1 Molecular and Cellular Cardiology Program, Veterans Affairs New York Harbor Healthcare System, and State University of New York Health Science Center, Brooklyn, New York 11209; and 2 Department of Molecular Pharmacology, Stanford University, Stanford, California 94305
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ABSTRACT |
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Investigation of the role of
individual protein kinase C (PKC) isozymes in the regulation of
Na+ channels has been largely limited by the lack of
isozyme-selective modulators. Here we used a novel peptide-specific
activator (V1-7) of
PKC and other peptide isozyme-specific
inhibitors in addition to the general PKC activator phorbol
12-myristate 13-acetate (PMA) to dissect the role of individual PKCs in
the regulation of the human cardiac Na+ channel hH1,
heterologously expressed in Xenopus oocytes. Peptides were
injected individually or in combination into the oocyte. Whole cell
Na+ current (INa) was recorded using
two-electrode voltage clamp.
V1-7 (100 nM) and PMA (100 nM)
inhibited INa by 31 ± 5% and 44 ± 8% (at
20 mV), respectively. These effects were not seen with the
scrambled peptide for
V1-7 (100 nM) or the PMA analog
4
-phorbol 12,13-didecanoate (100 nM). However,
V1-7-
and PMA-induced INa inhibition was abolished by
V1-2, a peptide-specific antagonist of
PKC. Furthermore,
PMA-induced INa inhibition was not altered by
100 nM peptide-specific inhibitors for
-,
-,
-, or
PKC. PMA
and
V1-7 induced translocation of
PKC from soluble to
particulate fraction in Xenopus oocytes. This translocation
was antagonized by
V1-2. In native rat ventricular myocytes,
PMA and
V1-7 also inhibited INa; this
inhibition was antagonized by
V1-2. In conclusion, the results
provide evidence for selective regulation of cardiac Na+
channels by
PKC isozyme.
protein kinase C; two-electrode voltage clamp; peptides; Xenopus oocyte; electrophysiology
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INTRODUCTION |
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CARDIAC
Na+ channels determine cell excitability and are
responsible for conduction velocity of the action potential. They are
the targets of several anti-arrhythmic drugs (13) and
kinases (12, 51). Both protein kinase A and protein kinase
C (PKC) have been implicated in the modulation of Na+
channels(12, 51). Two subfamilies of PKC isozymes can be
stimulated by the tumor-promoting drug 4-phorbol 12-myristate
13-acetate (PMA): the conventional PKC (cPKC) isozymes
-,
I-,
II-, and
PKC, which contain the Ca2+ binding domain
(C2-containing), and the novel PKC (nPKC) isozymes
-,
-,
-,
and
PKC, or C2-less isozymes (8).
The regulation of Na+ channels by PKC has been studied
using general PKC activators such as PMA (7) and
1-oleoyl-2-acetyl-sn-glycerol (34). In general,
activation of PKC by these non-isozyme-specific activators leads to a
reduction in Na+ current (INa) in
both brain and heart (25, 31, 32). The characterization of
the role of individual PKC isozymes in the regulation of ion channels
in general and Na+ channels in particular has been largely
limited by the lack of isozyme selective activators and inhibitors.
Identification of the particular isozyme(s) that mediates the
regulation of Na+ channels is essential for our better
understanding of the regulation of INa in
physiological and pathological settings. Recently, we have
demonstrated, using novel peptide activators and inhibitors of
individual isozymes (16, 54), that C2-containing isozymes and PKC play an important role in mediating PMA-induced inhibition of L-type Ca2+ channels. PKC activation has been associated
with the translocation of PKC isozymes from one intracellular
compartment to another (10, 27). This translocation event
is required for the functional PKC isozymes (40) and is
mediated, at least in part, by the binding of activated PKC isozymes to
the selective anchoring proteins (RACKs, or receptors for activated
C-kinase) that anchor them to different subcellular sites and
consequently activate them (28). Anchoring is required for
the proper function of individual PKC isozymes. Inhibition or
activation of anchoring will 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
(41). On the other hand, a peptide that binds PKC, opens
up PKC structure, exposes the catalytic site, and enables anchoring to
RACKs will be a PKC agonist (41). On the basis of this
rationale, 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 (19, 41). Using these
peptides, we examined the potential role of individual PKC isozymes in
the regulation of cloned human Na+ channels expressed in
Xenopus oocytes and of Na+ channels in rat
ventricular myocytes.
