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Departments of 1 Pediatrics, 2 Physiology and Neurosciences, and 3 Biochemistry, New York University Medical Center, New York, New York 10016
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Current evidence suggests that members of the
Kv4 subfamily may encode native cardiac transient outward current
(Ito).
Antisense hybrid-arrest with oligonucleotides targeted to Kv4 mRNAs
specifically inhibited rat ventricular
Ito, supporting
this hypothesis. To determine whether protein kinase C (PKC) affects
Ito by an action on these molecular components, we compared the effects of PKC activation on Kv4.2 and Kv4.3 currents expressed in
Xenopus oocytes and rat ventricular
Ito. Phorbol
12-myristate 13-acetate (PMA) suppressed both Kv4.2 and Kv4.3 currents
as well as native
Ito, but not
after preincubation with PKC inhibitors (e.g., chelerythrine). An
inactive stereoisomer of PMA had no effect. Phenylephrine or carbachol
inhibited Kv4 currents only when coexpressed, respectively, with
1C-adrenergic or
M1 muscarinic receptors (this
inhibition was also prevented by chelerythrine). The voltage dependence
and inactivation kinetics of Kv4.2 were unchanged by PKC, but small effects on the rates of inactivation and recovery from inactivation of
native Ito were
observed. Thus Kv4.2 and Kv4.3 proteins are important subunits of
native rat ventricular
Ito, and PKC
appears to reduce this current by affecting the molecular components of the channels mediating
Ito.
protein kinase C; potassium channel; transient outward current; antisense oligonucleotides
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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ACTIVATION OF protein kinase C (PKC) by
1-adrenoreceptor agonists,
purinergic agonists, and endothelin modulates ion channels in the heart
and plays a role in regulating cardiac contractility, ischemic
preconditioning, and protection from ischemia-reperfusion injury (23,
26). PKC has been shown to regulate the activity of a number of cardiac
ion channels, including voltage-gated potassium channels (13, 28),
sodium channels (25), and the L-type calcium channel (34). The
transient outward current
(Ito), which is
activated by membrane depolarization and which plays an important role
in determining the action potential duration and hence contractile force, has also been described to be modulated by PKC (2). Because
modulation of Ito
may be involved in the onset and/or prevention of arrhythmias,
we sought further to examine the effects of PKC on native
Ito and
specifically to explore whether these effects are due to actions on the
protein components of the channels mediating rat ventricular
Ito.
Recently, significant progress has been made in characterizing the molecular composition of Ito in different species and in different regions of the heart. With the use of Northern analysis (30), ribonuclease protection assays (14), immunohistochemistry (6), and single-cell in situ hybridization (7), it was found that, of 23 different K+ channel genes, expression of several occurred at significant levels in mammalian cardiac muscle; these include members of mammalian Shaker (Kv1 subfamily), Shab (Kv2 subfamily), Shaw (Kv3 subfamily), and Shal (Kv4 subfamily). Of these, Kv1.4, Kv3.4, and the Kv4s all inactivate rapidly and can be described as transient outward (A-type) currents (5, 30, 32, 33, 37). There is mounting evidence that members of the Kv4 subfamily are primary subunits of native Ito in rat, dog, and human ventricular myocytes (6, 14, 15, 30), with Kv4.2 being the main Kv4 subfamily member occurring in rat ventricle (15). There is also evidence for the existence of Kv4.3 (but not Kv4.1) in rat ventricle (15, 33). This contention relies on the distribution of mRNA, protein levels, electrophysiological similarities, and comparable pharmacological profiles [e.g., block by 4-aminopyridine (4-AP) and flecanide] (5, 6, 14, 15, 30, 33). However, more direct proof is lacking. We therefore tested the effect of antisense oligonucleotides specifically directed against Kv4.2 and Kv4.3 mRNAs and found a significant depression of rat ventricular Ito after 40- to 72-h culturing in the presence of these oligonucleotides. Our results therefore provide strong support for the concept that Kv4 proteins are key molecular components of rat ventricular Ito. Hence, we examined the effects of PKC activation on Kv4.2 and Kv4.3 currents expressed in Xenopus oocytes and compared these responses with those observed on rat ventricular Ito. Our results support the notion that the inhibition of Ito by PKC is due to effects on the Kv4 channel components responsible for native Ito.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In Vitro Transcription and Oocyte Injection
cDNAs were linearized and cRNA was synthesized in vitro with the following enzymes: Kv4.2 cloned from rat brain (EcoR I/T7), Kir2.1 (IRK1) cloned from mouse macrophage (Not I/T7) (a kind gift from Dr. L. Jan),1C-adrenergic receptor cloned
from human prostate (Not
I/T7) (a kind gift from Dr. J. Tseng-Crank), and M1 muscarinic receptor cloned from
porcine brain (Xba I/SP6) (a kind gift
from Dr. T. Kubo). Adult female Xenopus
laevis frogs (Nasco, Fort Atkinson, WI) were
anesthetized with 0.17% 3-aminobenzoic acid ethyl ester (Sigma, St.
Louis, MO), and unfertilized oocytes were harvested after abdominal
incision. Stage V-VI oocytes were stripped under a dissecting
microscope and defolliculated enzymatically with 1 mg/ml collagenase
(Sigma type I) in Ca2+-free ND96
solution (see below) for 30 min at room temperature. Defolliculated
oocytes were injected with 50 nl cRNA (~20 ng) using a 10-µl
micropipette (Drummond Scientific, Broomall, PA). Injected oocytes were
kept in 0.5× L-15 solution (see below) at 18-20°C and
used for recording 2-5 days after injection.
