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Identification of Gating Modes in Single Native Naⴙ
Channels From Human Atrium and Ventricle
Thomas Böhle, Mathias C. Brandt, Michael Lindner, Dirk J. Beuckelmann
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Abstract—The aim of the present study was to investigate the single-channel properties of different gating modes in the
native human cardiac Na⫹ channel. Patch-clamp experiments were performed at low noise using ultrathick-walled
pipettes. In 17 cell-attached patches containing only one channel, fast back and forth switching between five different
Na⫹-channel gating modes (F-mode, M1-mode, M2-mode, S-mode, and P-mode) was identified, but no difference in the
gating properties was found between normal and diseased cardiomyocytes from atrium or ventricle, respectively.
Hodgkin-Huxley fits to the ensemble-averaged currents yielded the activation-time (␶m) and inactivation-time (␶h)
constants. ␶m was comparably fast in the F-mode, M1-mode, M2-mode, and S-mode (0.15 ms) and slow in the P-mode
(0.3 ms). ␶h ranged from 0.35 ms (F-mode) to 4.5 ms (S-mode and P-mode). The mean open-channel lifetime (␶o) was
shortest in the F-mode and P-mode (0.15 ms) and longest in the S-mode (1.25 ms). The time before which half of the
first channel openings occurred (t0.5) was comparably short in the F-mode, M1-mode, M2-mode, and S-mode (0.3 ms)
and long in the P-mode (0.9 ms). It is concluded that (1) a single native human cardiac Na⫹ channel can be recorded
at low noise, (2) this channel can change its gating properties at a time scale of milliseconds, (3) lifetimes of the observed
gating modes are short ranging from milliseconds to seconds only, and (4) the gating modes are characterized by specific
activation and inactivation kinetics and differ at least in their mean open time and first latency. (Circ Res. 2002;91:421426.)
Key Words: patch clamp 䡲 human myocardial sodium channels 䡲 modal gating 䡲 channel activation
䡲 channel inactivation
lthough Na⫹ channels have been subject to intense
electrophysiological investigation in various tissues,1
little is known about the native human cardiac Na⫹ channel.
Previously published studies have been conducted in heterologous expression systems, where channel proteins are
removed from their physiological environment.2– 4 In these
experiments, the Na⫹ channel ␣-subunit (hH1) was expressed
alone, or coexpression with the ␤1-subunit was performed.
Other methods used the reconstituted Na⫹-channel protein
fused into planar lipid bilayers.5 A major disadvantage of
these methods is the artificial environment, in which electrophysiological experiments on the channel were performed. In
the present study, results on single native human cardiac Na⫹
channels are presented. Our approach may lead to important
insights into the physiological function of this channel
protein.
Using this approach, arrhythmias may be understood in
more detail. It has been shown that the LQT3 syndrome and
the Brugada syndrome are based on different mutations in the
gene (SCN5A, 3p21) encoding for hH1.6 – 8 In heterologous
expression systems, a distinct mutation (⌬KPQ) underlying
the LQT3 syndrome is associated with late Na⫹ currents.9
From fits of the results of these experiments to different
A
kinetic models, it was postulated that modal gating of the
channel protein may be the reason for the late currents.9,10
Hence, genetic defects may underlie different rates of switching between gating modes. Therefore, the analysis of modal
gating in native human cardiac Na⫹ channels may also help to
better understand pathophysiological processes.
For the characterization of different gating modes, it is
necessary to record at low noise from a patch, which contains
no more than one channel. By using thick-walled patch
pipettes11 with ultraclean tips,12 five distinct gating modes of
Na⫹-channel action (F-mode, M1-mode, M2-mode, S-mode,
and P-mode) have been described in ventricular cells from
adult white mice.13–15 They differ at least in activation and/or
inactivation kinetics, open-channel lifetime, and first latency.
In the present report, we identified these gating modes in
atrial and ventricular cells of normal and diseased human
myocardium. Durations of these modes were observed to be
short.