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MATERIALS AND METHODS |
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Preparation of Xenopus oocyte and cRNA injection. Mature female Xenopus frogs, purchased from Xenopus I (Ann Arbor, MI), were anesthetized with 1.5 mg/ml tricaine. Surgically removed ovarian lobes were dissected and treated for 1.5 h with 1.5 mg/ml collagenase type IA dissolved in Ca2+-free ND96 medium (in mM: 96 NaCl, 2 KCl, 2 MgCl2, and 5 HEPES, pH 7.4). Stage IV and V oocytes were selected. Plasmids encoding human cardiac Na+ channel hH1 subunit, pCDNA3.1+-SCN5A, were generously given by Dr. Robert S. Kass (Columbia University, New York, NY). Plasmids were first linearized with restriction enzymes, and in vitro transcription was carried out using the mMSSAGE mMACHINE (Ambion, Austin, TX). Each oocyte was injected with 50 nl of hH1 cRNA. The injected oocytes were stored at 18°C in Leibovitz's L-15 medium (GIBCO BRL, Gaithersburg, MD) supplemented with 50 U/ml penicillin/streptomycin. Currents were recorded from the third to the fourth day.
Isolation of cardiac myocytes. Cardiac myocytes were obtained from hearts of Wistar rats (200-250 g) by enzymatic dissociation as previously described (16, 54). Briefly, hearts were perfused with HEPES-buffered solution containing (in mM) 117 NaCl, 5.4 KCl, 4.4 NaHC03, 1.5 NaH2P04, 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 Mannhein, Indianapolis, IN) for 25-30 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 mM CaC12 and 0.5% BSA (pH 7.4). All solutions used for 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 for oocytes.
The composition of external solution for INa
recording is ND96 (20). V1- or C2-region-derived peptides
(100 nM) were injected individually or in combination, as indicated, in
a total volume of 50 nl (1/20 of oocyte volume). Proper diffusion of
the peptides into the cytoplasm is reached within 10-15 min as
previously reported (48). Ten to fifteen minutes after
injection of the antagonist peptide, oocytes were superfused with PMA
or 4-phorbol 12,13-didecanoate (4
PDD). For
V1-7 (
PKC
agonist peptide), the time course of INa was
recorded immediately after injection. The peptides
V1-7 [HDAPIGYD;
PKC agonist, also termed pseudo-
RACK (
-
RACK)]
(10),
V1-2 (EAVSLKPT;
PKC antagonist),
C2-4 (SLNPQWNET;
PKC antagonist),
C2-4 (SLNPEWNET;
PKC antagonist), and
V1-2 (EAVGLQPT;
PKC antagonist) were
synthesized at Genemed Synthesis (South San Francisco, CA). All
peptides used were >90% pure. All chemicals were purchased from Sigma
or otherwise indicated.
Solutions and drugs for rat ventricular myocytes. The composition of external solution for INa recordings was (in mM) 100 tetraethylammonium (TEA)-Cl, 15 NaCl, 5 CsCl, 0.1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 adjusted with CsOH). L- and T-type Ca2+ currents were blocked by CoCl2 (5 mM) and NiCl2 (1 mM), respectively. The internal solution contained (in mM) 135 CsOH, 135 L-aspartic acid, 1 MgCl2, 10 EGTA, 10 HEPES, 4 Mg-ATP, and 0.1 Na-GTP (pH 7.1~7.2 adjusted with CsOH). Peptides were added to the peptide solution at a concentration of 100 nM as previously described (16, 54).
Oocyte INa recordings.
The expressed INa was recorded with a
two-electrode voltage-clamp technique using a GeneCLAMP 500 amplifier (Axon Instrument, Foster City, CA). The volume of the
recording chamber was 0.3 ml, and the rate of perfusion was 0.3 ml/min.
Oocytes were impaled with electrodes filled with 3 M KCl in ND96
external solution. Oocytes with membrane potential more negative than
40 mV were used for current recording. For INa
(20) current-voltage (I-V) relations, oocytes
were depolarized from a holding potential of
130 mV to tests ranging
from
100 to 70 mV with increments of 5 mV. A depolarization pulse to
20 mV from a holding potential of
130 mV recorded the time course
for INa. Oocyte membrane capacitance was
calculated from the capacitance transient during a voltage step from
130 to
120 mV. The steady-state inactivation of
INa was obtained by using the double-pulse
protocol. Prepulse potentials ranging from
130 to 70 mV were applied
and then followed by a 5-ms interpulse interval at a potential of
130
mV. The membrane was then depolarized for 200 ms to test potentials of
20 mV. Steady-state inactivation was measured as the ratio of
I to Imax (I/Imax), where
Imax is the maximum current amplitude elicited during the test pulse to
20 mV after the most hyperpolarizing prepulse. The current ratio was plotted as a function of the prepulse potential. The curves 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. Current recording was done at
room temperature (22 ± 2°C).
