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PKC isozyme in
the regulation of cardiac Ca2+ channels
1 Molecular and Cellular Cardiology Program, Veterans Affairs New York Harbor Healthcare System, and 2 State University of New York Health Science Center, Brooklyn, New York 11209; and 3 Department of Molecular Pharmacology, Stanford University, School of Medicine, Stanford, California 94305
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Limited information is
available regarding the effects of protein kinase C (PKC) isozyme(s) in
the regulation of L-type Ca2+ channels due to lack of
isozyme-selective modulators. To dissect the role of individual PKC
isozymes in the regulation of cardiac Ca2+ channels, we
used the recently developed novel peptide activator of the 
PKC,
V1-7, to assess the role of PKC in the modulation of L-type
Ca2+ current (ICa,L). Whole cell
ICa,L was recorded using patch-clamp technique
from rat ventricular myocytes. Intracellular application of
V1-7 (0.1 µM) resulted in a significant inhibition of
ICa,L by 27.9 ± 2.2% (P < 0.01, n = 8) in a voltage-independent manner. The
inhibitory effect of V1-7 on ICa,L was
completely prevented by the peptide inhibitor of PKC, V1-2
[5.2 ± 1.7%, not significant (NS), n = 5] but
not by the peptide inhibitors of cPKC, 
C2-4 (31.3 ± 2.9%, P < 0.01, n = 6) or
C2-2 plus C2-4 (26.1 ± 2.9%, P < 0.01, n = 5). In addition, the use of a general
inhibitor (GF-109203X, 10 µM) of the catalytic activity of PKC also
prevented the inhibitory effect of V1-7 on
ICa,L (7.5 ± 2.1%, NS, n = 6). In conclusion, we show that selective activation of PKC
inhibits the L-type Ca channel in the heart.
calcium channels; protein kinase C; whole cell patch clamp; peptides; cardiac myocytes
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CARDIAC CALCIUM ION
CHANNELS play an important role in both physiological and
pathophysiological settings (24). In the heart, as in many
other tissues, the regulation of L-type Ca2+ current
(ICa,L) by protein kinase C (PKC) has been
studied extensively. However, conflicting observations regarding the
modulation of ICa,L by PKC activation still
remain (6, 19, 36, 39, 41), in part because there are
multiple PKC isozymes in the heart. The characterization of the role of
individual PKC isozymes in the regulation of Ca2+ channel
has been largely limited by the lack of isozyme-selective activators
and inhibitors. Identification of the particular isozyme(s) that
mediates the regulation of L-type Ca2+ channels will have
important therapeutic implications. In this regard, we have previously
demonstrated that C2-containing isozymes play an important role in
mediating phorbol myristate acetate (PMA)-induced inhibition of L-type
Ca2+ channels, based on the ability of C2 region-derived
peptide inhibitors for classic PKC isozymes (cPKC) to prevent the
inhibition of L-type Ca2+ channels by PMA in adult rat
ventricular myocytes (42). However, whether other
PKC isozymes are involved in the regulation of L-type Ca2+
channel activity in the heart is unknown. We recently developed a
peptide V1-7 that specifically activates the translocation and
function of a single PKC isozyme PKC (5). Using this
peptide, we were able to examine the PKC isozyme involvement in the
regulation of the L-type Ca2+ channel by direct activation
of endogenous PKC.
