Properties of voltage-gated Ca2+ channels in
rabbit ventricular myocytes expressing Ca2+ channel
1E cDNA
Michihiro
Tateyama1,
Shuqin
Zong2,
Tsutomu
Tanabe2, and
Rikuo
Ochi1
1 Department of Physiology, Juntendo University School of
Medicine, Tokyo 113-8421; and 2 Department of Pharmacology and
Neurobiology, Graduate School of Medicine, Tokyo Medical and Dental
University, Bunkyo-ku, Tokyo 113-8519, Japan
 |
ABSTRACT |
Using the whole-cell patch-clamp
technique, we have studied the properties of
1E
Ca2+ channel transfected in cardiac myocytes. We have also
investigated the effect of foreign gene expression on the intrinsic
L-type current (ICa,L). Expression of green
fluorescent protein significantly decreased the
ICa,L. By contrast, expression of
1E with
2b and
2/
significantly increased the total Ca2+ current, and in
these cells a Ca2+ antagonist, PN-200-110 (PN), only
partially blocked the current. The remaining PN-resistant current was
abolished by the application of a low concentration of Ni2+
and was little affected by changing the charge carrier from
Ca2+ to Ba2+ or by
-adrenergic stimulation.
On the basis of its voltage range for activation, this channel was
classified as a high-voltage activated channel. Thus the expression of
1E did not generate T-like current in cardiac myocytes.
On the other hand, expression of
1E decreased
ICa,L and slowed the
ICa,L inactivation. This inactivation slowing
was attenuated by the
2b coexpression, suggesting that
the
1E may slow the inactivation of
ICa,L by scrambling with
1C for
intrinsic auxiliary
.
green fluorescent protein; culture; L-type calcium channel; calcium
channel
-subunit; transfection
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INTRODUCTION |
IN MAMMALIAN CARDIAC
MYOCYTES, Ca2+ influx through L-type Ca2+
channels triggers excitation-contraction coupling. Functional L-type Ca2+ channel in cardiac myocytes is composed of at least
four subunits:
1C,
2/
, and
,
similar to the other high-voltage-activated (HVA) Ca2+
channels (11). Ion conducting pore, voltage sensor, and
the binding sites for dihydropyridine (DHP) are all located on
1C-subunit, while auxiliary subunits
and
2/
serve regulatory roles affecting the biophysical
and pharmacological properties of evoked Ca2+
current (13, 15, 19). In cardiac myocytes,
-subunit is considered to play a critical role in regulating L-type
channel function, since overexpression of cardiac
-subunit
(
2a) enhances the L-type current amplitude and alters
the inactivation kinetics (38). In addition, application
of the antisense oligonucleotide against
2-subunit
results in slowing of the decay of L-type current (35).
Recently, expression of
1E-,
1G-, and
1H-subunit has been reported in young rat atrial
myocytes (16).
1E gene has been suggested
to constitute part of the T-type current (3, 14, 21, 32, 33,
36), although major parts of the T-type current are thought to
consist of
1G (29),
1H
(6), and/or
1I (18). In
cardiac myocytes, application of antisense oligonucleotide against
1E-subunit resulted in suppression of the cardiac T-type current induced by insulin-like growth factor-1 (IGF-1) (4, 30). T-type current is not detected in ventricular myocytes of
normal rat and feline heart but is detected in those of hypertrophied hearts (20, 27). Moreover, properties of L-type current in hypertrophied hearts are reported to be altered, resulting in reduction
of the current amplitude and slowing of the inactivation. These
alterations might reflect the expression of
1E gene,
because
1E-subunit is a candidate for T-type current and
interacts with auxiliary subunits (25, 28). The latter
could cause deficiency of auxiliary subunits for
1C,
leading to the alteration of L-type current properties. In the present
study, we investigated the properties of voltage-gated Ca2+
channels in cultured adult ventricular myocytes expressing several exogenous cDNAs. In rabbit ventricular myocytes, neither
1E-subunit (26) nor T-type channel
(23) is expressed, so we have used these cells to
investigate whether the
1E channel expressed in cardiac
myocytes shows T-like channel character. We have also investigated
whether the expression of
1E-subunit could affect the
properties of intrinsic L-type current by the possible removal of
accessory subunits from the
1C-subunit.