Myocyte INa recordings.
Whole cell INa recording was performed using an
Axopatch 200B amplifier with a CV-203BU head stage and pCLAMP software
(Axon Instruments). Suction pipettes were made from borosilicate glass capillaries using a horizontal puller (Sutter Instrument, Novato, CA).
When filled with pipette solution, tips had resistances ranging between
0.8 and 1.2 M. The tip potential was compensated before the
formation of membrane seals. After a seal formed, transient application
of negative pressure ruptured the membrane. Hyperpolarizing voltage-clamp steps (to
10 mV from a holding potential of 0 mV) were
used to record cell capacitance, which was calculated by integrating
the area under the uncompensated capacitance transient and dividing
this area by the voltage step. Cell capacitance and pipette series
resistance were both compensated before the onset of the experiment. To
record INa time course, cells were depolarized to
25 mV for 50 ms from a holding potential of
90 mV
(24). All experiments were performed at room temperature
(22~24°C). Data acquisition, voltage protocols, and analysis were
performed using the pCLAMP suite of software (Axon Instruments,). We
allowed 5-8 min for INa to reach steady
state and also for peptides to properly enter the cell (16,
54). Therefore, the time 0 shown in Fig. 8 represents
about 5-8 min after formation of whole cell configuration.
Immunoprecipitation and Western blot.
Stage IV-V oocytes were treated with either 1) PMA (100 nM) or V1-7 (100 nM) or 2) PMA or
V1-7 plus
V1-2 (100 nM). Membranes were obtained from these oocytes 30 min after the above treatment and purified as previously described
(52). Briefly, 50-90 treated oocytes were homogenized
in 10% sucrose, 15 mM NaCl, 5 mM KCl, and 20 mM HEPES, pH 7.5, supplemented with proteinase inhibitor cocktail (37).
After centrifugation, membrane fractions from 20-50% sucrose
gradient interface were collected as particulate fractions, and pellet
fractions from 10% and 10-20% sucrose gradient interface were
collected as cytosolic fractions. Each fraction was homogenized and
solubilized in 2.5 ml of buffer (75 mM KCl, 75 mM NaCl, and 50 mM
Na-phosphate, pH 7.2, plus 2 mg/ml soybean lipids and 1% Triton X-100)
and centrifuged for supernatant collection.
Data analysis. Data acquired were stored and then analyzed off-line with pCLAMP 6 software (Axon Instruments). All values were measured as the difference between zero and the peak current. All measurements of INa changes were performed at 30 min to avoid potential time-dependent internalization of plasma membrane in oocytes reported after 30 min of exposure to phorbol esters (46). Microcal Origin v5.0 (Microcal Software) was used to generate figures and perform statistical analysis. Data are presented as means ± SE. Percent inhibition was calculated as the difference in the current amplitude caused by the intervention(s), divided by the control value. 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. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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PMA inhibited INa expressed in Xenopus oocytes.
To investigate whether, under our experimental conditions, PKC is
involved in the modulation of INa, we first used
a general PKC activator, PMA, and a general PKC inhibitor, calphostin
C. Figure 1 shows the effect of PMA in
the absence (Fig. 1, A and B) and presence of
calphostin C (Fig. 1D). Exposure of oocytes to PMA (100 nM)
resulted in a slow and time-dependent inhibition of peak
INa (Fig. 1A). The I-V
relations during control and PMA at 30 min are shown in Fig.
1B. PMA inhibited INa by 44.3 ± 8.2% at 20 mV (n = 8, P < 0.05 compared with control). The specificity of PMA effects on
INa was confirmed by comparing its effects to another phorbol ester, 4
PDD, which does not activate PKC (11, 44). PMA effects were not seen [2.9 ± 2.2%,
n = 5, P = not significant (NS)
compared with control] with its inactive analog, 4
PDD, at the same
concentration of 100 nM (Fig. 1C). PMA and 4
PDD effects at 30 min on the cell capacitance of oocytes are shown in the lower
part of Fig. 1, A and C, respectively. PMA and
4
PDD reduced oocyte cell capacitance by 17 ± 3.7%
(n = 6, P < 0.05) and 15 ± 3.6%
(n = 5, P < 0.05), respectively.
Calphostin C superfusion for 10-15 min before the onset of PMA
application completely blocked PMA inhibition of
INa (only 4.6 ± 3.3%, n = 4, P = NS compared with control). These experiments
indicate that PMA inhibition of INa is mediated
through PKC.