PKC activation has been associated with the translocation of PKC
isozymes from one intracellular compartment to another (4, 26). This translocation event is required for the functional PKC
isozymes (37) and is mediated, at least in part, by the binding of activated PKC isozymes to the selective anchoring proteins (receptors for activated C-kinase, RACKs) that anchor them to different
subcellular sites and the functional consequences of these interactions
(27). If anchoring is required for the proper function of
individual PKC isozymes, then inhibition or activation of anchoring
should alter function. Peptides that mimic either the PKC binding site
on RACKs or the RACK binding site on PKC are translocation inhibitors
of PKC that inhibit the function of the enzyme (28). On
the other hand, a peptide that binds PKC, opens up PKC structure,
exposes the catalytic site, and enables anchoring to RACKs should be a
PKC agonist (28). Based on this rationale design, peptide
inhibitors and activators of particular PKC isozymes have been
developed to inhibit and activate interaction of individual PKC
isozymes with their respective RACKs, thus altering their translocation
and function as well (16, 28). In the present study, we
used for the first time a novel peptide activator (V1-7)
(5) for PKC to assess the potential functional role of
PKC in the modulation of ICa,L.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolation of cardiac myocytes. Cardiac myocytes were obtained from hearts of Wistar rats (200-250 g) by enzymatic dissociation as previously described (42). Briefly, the heart was perfused with HEPES-buffered solution containing (in mM) 117 NaCl, 5.4 KCl, 4.4 NaHCO3, 1.5 NaH2PO4, 1.7 MgCl2, 20 HEPES, 11 glucose, 10 creatine, and 20 taurine. Hearts were then perfused with the same solution containing collagenase type B (1.0-2.0 mg/ml; Boehringer Mannheim, Indianapolis, IN) for 25 to 30 min. The softened ventricular tissues were removed, cut into small pieces, and mechanically dissociated by trituration. Cells were suspended in petri dishes containing HEPES buffer with 1 mmol/l CaCl2 and 0.5% BSA (pH 7.4). All solutions used in perfusion were gassed with 100% O2 and warmed to 37°C. After incubation for 30 min, a small aliquot of the medium containing single cells was transferred to a chamber mounted on the stage of an inverted microscope (Nikon, Tokyo, Japan). Rod-shaped, noncontracting cells with clear striations were used for the whole cell voltage clamp studies. All experiments were carried out at room temperatures (22-24°C).
Solutions and drugs.
The composition of external solution is (in mM) 132 NaCl, 5.4 CsCl, 1.8 CaCl2, 1.8 MgCl2, 0.6 NaH2PO4, 5 4-aminopyridine, 10 HEPES, and 5 dextrose (pH 7.4). Patch electrodes were filled with control internal
solution containing (in mM) 139.8 CsCl, 10 EGTA, 2 MgCl2,
0.062 CaCl2, 5 disodium creatine phosphate, 10 HEPES, 3.1 disodium ATP, 0.42 disodium GTP (pH 7.1). V1 or C2-region-derived peptides (V1-2, V1-7, or
C2-4, 0.1 µM) were intracellularly applied, individually or
in combination as indicated, with the pipette solution. For these
experiments, larger electrode tips (0.8-1.0 M
) were used to
ensure proper diffusion of the peptides into the cytoplasm within
10-15 min as previously reported (42). The Peptides
V1-7 [HDAPIGYD; PKC (85)],
V1-2 [EAVSLKPT; PKC (14-21)],
C2-4 [SLNPQWNET; PKC (218)],
C2-2 [MDPNGLSDPYVKL; PKC ()], and
C2-4 [SLNPEWNET; PKC (218)]
were synthesized at the Protein and Nucleic Acid Facility, Stanford
University, Stanford, CA. All peptides used were over 90% pure.
Peptides were dissolved in dimethyl sulfoxide (DMSO) and stored at
20°C. The maximal concentration of DMSO in the internal solution
was 0.05%. The same amount of DMSO was added to the control internal
solution (42). All chemicals were purchased from Sigma
Chemicals or otherwise indicated.
Electrophysiology.
The whole cell configuration of the patch-clamp technique was utilized
(9). Data were digitized at 5 kHz with an
analog-to-digital converter (Digidata 1200, Axon Instruments) and
stored on the hard disk of a computer for subsequent analysis. The
recordings were filtered with a low-pass corner frequency of 2 kHz.
