© 1999 American Heart Association, Inc.
Original Contribution |
Potential Molecular Basis of Different Physiological Properties of the Transient Outward K+ Current in Rabbit and Human Atrial Myocytes
From the Department of Medicine (Z.W., J.F., H.S., S.N.), Montreal Heart Institute, Department of Medicine, University of Montreal (Z.W., S.N.), and the Department of Pharmacology and Therapeutics (S.N.), McGill University, Montreal, Quebec, Canada, and the Department of Cell Biology (A.P., J.M.N.), Washington University, St. Louis, Mo.
Correspondence to Zhiguo Wang, PhD, Research Center, Montreal Heart Institute, 5000 Belanger East, Montreal, Quebec, Canada H1T 1C8.
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Abstract—The properties of the transient outward current (Ito) differ between rabbit and human atrial myocytes. In particular, rabbit Ito is known to recover more slowly than its human counterpart and to show much more frequency dependence. To assess the possibility that these physiological differences may reflect differing expression of K+ channel subunit gene products, we used a combination of whole-cell voltage-clamp, heterologous expression, pharmacological, antisense, and Western blot techniques. The inactivation of Ito in rabbit atrial myocytes was significantly slowed by hydrogen peroxide, with human Ito being unaffected. Use-dependent unblocking with 4-aminopyridine was not seen for rabbit Ito nor for Kv1.4 currents in Xenopusoocytes, whereas human Ito showed strong use-dependent unblock (as did Kv4 currents). Western blots indicated the presence of Kv4 proteins in both human and rabbit atrial membranes, but Kv1.4 was only detected in the rabbit. Antisense oligodeoxynucleotides directed against Kv4.3, Kv4.2, or Kv1.4 subunit sequences significantly inhibited Ito current density in cultured rabbit atrial myocytes, whereas only Kv4.3 antisense significantly inhibited Ito in human cells. Neither mismatch oligodeoxynucleotides nor vehicle altered currents in either species. We conclude that, unlike human atrial myocytes, rabbit atrial myocytes express Kv1.4 channel subunits, which likely contribute to a number of important physiological differences in Ito properties between the species. To our knowledge, these studies constitute the first demonstration of a functional role for Kv1.4 channels in cardiac membranes and provide insights into the molecular mechanisms of an important cardiac repolarizing current.
Key Words: electrophysiology • cardiac arrhythmia • action potential • antiarrhythmic drug
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Arapidly activating and inactivating K+ current, known in nerve as A-type current, is widely distributed in various tissues and organ systems of mammals. In cardiac cells, this current has been named the transient outward K+ current (Ito), and it is believed to play a major role in action potential repolarization.1 2 3 4 5 6 Ito is particularly important in early (phase 1) repolarization and influences the participation of other currents and membrane transport processes by influencing the voltage-time trajectory of the action potential. Changes in Ito contribute to action potential changes caused by heart disease,7 8 9 10 11 and Ito is a target for many antiarrhythmic drugs.12 13 14 15 16 Because of its important role in cardiac electrical activity, major efforts have been made to understand the biophysical properties and the molecular basis of Ito. Recent advances in molecular biology have resulted in the cloning of a large number of distinct cDNAs encoding K+ channel subunits. The heterologous expression of a variety of cloned K+ channel
-subunits, including Kv1.4,17 18
Kv3.3,19 Kv3.4,20 Kv4.1,21
Kv4.2,22 23 24 25 and Kv4.3,26 27 28 results in
rapidly activating and inactivating K+ currents
with characteristics similar to those of endogenous cardiac
Ito. The first cardiac clone found to encode an Ito-like current was Kv1.4.17 It was initially believed to be a strong candidate to underlie Ito in the heart; however, there are important discrepancies between the kinetics of Kv1.4 inactivation and those of native human Ito,17 and the distribution of Kv1.4 protein does not parallel that of Ito in the rat.29 30 Subsequent work has pointed to an important role of Shal-type genes, particularly Kv4.2 and Kv4.3, as a molecular basis for mammalian cardiac Ito.27 31 32 33 Thus, it is now widely accepted that Kv4 genes are important in encoding cardiac Ito and uncertain whether Kv1.4 participates at all in cardiac Ito.
The rabbit heart classically has a very important Ito1 and has served as a widely used model for the analysis of the role of Ito in cardiac repolarization. There are important differences between Ito in the rabbit and the corresponding current in humans,4 with human Ito strongly resembling that of other species, including rat,5 ferret,34 and dog.6 A striking difference is the slow reactivation rate of Ito in the rabbit1 4 compared with other species. The mechanisms underlying the unusual properties of rabbit Ito are unknown. Slow reactivation is typical of Kv1.4, apparently because of an essential role of C-type inactivation in determining the recovery rate.35 The physiological differences between human and rabbit Ito could be, at least in part, due to the unique participation of subunits encoded by Kv1.4 in the rabbit heart.
The purpose of the present study was to obtain further information about the potential basis for the differences between rabbit and human atrial Ito. A variety of techniques, including whole-cell patch clamp, pharmacological probes, antisense oligonucleotides, and detection of ion channel protein expression in cardiac cell membranes, were used to evaluate the hypothesis that differences in the molecular composition of rabbit and human Ito account for differences in physiological properties.
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Materials and Methods |
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Cell Isolation
Procedures for isolating cells from human tissues and from rabbit hearts were based on approaches described previously in detail.4 14 36 37 38 39 When cells were cultured, all procedures were conducted under strictly sterile conditions.
Specimens of human right atrial appendage were obtained from the hearts
of 26 patients (mean age, 62 years; range, 41 to 78 years) undergoing
aortocoronary bypass surgery. The atria were all grossly normal
and from patients without heart failure, atrial arrhythmias, or
electrocardiographic P-wave abnormalities. The procedure for obtaining
the tissue was approved by the Ethics Committee of the Montreal Heart
Institute. Tissue chunks of 
1 mm3 were
incubated in oxygenated (100% O2)
nominally Ca2+-free Tyrode's solution containing
(in mmol/L): NaCl 137,
KH2PO4 5,
MgSO4 8, dextrose 10, EGTA 0.1, HEPES 5, and
taurine 10 (pH 7.4 with NaOH, 36°C), along with 140 U/mL
collagenase (CLS II, Worthington Biochemical), 4 U/mL
protease (type XXIV, Sigma), and 0.1%. BSA Dispersed cells were
suspended in a storage solution containing (in mmol/L) KCl 20,
KH2PO4 10, dextrose 10,
glutamic acid 70, taurine 10, and EGTA 10 and 0.1% BSA (pH adjusted to
7.4 with KOH). Repeated exposures to fresh enzyme-containing solution
were made until the yield was maximal. For cell culture, the cell
suspension was centrifuged at 250 rpm for 5 minutes, and the
pellet removed for culture.
Rabbits weighing 1.4 to 2.3 kg were euthanized by cervical dislocation, and their hearts were rapidly removed, mounted on a Langendorff apparatus, and perfused with nominally Ca2-free Tyrode's solution containing collagenase (CLS II, Worthington, 0.2%) and 1% BSA (Sigma) for 15 minutes. Atrial tissue was then removed and placed in the storage solution, and cells were dissociated by mechanical agitation with a Pasteur pipette. Cells were used freshly for biophysical and pharmacological studies or concentrated by centrifugation at 250 rpm for cell culture.