We found that green fluorescent protein (GFP), which was used
as a marker for successful cDNA expression, decreased L-type Ca2+ current (ICa,L) amplitude
without affecting the biophysical properties of L-type channel.
Expression of
1E gene did not generate T-like current
but slowed the decay of ICa,L as expected for
the decrease of intrinsic
-subunit associated with the L-type channel.
 |
MATERIALS AND METHODS |
Plasmid construct.
The 7.7-kb Hind III/Xba I fragment from pSPCBII-2
was ligated with Hind III/Xba I-cleaved
pCDNA1/Amp (Invitrogen) to yield pBII-2E, encoding the
1E-subunit (37). The 4.7-kb Sal
I/Not I fragment from pBH17 was ligated with Sal
I/Not I-cleaved pSV SPORT 1 (GIBCO BRL) to yield
PSVCa
2b, encoding the cardiac
(
2b)-subunit (13). PKCR
2, encoding the
2/
-subunit, was described previously
(42). PCAGS65A, encoding a mutated form of GFP, was a gift
from Dr. C. Akazawa.
Isolation and culture of ventricular myocytes.
Single ventricular myocytes were isolated from the hearts of female
Japanese White rabbit (1.5 kg) or male guinea pig (300 g) by
collagenase treatment during Langendorff perfusion (1). Guinea pig myocytes were prepared and used for electrophysiology within
8 h after dissociation. Isolated rabbit myocytes were suspended in
medium 199 without glutamate, plated onto laminin-coated glass coverslips in 35-mm culture dishes, and allowed to attach for 2 h
at 37°C under an atmosphere of 5% CO2-95% air. Plated
cells were washed twice and maintained in medium 199 supplemented with 5% fetal bovine serum, 0.25 µg/ml amphotericin B, 0.1 mg/ml
streptomycin, and 100 U/ml penicillin. The culture medium was changed daily.
Transient transfection.
After the myocytes had been cultured for 7 days in 35-mm dishes, they
were transfected with expression plasmids encoding the
1E-subunit and GFP, with or without the
2b- and
2/
-subunits. Effectene
(Qiagen, Valencia, CA), which we found most effective for transient
transfection in primary cultured cardiac myocytes, was used as a
carrier. The plasmid DNAs were mixed with 25 µl of Effectene reagent
and added into the 35-mm dishes containing 1.6 ml of culture medium to
perform transfection according to the manufacturer's instructions. The
plasmid DNA mass ratio was adjusted to 1:1:1:0.1 for
1E:
2b:
2/
:GFP. Three or
four days after transfection, electrophysiological experiments were
performed, usually by selecting GFP-luminescent cells.
Electrophysiology.
Macroscopic currents were measured by using the whole cell variant of
the patch-clamp technique (10). Patch pipettes have resistances of 1-3 M
when filled with pipette solution
containing (in mM) 140 CsCl, 10 EGTA, 3 MgATP, and 5 HEPES, pH 7.3 with
CsOH. Ca2+ current (ICa) amplitude
was measured at room temperature (22-25°C) while cells were
bathed in the external solution consisting of (in mM) 135 tetraethylammonium chloride, 5.4 KCl, 10 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4 with Tris. Peak
ICa amplitudes were estimated as the maximal
inward deflection from holding current. Current amplitudes were
normalized to the cell capacitance, estimated by analyzing the charging
transients elicited by a 10-mV pulse, and were represented as current
densities. All recordings were made with an EPC-7 patch-clamp amplifier
(List-Electronik, Darmstadt, Germany); pCLAMP software (version 6; Axon
Instruments, Foster City, CA) was used for both command-pulse delivery
and data analysis.
Data analysis.
