From the Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan
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ABSTRACT |
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We cloned a cDNA (HAC4) that encodes the
hyperpolarization-activated cation channel (If
or Ih) by screening a rabbit sinoatrial (SA)
node cDNA library using a fragment of rat brain
If cDNA. HAC4 is composed of 1150 amino
acid residues, and its cytoplasmic N- and C-terminal regions are longer
than those of HAC1-3. The transmembrane region of HAC4 was most
homologous to partially cloned mouse If BCNG-3
(96%), whereas the C-terminal region of HAC4 showed low homology to
all HAC family members so far cloned. Northern blotting revealed that
HAC4 mRNA was the most highly expressed in the SA node among the
rabbit cardiac tissues examined. The electrophysiological properties of
HAC4 were examined using the whole cell patch-clamp technique. In COS-7
cells transfected with HAC4 cDNA, hyperpolarizing voltage steps
activated slowly developing inward currents. The half-maximal
activation was obtained at The hyperpolarization-activated cation channel
(If)1
was first described in rabbit heart sinoatrial (SA) node (1, 2). The
If current was characterized by activation by
hyperpolarizing voltage steps; mixed permeability for Na+
and K+; inhibition by extracellular Cs+, not by
Ba2+; and a positive shift in the
voltage-dependent activation curve by intracellular cyclic
nucleotide (1-6).
Based on the above physiological properties, the functional roles of
If in the SA node have been discussed in many
publications. If is one of the inward currents
that generate pacemaker depolarization (7-9). The pacemaker cells of
the SA node are coupled to surrounding atrial myocytes through gap
junctions. Since atrial myocytes have more negative resting membrane
potentials, they hyperpolarize pacemaker cells electrotonically (10).
Pacemaker cell If is activated under this
condition; therefore; the inward If current is
likely to limit the level of hyperpolarization of pacemaker cells
(1).
Recently, three full-length (mouse BCNG-1, -2, and -4; corresponding to
HAC2, -1, and -3, respectively) and one partial (mouse BCNG-3)
mammalian cDNA clones encoding If were
isolated from a mouse brain cDNA library (11-14), and one cDNA
clone was isolated from sea urchin testis (SPIH) (15). However, despite
the physiological significance of If in the SA
node, its molecular characteristics still remain unclear. Therefore, we
have screened a rabbit heart SA node cDNA library and isolated a
cDNA (HAC4). HAC4 is composed of 1150 amino acid residues and most
likely encodes If. In this study, we demonstrate
the amino acid sequence of HAC4, the distribution of HAC4 mRNA in
cardiac tissue, and the electrophysiological properties of HAC4
heterologously expressed in COS-7 cells.
Molecular Biology--
The cloning procedure was performed as
described previously (16). Briefly, cDNA templated by mRNA
isolated from rat brain was used as a DNA template for polymerase chain
reaction amplification. The 5' (P1) and 3' (P2) sequences were derived
from mouse HAC2 (13) and are as follows (N represents A/G/C/T): P1,
5'-ATG(C/T)TNTG(T/C)AT(T/C/A)GGNTA(T/C)GG-3'; and P2,
5'-AT(A/G)TA(A/G)TCNCCNGG(T/C)TG(A/G)AA-3'. Polymerase chain reaction
amplification was performed according to the following schedule: five
cycles at 94 °C for 1 min, 46 °C for 1 min, and 72 °C for 2 min, followed by 26 cycles at 94 °C for 1 min, 55 °C for 1 min,
and 72 °C for 2 min. The polymerase chain reaction products were
electrophoresed on a 1% agarose gel, excised, and purified with
QIAEX-II (QIAGEN Inc.) for subsequent subcloning and sequence
determination. Through this procedure, we identified a clone for a rat
homologue of mouse HAC2, pR1. A cDNA library was prepared from
rabbit heart SA node regions. Using pR1 as a probe, 5 × 104 phage clones of the cDNA library were hybridized
for the isolation of a new clone (washed with 2× SSC at 55 °C).