Cell Culture
Freshly isolated cells were plated at low density
(104 cells/cm2) onto
2x2-mm square glass coverslips coated with laminin (20 µg/mL). Cells
were maintained in Petri dishes (35 mm, Nunc) containing medium
199 with 10% FBS (GIBCO-BRL) supplemented with sodium penicillin G (1
U/mL) and streptomycin sulfate (1 µg/mL, GIBCO-BRL). Cultures were
incubated at 37°C in a humidified, 5%
CO2-enriched atmosphere.38 40
Cloning of cDNA Fragments From Rabbit Heart and Preparation of
Antisense Constructs
Because the genes of Kv1.4, Kv4.2, and Kv4.3 had not been
identified in rabbit hearts, cloning of cDNA sequences for these
constructs was required to design appropriate antisense oligomers.
Degenerate primer pairs for polymerase chain reaction (PCR) were
designed (Table 1
) on the basis of the
published cDNA sequences of Kv1.4, Kv4.2, and Kv4.3, targeting regions
with minimal homology to other channel sequences but with strong
conservation across species. Reverse transcription was performed for
first-strand cDNA synthesis from an RNA sample extracted from rabbit
heart. PCR was then conducted to amplify the desired cDNA fragments.
PCR products were subcloned into pGEM-T Easy vector (Promega) and
subjected to sequencing analysis. Antisense oligomers (20 to 21
bp) were designed on the basis of the cloned cDNA sequences. Human
constructs were based on previously published sequences, with regions
of 100% identity between rabbit and human chosen. The specificity of
antisense oligomers was confirmed by comparison with all other
sequences in GenBank with the use of the Basic Local Alignment Search
Tool (BLAST). As a control, mismatch oligomers were prepared that were
identical to each gene-specific antisense sequence with the exception
of 4 or 5 mismatch mutations. The oligodeoxynucleotide
sequences used are shown in Table 2.
Phosphorothioate oligodeoxynucleotides were synthesized
commercially by Research Genetics Inc.
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Exposure to Oligodeoxynucleotides
Oligodeoxynucleotide treatment was started 24 hours
after the onset of cell culture. Three groups of cultured cells were
studied in all series of experiments. One group of cells (referred to
as the control group) was exposed to the same volume of vehicle
(sterile distilled water) used for oligodeoxynucleotide
delivery, a second group was exposed to mismatch
oligodeoxynucleotides, and a third group was exposed to
antisense oligodeoxynucleotides. For each treatment, the
growth medium was removed, and the cells were washed twice with
serum-free medium and antibiotics. Medium 199 (1 mL) was mixed with
lipofectin (4 µmol/L final concentration, GIBCO-BRL) and vehicle
alone or vehicle containing antisense (0.5 µmol/L) or mismatch
oligodeoxynucleotides (0.5 µmol/L) and then
incubated at room temperature for 40 minutes before addition to the
cells. After 6 hours of incubation at 37°C, the medium was removed
and the cells were washed with fresh medium 199. The cells were then
incubated in growth medium (including antibiotics and
heat-inactivated serum) with vehicle, antisense, or
mismatch oligomers (1 µmol/L) but without lipofectin for another
18 hours before patch-clamp experiments were performed.
Competitive Reverse Transcriptase (RT)–PCR
RNA Purification
Cultured cells were washed twice with PBS solution containing
(in g/L) KCl 0.2, KH2PO4
0.2, NaCl 8, and
Na2HPO4 · 7
H2O 2.16) and then incubated in Trizol reagent
(GIBCO-BRL). Total RNA was extracted by the acidic
guanidinium-isothiocyanate method using chloroform and precipitated
with isopropanol. Isolated RNA was quantified from absorbance at 260 nm
by spectrophotometry, and the purity was confirmed by the ratio of
A260/A280.
Integrity of the total RNA was evaluated by ethidium bromide staining
of denaturing agarose gels. RNA samples were stored in DEPC-treated
double-distilled H2O at –80°C.
Primers
Degenerate primers were designed on the basis of published cDNA
sequences of Kv1.4, Kv4.2, and Kv4.3 with
oligonucleotide sequences chosen from cDNA regions with
minimal homology among clones. cDNA fragments were synthesized by
RT-PCR of mRNA from rabbit hearts, and the PCR products were
subcloned into pGEM-T easy vector (Promega) for sequencing. The
gene-specific primer (GSP) pairs were then designed on the basis of
cloned cDNA fragments for each channel. Specificity of the primer pairs
was verified by comparison with the entire GenBank database using
BLAST. Chimeric primer pairs were constructed with sequences homologous
to human cardiac -actin cDNA flanked at the 5' ends by GSPs, and an
8-nucleotide (GGCCGCGG) linker homologous to the 3' end
sequence of T7 promoter was conjugated to the 5' end of each forward
chimeric primer.
Synthesis of RNA Mimic
First-strand cDNA was synthesized by reverse transcription
and was used as template for subsequent PCR amplification with chimeric
primer pairs to obtain a cDNA mimic consisting of a 480-bp fragment of
human cardiac -actin flanked by a GSP sense sequence at the 5' and
antisense sequence at the 3' end. A second run of PCR was performed
with the T7 promoter sequence as a forward primer and a reverse GSP,
and the resulting products were cDNA fragments carrying a T7
promoter followed by a forward GSP, an -actin fragment, and then a
reverse GSP. The products were then gel purified with the Glassmax
(silica membrane for DNA binding) DNA Isolation Spin Cartridge System
(GIBCO-BRL). RNA mimics were generated by in vitro transcription with
purified cDNA mimics as templates using mMESSAGE mMACHINE
(Ambion). To remove any remaining cDNA templates, the reaction
products were incubated with RNase-free DNase 1 at 37°C for 20
minutes, followed by phenol/chloroform extraction and isopropanol
precipitation.
Reverse Transcription
RNA mimic samples with serial 10-fold dilutions were prepared
and added to a constant quantity of sample RNA (total RNA of 1 µg for
each reaction). RNAs were denatured by incubating at 65°C for 15
minutes. Reverse transcription was carried out in a 20-µL reaction
mixture containing reaction buffer (in mmol/L, Tris-HCl [pH 8.3]
10, KCl 50, and MgCl2 2.5), 1 mmol/L dNTPs
(Boehringer Mannheim), 3.2 µg random primer
p(dN)6 (Boehringer Mannheim), 5
mmol/L DTT, 50 units RNase inhibitor (GIBCO-BRL), and 200
units of Moloney murine leukemia virus RT (GIBCO-BRL).
First-strand cDNAs were synthesized at 42°C for 60 minutes, and the
remaining enzymes were inactivated by heating at 99°C for
5 minutes.
PCR Amplification
First-strand cDNA (10 µL) was used as a template for
amplification in a total volume of 50 µL of reaction mixture. The
reaction mixture contained (in mmol/L) Tris-HCl (pH 8.3) 10, KCl
50, MgCl2 1.5, and dNTPs 1; 0.5 µmol/L of
each GSP pair; and 2.5 units of Taq polymerase (GIBCO-BRL).