Site-Directed Mutagenesis
The tyrosine (Y) in position 592 of Kv4.2 was mutated to phenylalanine (F) using the QuikChange site-directed mutagenesis kit from Stratagene using the manufacturer's protocols. The following mutant oligonucleotide primer 5'-GT GAA CAA CCT TTC GTG ACC ACA GC-3' and its complement (each complementary to 1 of the 2 strands of the Kv4.2 cDNA) served to initiate Pfu DNA polymerase-mediated replication of the Kv4.2-containing recombinant pCDNA3 plasmid. The TTC sequence in this primer replaces the TAC codon in Kv4.2 encoding Y592, thus resulting in the synthesis of a mutated Kv4.2 cDNA. Mutated cDNA strands are selected after digestion of the original strands with Dpn I endonuclease. The point mutation was confirmed by sequencing.Electrophysiological Measurements in Oocytes
Oocytes were voltage-clamped using the two-microelectrode voltage-clamp technique. Microelectrodes were pulled from thin-walled glass capillaries (1.5-mm OD, World Precision Instruments) with tip resistance of 0.7-1.2 M
for the current-passing electrode and
1-5 M for the voltage-measuring electrode when filled with 3 M
KCl solution. Recordings were obtained using a Geneclamp 500 amplifier
(Axon Instruments, Foster City, CA) with data sampled at 5 kHz and
filtered at 1 kHz. Currents were elicited by depolarizing steps from
80 to +50 mV in 10-mV increments every 15 s from a holding
potential of 110 mV (to allow full recovery from inactivation). No leak subtraction was performed during the experiments. The recording
chamber was continually perfused (1 ml/min). To avoid contamination
with Ca2+-activated
Cl
currents, a low
Cl recording solution was
used (see below). All experiments were performed at room temperature
(20 ± 2°C).
Isolation of Cardiac Myocytes
Single ventricular myocytes were isolated from adult rat hearts using standard enzymatic techniques. Briefly, hearts were rapidly removed from pentobarbital sodium (50 mg/kg)-anesthetized male Wistar rats (weight 200-250 g) and perfused in a constant-pressure Langendorff system with a standard Tyrode solution (see below) for 5 min. The perfusate was oxygenated and maintained at 37°C. The hearts were perfused with Ca2+-free Tyrode solution for 5 min followed by perfusion with the same solution containing 0.114% (wt/vol) collagenase (Sigma type I) and 0.014% (wt/vol) protease (Sigma type XIV) for 9 min. The enzyme-containing solution was then washed out by perfusing with Ca2+-free Tyrode solution for 5 min, and the ventricles were separated from the rest of the heart. To eliminate the reported differences in Ito measured in epicardium and endocardium (19), myocytes were isolated only from epicardial sections by mechanical agitation in KB solution (see below) and were used for patch-clamp experiments up to 8 h after isolation.Culturing of Rat Ventricular Myocytes
Cells were isolated essentially as described above, except that all procedures were carried out under sterile conditions. The heart was perfused and digested as described but washed with Tyrode solution containing 100 µM Ca2+. The ventricles were separated, cut into small pieces, and incubated for 10 min at 34°C while bubbling with 100% O2. The cell suspension was filtered (200-µm mesh) and subjected to three rounds of centrifugation (50 g) and resuspended in Tyrode solution containing increasing Ca2+ concentration (500 µM and 1 mM) and finally in serum-free Eagle's minimum essential medium (1.8 mM Ca2+). Myocytes were immediately plated onto laminin (100 µg/ml)-coated glass coverslips and allowed to attach by incubation at 37°C under a water-saturated atmosphere with 5% CO2. After 4 h, the medium was changed to remove dead or unattached myocytes, and S-oligonucleotides (3 µM) were applied to some culturing dishes. S-oligonucleotides were supplemented every 24 h, and measurements were performed after 40-72 h of culturing.Design of Antisense Oligonucleotides
For these experiments, we designed phosphorothioate oligonucleotides (S-oligonucleotides) against two distinct regions of Kv4.2. The oligonucleotides were as follows: 5'-TGCAACACCGGCTGCCATGTTG-3' (22 nt; 59% GC content; targeted across the initiation codon) and 5'-CTCGCCTTAAGGGCCTGGGCTC-3' (22 nt; 68% GC content; targeted to a region just 5' of the termination codon). Extensive Genbank database searches indicate identity with Kv4.2 (RK5; accession number M59980) with little identity to other K+ channels. Similarity with the primary sequence of Kv4.3 (which is abundantly expressed in rat heart) and Kv4.1 (which is only expressed at very low levels; Ref. 14) was observed. The first of these oligonucleotides has an 81% identity with the Kv4.3 primary sequence and 73% with Kv4.1, whereas the second has a 42% identity with Kv4.3 and 38% with Kv4.1. We incubated rat ventricular myocytes with a combination of these two S-oligonucleotides (3 µM each). In some experiments, we used a combination of two control oligonucleotides that had no sequence similarity with Kv4.2 or Kv4.3 (5'-TGGTTCTCACACTGCCCATTGC-3'; 22 nt; 55% GC content and 5'-CTCGCCTTAAGGGCCTGGGCTC-3'; 22nt; 68% GC content).Electrophysiological Measurements in Isolated Myocytes
Cells were placed in a bath (~200 µl volume) on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan) and voltage clamped using an Axopatch 200A and pClamp software (Axon Instruments) in the whole cell configuration while superfused (1-2 ml/min) with a bath solution at room temperature (20 ± 2°C). Patch electrodes were made from thin-walled glass capillaries using a horizontal puller (Zeitz Instrumente Universal Puller, Augsburg, Germany) and heat polished. When filled with a pipette solution, electrode resistance ranged between 2 and 4 M. Cell capacitance and pipette series
resistance were both compensated. Whole cell currents were expressed as
picoamperes per picoFaradays to allow for variation in
cell size. Ito
was elicited by the voltage protocol described in the legends to Figs.