Five positive clones were isolated and sequenced. All of them were
identical. A representative clone, pIH1, was subjected to sequence
determination. Both strands of the cDNA sequence were determined by
the chain termination method (BigDye Terminator Cycle Sequencing,
Applied Biosystems, Inc.). The clone contains 3396 base pairs of
cDNA that comprises a large open reading frame. However, the
cDNA does not possess a termination codon in the C-terminal region.
Therefore, we performed 3'-rapid amplification of cDNA ends
(Marathon cDNA amplification kit, CLONTECH) and
obtained the C-terminal region of the clone by using proofreading
Taq polymerase (LA Taq, TaKaRa). We added the C-terminal
region amplified by the proofreading Taq polymerase to pIH1
by using a SacI restriction site. Northern blots, prepared with 2 µg of poly(A)+ mRNA isolated from the
indicated rabbit tissues (atrium does not contain the SA node region),
were probed in hybridization solution (Life Technologies, Inc.) and
50% formamide at 42 °C with a radiolabeled DNA fragment derived
from the coding region of pIH1 (corresponding to amino acids
707-1116), washed with 0.1× SSC and 0.1% SDS at 65 °C, and
exposed to x-ray film at Functional Expression and Electrophysiological
Measurements--
HAC4 cDNA and green fluorescent protein S65A
cDNA (a gift from Dr. K. Moriyoshi) were subcloned into independent
PCI vectors (Promega), and the mixture of vectors were transfected into
COS-7 cells using LipofectAMINE (Life Technologies, Inc.) following the
manufacturer's instructions. The vector amounts were 1.6 µg/35-mm dish for HAC4 and 0.4 µg/35-mm dish for green fluorescent protein. COS-7 cells (Riken) were cultured on coverslips in Dulbecco's modified
essential medium (Life Technologies, Inc.) supplemented with 10% fetal
calf serum (Life Technologies, Inc.) and antibiotics.
36-48 h after transfection, a coverslip was transferred to the
recording chamber on an inverted microscope (TMD300, Nikon), and
patch-clamp experiments were carried out in green fluorescent protein-positive cells using Axopatch 200B amplifier (Axon Instruments, Inc.). The data were directly recorded on the hard disk of an IBM-PC
compatible computer thorough an AD converter (Digipack 1200, Axon
Instruments, Inc.) and were analyzed using commercially available
software (pClamp6 and Clampex7, Axon Instruments, Inc.). The data
points represent the means ± S.E. The statistical difference was
evaluated using Student's unpaired t test.
The composition of the bathing solution was 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,
and 5 mM HEPES. The pH was adjusted to 7.4 with NaOH.
Appropriate amounts of KCl, CsCl, and BaCl2 were added in
some experiments. The pipette solution contained 130 mM
KCl, 5 mM HEPES, 5 mM EGTA, 5 mM
MgATP, and 5 mM disodium creatine phosphate. The pH was
adjusted to 7.4 with KOH. The final K+ concentration was
~145 mM. In some experiments, 0.3 mM cAMP was added to the pipette solution, and the pH was readjusted to 7.4 with
KOH. All patch-clamp experiments were carried out at 35 °C by
perfusing the bathing solution through a water jacket.