Reactions were hot-started at 94°C and continued for 3 minutes for
initial melting. The cycling profiles were 30-second denaturing at
94°C, 30-second annealing at 54°C, and 40-second extension at
72°C, for 30 cycles, followed by a final 5-minute period at
72°C.
Quantification of PCR Products
Densitometry was used for quantification of PCR
products.22 23 24 PCR products were visualized under
UV light with the use of a PDI Image System and a Nighthawk camera
after ethidium bromide staining of a 1.5% agarose gel. The density of
each band was then determined by Quantity One software. A DNA mass
marker (100 ng) was run in the gel to analyze the size and
quantity of PCR products. The density of the DNA mass ladder was
used to generate a standard curve by linear regression with
extrapolation to 0 for each experiment. The density of each sample band
was then converted to the absolute quantity by calibrating to the
standard curve. Based on the principle of competitive PCR, when the
target is coamplified with a known amount of the internal standard, the
logarithmic ratio of target over standard product concentration
will be linearly related to the initial amount of standard,
intercepting the horizontal axis (target/standard ratio of 1) at an
internal standard concentration equal to the concentration of the
initial target. Accordingly, plots of log (mimic/target concentration)
versus known log (mimic) concentration were constructed from the data
and fit by linear regression to determine the abundance of target
message present.
Whole-Cell Patch-Clamp Recording
Electrophysiological Recording From
Native Myocytes
Borosilicate glass microelectrodes (1-mm outer diameter)
were used, with tip resistances of 1 to 3 M
when filled with a
pipette solution containing (in mmol/L) KCl 20, potassium
aspartate 110, MgCl2 1, HEPES 5, EGTA 5, Mg-ATP
5, GTP 0.1, and Na2-phosphocreatine 5 (pH 7.3,
adjusted with KOH). Cells were superfused with modified Tyrode's
solution at 37°C containing (in mmol/L): NaCl 126, KCl 5.4,
CaCl2 1, MgCl2 1, HEPES 5,
and dextrose 10. Cd2+ (200 µmol/L) was
added to the superfusate to block
ICa. Any basal
acetylcholine-activated current was inhibited by including
atropine (1 µmol/L) in the superfusate. ATP-sensitive
K+ current (IKATP)
activation was prevented by including 5 mmol/L ATP in the pipette
and 10 µmol/L glyburide in the external solution.
INa was inactivated by using a
holding potential of –50 mV, by replacing extracellular
Na+ with choline, or both.
The electrodes were connected to a patch-clamp amplifier
(Axopatch 1-D, Axon Instruments) that applied voltage command pulses
with the use of pCLAMP6 software running on an IBM-compatible computer
interfaced with a digital/analog converter. Recordings
were low-pass filtered at 2 Hz. Junction potential offsets were
compensated before formation of the membrane-pipette seal. After
gigaseal formation (seal resistance>20 G), gentle suction was
applied to rupture the membrane for whole-cell recording. Cell
capacitance averaged 29.6±1.8 and 26.5±1.8 pF before and after
compensation, respectively, for human cells, and 49.6±3.2 and
43.5±2.4 pF for rabbit cells. Series resistance averaged 5.2±0.6 and
2.1±0.4 M before and after compensation in human atrial cells and
5.0±0.4 and 1.9±0.6 M in rabbit cells. Leak currents were minimal,
and no leakage correction was applied.
The amplitude of Ito was measured as the difference between the peak of Ito and the current level at the end of the pulse. IKur was measured as the amplitude of the current at the end of the test pulse relative to the 0-current level. Individual currents were normalized to the membrane capacity to control for differences in cell size.
Expression and Recording of Currents Encoded by Kv1.4,
Kv4.2, and Kv4.3
Kv1.4 was obtained as a kind gift from Dr Arthur Brown (Case
Western University, Cleveland, Ohio). It was subcloned into a
modified version of PCRII vector containing a small portion of the
3'-untranslated region of DRK1, including the poly
A+ tail, to allow expression in
Xenopus oocytes. Kv4.2 was subcloned into pRC-CMV vector.
cRNAs were prepared with the mMESSAGE mMACHINE kit (Ambion) using T7
RNA polymerase after linearization of the plasmid, according to the
manufacturer's protocols. cRNAs were dissolved in DEPC-treated sterile
water, stored at –80°C, and diluted immediately before injection.
Stage V through VI Xenopus oocytes were injected with 46 nL
of cRNA.
Approximately 48 hours after cRNA injection, 2-electrode voltage clamp
was performed on individual oocytes. Electrodes were filled with 3
mol/L KCl in agar and had resistances of 0.5 to 1.0 M when
measured in the bath solution containing (in mmol/L) NaCl 100, KCl
5, CaCl2 0.3, MgCl2 2, and
HEPES (pH 7.4) 10. The electrodes were connected to a GeneClamp-500
amplifier (Axon Instruments). The pClamp 6 suite of programs was used
for data acquisition and analysis. Records were digitized
at 5 kHz and filtered at 2 kHz. Experiments were conducted at room
temperature (22°C to 23°C).
The entire coding region of Kv4.3 was subcloned into pBK-CMV vector (Stratagene). Kv4.3-pBK-CMV plasmid was linearized with MluI before transfection to facilitate recombination of the plasmid DNA with the HEK chromosomal DNA. HEK cells were incubated in DMEM at 37°C in a CO2 incubator until the cells were 70% to 80% confluent. For each transfection, the cells (1x106) were incubated with the linearized DNA (1 µg) mixed with 1 µg of CD8 antigen and 18 µg of the liposome preparation lipofectamine (GIBCO-BRL) for 5 hours at 37°C in serum-free DMEM. Fresh DMEM with 20% FBS was then added to the cells without removing the transfection mixture. Seventy-two hours after the start of transfection, Dynabeads M-450 CD8 (Dynal) were added to the medium at a final bead concentration of 1.4x105 beads/mL to permit recognition of the transfected cells. Whole-cell patch-clamp recording was performed on the cells with beads attached (indicating gene expression) 40 minutes after magnetic bead exposure.
Western Blots
Peptides corresponding to unique sequences in the N terminus of
Kv1.4 (residues 13 to 37; CNSHMPYGYAAQARARERERLAHSR) and the C terminus
of Kv4.3 (residues 484 to 502; CLEKTTNHEFVDEQVFEES) were generated by
the Protein Chemistry Laboratory (Washington University Medical
Center). The peptides were coupled to the keyhole limpet hemocyanin
carrier protein and sent to Caltag (San Francisco, CA) for injection
into rabbits. Sera were screened using ELISAs, and antibodies were
subsequently affinity purified using the ImmunoPure Antigen/Antibody
Immobilization Kit No. 2 (Pierce). ELISA assays on the
affinity-purified antibodies revealed that each antibody detected only
the peptide against which it was generated.