1-9. In all cases, the membrane potential was corrected by
18 mV to account for the liquid junction potential (calculated
using Axoscope, Axon Instruments).
Previous studies indicate that rat ventricular myocytes contain
predominantly two depolarization-activated
K+ currents; a rapidly
inactivating Ito
that can be blocked by 4-AP and a 4-AP-insensitive sustained outward
current (IK)
that can be blocked by tetraethylammonium (TEA) (3). The 4-AP-sensitive Ito has been
shown to activate and inactive at more depolarized potentials than the
TEA-sensitive IK
(3). We confirmed that this was also the case under our experimental
conditions and consequently selected a holding potential of 58
mV, which mostly inactivated IK without
affecting the amplitude of
Ito (data not
shown).
Solutions and Chemicals
Solutions used for oocytes.
For the preparation and enzyme digestion of oocytes, a
Ca2+-free ND96 solution was
used containing the following (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.5 adjusted with NaOH), and 0.5× L-15 solution, 1:1 dilution of GIBCO's Leibovitz L-15 medium in filter-sterilized 50 mM HEPES buffer (pH 7.5 adjusted with NaOH) containing 50 U/ml nystatin
(GIBCO) and 0.1 mg/ml gentamycin (GIBCO). For the measurement of
membrane currents, a superfusion solution was used containing the
following (in mM): 96 sodium glutamate, 2 potassium glutamate, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.5 adjusted with NaOH) (low Cl
ND-96 solution).
Solutions used for isolated myocytes.
Normal Tyrode solution contained the following (in mM): 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 0.33 Na2PO4,
1.8 CaCl2, and 10 glucose (pH 7.4 adjusted with NaOH). KB solution (for storing myocytes) contained the
following (in mM): 20 taurine, 50 glutamic acid, 10 HEPES, 0.5 ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 3 MgSO4, 30 KH2PO4,
and 30 KCl (pH 7.2 adjusted with KOH). The superfusion solution for the
measurement of
Ito in myocytes contained the following (in mM): 135 choline chloride (to avoid Na+ currents), 5.4 KCl, 1.1 MgCl2, 1.8 CaCl2, 0.5 CdCl2 (to block Ca2+ currents), 10 HEPES, 1 ribose, 10 glucose, 0.001 ryanodine (to inhibit release of
Ca2+ from the sarcoplasmic
reticulum), and 0.01 atropine sulfate (to block acetylcholine-induced
K+ currents) (pH 7.4 adjusted with
NaOH). The pipette solution contained the following (in mM): 115 potassium aspartate, 5 KCl, 4 Na2ATP, 7 MgCl2, 5 EGTA, and 10 HEPES (pH
7.2 adjusted with KOH).
Chemicals.
Phorbol 12-myristate 13-acetate (PMA), 4-phorbol 12,13-didecanoate
(4-PDD), chelerythrine chloride, and staurosporine were purchased
from Calbiochem-Novabiochem International (La Jolla, CA). Each stock
solution (1 mM PMA, 1 mM 4-PDD, 6 mM chelerythrine, and 1 mM
staurosporine) was made in dimethyl sulfoxide (DMSO), and aliquots were
kept at 20°C. All test solutions were freshly made by
diluting the stock solution with bath solution just before the start of
each experiment. The final concentration of DMSO in each test solution
was <0.3%, and these concentrations of DMSO did not affect Kv4.2
currents in Xenopus oocytes or native
Ito in rat
ventricular myocytes. Carbachol,
L-phenylephrine hydrochloride, and all other reagents were purchased from Sigma.