Borosilicate glass electrodes (outer diameter, 1.5 mm) with resistances
of 0.8-1.0 M when filled were connected to a patch-clamp
amplifier (Dagan model 3900A). Junction potentials were always
compensated. To record ICa,L, all K+
currents were blocked with intracellular and extracellular
Cs+ and extracellular 4-aminopyridine. The fast Na current
was blocked by a prepulse to 50 mV from a holding potential of 80
mV (42). Cells were depolarized every 10 s from a
holding potential of 80 mV to a prepulse level of 50 mV for 100 ms
and subsequently to a test pulse of 10 mV for 300 ms. This test voltage
is based on the peak current of the current-voltage relationship
(I-V) for ICa,L.
ICa,L was measured as the peak inward current.
To obtain the I-V relationship, a series of test
pulses of 300-ms duration were applied with 10-mV increments from a
holding potential of 50 mV.
90 to 60 mV were applied for a duration of 2 s from a holding
potential of 80 mV, followed by a 5-ms interpulse interval at a
potential of 80 mV. Then the membrane was depolarized for 200 ms to a
test potential of 10 mV. Steady-state inactivation was measured as the
ratio of I/Imax, where
Imax is the maximum current amplitude elicited
during the test pulse at 10 mV after the most hyperpolarizing prepulse
to 90 mV. The current ratio was plotted as a function of the prepulse
potential. Voltage-dependent activation was estimated from peak
conductance according to Isenberg and Klockner formula (15)
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80 mV.
The membrane capacitance (Cm) was calculated
according to the equation: Cm = 
× Io/
Em, where is
the time constant for cell membrane charge, Io
is the maximum capacitative current, and Em
is the clamp voltage. The average Cm was
113.2 ± 5.3 pF (n = 45).
Five to seven minutes were allowed for ICa,L to
reach steady state. Therefore, the zero time shown in Figs. 1-6
represents about 5 min after the formation of whole cell configuration.
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Data analysis. Data are presented as means ± SE. Percent inhibition was calculated as the difference of the current amplitude by the intervention(s) over the control value. A Student's paired t-test was used to compare the data before and after interventions. Unpaired t-test or ANOVA was used to compare the data between groups. Figures 1, 4, and 5 were generated by Microcal Origin (version 5.0). P < 0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Sensitivity of L-type Ca2+ channels to
dihydropyridines.
The sensitivity of L-type Ca2+ channel currents was tested
with dihydropyridines. Cells were routinely depolarized from a holding potential of 80 to 50 mV for 100 ms, followed by a test pulse of 10 mV for 300 ms, every 10 s. The currents were markedly increased by
1 µM BAY K 8644 and inhibited by 2 µM nisoldipine (data not shown). These characteristics represent those of L-type
Ca2+ channels.
Effects of peptide activator of PKC, V1-7, on
ICa,L.
We have previously shown that PMA-induced inhibition of
ICa,L was attenuated by C2-region-derived
peptides that block the translocation and function of cPKC
(42). To further dissect the role of individual PKC
isozyme(s) in the modulation of cardiac L-type Ca2+
channels, we studied the effect of a novel peptide activator of PKC
V1-7 on ICa,L. This peptide is derived
from the regulatory V1-region of PKC and has been shown to
selectively activate the translocation of PKC by immunofluorescence
and Western blot analysis (5). Figure
1 shows the effects of V1-7 on
ICa,L. The time course of peak
ICa,L from one cell dialyzed with 0.1 µM
V1-7 is shown in Fig. 1A.
ICa,L was decreased by 32.2% from 0.96 to 0.65 nA 15 min after achieving a steady-state ICa,L.
Average ICa,L was decreased by 27.9 ± 2.2% from 0.87 ± 0.20 to 0.61 ± 0.13 nA (n = 8, P < 0.01). To exclude the possibility that the
inhibitory effect of V1-7 on ICa,L was
due to a significant current rundown, we performed similar experiments
with the pipette solution containing the same amount of DMSO (0.05%)
as used for the preparation of peptides. As evident from Fig.