Rat brain membrane proteins were prepared essentially as previously described.30 All procedures were performed at 4°C, and all solutions contained a mixture of protease inhibitors (in mmol/L, iodoacetamide 1, 1,10-phenanthroline 1, and pefabloc 0.5 and 1.4 µmol/L pepstatin). Briefly, brains were homogenized in 10 mL of Tris-HCl buffer (5 mmol/L Tris; pH 7.4) with 0.32 mol/L sucrose. After nuclei and debris were pelleted by centrifugation (1000g, 10 minutes), the supernatant was centrifuged at 100 000g for 1 hour. The pellet was resuspended in 20 mmol/L Tris-HCl containing 1 mmol/L EDTA (pH 7.4), centrifuged again (40 000g, 20 minutes), and resuspended in solubilization buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 10% glycerol, 120 mmol/L KCl, and 2% Triton X-100; pH 7.4). After incubation on ice for 1 hour, the final suspension was centrifuged at 78 000g for 2.5 hours to pellet insoluble material. Protein assays (Bio-Rad) were completed, and the samples were aliquoted and frozen at –20°C until used.
Human and rabbit heart membrane proteins were isolated using a protocol similar to that previously described.30 Rabbit atrial samples were harvested from anesthetized adult rabbits and placed on ice. Adult human atrial tissue samples from normal donor hearts not accepted for transplantation were procured from LifeBanc (Cleveland, OH) with consent for research use. Macroscopic and microscopic examination revealed no evidence of pathology or underlying cardiovascular disease. All procedures were performed at 4°C, and all solutions contained the mixture of protease inhibitors described, as well as 1 mmol/L benzamidine, 7.9 µmol/L aprotinin, and 0.15 µmol/L leupeptin. For the preparation of human membrane proteins, tissue samples (1 to 2 g) from individual donors were processed. Tissue samples were homogenized at 4°C in 10 mL TE buffer (containing 10 mmol/L Tris-HCl and 1 mmol/L EDTA; pH 7.4). After centrifugation (1000g, 10 minutes), the supernatants were retained and the pellets were resuspended to original volume in TE buffer, homogenized, and centrifuged. The supernatants were collected, pooled with the original supernatants, and centrifuged (40 000g, 10 minutes). The resulting pellets were resuspended in TE buffer containing 0.6 mol/L KI, incubated on ice (30 minutes), centrifuged (40 000g, 10 minutes), and then washed twice with TE buffer. The final pellets were resuspended in TE buffer containing 2% Triton X-100 and incubated on ice (1 hour) to solubilize membrane proteins. A final centrifugation (17 400g, 30 minutes) precipitated the insoluble material. After protein assays had been completed, samples were aliquoted and frozen at –20°C for subsequent analysis.
Rat brain membrane proteins and rabbit and human heart membrane proteins (15 to 30 µg) were fractionated by SDS-PAGE and then probed by Western blot analysis with the anti-Kv1.4 and anti-Kv4.3 antibodies.30 Membranes were fractionated on 10% polyacrylamide gels and transferred to Hybond polyvinylidene difluoride membrane (Amersham). The membranes were washed (3 minutes) in PBS and incubated (1 hour) in blocking buffer III (0.2% I-block blocking buffer [Tropix] in PBS with 0.1% Tween 20), followed by an overnight incubation at 4°C with either the anti-Kv1.4 or the anti-Kv4.3 antibody in blocking buffer. The next day, membranes were washed (10 minutes) and then incubated for 1 hour at room temperature with alkaline phosphatase-conjugated goat anti-rabbit IgG (Tropix) diluted 1:10,000 in blocking buffer III. After incubation, membranes were washed in blocking buffer III for 15 minutes and then for 2 minutes in assay buffer (0.1 mol/L diethanolamine with 1 mmol/L MgCl2, pH 9.8; Tropix). Bound antibodies were detected using Chemiluminescent Substrates for Alkaline Phosphatase Permit Light-Based Detection (CSPD; Tropix).
Data Analysis
Comparisons among groups were performed by ANOVA with
Scheffé's contrasts. A 2-tailed probability of 5% was taken to
indicate statistical significance. Group data are presented as
mean±SEM. Nonlinear curve fitting (the Marquardt procedure) was
performed using software for patch-clamp data acquisition and analysis
(Clampfit in pClamp6).
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Results |
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Comparison of Biophysical and Pharmacological Properties of Rabbit and Human Ito
Reactivation Kinetics
Figures 1A
and 1B illustrate the
recovery kinetics of Ito in rabbit and
human atrial cells, respectively. A double-pulse protocol was used to
assess Ito recovery, with 200-ms test
pulses after an identical conditioning pulse at a variable
interval. The results shown are mean±SEM for 12 rabbit and 14 human
cells. Recovery was biexponential in the rabbit, with mean recovery
time constants averaging 21.8±1.3 ms (16±2% of current) and 2780±57
ms (83±6% of current) at –80 mV. In human cells, recovery was
monoexponential with a mean time constant at –80 mV of
15.4±0.7 ms. As illustrated in Figure 1A and 1B,
Ito recovery slowed at less negative
holding potentials. The kinetic differences between
Ito in the 2 species are not due to
differences in voltage dependence of inactivation, as shown by the
results in Figure 1C. A 1000-ms prepulse from a holding
potential of –80 mV was followed by a 200-ms test pulse to +50 mV. In
7 rabbit and 6 human cells, the half-activation potential averaged
–33.3±2.1 mV in rabbit and –32.7±2.4 mV in human cells
(P=NS), indicating no difference in the voltage dependence
of Ito inactivation.
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Effects of 4-Aminopyridine (4AP) on
Ito
4AP is a classical Ito
blocker.1 2 3 15 As illustrated by the data in Figure 2, the responses of human and rabbit
Ito to 4AP are quite different. Figure 2A shows Ito elicited in a rabbit
cell in the presence of 2 mmol/L 4AP on depolarization for 100 ms
from –60 to +40 mV. Results are shown during 2 consecutive pulses (P1
and P2) at 1 Hz after a long quiescent period (>1 minute). Virtually
no time-dependent current is elicited by either the first or second
pulse. Figure 2B shows data obtained in a human cell with an
identical protocol. During the first pulse, there is a small but
measurable Ito, and the second pulse
elicits a relatively large Ito. Figure 2C shows mean data for current in the presence of 4AP relative
to control current (4AP/Control) as a function of pulse number during
pulsing at 1 Hz after a >1-minute quiescent period in 6 human and 5
rabbit cells. In rabbit cells, strong inhibition is present during
all pulses, whereas in human cells there is prominent use-dependent
unblocking. Figure 2D shows results obtained with the same
protocol applied to Kv1.4 channels expressed in Xenopus
oocytes. As was the case for rabbit Ito,
little 4AP unblocking is observed. In contrast, when Kv4.3 is expressed
in HEK cells and the same protocol applied, clear
Ito unblocking is seen (Figure 2E).
Figure 2F shows mean data for 4AP block obtained in 15 oocytes
for Kv1.4, 4 cells for Kv4.3, and 8 oocytes for Kv4.2 and indicates
that the absence of use-dependent unblocking of 4AP is typical of Kv1.4
channels.