cDNA Cloning of HAC4--
The transmembrane region of rat HAC2
was amplified by the polymerase chain reaction method with a rat brain
cDNA mixture and was used to screen a rabbit SA node cDNA
library. The most 5' methionine codon in the positively hybridizing
clone (pIH1) initiated an open reading frame that did not contain a
termination codon in the 3'-terminal region. 3'-Rapid amplification of
cDNA ends was performed to obtain the 3'-terminal region, and pIH1
lacked 103 base pairs of the 3'-coding region. The 3'-terminal region was amplified by proofreading Taq polymerase and attached to
pIH1. The sequence (HAC4) (Fig. 1)
predicts a protein of 1150 amino acid residues with six transmembrane
domains, a pore region, and a cyclic nucleotide-binding domain. HAC4
shows 85-90 and 80-96% identities to HAC1-3 in the transmembrane
region and the cyclic nucleotide-binding domain, respectively. HAC4 is
most related to mouse BCNG-3. Although full-length mouse BCNG-3
cDNA has not been cloned, the partial sequence of mouse BCNG-3 was
96% homologous to the transmembrane region of HAC4. The predicted N-
and C-terminal regions of HAC4 are notably longer than those of the
rest of the HAC clones. The last five amino acid residues
(corresponding to positions 1146-1150) at the C terminus were
conserved in all HAC clones. The amino acid sequence corresponding to
positions 1045-1055 was also conserved in all HAC clones. The rest of
the C-terminal regions were remarkably diverse.
Expression Patterns of HAC4 mRNA--
To examine the
expression of HAC4 mRNA, we performed Northern blotting (Fig.
2). Since the C-terminal regions are
diverse among HAC family members, we synthesized a radiolabeled DNA
fragment derived from the coding region corresponding to amino acids
707-1116. It is clear from Fig. 2 that HAC4 was the most highly
expressed in the SA node among the cardiac tissues examined. Unlike
HAC1-3, HAC4 was not significantly expressed in brain. The size of the mRNA for HAC4 was estimated to be 7.1 kilobases, although
presumably a partially processed transcript was detected.
Voltage-dependent Gating of HAC4--
Fig.
3A shows representative
current traces of HAC4 expressed in COS-7 cells. When a voltage step
was more negative than
The voltage dependence of current activation was further analyzed by
measurements of outward tail currents (Fig. 3C). Under control conditions, hyperpolarizing voltage steps to Ion Pore Properties of HAC4--
The If
current of the SA node and other HAC clones is inhibited by
extracellular Cs+ and divalent cations (3, 7, 12, 13, 15,
17, 18). We examined the effects of Cs+ and
Ba2+ on HAC4 currents as shown in Fig.
4A. The magnitude of the block was estimated by measuring the amplitude of time-dependent
currents in the presence and absence of blockers. At
It is well known that both Na+ and K+ permeate
If in native pacemaker cells (3, 5, 6). In Fig.
4B, we examined the ion selectivity of HAC4 by measuring the
reversal potential, which was determined by intersections between the
I-V curve of initial tail currents (closed
symbols in the I-V diagrams) and the I-V curve of late tail currents (data not plotted). Reversal potentials were
Besides Na+ and K+, Li+ also
permeates If (17). In Fig. 4C, we
compared the permeability of K+, Na+, and
Li+ through HAC4. When 140 mM
Na+]o was replaced with 140 mM
Li+]o in the presence of 5 mM
K+]o, the amplitude of the outward tail current
dramatically increased. Under these conditions, the reversal potential
shifted to In this study, we have cloned a cDNA (HAC4) and demonstrated
that HAC4 encodes If. HAC4 is composed of 1150 amino acid residues. Both the cytoplasmic N- and C-terminal regions of
HAC4 are longer than those of HAC1-3. We could not exclude the
possibility that the N terminus of HAC4 was not complete because we
have not obtained a clone that has an in-frame stop codon in front of
the assigned start codon. However, the N-terminal region contained
enough length and showed weak but significant homology compared with
other HAC clones. Moreover, the cDNA of HAC4 generated robust
functional If currents when expressed in COS-7
cells. These findings might support the idea that the cDNA of HAC4
contains the entire coding sequence. The C-terminal region of HAC4
showed remarkably low homology compared with other HAC clones. The
amino acid sequence of the transmembrane region of HAC4 was 89%
homologous to HAC1 (mouse BCNG-2), 86% to HAC2 (mouse BCNG-1), 85% to
HAC3 (presumably a splicing variant of mouse BCNG-4), and 97% to mouse
BCNG-3 (14). The partial sequence of the N-terminal region (82 amino
acid residues) of mouse BCNG-3 was also homologous to HAC4 (89%).