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Peptide activator of PKC,
V1-7, inhibited
INa expressed in Xenopus oocytes.
To selectively activate
PKC, we used a novel peptide,
V1-7,
also termed pseudo-
RACK (
-
RACK), which is derived from the regulatory V1 region of
PKC and was previously shown to
selectively activate the translocation of
PKC (10).
This is the first and only available agonist peptide activator of one
single PKC isozyme. Figure 2
shows the effect of this
PKC agonist,
V1-7, and its scrambled peptide (negative control) on INa.
Recording of INa began immediately after
injection of
V1-7 (100 nM) or its scrambled peptide (100 nM).
Figure 2A illustrates the time course of
V1-7 inhibition of INa from one oocyte.
V1-7
at 30 min reduced oocyte cell capacitance by 3.9 ± 3.0% (Fig.
2A, bottom; n = 5, P = NS). Figure 2B shows the I-V
relations of INa during control and 30 min after
the injection of
V1-7.
V1-7 inhibited peak
INa by 30.5 ± 4.5% (n = 15, P < 0.05 compared with control). Figure
2C shows the lack of effect of
V1-7 scrambled
peptide on INa I-V relations
(3.2 ± 2%, n = 5, P = NS
compared with control). However, PMA superfusion of seven other oocytes
preinjected with
V1-7 scrambled peptide resulted in
INa inhibition by 40.1 ± 6.3%
(n = 7, P < 0.05 compared with
V1-7 scrambled peptide).
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PMA inhibition of INa was not altered by
peptide-specific antagonists of -,
-,
-, and
PKC isozymes.
Figure 4A shows the
I-V relations of INa during control
and 30 min after PMA superfusion of oocytes injected with
C2-4
peptide; INa was decreased by 44.9 ± 7.9%
(n = 6, P = NS compared with PMA
alone). Figure 4B shows the I-V relations of
INa during control and 30 min after PMA
superfusion of oocytes injected with
C2-4 peptide;
INa was decreased by 43.6 ± 6.9%
(n = 5, P = NS compared with PMA
alone). Figure 4C shows the I-V relations of
INa during control and 30 min after PMA
superfusion of oocytes injected with
V1-1 peptide;
INa was decreased by 44.5 ± 8.2%
(n = 5, P = NS compared with PMA
alone). Figure 4D shows the I-V relations of INa during control and 30 min after PMA
superfusion of oocytes injected with
V1-2 peptide;
INa was decreased by 43.9 ± 7.0% (n = 7, P = NS, compared with PMA
alone). These results demonstrate that
-,
-,
-, and
PKC
isozymes did not alter PMA-induced INa inhibition. A summary of all the above results is shown in Fig. 5.
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PMA and V1-7 did not alter steady-state inactivation of
INa expressed in Xenopus oocytes.
The effects of PMA and
V1-7 on steady-state inactivation of
INa were also investigated using the
double-pulse protocol as indicated in MATERIALS AND
METHODS. Figure 6 shows the
averaged normalized data plotted against the prepulse potentials for
PMA (A) and
V1-7 (B). The curves in Fig.
6 were obtained by fitting the data points with the Boltzmann
distribution described in MATERIALS AND METHODS. The
inactivation curves were nearly identical between control and either
PMA or
V1-7 in Fig. 6, A or B,
respectively, suggesting that PMA or peptide
V1-7 did not
change the kinetics of voltage-dependent inactivation of
INa. For the control group (n = 6), V0.5 was
80 ± 4.3 mV and
k was 4.4 ± 0.7 mV, whereas for the PMA group
(n = 6), V0.5 was
84 ± 6.8 mV and k was 4.3 ± 0.9 mV. Similarly, for the
control group (n = 5), V0.5 was
79.7 ± 3.8 mV and k was 4.5 ± 0.5 mV, whereas
for the
V1-7 group (n = 5),
V0.5 was
82.6 ± 6.4 mV and k
was 4.4 ± 0.8 mV.
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PMA and V1-7 induced translocation of
PKC in Xenopus
oocytes.
To demonstrate that the functional inhibition of
INa is associated with biochemical translocation
of
PKC from the cytosol to the membrane, we performed Western blot
assays on oocytes treated with
PKC activators and/or inhibitors.
Figure 7 shows the translocation of
PKC in oocytes by PMA (100 nM) and
V1-7 (100 nM) and its inhibition by
V1-2 (100 nM). Figure 7, lanes 2 and
4, show that
PKC was translocated from the soluble to the
particulate fraction by PMA and
V1-7, respectively. Figure 7,
lanes 6 and 8, show that
V1-2 antagonized
PMA- and
V1-7-induced
PKC translocation from the soluble to
the particulate fraction, respectively. Similar results were obtained
in a total of four independent experiments.