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Effects of H2O2
The inactivation of Kv1.4, but not Kv4.2 or Kv4.3, has been
shown to be substantially slowed in response to oxidative
stress.27 41 42 We therefore compared the response of
rabbit and human Ito to the addition of
H2O2 in the
superfusate. Typical results are displayed in Figure 3A. Oxidative stress resulting from
0.01% H2O2 dramatically
slowed the inactivation of Ito in rabbit
myocytes (Figure 3A, left). In contrast, human
Ito was not significantly affected by
exposure to H2O2 (Figure 3A, right) under the same conditions. Average kinetics before
and after H2O2 in 10 rabbit
cells and 8 human cells are shown in Figure 3B. Overall, the
rapid and slow inactivation time constants of rabbit
Ito increased from 21.1±3.0 and
249.2±30.3 ms under control conditions to 34.7±5.1 and 364.8±45.3 ms
(P<0.001 for each) after exposure to 0.01%
H2O2 in 10 cells. In
addition, the proportion of slowly inactivating
Ito in rabbit cells was increased by
H2O2 from 24±6% to
63±6% of total current (P<0.01). For human
Ito, the rapid and slow phases of
Ito inactivation had time constants of
7.1±1.0 and 22.2±2.3 ms before and 5.1±1.2 and 19.0±2.5 ms (n=6
cells, P=NS) after exposure to
H2O2, respectively. The
proportion of slowly inactivating current in human cells averaged
44±16% before and 41±8% after (P=NS) exposure to
H2O2.
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Detection of Kv1.4 and Kv4.3 Proteins in Rabbit and Human
Myocardial Membranes
Western blots of fractionated rat brain membrane proteins with the
anti-Kv4.3 antibody revealed a prominent protein band at 75 kDa
(Figure 4A, lane B). The band was
eliminated when the antibody was preincubated with the peptide against
which it was generated. In Western blots of fractionated rabbit and
human heart membrane proteins, prominent bands at 75 kDa were also
detected with this antibody (Figure 4A, lanes RA and HA). There
is also a broad, intense band at 55 to 60 kDa in the blot of the
rabbit atrial membrane proteins; this corresponds to rabbit IgG, also
detected by the goat anti-rabbit (IgG) secondary antibody. Although no
attempt was made to quantify these blots, it is of interest to note
that the expression levels of the Kv4.3 protein in rabbit and human
heart are similar.
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Western blots of fractionated rat brain membrane proteins with the
anti-Kv1.4 antibody revealed a prominent protein band at 95 kDa (Figure 4B
, lane B), and this band was eliminated when the antibody was
preincubated with the peptide against which it was generated. In
Western blots of fractionated rabbit heart membrane proteins, a
prominent band at 95 kDa was detected with this antibody (Figure 4B, lane RA). As in the blots with the anti-Kv4.3 antibody,
rabbit IgG was also detected in this blot. In contrast to these
results, however, no bands were detected with the anti-Kv1.4 antibody
in immunoblots of fractionated human heart membrane
proteins probed with the same antibody under identical experimental
conditions (Figure 4B, lane HA). When the film was exposed for a
considerably longer time (30 minutes), a faint band at 65 kDa was
detected with the anti-Kv1.4 antibody (Figure 4B, lane HA').
Although this band was eliminated when the antibody was preincubated
with the peptide against which it was generated, the low molecular
mass (65 kDa) suggests that this is not Kv1.4, which has a
predicted molecular mass of 85 kDa. Similar results were obtained with
all 3 preparations of each tissue type (each from a different subject)
studied.
Effects of Antisense Oligodeoxynucleotides on Rabbit
and Human Ito
The above results point to a potentially important role for Kv1.4
in rabbit but not human heart, and the potential participation of Kv4
proteins in both species. This possibility was further assessed by the
antisense experiments illustrated in Figures 5 (for rabbit) and
6 (for human cardiomyocytes).
Results in rabbit cells exposed to mismatch
oligodeoxynucleotides differed from those of rabbit cells
exposed to antisense oligodeoxynucleotides directed against
Kv1.4, Kv4.2 and Kv4.3 sequences, all of which reduced
Ito amplitude (Figure 5). Human
cells, unlike those of rabbits, did not appear to be appreciably
affected by Kv1.4 and Kv4.2 antisense
oligodeoxynucleotides; however, exposure to Kv4.3 antisense
produced significant reductions in Ito
(Figure 6).
|
|
Mean data for all cells studied (rabbit, 57, 45, and 59 control cells
for Kv1.4, Kv4.2, and Kv4.3, respectively; 34, 29, and 37 cells for
Kv1.4, Kv4.2, and Kv4.3 mismatch; and 54, 49, and 57 cells for Kv1.4,
Kv4.2, and Kv4.3 antisense; human, 48, 42, and 55 control cells for
Kv1.4, Kv4.2, and Kv3.4, respectively; 32, 34, and 41 cells for Kv1.4,
Kv4.2, and Kv4.3 mismatch; and 52, 48, and 51 cells for Kv1.4, Kv4.2,
and Kv4.3 antisense) are shown in Figure 7. In rabbit myocytes, there was a
45±2% decrease (P<0.01, compared with vehicle control)
produced by Kv1.4 antisense at +60 mV, 40±2% by Kv4.2 antisense
(P<0.01), and 68±3% by Kv4.3 antisense
(P<0.01). In human cells, however, only Kv4.3 antisense
produced a significant reduction in Ito
expression.
|
None of the antisenses significantly altered the kinetics of Ito. For example, on depolarization to +40 mV, the fast and slow inactivation time constants in rabbit cells averaged 26.4±2.3 and 261.2±40.8 ms, respectively, under control conditions (n=31), and 24.3±3.9 and 246.7±38.4 ms in the presence of Kv1.4 antisense (n=29), 26.3±4.7 and 281.5±41.1 ms in the presence of Kv4.2 antisense (n=25), and 27.2±3.4 and 282.3±42.1 ms in the presence of Kv4.3 antisense (n=23). In human cells, the inactivation time constants averaged 7.4±1.7 and 29.4±3.2 ms under control conditions (n=20) and 7.3±2.0 and 27.2±3.1 ms after Kv4.3 antisense (n=22).
The lack of effect of mismatch oligodeoxynucleotides on
Ito in rabbit and human cells argues
against nonspecific effects of oligodeoxynucleotides. To
verify further the specificity of antisense effects, we evaluated
possible effects on IKur in human atrium
and ICa in rabbit atrium. The results
(Figure 8) show that
ICa was not altered by any of the antisense
molecules in the rabbit, nor was IKur in
human cells affected by Kv4.3 antisense.
|
Finally, we performed additional experiments to determine the effects
of antisense on the concentration of various mRNA species in rabbit
atrial myocytes. In each experiment, separate groups of cells from 1
heart were exposed to vehicle, antisense
oligodeoxynucleotides against 1 ion channel mRNA species,
or mismatch oligodeoxynucleotides. At least 3 experiments
were done with each construct. Figure 9A
shows typical gels for competitive RT-PCR of Kv4.3 mRNA in cells
exposed to vehicle (left), Kv4.3 antisense (middle), and mismatch
(right) sequences. The point of identity shifted to the right in the
antisense-treated group, indicating a decline in Kv4.3 mRNA expression.