The peak conductance of Ca2+ channels (G) was
calculated as G = ICa/(ECa
Em), where ECa is the
apparent reversal potential and Em is the test
potential. G, normalized to maximal G
(Gmax), was plotted against
Em and then fitted by the Boltzmann equation of
the form G = Gmax/[1 + exp(Em
V1/2,act)/k], where
V1/2,act is the voltage evoking 50% activation
and k is the slope factor. In addition, steady-state
inactivation was determined from the channel availability after 1-s
prepulses. The current amplitudes (I) elicited by
depolarizing pulses to 30 mV from various prepulse potentials
(V) were normalized to the maximum test current amplitude (Imax). Individual experimental points in each
group were fitted to the Boltzmann equation
I/Imax = 1/[1 + exp(V
V1/2,inact)/k], where
V1/2,inact is the midpoint of inactivation.
Kinetics of Ca2+ current inactivation
(
inact) were estimated by fitting the trace elicited by
1-s depolarizing pulses to a single exponential function, except for
the L-type current trace, which was fitted to a double exponential
function. On the other hand, the activation kinetics
(
act) were estimated as the time from 10% to 90% of
peak (
10-90).
Data are expressed as means ± SE. Statistical analysis was
carried out with Bonferroni's multiple t-test, and
differences at P < 0.05 were deemed significant.
 |
RESULTS |
Expression of GFP in ventricular myocytes.
Culture of adult rabbit ventricular myocytes results in an ~70%
decrease of the inward rectifier K+ current (data not
shown) but little change in the density of ICa,L, as reported previously (23).
Exogenous cDNAs were expressed in cultured cardiac myocytes with an
efficiency of 1-10%, as assessed from the number of
GFP-luminescent cells. An example of a GFP-expressing luminescent
myocyte is shown in Fig. 1.
Significant changes in cell capacitance were not observed with the
expression of exogenous cDNAs; the mean value of the cell capacitance
is ~45 pF in each group. Figure
2A, left, depicts a
typical ICa,L elicited in the presence of 10 mM
Ca2+. PN-200-110 (PN; 10 µM), a DHP-type L-type
Ca2+ channel blocker, almost completely suppressed the
ICa,L (Fig. 2A, left,
closed circle). ICa,L was first detected at
depolarization to
20 mV from the holding potential and reached a peak
at 30 mV (Fig. 2B, open circles). A typical
ICa,L trace obtained from GFP-luminescent cells
is represented in Fig. 2A, right. Average peak
ICa,L in GFP-transfected cells
(n = 11) was ~60% of that in the control
(n = 13; Fig. 2B). Voltage-dependent
activation, steady-state inactivation, and time courses of
ICa,L in GFP-transfected cells are all similar
to those in the control (Fig. 2C and Table 1). The pharmacological properties of
ICa,L were also not affected by GFP expression.
PN decreased the magnitude of ICa,L to <5% in
GFP-transfected myocytes (Fig. 2A, right, closed
square; Table 1). From these results, transfection and expression of
GFP decreased ICa,L amplitudes
(P < 0.05) without affecting L-type channel function and without yielding a PN-resistant component of the Ca2+
current.

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Fig. 1.
Cardiac myocyte transfected with the expression plasmid
encoding green fluorescence protein (GFP). A: image of cardiac myocyte
obtained by bright-field microscopy. B: fluorescent image of the same
cell.
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Fig. 2.
Effects of GFP expression on L-type Ca2+
current (ICa,L). Expression of GFP decreased the
density of ICa,L but did not shift the
current-voltage (I-V) curve. A: typical traces of
Ca2+ current (ICa) recorded from
control (left) and GFP-expressing cells (right)
in the absence (open symbols) and presence (filled symbols) of 10 µM
PN-200-110 (PN). B: I-V relationship for whole
cell ICa,L in control (circles;
n = 13) and GFP-expressing cells (squares;
n = 11) in the absence (open symbols) and presence
(filled symbols) of 10 µM PN. Expression of GFP significantly
decreased peak ICa,L. C: voltage
dependence of ICa,L in control
( ) and GFP-expressing cells ( ).
Activation curve (I/Imax) of
ICa,L, obtained from I-V
relationship, was not affected by GFP expression. Steady-state
inactivation (G/Gmax) was determined
as channel availability after 1-s prepulses. Channel availability in
cells expressing GFP was not different from that of control (cf. Table
1). Each symbol represents the mean ± SE.