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PMA and V1-7 inhibited INa in rat ventricular
myocytes.
We next tested whether
PKC also modulates INa
in native cardiac myocytes. INa was recorded
from rat ventricular cells, and the effects of
PKC activation were
investigated. Figure 8A shows the time course of the effect of 100 nM PMA on
INa in the absence or presence of intrapipette
V1-2 (100 nM). PMA inhibited INa by
53 ± 5.9% (n = 5, P < 0.05).
However, in the presence of
V1-2, PMA-induced
INa inhibition was reduced to 22.4 ± 7%
(n = 4, P < 0.05 compared with PMA
alone). Figure 8B shows the time course of the effect of
V1-7 (100 nM) on INa in the absence and
presence of intrapipette
V1-2 (100 nM).
V1-7 inhibited
INa by 34.3 ± 5.4% (n = 6, P < 0.05). This inhibition of
INa by
V1-7 was completely abolished by
V1-2 (5.4 ± 1.4%, n = 4, P > 0.05). All together, the data obtained in native
cardiac myocytes indicate that selective
PKC activation also
inhibited INa. These results are similar to
those obtained in Xenopus oocytes.
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DISCUSSION |
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The present study is the first to show that PKC is involved in
PMA-induced inhibition of the cloned human INa
expressed in Xenopus oocyte and INa
recorded from rat ventricular myocytes. It is evident that
identification of the particular isozyme(s) that mediates the
regulation of Na+ channels is of important therapeutic implications.
Regulation of INa by PKC. The characterization of the role of individual PKC isozymes in the regulation of ion channels in general and Na+ channels in particular has been largely limited by the lack of isozyme selective activators and inhibitors. While several previous studies implicated PKC in the regulation of Na+ channels, the role and the identity of the isozyme(s) responsible for this regulation remain largely unexplored. Heterologously expressed rat brain (rBIIA) (7, 32) and human cardiac Na+ channel currents (hH1) (31) were reduced upon PKC activation. Although both rBIIA and hH1 contain consensus sites for phosphorylation by PKC, most of the sites are not conserved between these two isoforms. In one study (31), elimination of conserved consensus PKC sites in the hH1 interdomain III-IV linker, which contains the putative PKC site (Ser-1503), does not completely eliminate the PMA-induced INa inhibition, implying that other phosphorylation site(s) may exist.
Our present findings in Xenopus oocytes showing that INa is inhibited by PMA, a general activator of PKC, are consistent with previous studies using a heterologous expression system (7, 31, 34, 35). In addition, the use of a novel peptide-specific activator ofProposed mechanism of peptide V1-7 action.
Peptide
V1-7 is the first isozyme-selective PKC activator that
induces
PKC translocation from the cytosol to the particulate fraction (10). 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 (10). The evidence that a peptide translocation activator for
PKC,
V1-7, functionally inhibited
INa 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. 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 (22). The
molecular mechanisms by which PKC regulates cardiac Na+
channels are not completely defined.
Physiological and pathophysiological significance of the regulation of INa channels by PKC. In the last few years, research in the general area of signal transduction has advanced significantly. As a result, PKC has emerged as a key component along signal transduction pathways. PKC has been involved in the modulation of ion channels (11, 21, 33, 45, 47, 54), inotropic and chronotropic effects (5, 11, 23, 26, 53), gene expression (6, 38), secretion of cardiac factors (17, 36), hypertrophy (14, 39), ischemia, and infarction (43). It therefore becomes critical to characterize and gain insight on how PKC and, most importantly, its multiple isozymes regulate cardiac ion channels, in both physiological and pathological settings. In the heart, Na+ channels determine excitability and conduction velocity of the action potential (1, 2, 15, 30, 42) and, thus, constitute the key elements in the genesis of arrhythmias. The ability to dissect the individual role of PKC isozymes in the regulation of Na+ channels may provide functional information that will help in the design of isozyme-targeted therapeutics.
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ACKNOWLEDGEMENTS |
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This study was supported by Veterans Administration Medical Research Funds Merit Grant and REAP Grant (to M. Boutjdir) and by National Heart, Lung, and Blood Institute Grants HL-55401 (to M. Boutjdir) and HL-52141 (to D. Mochly-Rosen).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Boutjdir, Research and Development Office (151), Veterans Affairs 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 14 December 2000; accepted in final form 19 June 2001.
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