Figure 9B illustrates the graphical solution of mRNA
concentration with the use of data for Kv4.3. Figures 9C through
9E show that antisense reduced the mRNA concentration of the
corresponding species significantly compared with vehicle or mismatch
oligodeoxynucleotide–treated cells. To exclude antisense
effects on other molecular species, we measured mRNA concentrations of
the nontargeted molecular species in response to antisense. No
nonspecific effects were seen: with Kv1.4 antisense, Kv4.2 mRNA
concentrations were 6.3±1.1 (control) versus 6.4±1.5 amol/µg RNA
(antisense-treated cells) and Kv4.3 mRNA concentrations were 12.5±2.2
(control) versus 12.6±2.7 (antisense) amol/µg RNA; with Kv4.2
antisense, concentrations of Kv1.4 mRNA were 8.4±2.0 (control) and
8.2±2.6 (antisense) amol/µg and of Kv4.3 mRNA were 12.5±2.2
(control) and 12.1±2.4 (antisense) amol/µg; and with Kv4.3
antisense, mRNA concentrations for Kv1.4 mRNA were 8.4±2.0 (control)
and 8.2±1.2 (antisense) amol/µg and for Kv4.2 mRNA were 6.3±1.1
(control) and 5.5±1.8 (antisense) amol/µg. We were unable to study
the effects of antisense on mRNA concentrations in human atrial cells
because of the very small number of cells available in the small
specimens obtained.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we obtained several lines of evidence suggesting that, whereas Kv1.4 subunits do not appear to play a role in human atrial Ito, they are importantly involved in rabbit cardiac Ito. This conclusion is suggested by the effect of oxidative stress, the lack of use-dependent unblocking of 4AP, and the effect of Kv1.4 antisense on rabbit Ito, with human Ito failing to respond to oxidative stress or Kv1.4 antisense and showing prominent 4AP unblocking. Furthermore, Kv1.4 protein was demonstrated in rabbit heart membranes and was clearly absent from the human heart.
Previous Studies on the Molecular Mechanisms of
Ito
A variety of cDNA clones encoding K+ channel
subunits, including Kv1.4,17 18 Kv3.3,19
Kv3.4,20 Kv4.1,21 Kv4.2,22 23 24 25
and Kv4.3,26 27 28 carry currents resembling
Ito on heterologous expression. Transcripts
for many of these clones are detectable in mammalian
hearts,43 but protein expression for
Ito subunits may not match that of
mRNA.30 44 Direct evidence for the subunit
composition of native Ito is limited.
Studies of protein expression suggest that members of the
Shal (Kv4) subfamily of K+ channel
subunits are likely involved in forming rat,30
dog,27 and human27
Ito. Recent studies using antisense
techniques showed that Ito in the rat is
reduced by exposure to Kv4.2 or Kv4.3 antisense.31
The expression of dominant negative Kv4.2 constructs strongly reduces
Ito expression in rats32
and mice.33
In the present studies, we found a variety of types of evidence for differences in the molecular composition between rabbit and human Ito. Rabbit Ito showed little use-dependent unblocking of 4AP, whereas human Ito showed prominent unblocking with repeated depolarizing pulses. A lack of 4AP unblocking was also found to typify currents carried by Kv1.4, whereas currents carried by Kv4.2 and 4.3 showed prominent unblocking. Exposure to H2O2 clearly slowed inactivation of rabbit Ito but had no appreciable effect on human Ito. Kv1.4 currents are known to be highly sensitive to changes in redox potential, whereas those carried by Kv4.2 and Kv4.3 are not.27 41 42 45 Western blots demonstrated the presence of Kv1.4 and Kv4.3 protein in rabbit atrial and ventricular membranes, but only Kv4.3 was found in human atrium and ventricle. Finally, antisense oligodeoxynucleotides directed against Kv1.4, Kv4.2, and Kv4.3 all reduced Ito currents in cultured rabbit myocytes, but only Kv4.3 antisense significantly altered human Ito.
Potential Significance
Ito is an important repolarizing
current in the heart. Abnormalities in Ito
have been detected in a variety of cardiac pathologies, including
ventricular failure,46 47 myocardial
infarction,48 impaired metabolic
function,49 and atrial fibrillation.50 51
Furthermore, differences in regional expression of
Ito appear to be important in the
generation of the T wave,52 in explaining the
electrophysiological response to myocardial
ischemia,53 in proarrhythmic reactions to
antiarrhythmic drugs,54 and in the electrocardiographic
response to physiological perturbations such as
hypothermia.55 It is therefore important to understand the
molecular basis of the Ito channel. The
present studies are the first of which we are aware to provide
direct evidence for a role of Kv1.4 in forming native
Ito channels, in terms of data obtained
with Western blot, pharmacological, and antisense approaches.
Preliminary results have been published that indicate, on the basis of
immunohistochemical and biophysical evidence, that Kv1.4 may encode a
portion of Ito in ferret left
ventricular endocardium.56 The
involvement of Kv1.4 subunits in forming rabbit
Ito channels may account for their
typically very slow reactivation kinetics.4
Significant differences have been observed in the reactivation kinetics of Ito among various ventricular regions of the human heart.57 58 Whereas subepicardial cells recover rapidly from inactivation and show little frequency dependence, much like human atrial cells, subendocardial cells show an important slow phase of recovery and strong frequency dependence, much like rabbit atrial myocytes. It is thus quite conceivable that Kv1.4 channel subunits play a significant role in contributing to transmural repolarization gradients in the human ventricle. As mentioned above, preliminary data suggest this to be the case in ferret ventricle. Differences in the pharmacological and physiological properties of Kv1.4 and Kv4.3 subunits may have important potential implications for understanding interspecies and intertissue differences in Ito regulation.
Potential Limitations
None of the antisense molecules studied produced complete
inhibition of Ito in either rabbit or human
atrium. This observation is compatible with findings in previous
studies of antisense oligodeoxynucleotide effect on
K+ channel function.34 38
Possible explanations include incomplete inhibition of target subunit
protein production by antisense and the existence of
as-yet-unidentified subunits contributing to macroscopic currents.
A second issue relates to the nature of the contribution of Kv1.4
subunits to overall current in the rabbit. If Kv1.4 subunits existed in
separate, homotetrameric channels from those involving Kv4 subunits,
one might have expected to see a change in channel kinetics with
antisense exposure and to see use-dependent unblocking of a portion of
current (that portion carried by Kv4 channels) in the presence of 4AP.
Neither of these phenomena was seen: antisense did not alter kinetics
of Ito in the rabbit, nor was a component
of 4AP unblocking seen. One possible explanation of this observation
would be the coassembly of Kv1.4 and Kv4 subunits as heterotetramers.
The experiments necessary to determine whether Kv1.4 and Kv4 subunits
can coassemble have not, to our knowledge, been performed; however, the
observation that a Kv4 dominant negative construct did not inhibit
coexpressed Kv1.4 channels32 makes coassembly
unlikely. The final limitation is that our study is limited to
-subunits that encode 4AP-sensitive transient outward channel
subunits. The potential role of other subunits, including ß peptides,
cannot be assessed from the present experiments.
|
Acknowledgments |
|---|
This work was supported by the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, the Fonds de la Recherche en Santé du Québec, NIH (grant NHLBI RO1-HL 34161), and the Fonds de la Recherche de l'Institut de Cardiologie de Montréal. Dr Wang is a research scholar of the Heart and Stroke Foundation of Canada. Dr Pond was supported by a postdoctoral fellowship from NIH. The authors thank Nathalie Talbot and XiaoFan Yang for their excellent technical assistance, Diane Campeau for secretarial help with the manuscript, and Dr David Van Wagoner for providing the human heart samples.