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Expression of
1E gene in ventricular myocytes.
Expression of
1E-,
2b-, and
2/
-subunits increased the total
ICa compared with that in the control and in
cells transfected with GFP alone (P < 0.05; Table 1).
This increase was caused by the appearance of a large PN-resistant
Ca2+ current (Fig.
3A, left, closed
circle). Further addition of 100 µM Ni2+ markedly
inhibited this current (Fig. 3A, left, closed
square). Changing the charge carrier from Ca2+ to
Ba2+ resulted in a current-voltage (I-V)
relationship shift toward negative potentials, no increase of the
current amplitude, and no slowing of the current decay (Fig.
3B). These results indicated that the channel consisted of
the
1E-subunit (ICa,E; Refs.
37 and 39) is responsible for the PN-resistant inward
current. For comparison, T-type current (ICa,T)
was recorded from freshly dissociated guinea pig ventricular myocytes.
Figure 3A, right, represents the typical
ICa,T in the presence of 10 mM Ca2+,
which rapidly activates and inactivates. ICa,T
was clearly observed at a test potential of
40 mV and reached a peak
at approximately
20 mV (Fig.
4A, bottom right).
ICa,T was not inhibited by PN (Fig.
3A, left, closed circle), where the peak
amplitude of ICa,T was 2.2 ± 0.2 and
2.0 ± 0.4 pA/pF (n = 12) in the absence and presence of 10 µM PN, respectively. ICa,T was
partially or almost completely inhibited by 0.1 or 1 mM
Ni2+, respectively (Fig. 3A, right,
closed or open squares, respectively).

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Fig. 3.
Properties of 1E current
(ICa,E) expressed in rabbit ventricular myocytes
and native T-type current in guinea pig ventricular myocytes.
A: effect of PN and Ni2+ on total
ICa. Left: Ca2+ current
obtained by 50-ms depolarization to +20 mV from a holding potential of
80 mV in
1E 2b 2/ -transfected
cells. Right: T-type Ca2+ current
(ICa,T) obtained by a depolarization to 20 mV
for 50 ms from a holding potential of 80 mV in freshly isolated
guinea pig ventricular myocytes. Bath solution was tetraethylammonium
solution containing 10 mM Ca2+. Currents were recorded in
the absence ( ) or presence ( ) of
PN-200-110 (10 µM) and in the presence of PN plus 100 µM
Ni2+ ( ) or PN plus 1 mM Ni2+
( ). B: effect of exchange of
Ca2+ to Ba2+ on PN-insensitive currents.
Left: Ca2+ and Ba2+ current traces
in the presence of PN (10 µM) were superimposed.
inact = 22.1 ± 4.7 and 19.0 ± 2.7 ms
for Ca2+ and Ba2+, respectively, at 20-mV test
potential. Right: I-V relationship of
ICa,E with Ca2+ ( )
or Ba2+ ( ) as a charge carrier in
1E 2b 2/ -transfected
cells obtained in the presence of 10 µM PN (n = 6).
Each symbol represents the mean ± SE.
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Fig. 4.
Properties of ICa,E expressed in rabbit
ventricular myocytes and ICa,T in guinea pig
ventricular myocytes. A: I-V relationships
obtained from 1E 2b 2/ -
(n = 12), 1E 2b-
(n = 6), and 1E-transfected myocytes
(n = 11) and from guinea pig ventricular myocytes
(T-type; n = 12) before ( ) or after
( ) application of PN (10 µM). B: voltage
dependence of activation and steady-state inactivation of
ICa,E and ICa,T.
Left: steady-state activation curves obtained by plotting
the peak Ca2+ channel conductance (G) normalized
to the maximal conductance (Gmax) at each
test-potential: 1E ( , n = 10), 1E 2b ( ,
n = 8);
1E 2b 2/
( , n = 11), and T-type channel
( , n = 12). In each case, the data were
well fit by a Boltzmann equation (see values in Table 1). Each symbol
represents the mean ± SE. Right: steady-state
inactivation (I/Imax) was measured
with same protocol described in text: 1E
( , n = 4),
1E 2b ( , n = 4), 1E 2b 2/
( , n = 8), and T-type channel
( , n = 8). Data were well fit by a
Boltzmann equation (see values in Table 1). Each symbol represents the
mean ± SE.