Data Analysis
Data were analyzed using the pClamp suite of software (Axon Instruments) and Origin for Windows (Microcal Software, Northampton, MA) software. Steady-state inactivation curves and recovery from inactivation curves were obtained by using standard two-pulse protocols. Curve fitting was performed using a Boltzmann equation {normalized current = 1/[1 + e(V V1/2)/k],
where V is prepulse potential,
V1/2 is
half-maximal inactivation potential, and
k is slope factor} for
steady-state inactivation curves and a first-order exponential function
(normalized current = 1 et/
,
where t is recovery time and 
is
time constant of recovery from inactivation) for the recovery from
inactivation. The time constants of inactivation of expressed currents
and native Ito were obtained by fitting the current traces with either first-order (for myocytes) or second-order (for oocytes) exponential functions. All
data are expressed as means ± SE. Comparisons between the data
before and after application of drugs were performed by a paired t-test. Differences at
P < 0.05 were considered
significant.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effects of Kv4.2 Antisense Oligonucleotides on Rat Ventricular Ito
To test the previously proposed hypothesis that members of the Kv4 subfamily are components of the membrane channels responsible for native cardiac Ito (6, 14, 15), we used antisense oligonucleotides complementary to Kv4 transcripts (see METHODS for details) and tested their effects on the heterologously expressed currents produced by Kv4.2 and Kv4.3 proteins, the two Kv4 family members expressed in rat heart (14). The effect of these oligonucleotides against Kv4.2 currents was first tested using oocytes coinjected (50 nl total injection volume) with Kv4.2 and Kir2.1 cRNAs (0.3-0.7 µg/µl solution). These oocytes were also injected with both Kv4 antisense oligonucleotides (1 pmol each) or H2O to keep the injection volume constant. Two days after injection, oocytes were subjected to two-microelectode voltage clamping. The inwardly rectifying Kir2.1 currents were predominately observed during negative-clamp steps, whereas the voltage-dependent Kv4.2 currents were seen during depolarizing pulses where little Kir2.1 currents are observed (Fig. 1A). In oocytes injected with Kv4 antisense oligonucleotides, Kir2.1 currents were unaffected, whereas Kv4.2 currents were significantly inhibited (Fig. 1A). Peak Kv4.2 current at +60 mV was 2,068 ± 435 nA (n = 6) in control oocytes and 453 ± 69 nA (n = 6, P < 0.05) in antisense oligonucleotide-injected oocytes (Kir2.1 current amplitudes at160 mV were 5,615 ± 882 and 5,397 ± 711 nA, respectively). We also examined the effects of these antisense
oligonucleotides on Kv4.3 currents and found them also to be inhibited.
In the absence of these oligonucleotides, the average peak Kv4.3
current at +20 mV was 331 ± 41 nA, whereas in oocytes coinjected
with antisense oligonucleotides, the current amplitude was 52 ± 4 nA (Fig. 1B;
n = 6, P < 0.05). Injection of a
combination of control oligonucleotides with no sequence similarity or
complementarity with Kv4 transcripts (see
METHODS; 1 pmol each) had no effect on
Kv4.2 currents at +60 mV (1,667 ± 296 nA;
n = 6). Thus we conclude that our antisense oligonucleotides specifically inhibited expression of Kv4 channel proteins.
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To examine the effect of the Kv4 specific antisense oligonucleotides on
native Ito, adult
rat ventricular myocytes were placed in culturing conditions in the
presence or absence of these antisense oligonucleotides (3 µM each).
Ito amplitude was
measured as the peak current during a depolarization pulse from a
holding potential of 58 mV. At 2 days after culturing in the
presence of antisense oligonucleotides, the
Ito amplitude (at
+42 mV; defined in this figure as the difference between peak current
and quasi-steady-state current at the end of the depolarization pulse)
was found to be significantly smaller compared with time-matched
control myocytes (Fig.
2A). The
current-voltage relationship shows a reduced
Ito amplitude in
Kv4 antisense-treated myocytes at all potentials studied (Fig.
2B). When the
Ito is defined as
the total peak current amplitude, there was a similar inhibition by
antisense oligonucleotides (9.3 ± 1.7 vs. 19.0 ± 4.6 pA/pF in
control; n = 7, P < 0.05). In a separate series of
experiments (after 3 days in culture), we tested the specificity of the
Kv4 antisense oligonucleotides. In these experiments, the peak
Ito amplitude was
similarly decreased (7.3 ± 0.7 vs. 15.1 ± 2.7 pA/pF in control;
P < 0.05). However, other membrane
currents were unaffected by antisense oligonucleotides. Neither the
holding current at 58 mV (0.35 ± 0.37 vs. 0.46 ± 0.27 pA/pF in control; n = 7) nor the peak
inward rectifier K+ current
(IK1)
at the beginning of a hyperpolarizing pulse to 120 mV
(10.1 ± 1.61 vs. 13.8 ± 1.16 pA/pF in control)
were affected by these oligonucleotides. Control antisense
oligonucleotides (see METHODS)
had no effect on peak
Ito (12.0 ± 1.48 vs. 15.1 ± 2.7 pA/pF in control,
n = 7). These results provide direct
evidence that Kv4 channel proteins are key components of the
channels mediating rat ventricular
Ito (see
DISCUSSION).
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PMA Specifically Suppresses Kv4.2 and Kv4.3 Currents
The effect of PMA on Kv4.2 currents expressed in a Xenopus oocyte was examined. A marked suppression of the peak current occurred after application of PMA (10 nM; 29.45% of initial current after 30 min of drug application, n = 10) (Fig. 3A). This inhibition occurred at all test potentials examined (Fig. 3B, left). Normalization of the current-volage relationships (I/Imax) did not reveal any significant shift in the voltage dependence of activation (Fig. 3B, right). PMA inhibited Kv4.2 currents after a short delay following its application and in a dose-dependent manner (to 62.2 ± 3.4, 29.4 ± 5.1, and 9.1 ± 1.7% of initial current after 30 min by 1, 10, and 100 nM PMA, respectively, n = 6) (Fig. 3C). PMA also inhibited Kv4.3 currents (Fig. 3D). Application of 10 nM PMA suppressed Kv4.3 currents by 27.5 ± 10.6% after 30-min incubation (n = 9).
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Figure 4 shows that the inhibition of Kv4.2 and Kv4.3 currents by PMA is not a general phenomenon of the oocyte expression system. Oocytes were coinjected with Kv4.2 and Kir2.1 cRNA (see above). The differential effect of PMA (10 nM) on Kv4.2 and Kir2.1 was examined, exploiting the different voltage and time dependence of these currents (in oocytes, Kir2.1 only activates at hyperpolarized potentials, whereas Kv4.2 currents activate at depolarized potentials, where little Kir2.1 current flows). As reported previously for Kir2.1 currents expressed in oocytes (21), we found little effect of PMA on Kir2.1 currents over the time course in which Kv4.2 currents were significantly depressed (Fig. 4). These results therefore suggest that the PMA-induced inhibition of Kv4.2 and Kv4.3 currents was not due to some general nonspecific effect of phorbol esters on membrane currents.