Received September 8, 1998; accepted January 3, 1999.
|
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 G.-L. Wang, G.-X. Wang, S. Yamamoto, L. Ye, H. Baxter, J. R Hume, and D. Duan Molecular mechanisms of regulation of fast-inactivating voltage-dependent transient outward K+ current in mouse heart by cell volume changes J. Physiol., October 15, 2005; 568(2): 423 - 443. [Abstract] [Full Text] [PDF]
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Z. Ren and C. M. Baumgarten Antagonistic regulation of swelling-activated Cl- current in rabbit ventricle by Src and EGFR protein tyrosine kinases Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2628 - H2636. [Abstract] [Full Text] [PDF]
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F. G. Akar, R. C. Wu, G. J. Juang, Y. Tian, M. Burysek, D. DiSilvestre, W. Xiong, A. A. Armoundas, and G. F. Tomaselli Molecular mechanisms underlying K+ current downregulation in canine tachycardia-induced heart failure Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2887 - H2896. [Abstract] [Full Text] [PDF]
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J. Rose, A. A. Armoundas, Y. Tian, D. DiSilvestre, M. Burysek, V. Halperin, B. O'Rourke, D. A. Kass, E. Marban, and G. F. Tomaselli Molecular correlates of altered expression of potassium currents in failing rabbit myocardium Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2077 - H2087. [Abstract] [Full Text] [PDF]
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G. M. Jang, L. E.-C. Leong, L. T. Hoang, P. H. Wang, G. A. Gutman, and B. L. Semler Structurally Distinct Elements Mediate Internal Ribosome Entry within the 5'-Noncoding Region of a Voltage-gated Potassium Channel mRNA J. Biol. Chem., November 12, 2004; 279(46): 47419 - 47430. [Abstract] [Full Text] [PDF]
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 P. Zhabyeyev, T. Asai, S. Missan, and T. F. McDonald Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1396 - C1403. [Abstract] [Full Text] [PDF]
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R. A. Bassani, J. Altamirano, J. L. Puglisi, and D. M. Bers Action potential duration determines sarcoplasmic reticulum Ca2+ reloading in mammalian ventricular myocytes J. Physiol., September 1, 2004; 559(2): 593 - 609. [Abstract] [Full Text] [PDF]
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S. G. Birnbaum, A. W. Varga, L.-L. Yuan, A. E. Anderson, J. D. Sweatt, and L. A. Schrader Structure and Function of Kv4-Family Transient Potassium Channels Physiol Rev, July 1, 2004; 84(3): 803 - 833. [Abstract] [Full Text] [PDF]
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F. G. Akar, R. C. Wu, I. Deschenes, A. A. Armoundas, V. Piacentino III, S. R. Houser, and G. F. Tomaselli Phenotypic differences in transient outward K+ current of human and canine ventricular myocytes: insights into molecular composition of ventricular Ito Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H602 - H609. [Abstract] [Full Text] [PDF]
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H. Li, W. Guo, K. A. Yamada, and J. M. Nerbonne Selective elimination of IK,slow1 in mouse ventricular myocytes expressing a dominant negative Kv1.5{alpha} subunit Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H319 - H328. [Abstract] [Full Text] [PDF]
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S. Zicha, I. Moss, B. Allen, A. Varro, J. Papp, R. Dumaine, C. Antzelevich, and S. Nattel Molecular basis of species-specific expression of repolarizing K+ currents in the heart Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1641 - H1649. [Abstract] [Full Text] [PDF]
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 R. F. Bosch, C. R. Scherer, N. Rub, S. Wohrl, K. Steinmeyer, H. Haase, A. E. Busch, L. Seipel, and V. Kuhlkamp Molecular mechanisms of early electrical remodeling: transcriptional downregulation of ion channel subunits reduces ICa,L and Ito in rapid atrial pacing in rabbits J. Am. Coll. Cardiol., March 5, 2003; 41(5): 858 - 869. [Abstract] [Full Text] [PDF]
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 N. lost, L. Virag, A. Varro, and J. Gy. Papp Comparison of the Effect of Class IA Antiarrhythmic Drugs on Transmembrane Potassium Currents in Rabbit Ventricular Myocytes Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2003; 8(1): 31 - 41. [Abstract] [PDF]
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I. Moreno, R. Caballero, T. Gonzalez, C. Arias, C. Valenzuela, I. Iriepa, E. Galvez, J. Tamargo, and E. Delpon Effects of Irbesartan on Cloned Potassium Channels Involved in Human Cardiac Repolarization J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 862 - 873. [Abstract] [Full Text] [PDF]
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D. Dong, Y. Duan, J. Guo, D. E Roach, S. L Swirp, L. Wang, J.P Lees-Miller, R.S Sheldon, J. D Molkentin, and H. J Duff Overexpression of calcineurin in mouse causes sudden cardiac death associated with decreased density of K+ channels Cardiovasc Res, February 1, 2003; 57(2): 320 - 332. [Abstract] [Full Text] [PDF]
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S. Wang, M. J Morales, Y.-J. Qu, G. C L Bett, H. C Strauss, and R. L Rasmusson Kv1.4 channel block by quinidine: evidence for a drug-induced allosteric effect J. Physiol., January 15, 2003; 546(2): 387 - 401. [Abstract] [Full Text] [PDF]
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W. Han, L. Zhang, G. Schram, and S. Nattel Properties of potassium currents in Purkinje cells of failing human hearts Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2495 - H2503. [Abstract] [Full Text] [PDF]
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W. Han, W. Bao, Z. Wang, and S. Nattel Comparison of Ion-Channel Subunit Expression in Canine Cardiac Purkinje Fibers and Ventricular Muscle Circ. Res., November 1, 2002; 91(9): 790 - 797. [Abstract] [Full Text] [PDF]
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R. Kaprielian, R. Sah, T. Nguyen, A. D. Wickenden, and P. H. Backx Myocardial infarction in rat eliminates regional heterogeneity of AP profiles, Ito K+ currents, and [Ca2+]i transients Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1157 - H1168. [Abstract] [Full Text] [PDF]
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 B. J. Padanilam, T. Lu, T. Hoshi, B. A. Padanilam, E. F. Shibata, and H.-C. Lee Molecular Determinants of Intracellular pH Modulation of Human Kv1.4 N-Type Inactivation Mol. Pharmacol., July 1, 2002; 62(1): 127 - 134. [Abstract] [Full Text] [PDF]
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Y. Xu, P. H. Dong, Z. Zhang, G. U. Ahmmed, and N. Chiamvimonvat Presence of a calcium-activated chloride current in mouse ventricular myocytes Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H302 - H314. [Abstract] [Full Text] [PDF]