Therefore, HAC4 may be the rabbit homologue of mouse BCNG-3. It is
difficult, however, to conclude this matter because only a partial
sequence of mouse BCNG-3 has been determined (506 amino acids), and in particular, the amino acid sequence of the C-terminal region of mouse
BCNG-3 has not been determined.
Although HAC4 and mouse BCNG-3 are highly homologous, their expression
patterns seem different; in mouse, BCNG-3 is expressed in cardiac
tissue, but not in the SA node. BCNG-3 is also expressed in mouse brain
and skeletal muscle (12). In contrast, HAC4 mRNA is highly
expressed in rabbit heart SA node. In other parts of cardiac tissues,
brain, and skeletal muscle, HAC4 signals are not significant. We do not
have ready explanations for this difference. In Northern blotting, we
used the sequence that corresponds to amino acids 707-1116. Because
the amino acid sequence of the corresponding region of mouse BCNG-3 had
not been determined, we could not exclude the possibility that the
C-terminal region was not homologous between HAC4 and mouse BCNG-3.
Another possibility may be that the different expression pattern is
simply due to the difference of species.
In accordance with the distribution of HAC4 mRNA, the
electrophysiological properties of HAC4 closely resembled those of the If current reported in native pacemaker cells
isolated from rabbit SA node. In native pacemaker cells, particularly
during The functional role of If in the SA node is to
generate pacemaker depolarization (7, 8, 9) and to limit the
hyperpolarization of pacemaker cells caused by electronic coupling with
atrial myocytes (1, 10). The significance of If
in the generation of pacemaker potential still appears to be a matter
of debate (17, 20-23). Targeting of the HAC gene should provide a clue
to address this question. To conduct such an experiment, it is
essential to determine the molecular characteristics of
If in the SA node. In this study, we have
demonstrated that HAC4 forms If in the SA node.
This molecular identification of HAC4 in the SA node would facilitate
the understanding of the physiological function of
If.
87.2 ± 2.8 mV under control
conditions and at
64.4 ± 2.6 mV in the presence of
intracellular 0.3 mM cAMP. The reversal potential was
34.2 ± 0.9 mV in 140 mM Na+o
and 5 mM K+o versus 10 mM Na+i and 145 mM
K+i. These results indicate that HAC4 forms
If in rabbit heart SA node.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C with an intensifying screen for
84 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence of HAC4 and comparison
with other HAC channels. HAC1-3 were cloned from mouse, whereas
HAC4 was from rabbit. Alignments were generated by eye;
dashes represent gaps introduced to optimize the alignment.
The six predicted transmembrane domains (S1-S6), the pore
region (P), and the cyclic nucleotide-binding domain
(CNBD) are underlined. Amino acid residues that
are conserved between HAC4 and the rest of the HAC family are
boxed. The asterisks indicate stop codons. Amino
acid numbers for the full-length coding sequences are given on the
right.
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Fig. 2.
Northern blot analyses of HAC4 mRNA
distribution. Poly(A)+ mRNA (2 µg), isolated
from the indicated tissue sources, was loaded in each lane. Sizes
(kilobases) are indicated to the left. HAC4 mRNA (top
arrow) was mainly expressed in the SA node, but not clearly in the
atrium, ventricle, forebrain, or cerebellum. Glyceraldehyde-3-phosphate
dehydrogenase (bottom arrow) was used as a control.
60 mV, slowly activating inward currents were
activated. Such a current was not observed when COS-7 cells were
transfected with an empty vector (data not shown). The current
activation appeared to be the sum of two exponential time courses. For
example, time constants of current activation were 384 ± 71 and
2275 ± 319 ms at
110 mV (n = 6). These values
were similar to those reported for the If
current of rabbit SA node cells (17). Fig. 3B shows the
current-voltage (I-V) relationship. The closed
circles indicate the amplitude of the initial current measured at
the beginning of hyperpolarizing pulses. The open circles
are the steady-state I-V relationship measured at the end of
pulses. It is clear from Fig. 3B that the threshold of
current activation was between
60 and
70 mV.