Materials and Methods
Cell Isolation
Right atrial appendage specimens were obtained from patients
suffering from coronary heart disease or aortic-valve disease under-
Original received March 11, 2002; revision received July 15, 2002; accepted August 2, 2002.
From the University of Cologne, Department of Medicine III, Cologne, Germany.
Correspondence to Dr Thomas Böhle, LFI 4.103, Department of Medicine III, University of Cologne, Joseph-Stelzmann-Str. 9, D-50924 Cologne,
Germany. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000033521.38733.EF
421
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Circulation Research
September 6, 2002
going heart surgery. The procedure for isolation of cardiomyocytes
from human atrial appendages has been described in detail by Brandt
et al.16 Single cells of nonfailing (not suitable for transplantation)
and terminally failing (ischemic/dilated cardiomyopathy) ventricles
were isolated according to the procedure described by Beuckelmann
et al.17 The experimental protocol was designed to conform with the
recommendations from the Declaration of Helsinki and approved by
the ethical committee of the University of Cologne.
Solutions and Temperature
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The cardioplegic solution used for transport of the tissue was
composed of (in mmol/L) NaCl 15, KCl 9, MgCl2 4, histidine
hydrochloride 18, histidine 180, tryptophan 2, mannitol 30, CaCl2
0.015, glutaric acid 1, and KOH 2. The modified Tyrode’s solution
for cell isolation and storage contained (in mmol/L) NaCl 135, KCl
4, MgCl2 1, CaCl2 0.1, NaH2PO4 0.33, HEPES 10, and glucose 10
(pH 7.3). The experiments were performed at 23⫾1°C in a bath
solution that contained (in mmol/L) KCl 230, CsCl 20, MgCl2 1,
EGTA 10, and HEPES 5 (pH 7.3) and a pipette solution that
contained (in mmol/L) NaCl 255, CaCl2 2.5, KCl 4, and HEPES 5
(pH 7.3). The elevated Na⫹ concentration was used to enhance
unitary current amplitudes.18
Patch Pipettes
For low-noise recordings,11 patch pipettes were prepared from
thick-walled borosilicate glass tubing (Hilgenberg) with an external
diameter of 2.0 mm and an internal diameter of 0.25 mm. The patch
pipettes were inserted into a pipette holder of small dimension made
completely of silver.11 Pipette tips were prepared only seconds
before starting gigaohm seal formation by breaking off the final tip
region at the glass bottom of the bath chamber.12 Gigaohm seals
were formed after touching the cell membrane by application of
slight suction.
Data Acquisition and Analysis
Unitary Na⫹-channel currents were recorded at a sampling rate of
100 kHz in the cell-attached patch configuration with an Axopatch
200B amplifier (integrating head stage, intrinsic noise 0.045 pA rms
at 5 kHz, Axon Instruments, Inc). Analog filtering was performed
with an intrinsic filter at a bandwidth of 10 kHz. When data needed
further filtering for the analysis, an offline gaussian filter algorithm
was used. The holding potential was ⫺120 mV, duration of prepulses
was 20 ms, and 4-ms pulses were applied at a rate of 10 Hz.
Capacitive transients were compensated for carefully via compensation circuits containing two exponentials. Leak and residual capacitive currents were removed by subtracting averaged blank traces,
which were formed exclusively from traces in the neighborhood of
the actual sweep. None of the patches in the present study showed
any sign of containing more than one active Na⫹ channel. This was
verified by inspecting several thousands of consecutive traces at
pulses to ⫺40 mV or more positive potentials to exclude the
existence of any superimposition of opening events. Histograms of
the open-channel lifetime were constructed by using the baseline
method.11 Herein single-channel open times were calculated from
the distribution of the opening-induced gaps in the middle of the
baseline noise. In contrast to usual open-time histograms, the
distribution of dwell times measured at the baseline contains both
channel events (to) and noise events (tn). For separating the to from
the tn distribution, an exponential can be fitted to the tn distribution,
and the corresponding tn bins can be clipped for fitting the to
distribution. To improve resolution, amplitude histograms were
formed by eliminating the transition points with the variance-mean
technique. 19 For curve-fitting, a derivative-free LevenbergMarquardt routine20 was used. Recording was performed on a
PC-80486 with P-clamp software (Axon Instruments, Inc). Analysis
was performed on a Pentium personal computer with ISO2 software
(MFK Computer) after converting the data with PCV software (MFK
Computer).
