hyperpolarization-activated
If due to screening of positive charges
near the external pore of the If
channel51 However, the
hyperpolarization-activated inwardly
rectifying currents observed in our experiments are unlikely due to
If or other cationic inward rectifiers
because (1) 10 mmol/L Cs+ and 2 mmol/L
Ba2+ were included in the external bath solutions
and the nonpermeable large cation NMDG was the only major cation in the
internal pipette solutions, effectively precluding possible
contamination from If and other cationic
inward rectifiers, (2) the I-V relationship was not altered
by replacement of cations with NMDG and shifted as expected for a
Cl- selective channel when intracellular
aspartate was substituted for Cl-, and the
measured reversal potentials of ICl.ir
closely corresponded to the predicted value of
ECl, (3) substitution of
[Cl-]o by small anions
such as I- or
Asp- shifted the reversal potential of
ICl.ir to positive potentials,
consistent with a channel with a relative anion permeability of
Cl->I->>Asp-,
and (4) ICl.ir in cardiac myocytes was
blocked by extracellular Cd2+, an effective
blocker of ICl.ir in many noncardiac cells.
Recently, Clemo et al35 have reported a nonspecific
cationic inward rectifier current
(ICir.swell) that is also regulated by cell
volume in rabbit ventricular myocytes and
ventricular myocytes isolated from dog with
tachycardia-induced congestive heart
failure.36 However,
ICir.swell differs from
ICl.ir in mouse and ventricular
myocytes in that ICir.swell (1) does not
show time-dependent activation at hyperpolarization
potentials (see Figure 1 in Reference 35 ), (2) is not
sensitive to changes in bath Cl-
concentrations, (3) is not sensitive to 9-AC, and (4) is dependent
on extracellular cations and can be abolished by NMDG replacement for
extracellular cations. Our results, therefore, clearly exclude the
possibility that ICl.ir is the same as
ICir.swell.
The properties of ICl.ir described in the
present study are consistent with currents generated by
ClC-2 channels, including the cardiac form ClC-2, when expressed in
Xenopus oocytes19 20 or mammalian cell
lines.46 Furthermore, we found that ClC-2 transcripts were
present in both atrial and ventricular tissue and
isolated single myocytes of mouse and guinea pig heart. Our results,
therefore, provide the first compelling evidence for the functional
expression of a novel Cl--dependent inward
rectifier that may be encoded by the ClC-2 gene in native mammalian
cardiac cells and support a potentially important role of ClC-2 in
cardiac function.
Possible Functional Role and Significance. In general, the
physiological role of ClC-2 channels remains
uncertain because most studies have been carried out only on
recombinant ClC-2 channels.9 18 19 20 The volume sensitivity
of the channel suggests some role in cell volume regulation. Volume
regulatory mechanisms are critical in maintaining structural integrity
and proper cellular functions of living cells. Cardiac myocytes, like
other mammalian cells, are able to use a variety of mechanisms to
precisely maintain their size in the face of osmotic perturbations. It
is now well known that the outwardly rectifying
ICl.vol plays a role in volume regulation
of cardiac and noncardiac cells.52 53 54 55 56 However, ClC-2
channels differ from the typical ICl.vol
investigated in cardiac and noncardiac cells in terms of their anion
selectivity, pharmacology, and rectification
properties.5 9 10 18 19 20 In fact,
ICl.vol in heart, and possibly in other
tissues as well, may be encoded by
ClC-3.10 Furukawa et al19
recently found that ClC-2G (ClC-2), when expressed in
Xenopus oocytes, contributes to volume regulation in the
face of osmotic perturbations. However, in human intestinal T84 cells,
it was found that only the TMX-sensitive outwardly rectifying
ICl.vol was involved in volume regulation
but not the Cd2+-sensitive inwardly rectifying
ClC-2–like Cl- current.33 How
ClC-2 channels are involved in the volume regulation of cardiac cells
and their relationship to ClC-3 and other Cl-
channels involved in volume regulation, such as CFTR
channels,57 needs further investigation.
Considering the well-established physiological significance of cationic inward rectifiers like Kir58 59 and If21 in the regulation of resting membrane potential, action potential duration, and pacemaker activity, it is conceivable that anionic inward rectifiers may also play a significant role in cardiac electrical activity. Under physiological conditions, the Cl- equilibrium potential (ECl) is more positive (-65 to -30 mV) than the resting membrane potential.60 61 62 At negative membrane potentials, activation of ClC-2 channels would promote Cl- efflux and the generation of significant inward current because of their inwardly rectifying properties, thus potentially contributing to the regulation of resting membrane potentials. In the present study, given that functional ICl.ir could only be demonstrated in a small percentage of isolated mouse and guinea pig atrial and ventricular myocytes, it may be that the physiological significance of these channels in these cell types only becomes prominent under some pathological conditions (ischemia or hypoxia).63 It is possible that ICl.ir may normally play a much more prominent role in pacemaker cells in the sinoatrial or atrioventricular nodal regions of the heart, in a manner analogous to the physiological role of If channels and their known tissue distribution pattern in heart.22 64 65 Future studies should be performed to examine functional and molecular expression of ClC-2 in nodal regions of the heart. Finally, additional functional significance of ClC-2 expression in some types of cardiac cells may be related to the formation of heteromultimers with other ClC Cl- channel family subunits66 to form unique, yet to be characterized, Cl- channel subtypes.
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Acknowledgments |
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This study was supported by a grant-in-aid from the American Heart Association (D.D.). F.B. was supported by a fellowship from the Western States Affiliate of the American Heart Association, and B.H. and J.R.H were supported by NIH grant HL52803.
Received January 17, 2000; accepted February 1, 2000.
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