1B, ICa,L was decreased by 5.1% from
0.97 to 0.92 nA at about 20 min after achieving a steady-state level of
ICa,L recordings. The average percent decrease
in ICa,L recorded over a period of 25 min was
6.9 ± 1.5% [from 1.22 ± 0.26 to 1.17 ± 0.27 nA,
n = 5, not significant (NS)], which is similar to that
in the cells without DMSO (data not shown) but significantly smaller
compared with that in cells dialyzed with V1-7
(P < 0.001). The data indicate that the concentration
of DMSO used had no significant effect on ICa,L,
and current rundown was minimum and distinct from the inhibitory effect
of V1-7 on ICa,L. Taken together, these
results demonstrate the ability of the novel peptide V1-7 to
activate one single PKC isozyme, PKC, and thus alter
Ca2+ channel function.
V1-7 in six cells. V1-7 significantly inhibited ICa,L at all voltages tested
(0.82 ± 0.15 to 0.59 ± 0.11 nA at 10 mV, n = 6, P < 0.01). The voltage dependence for activation of ICa,L was not changed by
V1-7.
The effects of V1-7 on the steady-state inactivation and
activation were examined. The steady-state inactivation of
ICa,L was obtained by the double-pulse protocol
as described in the MATERIALS AND METHODS. Figure
3A shows the averaged
normalized data plotted against the prepulse potentials during the
control and in the presence of V1-7. The curves in Fig. 3
were obtained by fitting the data points with Boltzmann distribution of
the form finf(V) = 1/{1 + exp[(Vm V0.5)/k]}, where
finf(V) is the steady-state
inactivation parameter, Vm is membrane voltage,
V0.5 is the half-maximum inactivation potential,
and k is the slope factor. During control,
V0.5 was 28.1 ± 0.5 mV and k
was 9.3 ± 0.4 mV. For V1-7 group,
V0.5 and k were 26.4 ± 0.3 mV
and 7.6 ± 0.3 mV, respectively. The inactivation curves were
nearly identical (NS, n = 5), indicating that the
peptide V1-7 did not change the kinetics of voltage-dependent
inactivation of ICa,L.
The voltage-dependent activation was estimated from peak conductance as
described in the MATERIALS AND METHODS. Figure
3B shows the averaged normalized data plotted against the
membrane potentials during control and in the presence of V1-7.
The curves were fit by the Boltzmann equation
dinf(V) = 1/{1 + exp[(V0.5 Vm)/k]}.
V0.5 and k value were 3.6 ± 0.6 mV and 8.1 ± 0.5 mV, respectively, for control, and
5.0 ± 0.5 mV and 6.9 ± 0.4 mV, respectively, for
V1-7 group. Again, these two curves were nearly identical (NS,
n = 6). There was no significant shift in the
steady-state activation and inactivation curves by V1-7. These
results demonstrate that the peptide activator of PKC, V1-7,
inhibits ICa,L without changing voltage
dependence of the activation and inactivation of
ICa,L.
To further evaluate the selectivity of the peptide V1-7 on
ICa,L and its mechanism in the regulation of
ICa,L by PKC, we studied the effect of PKC
activator V1-7 on ICa,L in the presence of peptide inhibitors of PKC (Fig. 4). We
first used the peptide inhibitor of PKC V1-2, which has been
shown to selectively inhibit the translocation of PKC
(8). Figure 4A shows the effect of V1-7
on ICa,L in the presence of V1-2 from
one cell dialyzed with both peptides V1-7 (0.1 µM) and
V1-2 (0.1 µM). The peak ICa,L was
decreased by 6.2% after 20 min of recording, indicating that
V1-7 failed to significantly inhibit
ICa,L in the presence of V1-2. The
average percent decrease was 5.2 ± 1.7% (from 1.20 ± 0.27 to 1.16 ± 0.28 nA, n = 5, NS), which is not
significantly different from that in DMSO group (n = 5, NS). The data show that the inhibitory effect of the peptide activator
V1-7 on ICa,L was completely prevented
by the peptide inhibitor V1-2, indicating that V1-7
inhibits ICa,L by functionally activating the
translocation of PKC.