Gating modes were selected by reidentifying blocks of traces from
the average-of-interval plots. In these diagrams, the time course of an
Figure 1. Method for the separation of different gating modes
(ventricular cell from a nonfailing human heart, prepulse potential ⫺120 mV, pulses to ⫺40 mV; empty sweeps were excluded). A, Narrow part of the average-of-interval plot, in which
switching from one gating mode to another was identified (each
square dot represents the averaged current of an individual
sweep of 4-ms duration; the narrow line indicates zero current,
filter 10 kHz). B, Sixteen consecutive sweeps before and after
switching from the F-mode to the S-mode (the arrow indicates
beginning of the test pulse; the amplitude of short openings
may be reduced by the filter of 5 kHz). C, Ensemble-averaged
currents formed from the respective traces in panel B (the arrow
indicates beginning of the test pulse, filter 5 kHz).
experiment is illustrated by plotting the averaged current of each
individual sweep as a dot. The method of separating different gating
modes is illustrated in Figure 1.
Figure 1A depicts a narrow part of an average-of-interval plot, in
which switching between two different gating modes is detected. In
Figure 1B, 16 consecutive sweeps in the F-mode (left) and 16 sweeps
after switching to the S-mode (right) are illustrated. Both before and
after the switch, empty sweeps, ie, traces without openings were
excluded. Next (Figure 1C), ensemble-averaged currents of each
suspected gating mode were formed from the traces in panel B.
Different blocks of traces in each suspected gating mode were
combined to increase the number of openings and to yield smooth
ensemble-averaged currents. This is demonstrated later in Results
(see Figure 3). The mode-specific kinetics of the currents were then
fitted (see also Figure 3) with a Hodgkin-Huxley model of the type
Am3h.21 In this model, A represents a scaling factor, and m and h
denote parameters describing activation and inactivation, respectively, of the Na⫹ channel. m is raised to the third power as activation
is assumed to appear in several steps, ie, several conformational
changes of the Na⫹-channel protein are necessary for activation. This
fitting procedure yields the activation-time constant (␶m) and the
inactivation-time constant (␶h). We have chosen only one inactivation gate (h) rather than two (h, j), as often used in the more common
Am3hj scheme, because we believe that h and j may represent two
different gating modes. In contrast, fitting one gate to our ensembleaveraged current represents fitting of a single gating mode. The
gating modes were further differentiated by the mean open-time
Böhle et al
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Figure 2. Comparison of single Na⫹-channel currents of cells
isolated from different regions of human hearts, which have
been either healthy or in different disease states (prepulse
potential ⫺120 mV, pulses to ⫺40 mV). A, Selected single Na⫹channel currents of an atrial cell from a patient with aortic-valve
incompetence (left) and a ventricular cell from a nonfailing
human heart (right). In both patches, long and short openings
were observed (the arrows indicate beginning of the test pulses;
the amplitude of short openings may be reduced by the filter of
5 kHz). B, Ensemble-averaged currents (shown here for the
F-mode) of an atrial cell from a patient with coronary heart disease (left, 253 sweeps, filter 10 kHz) and a ventricular cell from
a nonfailing human heart (right, 71 sweeps, filter 5 kHz), which
have been constructed according to the method illustrated in
Figure 1. The currents were fitted with a Hodgkin-Huxley model
of the type Am3h (see Materials and Methods), yielding the
activation-time (␶m) and inactivation-time (␶h) constants (the
arrows indicate beginning of the test pulses; empty sweeps
were excluded). C, Amplitude histograms from single Na⫹channel current recordings of an atrial cell from a patient with
coronary heart disease (left, 752 sweeps) and of a ventricular
cell from a nonfailing human heart (right, 893 sweeps). Each distribution was fitted with the sum of two gaussian curves, the
peak of the baseline noise is truncated, and the mean amplitude
of the open-channel current is indicated (filter 10 kHz).
constant (␶o) and the time before which half of the first channel
openings occurred (t0.5), as is also illustrated in Results (see Figures
4 and 5).