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PMA or 1C- and
M1 Receptors Inhibit Kv4.2 and Kv4.3
Currents by Activation of PKC
-PDD
(Fig. 5). There was no significant effect
of 4-PDD (10 nM) on Kv4.2 currents (91.9 ± 3% of initial
current remaining after 30 min, n = 4)
(Fig. 5B), whereas the same
concentration of PMA decreased the current to 29.4 ± 5% of control
(n = 4) (Fig. 5A). Furthermore, when oocytes were
preincubated with the PKC-selective inhibitor chelerythrine (20 µM)
for 20 min, PMA (in the presence of chelerythrine) had little effect on
Kv4.2 currents during a 30-min incubation period (94.4 ± 16.5% of
initial current remained, n = 4) (Fig.
5C). Another PKC inhibitor,
staurosporine (2 µM, 20-min preincubation), also prevented the effect
of PMA (82.5 ± 2.8% of initial current remaining after 30 min,
n = 3). Similarly, effects of PMA on
Kv4.3 currents were prevented by PKC inhibitors (data not shown). These
results indicate that the effect of PMA on Kv4.2 and Kv4.3 currents is
mediated by activation of PKC.
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PKC activation has been shown to lead to inhibition of Kv1.2 channels via protein tyrosine kinase (PTK) phosphorylation (24). Because Kv4.2 contains a single consensus sequence for phosphorylation by PTK, we performed a point mutation of the tyrosine residue at position 592 of Kv4.2 to phenylalanine [Kv4.2(Y592F)]. Currents expressed by Kv4.2(Y592F) proteins were indistinguishable from those expressed by Kv4.2 proteins. PMA (10 nM) caused an inhibition of Kv4.2(Y592F) currents (38 ± 2.7% of preapplication values after 30 min) similar to that of wild-type Kv4.2 currents (Fig. 5, D and E). Furthermore, Kv4.3 (which does not have any PTK consensus sites) is also inhibited by PKC activation. These results therefore suggest that under these specific experimental conditions, PKC inhibits Kv4.2 and Kv4.3 channels independently of PTK phosphorylation at PTK consensus sites.
Physiologically, activation of PKC is induced by receptor-mediated
signal pathways such as stimulation of
1-adrenoceptors in heart (29)
and muscarinic acetylcholine receptors
(M1,
M3, and
M5) in other tissues. We
coinjected oocytes with Kv4.2 cRNA and
1C-adrenergic receptor cRNA (in
a 1:1 molar ratio). The recorded currents were indistinguishable from
the currents measured in oocytes injected with Kv4.2 transcripts alone.
In these oocytes, the
1-adrenoceptor agonist
phenylephrine (10 µM) caused a potent time-dependent
suppression of Kv4.2 currents (to 36.2 ± 3.5% of initial current
after 30-min incubation, n = 4) (Fig.
6, B and C). In contrast, no significant
effect on Kv4.2 currents was observed in the oocytes lacking the
receptor (the injection volume was kept constant) (Fig. 6,
A and
C). Furthermore, when oocytes
expressing both the Kv4.2 channel and
1C-adrenergic receptor were
preincubated with chelerythrine (20 µM, for 20 min) and then exposed
to phenylephrine in the presence of chelerythrine, the inhibitory
effect of phenylephrine was largely prevented (78.8 ± 7.1% of
initial current remained after 30 min,
n = 3) (Fig.
6C). As an alternative means to
increase activated cellular PKC, we coexpressed Kv4.2 channels and
M1 muscarinic receptors in
oocytes. Carbachol (100 µM) caused a potent inhibition of Kv4.2
current in the presence of the muscarinic receptor (to 34.6 ± 2.8%
of initial current after 30-min incubation,
n = 6), whereas no
significant effect was observed in the absence of the receptor (103.6 ± 1.2% of initial current remained,
n = 6). The inhibitory effect of
carbachol was also largely prevented by chelerythrine (64.1 ± 0.6%
of initial current remaining after 30 min,
n = 4). Similarly, Kv4.3 currents were
inhibited by carbachol when coexpressed with
M1 receptors (data not shown).
These results demonstrate that inhibition of Kv4.2 and Kv4.3 currents
occurred by receptor-mediated PKC activation.
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We explored the possibility that PMA may affect Kv4.2 current kinetics.
Standard two-pulse voltage-clamp protocols were used to assess the
steady-state inactivation and the time course of recovery from
inactivation of Kv4.2 currents. As shown in Fig. 7A, there
was no apparent change in steady-state inactivation of Kv4.2 currents
(V1/2 = 66 ± 2.1 and 68.6 ± 1.6 mV;
k = 6.3 ± 0.3 and 7.2 ± 0.5 mV; before and 30 min after the application of 10 nM PMA, respectively,
n = 7). Similarly, the time
course of recovery from inactivation was also not significantly changed by PMA (exponential time constant = 137.2 ± 21.9 ms before and 102.1 ± 24.9 ms after the application of 10 nM PMA;
n = 4; Fig. 7B). Similar results were obtained
with phenylephrine (10 µM) in oocytes expressing
1C-receptors and Kv4.2 currents
(data not shown).