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ICa,E was detected in 78% (15/19) of the
1E
2b
2/
-transfected and
GFP-luminescent cells. ICa,E was also observed
in
1E- or
1E
2-transfected
and GFP-luminescent cells with an efficiency of 45% (9/20) or 60%
(9/15), respectively. No PN-insensitive currents were recorded from the
1E
2b-transfected but GFP-negative cells (n = 10), which largely supports the statement that the
GFP is a good marker to detect cells with successful expression of the
1E channel. ICa,E in
1E- and
1E
2b-transfected
cells were qualitatively similar to those recorded from
1E
2b
2/
-transfected
cells, although they were substantially smaller in amplitude (Fig.
4A and Table 1).
The parameters of voltage-dependent activation and inactivation of
1E channel were quantified by I-V
relationship and steady-state inactivation curve, respectively (Fig.
4B). On the basis of the voltage dependence of activation,
ICa,E expressed in cardiac myocytes was
classified as an HVA current (Fig. 4B, left, open
circles; Table 1). The voltage for half-maximal inactivation
(V1/2,inact) of
1E channel was
not much different from that expressed in Xenopus oocytes
(Fig. 4B, right; Ref. 37). However,
ICa,E decayed more rapidly than previously
reported for heterogeneously expressed rabbit
1E channel
(Fig. 3A, left; Refs. 22 and 37). At
20 mV,
inact of ICa,E was
~20-30 ms, which was substantially shorter than that of
1E channel expressed in Xenopus oocytes
(37) or HEK-293 (22) cells, although it was
widely variable among cells transfected with
1E alone
(Table 1).
Modulation of Ca2+ channel by
isoproterenol.
The
-adrenergic agonist isoproterenol (Iso; 1 µM) potentiated the
L-type Ca2+ channel (Fig. 5).
Iso increased ICa,L amplitude 2.5-fold, shifted the I-V curve toward negative potentials, and slowed the
decay of ICa,L (Fig. 5, A,
top, and B). These changes induced by Iso were
also observed in GFP-expressing cells; Iso increased
ICa,L amplitude 2.2-fold (n = 4;
Fig. 5B). However, Iso basically had no effect on
ICa,E (Fig. 5A, bottom,
and B). The maximal amplitude of
ICa,E was 11.9 ± 3.2 and 11.8 ± 2.5 pA/pF (n = 4) in the absence and presence of Iso,
respectively.

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Fig. 5.
Effects of isoproterenol (Iso) on
ICa,E and ICa,L.
A: ICa traces obtained by
depolarization to 10, 0, 10, and 20 mV for 50 ms from a holding
potential of 80 mV in time-matched culture cells before
(top) or after (bottom) application of Iso (1 µM). B: I-V curves of control, GFP-expressing,
and 1E 2b 2/ -expressing
cells observed before ( ) or after ( )
application of Iso. Ordinate represents current density, and abscissa
represents test potential. Each symbol represents the mean ± SE.
All data for
1E 2b 2/ -expressing cells
were obtained in the presence of 10 µM PN.
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Prolongation of ICa,L inactivation by
1E
expression.
ICa,L, designated as a PN-sensitive current, was
decreased by the expression of
1E-subunit. Moreover,
inactivation of ICa,L was remarkably slowed in
cells transfected with
1E-subunit (Fig. 6A). We have quantified the
degree of inactivation slowing by comparing r50 (ratio of
current remaining after 50 ms to that at the peak × 100%). At 20 mV, r50 was 23.5 ± 4.0% (n = 12) in the control but was 53.2 ± 5.7% (n = 9) in cells
expressing
1E-subunit. Furthermore, the degree of
inactivation slowing of ICa,L was found to
depend on the expression level of
1E-subunit, where
r50 was 70.0 ± 4.1% (n = 4) in cells
with ICa,L/total ICa
<40%, while r50 was 40 ± 3% (n = 5) in ICa,L/total ICa
>40% (Fig. 6B, open circles). Thus, the less
ICa,L shares in total
ICa, the slower the ICa,L
decays. This inactivation slowing of ICa,L was
attenuated substantially by the coexpression of
2b-subunit (Fig. 6A), where r50
was 36 ± 8% (n = 4) in cells with
ICa,L/total ICa <40%
(Fig. 6B, closed circles). Therefore, the inactivation rate
of ICa,L is regulated by
2b,
which might be occupied by the coexpressed
1E-subunit.