Results
Figure 2 compares single Na⫹-channel currents of cells
isolated from different regions of human hearts. These organs
were either normal or had various diseases.
No obvious differences were found in the characteristics of
single-channel currents (Figure 2A), obtained from an atrial
cell of a patient suffering from aortic valve incompetence
(left) and a ventricular cell from a nonfailing human heart
(right). In both patches, long and short openings were
Human Naⴙ-Channel Gating Modes
423
observed. The amplitude of short openings may be reduced
due to filtering.
Ensemble-averaged currents (Figure 2B; illustrated for the
F-mode; left, atrial cell of a patient with coronary heart
disease; right, ventricular cell from a nonfailing human
heart), which have been constructed according to the method
illustrated in Figure 1, reveal no obvious differences. Subsequently, the currents were fitted with a Hodgkin-Huxley
model of the type Am3h (see Materials and Methods for
details). The activation-time constant ( ␶ m ) and the
inactivation-time constant (␶h) are indicated in both fits.
To define the mean single-channel current amplitude at
⫺40 mV, amplitude histograms were built. Figure 2C illustrates graphics from recordings of single Na⫹-channel currents from an atrial cell of a patient with coronary heart
disease (left) and from a ventricular cell from a nonfailing
human heart (right). Each distribution could be fitted with the
sum of two gaussian curves, and the peak of the baseline
noise is truncated. In both cell types, the mean amplitude of
the open-channel current is essentially the same, ⫺2.368 pA
in the atrial cell and ⫺2.309 pA in the ventricular cell.
Furthermore, the single-channel conductance was not altered
by mode switches (not illustrated).
Figure 3 shows ensemble-averaged currents of single
Na⫹-channel openings at test pulses to ⫺40 mV in the
F-mode, M1-mode, M2-mode, S-mode, and P-mode, respectively. Gating modes were reidentified according to the
method illustrated in Figure 1. Macroscopic kinetics of
the Na⫹ current are characteristic in each mode.13,15 In the
F-mode, fast activation is followed by fast inactivation; in the
M1-mode, fast activation is followed by fast to intermediate
inactivation; in the M2-mode, fast activation is followed by
intermediate to slow inactivation; in the S-mode, fast activation is followed by slow inactivation; and in the P-mode, slow
activation is followed by slow inactivation.
The mode-specific kinetics of the currents were fitted with
a Hodgkin-Huxley model of the type Am3h (see Materials and
Methods for details). The magnitude of the parameters,
activation-time constant (␶m) and inactivation-time constant
(␶h), is indicated for each mode. The kinetics of activation in
the F-mode, M1-mode, M2-mode, and S-mode are not fully
resolved, but the data show that in the P-mode ␶m is slower by
a factor of about two. Comparison of the first latency yields
a respective factor of about three (see Figure 5). More
accurate comparison is possible for ␶h. Compared with the
F-mode, the inactivation-time constant is nearly 2 times
slower in the M1-mode, more than 4 times slower in the
M2-mode, and ⬇13 times slower in the S-mode and the
P-mode, respectively.
Figure 4 shows histograms of the open-channel lifetime in
single-channel patches at test pulses to ⫺40 mV in the F-mode,
M1-mode, M2-mode, S-mode, and P-mode, respectively. Gating
modes were reidentified from the average-of-interval plots and
represent the same traces as those of Figure 3.