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The effect of PMA on other kinetic properties, such as the time to peak
and the rate of inactivation of Kv4.2 currents, were also investigated.
The time to peak and the rate of inactivation of Kv4.2 currents were
largely unaffected by PMA (Fig. 7, C
and D). The decay of Kv4.2 currents
was best fitted with a sum of two exponential functions (fast and slow
inactivation components). Both components depended to some extent on
the applied voltage [although this voltage dependence is much
smaller than that of other channels such as Kv3.3, Kv3.4 (37), and
Kv1.4 (12)]. PMA (10 nM, 30-min application) had no effect on the
time constants of either the fast (Fig.
7D) or the slow (191 ± 6.1 vs.
198 ± 33 ms in control, n = 6)
components of inactivation at any of the potentials studied. Similarly,
the time to peak of Kv4.2 currents was unaffected by PMA (Fig.
7C). Similarly, the kinetics of
Kv4.2(Y592F) currents were largely unaffected by PMA. There were no
effects of PMA on the time constants of inactivation (the fast and slow time constants were 39.5 ± 2.5 and 220.7 ± 8.60 ms,
respectively, after 30-min PMA vs. 32.5 ± 0.47 and 197.6 ± 1.31 ms, respectively, for control) or the midpoint of steady-state
inactivation (67.1 ± 0.49 vs. 70.8 ± 0.43 mV in
control), whereas the recovery from inactivation occurred slightly
faster in the presence of PMA (107.0 ± 2.36 vs. 143.8 ± 0.55 ms in control).
These results demonstrate that PKC activation does not reduce Kv4.2 currents by voltage shifts of activation or inactivation variables, nor by changes in channel availability during recovery from inactivation.
PMA-Induced Modulation of Ito in Rat Ventricular Myocytes
To perform a systematic comparison of the effects of PKC on rat ventricular Ito and its molecular components (Kv4.2 and Kv4.3), we reexamined the effects of PMA on the electrophysiological properties of Ito in adult rat epicardial ventricular myocytes.Application of 100 nM PMA caused a marked suppression of
Ito (to 62.5 ± 6.1% of initial current at +42 mV after 8 min,
n = 6) (Fig.
8, A and
D). As was the case for Kv4
currents, PMA inhibited native rat ventricular
Ito at all test
potentials examined and did not cause any significant shift in voltage
dependence (Fig. 8B). The inactive
stereoisomer 4-PDD had no significant effect on native cardiac
Ito (92.9 ± 0.3% of initial current remaining after 8 min,
n = 6) (Fig. 8,
C and
D), and chelerythrine (20 µM, 10-min preincubation) blocked the effect of PMA when simultaneously applied with the phorbol ester (99.4 ± 1.4% of initial current remaining after 8 min, n = 6) (Fig.
8D), suggesting that the effects of
PMA on Ito are
mediated by activation of PKC.
|
We also examined the effect of PMA on the steady-state inactivation and
the time course of recovery from inactivation of native Ito. As was the
case for Kv4.2 currents in oocytes, there was no change in the voltage
dependence of steady-state inactivation of native
Ito
(V1/2 = 33.1 ± 2.3 and 35.3 ± 2.7 mV;
k = 4.0 ± 0.2 and 4.8 ± 0.5 mV
before and 8 min after application of PMA, respectively,
n = 3; Fig.
9A).
However, PMA caused a 48% increase in the time constant of recovery
from inactivation (29.2 ± 5.9 and 43.1 ± 5.2 ms before and 8 min after application of PMA, respectively, n = 3; the differences were
statistically significant, P < 0.05; paired t-test) (Fig.
9B).
|
The effects of PMA on the time to peak and the rate of inactivation of native Ito during depolarizing pulses to various potentials were also investigated. The current decay during depolarizing pulses could best be described by a single exponential function, which was relatively independent of the test potential (Fig. 9D). PMA (10 nM) significantly increased the inactivation time constant at all potentials studied (Fig. 9D) and also increased the time to peak of Ito (Fig. 9C).