To further confirm that the effect of the peptide V1-7 on
ICa,L is due to activation of PKC, we used a
peptide inhibitor of cPKC, C2-4. This peptide has been
previously shown to inhibit the translocation and function of cPKC.
Figure 4B shows the time course of peak
ICa,L from a typical cell. Application of both peptides V1-7 (0.1 µM) and C2-4 (0.1 µM) into the
pipette solution resulted in a marked decrease in
ICa,L. The average inhibition was 31.3 ± 2.9% (0.74 ± 0.11 to 0.51 ± 0.09 nA, n = 6, P < 0.01). We also used peptides C2-2 (0.1 µM) and C2-4 (0.1 µM) previously shown to antagonize PMA
effects on IBa (42) to examine
changes in the effect of V1-7 on ICa,L.
Figure 4C shows the time course of peak
ICa,L from one cell dialyzed with V1-7,
C2-2, and C2-4 together. Similarly, the
combination of both C2-2 and C2-4 did not prevent the
inhibitory effect of V1-7 on ICa,L. The
average inhibition was 26.1 ± 2.9% (0.82 ± 0.15 to
0.61 ± 0.10 nA, n = 5, P < 0.01). These results indicate that the peptide V1-7 did not
interfere with cPKC and that V1-7 inhibitory effect on
ICa,L was not mediated through cPKC. Taken
together, the results suggest that the peptide V1-7 inhibits
ICa,L by activating the translocation of PKC
not cPKC in rat ventricular myocytes.
The peptide activator and inhibitors used were derived from the
regulatory region of the corresponding PKC isozymes and influence only
the regulatory activity of PKC isozymes. The blockade of the
translocation of PKC isozymes would prevent the function of PKC
isozymes. To demonstrate that the catalytic activity is also necessary
for the function of PKC isozymes, we examined the effects of
V1-7 on ICa,L in the presence of a
non-isozyme-selective inhibitor of the catalytic activity of PKC,
GF-109203X. GF-109203X was applied to the external solution 5 min after
the formation of whole cell configuration. Figure
5 shows a representative time course of peak ICa,L from one cell. Pipette
application of V1-7 (0.1 µM) in the presence of GF-109203X
(10 µM) caused a small but not significant decrease in
ICa,L (6.9%). The average percent inhibition by
V1-7 in the presence of GF-109203X was 7.5 ± 2.1% (from
1.06 ± 0.09 to 1.00 ± 0.09 nA, n = 6, NS).
The inhibitory effect of the peptide V1-7 on
ICa,L was completely reversed by the PKC
inhibitor, GF-109203X, suggesting that both regulatory and catalytic
activity are necessary for the full function of PKC isozymes in the
intact cell.
Figure 6 summarizes the effect of
V1-7 alone, DMSO alone, V1-7 plus V1-2,
V1-7 plus C2-4, V1-7 plus
C2-2/C2-4, and V1-7 plus GF-109203X on
ICa,L. Percent inhibition of
ICa,L induced by V1-7 alone or
V1-7 plus C2-4 or V1-7 plus
C2-2/C2-4 is significantly different from that in DMSO group.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We have shown, for the first time, that a novel peptide
V1-7, which selectively activates translocation and function of
PKC, inhibited ICa,L in adult rat ventricular
myocytes. The inhibitory effect of V1-7 on
ICa,L was completely abolished by the peptide inhibitor of PKC, V1-2, but not affected by the peptide
inhibitor of cPKC, C2-4, or C2-2 and C2-4. The
inhibitor of the catalytic activity of PKC, GF-109203X, completely
prevented the effect of V1-7 on ICa,L.
These observations indicate that PKC activation inhibits
ICa,L in rat ventricular myocytes, and selective
activation and inhibition of individual PKC isozyme can be achieved in
the cardiac myocyte.