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Fig. 3.
Voltage-dependent gating of
HAC4. A, a representative current recording of the HAC4
current. The pulse protocol is shown in the top. The dotted
line indicates zero current level. The initial current and the
steady-state current were measured at the times indicated ( and
,
respectively). B, I-V relationships measured at
and
. The data points were averaged from 6 to ~10 cells.
C, expanded traces of the outward tail current. Two sets of
current traces were recorded in different cells. The voltage of
conditioning pulse is indicated at the corresponding tail current. The
amplitude of the tail current (Itail) was the
difference between the peak current measured at the time indicated by
the marks and the steady-state current at the end of the 0-mV voltage
step. D, voltage-dependent activation curves
measured in the presence (
; n = 7) and absence (
;
n = 10) of 0.3 mM cAMP.
Abscissa, voltage of conditioning hyperpolarization;
ordinate, Itail normalized by the
maximal value in each experiment (Imax).
Vh and S (see "Results") were
measured in each experiment, and the fitted lines were drawn using
their average values. Vh was
87.4 ± 2.8 mV
(control) and
64.4 ± 2.6 mV (0.3 mM cAMP;
p < 0.01). S was 9.6 ± 0.8 mV
(control) and 7.9 ± 0.4 mV (0.3 mM cAMP;
p > 0.05). In all experiments, the extracellular
potassium concentration was 5 mM.
40 and
50 mV
did not elicit the outward tail current. When the voltage step was
60
mV, a miniature tail current was recorded. The amplitude of the tail
current increased as the conditioning voltage became more negative and
saturated at
140 mV. It is well known that intracellular cAMP
produces a positive shift in the voltage dependence of the activation
of the If and HAC1 currents in a
dose-dependent manner. The maximal effect was obtained at
~0.1 mM cAMP (4, 13). In accordance with these reports,
an obvious tail current was elicited by hyperpolarization to
50 mV in
the presence of intracellular 0.3 mM cAMP (Fig.
3C, right panel). In Fig. 3 (C and
D), the outward tail current (Itail)
measured at 0 mV was normalized by the maximal value
(Imax) in the presence (
) and absence (
)
of 0.3 mM cAMP. In each experiment, we fitted the Boltzmann
function to the data points:
Itail/Imax = 1/(1 + exp((Vm
Vh)/S)),
where Vm is the test potential,
Vh is the membrane potential for half-maximal
activation, and S is the slope factor. Under control
conditions, Vh was
87.4 ± 2.8 mV, and
S was 9.6 ± 0.8 mV (n = 10). In the
presence of 0.3 mM cAMP, the value of Vh
shifted toward the positive direction (
64.4 ± 2.6 mV
(n = 7); p < 0.01), whereas
S did not change significantly (7.9 ± 0.4 mV
(n = 7); p > 0.05). The shift in
Vh was 23 mV, which was clearly larger than those in
other mammalian clones such as mouse BCNG-1 (corresponding to HAC2; 2 mV) (12) or HAC1 (12 mV) (13).
90 mV, 3 mM Cs+ partially blocked the HAC4 current. The
magnitude of the block was 70.8 ± 5.4%, which was smaller than
reported for mouse BCNG-1 and HAC1. The outward tail current was also
suppressed by extracellular Cs+. 3 mM
Ba2+ blocked only 9.6 ± 2.0% of the HAC4
current.
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Fig. 4.
Ion pore properties of HAC4.