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Fig. 6.
Effect of 1E-subunit expression on
inactivation rate of ICa,L. A:
representative Ca2+ current traces obtained by 50-ms
depolarizations ranging between 10 and +20 mV from a holding
potential of 80 mV in cells expressing 1E
(left) and 1E 2b
(right). Top: total ICa.
Middle: ICa,E
(ICa in presence of 10 µM PN).
Bottom: ICa,L (total
ICa PN-resistant
ICa). Decay of ICa,L was
much slower in 1E-transfected cells than in cells
transfected with 1E and 2b. B:
inactivation kinetics of ICa,L in cells
transfected with 1E-subunit depend on the expression
level of 1E. r50 (ratio of current remaining
after 50 ms to that at the peak × 100%) at +20 mV was plotted
against the percentage of ICa,L in the total
ICa: ,
1E-expressed cells; , 1E-
and 2b-expressed cells. Expression of
1E-subunit caused reduction of
ICa,L inactivation, which was attenuated by the
coexpression of 2b.
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 |
DISCUSSION |
GFP construct, which contains membrane-sorting signal, was used as
a marker for detecting myocytes expressing the exogenous cDNAs. To
avoid the possible unknown effect of GFP tagging to channel subunits
(38), we expressed marker GFP together with channel
subunits (
1E,
2b, and
2/
). The molar ratio of GFP was reduced to 1/10
compared with channel subunits to increase the percentage of channel
availability in GFP-positive cells. The validity of the use of GFP was
verified by the fact that no ICa,E was detected
in 10 GFP-negative cells transfected with
1E
2b. The expression of GFP significantly
decreased ICa,L amplitude with little effect on
channel biophysical properties. (Fig. 2 and Table 1). This finding
indicates that GFP competes with endogenous protein, including
1-,
-, and
2/
-subunits, during the
processes of transcription, translation, and membrane insertion, which
may lead to reduction of ICa,L. HVA
1-subunits could generate T-like current in some
conditions (21) and could exhibit different character,
depending on the expression environment (2, 34, 40, 41).
This might be caused by cell-specific Ca2+-associated
proteins such as ryanodine receptor or calmodulin, which alter the
availability of channels (24), or by
Ca2+-dependent inactivation and facilitation of
Ca2+ channel (17, 43). We thus examined
whether
1E channel expressed in cardiac myocytes
generates T-like current. Expression of
1E-subunit produced the PN-resistant Ca2+ current that was abolished
by 100 µM Ni2+ (Fig. 3A, left).
Earlier studies demonstrated that
1E channel is
insensitive to DHPs but sensitive to lower concentrations of Ni2+ (IC50 = 30 µM; Refs.
37 and 39). Changing the charge carrier from
Ca2+ to Ba2+ caused no increase of current
amplitude, a finding that is also agreeable with the previous report
for the characteristic of
1E channel (3,
37). In addition, Iso potentiated the
ICa,L in cells expressing GFP, but the
PN-resistant current was insensitive to
-adrenergic modulation.
These pharmacological properties are similar to those of T-type current
of rabbit sinoatrial cell (9). However, the biophysical
properties are quite different from those of T-type current.
V1/2,act of the
1E channel (5 mV)
was far more positive than that for T-type channel (
33 mV) in 10 mM
Ca2+. The voltage range that the
1E channel
activated was similar to the range of
1E channel
expressed in Xenopus oocytes and HEK-293 cells (25,
37) but different from that of T-type channel (Fig. 4B). A subtype of
2/
-subunit
(
2/
-3) is known to shift the voltage dependence of
1E channel activation toward negative potentials; however, this subtype is not expressed in heart (15).