In our previous study (42), we have shown that PMA-induced
inhibition of ICa,L was almost completely
abolished by the combination of the two C2-region-derived peptides
C2-2 and C2-4, suggesting that only cPKC mediates the
inhibitory effect of PMA on ICa,L. Surprisingly,
we found in the present study that activation of PKC, one of novel
PKC isozymes, inhibited ICa,L in rat
ventricular myocytes. This discrepancy may be due to the use of the
peptide V1-7, a single PKC isozyme activator. Endogenous PKC
exists as a broad range of isozymes with distinct tissue distribution,
Ca2+ sensitivity, and substrate specificity (3,
34). It is therefore possible that different isozymes interact
differently with the L-type Ca2+ channel and the response
to PKC activation by PMA depends on the specific isozyme(s) involved.
It appears that the effect of PKC on ICa,L is
more evident in the absence of multiple PKC isozyme involvement, and
activation of endogenous PKC by peptide V1-7 is sufficient
to inhibit ICa,L. Since V1-7 is
currently the only PKC isozyme-selective activator available,
we were unable to determine the effect of selective activation of any
other individual PKC isozyme(s) on ICa,L. It is
possible that activation of different PKC isozymes may serve different
functions in the regulation of cardiac L-type Ca2+
channels. Another possible explanation for this discrepancy between the
previous study and the present study could be the experimental conditions. For example, in our previous study (42), PMA
effects, in the presence of different peptides, were studied on
IBa, not ICa,L as it is
the case in the present study. The possibility that Ba or Ca ions could
differentially affect PMA inhibition of Ca channels by an as yet
unknown mechanism cannot be excluded. Indeed, PMA inhibition of
ICa,L is more pronounced (51.4 ± 3.0%) than that of IBa (40.5 ± 7.4%).
Furthermore, PMA differentially inhibited ICa,L
and IBa in the presence of peptides C2-2
and C2-4. The inhibition of ICa,L was
19.9 ± 1.7% (n = 4, data not shown) in the
present study compared with 8.4 ± 5.5% (n = 3)
in our previous study (42). Altogether these data support
the idea that Ba and Ca ions may differentially modulate PMA effects
and isozyme activity by an unknown mechanism.
The peptide C2-4 is derived from the C2 region of PKC and
has been shown to inhibit the translocation of cPKC. This peptide did
not alter the inhibitory effect of peptide V1-7 on
ICa,L. Similarly, both C2-2 and
C2-4 peptides, which are derived from the C2 region of PKC,
did not change the inhibitory effect of peptide V1-7 on
ICa,L. Together, the data suggest that the
effect of the peptide V1-7 on ICa,L was
due to the selective activation of PKC.
PKC modulating of ICa,L.
Several lines of evidence suggest that PKC is involved in the
regulation of ICa,L in the heart.
Ca2+ channels are affected by agents that directly activate
PKC or by receptor systems that activate PKC through a second messenger cascade (21). In cardiac myocytes, the effect of PMA on
ICa,L has been reported to be inhibition
(36, 39, 43), no effect (41), stimulation
(6), or stimulation followed by inhibition (19,
39). The effect of the receptor-mediated PKC activation on
ICa,L is also somewhat inconclusive (7,
10, 11, 19, 23). These conflicting reports may be, in part, due
to the existence of different PKC isozymes in different species and
tissues in addition to variations in the experimental settings.
V1-7 is the first isozyme-selective PKC activator that
induces PKC translocation from the cytosol to the particulate fraction in cardiac myocytes. The molecular basis underlying the action
of the peptide V1-7 on PKC has not been fully explored. It
has been suggested that this peptide acts by interfering with the
intramolecular interaction within PKC between the RACK-binding site
and the pseudo-RACK site, thereby mimicking the conformational change
and dissociation of this intramolecular interaction that occurs upon
activation of PKC, rendering PKC more accessible to its anchoring
protein (5).
Various cardiac ion channels have been shown to be modulated by PKC
(2, 13, 14, 40). Phosphorylation of ion channel proteins
is the key mechanism in signal transduction pathways that alter channel
properties and influence excitability and thus the physiological
function of excitable cells (20). The most common
mechanism of cardiac ion channel phosphorylation involves the
phosphorylation of serine and threonine residues by cAMP-dependent PKA
and PKC. Such effects are reversed by protein phosphatase-catalyzed dephosphorylation (21). The molecular mechanisms of PKC
regulation of Ca2+ channels are not completely defined.