| |
DISCUSSION |
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|
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Kv4 Proteins Are Key Molecular Components of Rat Ventricular Ito
Previous data have suggested that members of the Kv4 subfamily are important molecular components of Ito. These currents share the property of rapid inactivation, and unlike the currents expressed by the Kv1.4 (12) and Kv3.3 and Kv3.4 (37), recovery from inactivation is fast (5). In both Kv4 currents and native Ito, activation and steady-state inactivation occur at more negative potentials than Kv3 subfamily members (37) and are blocked by 4-AP in the millimolar range but are insensitive to external TEA (5). These similarities, together with the expression patterns of the Kv4.2 and Kv4.3 mRNA and protein in heart tissue (6, 14, 15, 33), lend strong credence to the suggestion that Kv4 subfamily proteins are primary molecular components of native ventricular Ito. However, more direct experiments, such as the deletion of the cloned channel using in vitro antisense approaches or in vivo Kv4 gene knockout in transgenic animals, have not been performed to date. Here we provide direct evidence that Kv4 proteins are important molecular components of Ito in adult rat ventricular myocytes. Using antisense oligonucleotides that specifically eliminate Kv4.2 and Kv4.3 transcripts, we were able to produce a large and significant reduction of adult rat ventricular Ito. This conclusion was recently confirmed in experiments published while this manuscript was under review. Fiset et al. (18) showed a similar inhibition of Ito in ventricular myocytes from 14-day-old rats by antisense Kv4.2 and Kv4.3 oligonucleotides, and Johns et al. (22) found that dominant negative constructs against Kv4 proteins also inhibit Ito.We did not expect to see a complete reduction in Ito amplitude, since a dynamic situation is likely to occur under these experimental conditions (the rates of mRNA formation and degradation are unknown factors, as is the optimal concentration of oligonucleotide concentration to be used). Furthermore, because antisense oligonucleotides are not expected to affect preexisting channel proteins, the natural turnover rates of these proteins may determine the magnitude of any residual current. The turnover rate of native Kv4 proteins, however, is largely unknown. Thus a likely reason for the residual Ito after antisense oligonucleotide treatment is that the incubation time (40-72 h) was insufficient to deplete the preexisting membrane channel protein levels. However, we chose not to study the effects of a more prolonged exposure (>3 days) to oligonucleotides because of the confounding electrophysiological changes that have been described to occur under prolonged culturing conditions (20). It is interesting in this regard that our antisense experiments or those of Fiset et al. (18) using different oligonucleotides and cellular systems or the dominant negative studies of Johns et al. (22) produced a similar degree of inhibition of Ito. An alternative and obvious explanation for the residual Ito after treatment with antisense oligonucleotides is that channel proteins encoded by other genes may contribute to Ito in rat ventricular myocytes. Future experiments using antisense approaches will be able to address this question more fully. However, this is unlikely since the Ito current remaining in antisense-treated myocytes is very similar to the Ito in untreated myocytes.
Although our experiments provide compelling evidence in favor of the
hypothesis that Kv4 channel proteins are key components of native rat
ventricular Ito,
there was a significant difference between Kv4 currents and native
Ito in the
voltage threshold at which currents could be detected (Kv4.2 and Kv4.3
currents activated around 40 to 50 mV while activation of
native Ito could
only be detected at potentials beyond 30 mV). Similarly, the
midpoints of steady-state inactivation were more negative in Kv4.2 and
Kv4.3 currents
(V1/2 around
65 to 55 mV) than for native
Ito (around 35 mV). One possible explanation for this discrepancy is the presence of Cd2+ in the bath
solution (which was used to block
Ca2+ currents) for the measurement
of native Ito. It
is known that divalent cations such as
Cd2+ shift the voltage dependence
of activation and inactivation of Ito in a positive
direction (1). In concordance with this, we found that both the
midpoints of steady-state activation and inactivation of Kv4.2 currents
in oocytes were shifted to the right by ~15 mV with 500 µM
Cd2+ in the presence of 1 mM
Mg2+ and 1.8 mM
Ca2+ (our preliminary data). Other
differences of the composition of the solutions (ionic strength, other
cations, etc.) or differences between frog oocyte and mammalian cell
membrane compositions may also account for the differences in the
voltage dependence of these currents.
There were also important differences in the inactivation process
during sustained depolarizing pulses. The inactivation time constant of
Kv4.2 and Kv4.3 currents could best be described by a sum of two
exponential functions (see also Refs. 32, 33), whereas native
Ito could best be
fitted by a single exponential function (see also Ref. 3). Furthermore,
recovery from inactivation was slower in Kv4.2 and Kv4.3 currents than
in native Ito
(compare Figs. 7B and
9B; see also Ref. 33). The reasons for
these differences are presently unclear. It is possible that the
presence of other unknown accessory molecular components of
Ito in native
myocytes may alter the voltage dependence and its regulation by PKC. A likely candidate for such a regulatory protein is the putative accessory -subunit that may be present in native cells (31, 32) and
that alters Kv4.2 or Kv4.3 currents to resemble native Ito (i.e., a more
rapid development and recovery from inactivation and a loss of voltage
dependence of inactivation) when coexpressed in oocytes (31-33).
Modulation of Kv4 Currents by PKC
It has become increasingly apparent that phosphorylation/dephosphorylation reactions are key modulators of membrane ion channels. PKC can be activated in vivo by receptor-mediated pathways as well as by phorbol esters or diacylglycerol (DAG). In cardiac tissue, numerous agonists (e.g.,1-adrenergic, purinergic,
endothelin, angiotensin II, or thrombin) are known to stimulate PKC
(29). It has been demonstrated that PKC activation can increase or
decrease the activity of cloned voltage-dependent
K+ channels, such as Shaker H4,
Kv1.4, and Kv3.4 (13, 27, 28). In the present study, we provide
evidence that Kv4.2 and Kv4.3 currents are decreased when PKC is
activated either using phorbol esters or by receptor-mediated
intracellular pathways. The evidence can be summarized as follows: a
reduction of Kv4.2 and Kv4.3 currents that was observed with PMA (but
not with the inactive stereoisomer 4-PDD) was prevented by PKC
inhibitors (chelerythrine and staurosporine). Furthermore, stimulation
of 1-adrenoceptors or
M1 receptors, both of which
activate PKC (4, 29), also led to a decrease of these currents. The
agonist-induced suppression of Kv4 currents was largely, but not
completely, prevented by chelerythrine. A possible explanation for this
incomplete inhibition is that
1-adrenergic or muscarinic
receptor activation activated additional signal transduction pathways
that may have also influenced these currents independently of PKC
activation. One interesting candidate is Ca2+. It is well established that
stimulation of 1-adrenoceptors or M1 receptors induces production
of DAG and inositol 1,4,5-trisphophate by activation of phospholipase
C. DAG, in turn, activates PKC, whereas inositol 1,4,5-trisphosphate
increases cytosolic Ca2+ levels by
triggering the release of Ca2+
from intracellular stores. Preliminary results (Nakamura, unpublished observations) support an independent role for intracellular
Ca2+ in inhibiting Kv4 currents.