A, superimposed current traces recorded in the presence and
absence of 3 mM Cs+ (left panel,
top trace) and 3 mM Ba2+
(bottom trace). The holding potential was 40 mV. The HAC4
current was activated by the voltage step to
90 mV. The magnitudes of
the block are summarized in the right panel
(n = 5 and 6). B, effect of extracellular
K+ on the HAC4 current. The current traces were recorded in
the same cell. The external Na+ concentration was 140 mM in all experiments. The pulse protocol is shown in the
bottom. The initial I-V relationship was measured at the
time indicated by the arrow. Note that the I-V
curves of outward currents overlap each other. C,
replacement of extracellular charge carriers. In 140 mM
Na+ and 140 mM Li+ solutions, the
K+ concentration was 5 mM. Three sets of
current traces were recorded in the same cell. It should be noted that
the outward tail currents in 140 mM Li+
solution are clearly larger than those in 140 mM
Na+ solution. The pulse protocol was same as described for
B. The dotted lines indicate the zero current
level.
34.2 ± 0.9 mV (n = 8) in 5 mM
K+o and
25.5 ± 0.9 mV (n = 7) in 20 mM K+o. The permeability ratio
for Na+ and K+
(PNa/PK) was calculated
from the following equation: Erev = (RT/F)·ln(([K+]o + PNa/PK[Na+]o)/([K+]i + PNa/PK[Na+]i)),
where PNa is the permeability for
Na+; PK is the permeability for
K+; Erev is the reversal potential;
and R, T, and F are the usual thermodynamic parameters. The
PNa/PK ratios were
0.25 ± 0.01 in 5 mM K+o and
0.26 ± 0.01 in 20 mM K+o. It is
also clear from Fig. 4B that extracellular K+
increased the inward Na+ conductance in a
concentration-dependent manner. In the absence of
extracellular K+, no obvious time-dependent
current was activated during the hyperpolarization. These properties
are in good agreement with those of the If
current in native pacemaker cells (5).
63.7 ± 1.8 mV (n = 3). When we
estimated PLi/PK by
ignoring intracellular Na+, the
PLi/PK ration was 0.06. The time course of the decay of the outward tail current became slower
in 140 mM Li+o solution. In 140 mM K+o, the reversal potential was
2.6 ± 0.1 mV (n = 3). These properties were
similar to those reported for If in native pacemaker cells (17).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic stimulation, the separation of
If and delayed rectifier K+
(IdK) currents was difficult because the
activation of If was overlapped by the
deactivation of IdK (1, 19). In this study, there was no time-dependent current in COS-7 cells other
than the heterologously expressed HAC4 current, which enabled us to measure voltage-dependent gating of
If more precisely. The threshold of current
activation was between
60 and
70 mV under control conditions and
between
40 and
50 mV in the presence of intracellular 0.3 mM cAMP. The Vh of HAC4 (
87.4 mV) was
more positive than that of HAC1 (
103 mV) or mouse BCNG-1 (HAC2;
100
mV). In HAC4, the positive shift in Vh was 23 mV in
the presence of 0.3 mM cAMP, which was clearly larger than
that in HAC1 (12 mV by 0.1 mM cAMP) or mouse BCNG-1 (2 mV
by 3 mM cAMP). In native pacemaker cells, the activation
curve of If was considerably different from cell
to cell (3, 8). This variety may be explained in two ways.
Intracellular cAMP concentration may be different from cell to cell;
alternatively, If in the SA node may be a
heteromultimer composed of different HAC subunits, and their
composition may be different from cell to cell, although we do not have
information on the expression level of other HAC clones in rabbit SA
node. In the latter case, it appears reasonable to expect that the
heteromultimer may possess a property intermediate between those of
HAC4 and other HAC family members.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. K. Ono for critically reading our manuscript. We thank M. Fukao and K. Tuji for technical support.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to M. T., A. N., and H. O.) and by a Japan Heart Foundation research grant (to T. M. I).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB022927.
These two authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 81-75-753-4351; Fax: 81-75-753-4349; E-mail: ohmori{at}med.kyoto-u.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: If, hyperpolarization-activated cation channel(s); SA, sinoatrial.
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