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G. Schram, M. Pourrier, P. Melnyk, and S. Nattel Differential Distribution of Cardiac Ion Channel Expression as a Basis for Regional Specialization in Electrical Function Circ. Res., May 17, 2002; 90(9): 939 - 950. [Abstract] [Full Text] [PDF]
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R. F Bosch and S. Nattel Cellular electrophysiology of atrial fibrillation Cardiovasc Res, May 1, 2002; 54(2): 259 - 269. [Full Text] [PDF]
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Z. Kassiri, C. Zobel, T.-T. T. Nguyen, J. D. Molkentin, and P. H. Backx Reduction of Ito Causes Hypertrophy in Neonatal Rat Ventricular Myocytes Circ. Res., March 22, 2002; 90(5): 578 - 585. [Abstract] [Full Text] [PDF]
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W. Guo, H. Li, F. Aimond, D. C. Johns, K. J. Rhodes, J. S. Trimmer, and J. M. Nerbonne Role of Heteromultimers in the Generation of Myocardial Transient Outward K+ Currents Circ. Res., March 22, 2002; 90(5): 586 - 593. [Abstract] [Full Text] [PDF]
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J. M. Nerbonne, C. G. Nichols, T. L. Schwarz, and D. Escande Genetic Manipulation of Cardiac K+ Channel Function in Mice: What Have We Learned, and Where Do We Go From Here? Circ. Res., November 23, 2001; 89(11): 944 - 956. [Abstract] [Full Text] [PDF]
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H. Li, W. Guo, H. Xu, R. Hood, A. T. Benedict, and J. M. Nerbonne Functional expression of a GFP-tagged Kv1.5 alpha -subunit in mouse ventricle Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H1955 - H1967. [Abstract] [Full Text] [PDF]
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N. Decher, O. Uyguner, C. R Scherer, B. Karaman, M. Yuksel-Apak, A. E Busch, K. Steinmeyer, and B. Wollnik hKChIP2 is a functional modifier of hKv4.3 potassium channels: Cloning and expression of a short hKChIP2 splice variant Cardiovasc Res, November 1, 2001; 52(2): 255 - 264. [Abstract] [Full Text] [PDF]
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H. Wang, H. Han, L. Zhang, H. Shi, G. Schram, S. Nattel, and Z. Wang Expression of Multiple Subtypes of Muscarinic Receptors and Cellular Distribution in the Human Heart Mol. Pharmacol., April 16, 2001; 59(5): 1029 - 1036. [Abstract] [Full Text]
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T. González, M. Longobardo, R. Caballero, E. Delpón, J. Tamargo, and C. Valenzuela Effects of Bupivacaine and a Novel Local Anesthetic, IQB-9302, on Human Cardiac K+ Channels J. Pharmacol. Exp. Ther., April 13, 2001; 296(2): 573 - 583. [Abstract] [Full Text]
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R. Caballero, E. Delpón, C. Valenzuela, M. Longobardo, T. González, and J. Tamargo Direct Effects of Candesartan and Eprosartan on Human Cloned Potassium Channels Involved in Cardiac Repolarization Mol. Pharmacol., April 1, 2001; 59(4): 825 - 836. [Abstract] [Full Text]
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M. A. Allessie, P. A. Boyden, A. J. Camm, A. G. Kleber, M. J. Lab, M. J. Legato, M. R. Rosen, P. J. Schwartz, P. M. Spooner, D. R. Van Wagoner, et al. Pathophysiology and Prevention of Atrial Fibrillation Circulation, February 6, 2001; 103(5): 769 - 777. [Full Text] [PDF]
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L. Yue, Z. Wang, H. Rindt, and S. Nattel Molecular evidence for a role of Shaw (Kv3) potassium channel subunits in potassium currents of dog atrium J. Physiol., September 15, 2000; 527(3): 467 - 478. [Abstract] [Full Text] [PDF]
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H. Wang, H. Shi, L. Zhang, M. Pourrier, B. Yang, S. Nattel, and Z. Wang Nicotine Is a Potent Blocker of the Cardiac A-Type K+ Channels : Effects on Cloned Kv4.3 Channels and Native Transient Outward Current Circulation, September 5, 2000; 102(10): 1165 - 1171. [Abstract] [Full Text] [PDF]
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 A. Vincent, N. J. Lautermilch, and N. C. Spitzer Antisense Suppression of Potassium Channel Expression Demonstrates Its Role in Maturation of the Action Potential J. Neurosci., August 15, 2000; 20(16): 6087 - 6094. [Abstract] [Full Text] [PDF]
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T W Claydon, M R Boyett, A Sivaprasadarao, K Ishii, J M Owen, H A O'Beirne, R Leach, K Komukai, and C H Orchard Inhibition of the K+ channel Kv1.4 by acidosis: protonation of an extracellular histidine slows the recovery from N-type inactivation J. Physiol., July 15, 2000; 526(2): 253 - 264. [Abstract] [Full Text] [PDF]
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J. M Nerbonne Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium J. Physiol., June 1, 2000; 525(2): 285 - 298. [Abstract] [Full Text] [PDF]
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W. Guo, H. Xu, B. London, and J. M Nerbonne Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes J. Physiol., December 15, 1999; 521(3): 587 - 599. [Abstract] [Full Text] [PDF]
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H. Xu, D. M. Barry, H. Li, S. Brunet, W. Guo, and J. M. Nerbonne Attenuation of the Slow Component of Delayed Rectification, Action Potential Prolongation, and Triggered Activity in Mice Expressing a Dominant-Negative Kv2 {alpha} Subunit Circ. Res., October 1, 1999; 85(7): 623 - 633. [Abstract] [Full Text] [PDF]
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T. Kiyosue and M. Arita Altered expression of cardiac K+ channel genes during sub-acute and healing phases of myocardial infarction Cardiovasc Res, October 1, 1999; 44(1): 13 - 16. [Full Text] [PDF]
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D. McKinnon Molecular Identity of Ito : Kv1.4 Redux Circ. Res., March 19, 1999; 84(5): 620 - 622. [Full Text] [PDF]
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M. Song, G. Helguera, M. Eghbali, N. Zhu, M. M. Zarei, R. Olcese, L. Toro, and E. Stefani Remodeling of Kv4.3 Potassium Channel Gene Expression under the Control of Sex Hormones J. Biol. Chem., August 17, 2001; 276(34): 31883 - 31890. [Abstract] [Full Text] [PDF]
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H. Wang, B. Yang, Y. Zhang, H. Han, J. Wang, H. Shi, and Z. Wang Different Subtypes of alpha 1-Adrenoceptor Modulate Different K+ Currents via Different Signaling Pathways in Canine Ventricular Myocytes J. Biol. Chem., October 26, 2001; 276(44): 40811 - 40816. [Abstract] [Full Text] [PDF]
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Z. Kassiri, C. Zobel, T.-T. T. Nguyen, J. D. Molkentin, and P. H. Backx Reduction of Ito Causes Hypertrophy in Neonatal Rat Ventricular Myocytes Circ. Res., March 22, 2002; 90(5): 578 - 585. [Abstract] [Full Text] [PDF]
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W. Guo, H. Li, F. Aimond, D. C. Johns, K. J. Rhodes, J. S. Trimmer, and J. M. Nerbonne Role of Heteromultimers in the Generation of Myocardial Transient Outward K+ Currents Circ. Res., March 22, 2002; 90(5): 586 - 593. [Abstract] [Full Text] [PDF]
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