Nevertheless, the results obtained with
1-receptors suggest a
functional role for signal transduction-mediated modulation of
Ito amplitude in
heart muscle.
A reduction in current amplitude could be mediated by a slower recovery from inactivation, which would cause cumulative inactivation (as is the case for Ito at fast stimulation rates) (17). However, our results demonstrate that PMA had no significant effect on the time constant of recovery from inactivation of Kv4.2 currents and only moderately affected that of native Ito. Another possibility is that changes in the voltage dependence of steady-state inactivation may have led to a reduction of current amplitudes. However, we found the voltage dependence of steady-state inactivation of Kv4.2 currents and native Ito to be unchanged by PMA or by receptor stimulation. Therefore, these results suggest that reduction of these currents by PKC activation is likely due to direct inhibition of channel activity, probably by reduction in the number of functional channels or in the average single-channel conductance. The mechanisms by which activation of PKC results in inhibition of Kv4 channels expressed in Xenopus oocytes (or Ito in ventricular myocytes) remain to be established. It is not known at present whether the inhibition results from PKC phosphorylation of Kv4 proteins or if it is mediated by other signal transduction enzymes that by themselves are activated by PKC. However, we were not able to obtain evidence in favor of a PKC-dependent PTK-mediated effect as seen for the inhibition of Kv1.2 currents (24). We can rule out the possibility that PMA caused a general decrease of currents in the oocyte expression system. We (present study) and others (21) found that Kir2.1 currents in oocytes are unaffected by PMA over the time course when Kv4 currents are inhibited. However, the exact mechanism remains to be elucidated; for example, it is not known whether the inhibition of Kv4 channel activity results from specific protein internalization or from inactivation of channels at the membrane.
Modulation of Native Ito in Rat Ventricular Myocytes by PKC
In the heart, there is increasing evidence that activation of PKC modulates various types of ion channels and causes changes of cardiac function. For example, activation of PKC has been shown to increase delayed rectifier K+ currents in guinea pig ventricular myocytes (35), to modify mechanical behavior, and to increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes (16). Previous studies also suggested that rat ventricular Ito is modulated by PKC (2). In the present study, we extended this study by performing a detailed analysis of the effects of PKC activation on native rat ventricular Ito. We show that PMA, but not the inactive stereoisomer 4-PDD, reduces
Ito. Furthermore, the effect of PMA was largely prevented by the PKC inhibitor
chelerythrine. These results suggest that the effects of the phorbol
ester were also mediated by activation of PKC in heart cells. Our
results are in agreement with a previous study using rat ventricular
myocytes showing an inhibition of
Ito by PMA (2)
but contradict the findings of another study where phorbol esters were
described to be without effect on rat ventricular
Ito (36). We
found that if PMA is not made up freshly before each experiment, its
effect was significantly reduced (data not shown). Furthermore, cells from different areas in the heart may respond differently to PKC (we
only used epicardial myocytes for reasons of consistency). These
possible differences may account for some of the differences observed
in rat ventricle. However, in rabbit atria, PMA has been described to
increase Ito
amplitude (8). Although no obvious explanation is at hand, it should be
noted that rabbit
Ito differs in
several electrophysiological aspects from currents in rat, ferret, and
human (2, 9-11, 17). One interesting possibility, which would be
consistent with the conclusion of this study that the effects of PKC on
Ito are mediated
by its effects on Kv4 subunits, is therefore that the molecular
components of Ito
in rabbit atria and rat ventricles differ and that they are
differentially regulated by PKC activation. This discrepancy may be
resolved when more information becomes available regarding the
molecular components of
Ito in different
species and in different regions of the heart.
Interestingly, we observed a moderate increase in the inactivation time constant of native Ito (but not of Kv4.2 currents) in the presence of PMA. We also observed a small but significant slowing of the time course of recovery from inactivation of native Ito. These subtle differences in regulation of Kv4.2 currents and native ventricular Ito by PKC may well be explained by the presence of (as yet unknown) regulatory subunits (see above). The modulation of inactivation kinetics of native Ito by PKC activation, which has not been reported previously, may be important in modulation of receptor-mediated refractoriness after cardiac excitation.
In summary, we found that Kv4 gene products are important molecular components of native rat ventricular Ito. PKC activation, both by phorbol esters and receptor-mediated pathways, suppressed Kv4.2 and Kv4.3 currents expressed in Xenopus oocytes and native Ito in adult rat epicardial myocytes. These results therefore suggest that the PKC-mediated inhibition of Ito is due to effects on the channel protein components responsible for native Ito in rat ventricle.
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported in part by the Seventh Masonic District Association, Inc., Uehara Memorial Foundation, National Science Foundation Grant IBN9630832, and National Institute of Neurological Disorders and Stroke Grant NS-30989.
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FOOTNOTES |
|---|
Address for reprint requests: T. Y. Nakamura, Pediatric Cardiology, TH501, New York University Medical Center, New York, NY 10016.
Received 20 March 1997; accepted in final form 10 June 1997.
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Discussion
References
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)
and in presence of antisense oligonucleotides (
)
(n = 7/group).
C: recordings from cultured myocytes
(3 days) of Ito
(at +42 mV) and inward rectifier K+ current
(IK1) (at
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