Phosphorylation of channel proteins themselves may be the structural
basis for PKC-mediated events. There is evidence that the
dihydropyridine-sensitive Ca2+ channel protein is the
substrate for PKC phosphorylation (31, 32, 35). Puri et
al. (35) provided biochemical evidence that
1C (cardiac/brain) and 2a
subunits of L-type Ca2+ channels can be
individually phosphorylated stoichiometrically by PKC. Although the
data from the present study showed functional regulation of Ca channels
by PKC, these data cannot determine the site of PKC action.
Activation of PKC isozyme by PMA in cells triggers a redistribution and
translocation of PKC isozymes from cytosol to the particulate cell
fraction, where they are thought to regulate the activity of various
proteins by phosphorylation (17, 38). Translocation
of PKC isozymes also occurs after treatment of cells with
hormones or agonists that stimulate the accumulation of diacylglycerol (20, 30). Immunofluorescence studies demonstrate that PMA treatment causes the translocation of PKC isozymes to distinct cellular
loci in cardiac cells (4, 29). The ability of PKC translocation inhibitors to selectively inhibit the function of particular isozymes indicates that translocation is required for PKC
function (16, 42). The evidence that a peptide
translocation activator for PKC V1-7 functionally inhibited
ICa,L suggests that translocation activators
should be agonists of PKC function, independent of the amount of second
messengers that normally activate PKC. This finding further suggests
that the translocation of PKC isozymes is essential for the full
function of endogenous PKC activation in the intact cell.
In the present study, as many previous studies, we used whole cell
patch-clamp technique to record ICa,L. A
time-dependent decline of whole cell ICa,L is
commonly reported and is referred to as rundown (1). To
minimize the rundown process, we routinely used freshly prepared
pipette solution containing 5 mM ATP plus 10 mM EGTA for each cell. In
addition, the inhibitory effect of PKC activation of
ICa,L was larger than could be accounted for by
spontaneous rundown (see Fig. 1B with DMSO), and a
steady-state level of inhibition is always obtained.
Furthermore, the effect of the PKC agonist, V1-7, was
reversed by the PKC antagonist, V1-2. Therefore, the rundown
is unlikely to account for the inhibitory effects of V1-7 on
ICa,L.
Significance of our findings.
The present study is the first to demonstrate that PKC activation
inhibits cardiac L-type Ca2+ channels. The results suggest
that a translocation peptide activator can functionally modulate a
single PKC isozyme activity. Full understanding of the PKC regulation
of Ca2+ channels depends on the development of new
isozyme-selective activators and inhibitors. The present findings are
relevant not only to physiological but pathological settings. In this
regard, PKC activation has been associated with ischemic
preconditioning, making it a potential mediator for endogenous
cardioprotective mechanisms (5, 8, 12, 22, 33). It is
attractive to propose that PKC attenuates the deleterious increase
in intracellular Ca2+ that occurs during prolonged ischemia
and after reperfusion by depressing Ca2+ influx through
inhibiting the L-type Ca2+ channels (25).
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ACKNOWLEDGEMENTS |
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We thank the animal laboratory staff for their assistance.
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FOOTNOTES |
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This study was supported by Veterans Administration Medical Research Funds (to M. Boutjdir) and National Heart, Lung, and Blood Institute Grants HL-55401 (to M. Boutjdir) and HL-52141 (to D. Mochly-Rosen).
Address for reprint requests and other correspondence: M. Boutjdir, Research and Development Office (151), VA New York Harbor Healthcare System, 800 Poly Place, Brooklyn, NY 11209 (E-mail: mohamed.boutjdir{at}med.va.gov).
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 23 March 2000; accepted in final form 12 June 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Belles, B,
Malecot CO,
Hescheler J,
and
Trautwein W.
Rundown of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium.
Pflügers Arch
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