Therefore, it can be concluded that the
1E channel does
not represent T-like current, suggesting that the
1E
gene is not responsible for the cardiac T-type channel.
1E channel expressed in cardiac myocytes was almost
completely inhibited by 100 µM Ni2+, which is
different from that expressed in oocytes (37) and GH3 cells (IC50 = 140 µM; Ref.
39). Moreover, ICa,E in cardiac myocytes decayed more rapidly than in oocytes, GH3, or
HEK-293 cells, where the expressed
1E current remained
>50% at 160 ms after depolarization (22). On the other
hand, the decay was rather similar to that observed in neuronal R-type
currents (
inact = 20 ms; Ref. 31).
These differences among ICa,E could be caused by
the differences of
-subunit coexpressed. For example, coexpression of
2b accelerated the decay of
ICa,E and shifted the steady-state inactivation
curve toward negative potentials (Fig. 4B, right; Table 1), while
2a, a splicing variant of
2b, prolonged the decay and shifted the curve toward
positive potentials (28).
Expression of
1E-subunit decreased
ICa,L and altered inactivation properties of
ICa,L. The current decrease is explicable by the
aforementioned competition between endogenous channel subunits and
exogenous GFP and
1E-subunit during the channel
expression. Furthermore, it may be caused by the reduced availability
of auxiliary subunits to
1C, especially the
-subunit,
which is necessary for membrane targeting of the
1C-subunit (5, 7) and to alter the L-type
channel activation and inactivation (19). The reduced
availability of
-subunit for
1C was supported by the fact that properties of ICa,E were not much
different among cells expressing
1E,
1E
2b, and
1E
2b
2/
. These results
suggest that exogenously expressed
1E-subunit interacts
with and scrambles for intrinsic
- and
2/
-subunits. Furthermore,
ICa,L inactivation was markedly slower in cells
with a robust expression of
1E-subunit, and the
inactivation became faster with the coexpression of
2b-subunit. Thus
-subunit regulates the inactivation
of L-type channel also in ventricular myocytes (8, 35,
38). Inactivation of L-type channel in cardiac myocytes was
accelerated by
2b, while it was slowed by overexpression
of
2a (38). This may suggest that inactivation of L-type channel may be largely regulated by
2b-subunit in heart. In addition, deficiency of
-subunit for
1C could affect the L-type channel
function and cardiac contractility (12). On the other
hand, coexpression of
2b-subunit increased
ICa,L little, though it did increase
ICa,E. This is probably because of the
competition between exogenous cDNA and endogenous genes during the
process of transcription and translation. It is interesting to
speculate that the slowing of inactivation of L-type current observed
in this study may represent the state in the hypertrophied heart
(20, 27). This idea is reinforced by the previous
observation that the IGF-1-induced Ca2+ current is
inhibited by the
1E-specific antisense oligonucleotide (30). If this is the case,
1E channel may
be a key element to consider for the treatment of cardiac hypertrophy.
Further rigorous study will be necessary to prove this hypothesis in
atrial myocytes.
 |
ACKNOWLEDGEMENTS |
We thank Y. Mori for pSPCBII-2, F. Hofmann and V. Flockerzi for
pBH17, C. Akazawa for pCAGS65A, and M. T. Akuzawa for technical assistance.
 |
FOOTNOTES |
This research was supported by grants from the Ministry of Education,
Science, Sports and Culture (to R. Ochi and T. Tanabe), Japanese
Vehicle Corporate (to R. Ochi), and CREST, Japan Science Technology
Cooperation (to T. Tanabe).
Address for reprint requests and other correspondence: T. Tanabe, Dept. of Pharmacology and Neurobiology, Graduate School of
Medicine, Tokyo Medical and Dental Univ., Yusima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail:
t-tanabe.mphm{at}med.tmd.ac.jp).
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.
Received 24 May 2000; accepted in final form 22 August 2000.
 |
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