All histograms of the open-channel lifetime were formed
by making use of the baseline method (see Materials and
Methods for details).11 In the F-mode, M1-mode, M2-mode,
and P-mode, respectively, only one mean open time was
found. The F-mode (␶o⫽0.15 ms) and the P-mode (␶o⫽0.14
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Circulation Research
September 6, 2002
Discussion
Figure 3. Hodgkin-Huxley fit (Am3h) of ensemble-averaged currents in the F-mode (71 sweeps), M1-mode (89 sweeps),
M2-mode (142 sweeps), S-mode (94 sweeps), and P-mode (102
sweeps). The activation-time constant (␶m) and the inactivationtime constant (␶h) are indicated (ventricular cell from a nonfailing
human heart, prepulse potential ⫺120 mV; the arrow indicates
beginning of the pulse to ⫺40 mV; empty sweeps were
excluded, filter 5 kHz).
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ms) are dominated by a short mean open time. The M1-mode
(␶o⫽0.28 ms) and the M2-mode (␶o⫽0.40 ms) are dominated
by intermediate mean open times. In the S-mode, two mean
open times were found. One was long (␶o1⫽1.24 ms) and the
other was short (␶o2⫽0.12 ms). These are addressed later (see
Discussion).
Figure 5 shows histograms of the cumulative first latency
in one-channel patches at test pulses to ⫺40 mV in the
F-mode, M1-mode, M2-mode, S-mode, and P-mode, respectively. Gating modes represent the same traces as those in
Figures 3 and 4.
Cumulative first-latency distributions for the F-mode, M1mode, M2-mode, and S-mode differ only slightly. The time
before which half of the first channel openings had occurred
(t0.5) in each of these modes is ⬇0.3 ms. The time course of
the cumulative first latency in the P-mode was much slower,
yielding t0.5 of ⬇0.9 ms. In this mode, t0.5 may be even larger,
because late first channel openings may have been cut off by
the pulse duration of only 4 ms.
Because only traces with openings were reidentified from
the average-of-interval plots, the probability of nonempty
traces is zero in all plots.
To our knowledge, this is the first description of the gating
characteristics of single native human cardiac Na⫹ channels
in atrium and ventricle. We looked for distinct Na⫹-channel
gating modes (F-mode, M1-mode, M2-mode, S-mode, and
P-mode), which have previously been characterized in myocardial cells from adult white mice.13 For this purpose,
patches containing only a single Na⫹ channel were investigated. In previous studies,13–15 gating modes were identified
from the long time course of the averaged current per trace
(average-of-interval plots). There it was found that modal
gating may happen in at least two different ways: (1) The
lifetime of a specific mode may be rather long, ie, about some
seconds to several minutes or (2) the lifetime of a specific
mode may be rather short, ie, less than about a few seconds.
The second possibility was derived from the fact that in some
experiments, from only one large block of traces of the
average-of-interval plots, two different mean open-time constants were found. We suggested that fast back and forth
switching between two different gating modes was present in
these recordings. Both in (1) and (2), the time course of
switching itself was not resolved. We observed that it
appeared from one pulse to the next, which means that at a
pulse rate of 10 Hz, switching may have happened faster than
100 ms. Sometimes switching even appeared within one
trace, indicating a time course of less than a few milliseconds.
The gating modes in the present report were also identified
from the average-of-interval plots. Because only fast back
and forth switching between gating modes according to type
2 was observed, a new method had to be found to resolve
different gating modes. The results indicate that identification
of relatively small blocks of sweeps inherent in individual
gating modes and their subsequent combination facilitates the
analysis of characteristics of human Na⫹-channel gating
modes.
We found that the mean open time was short and nearly
identical in the F-mode (␶o⫽0.15 ms) and in the P-mode
(␶o⫽0.14 ms). It was prolonged in the M1-mode (␶o⫽0.28
ms) and in the M2-mode (␶o⫽0.40 ms). In the S-mode, two
different mean open-time constants were found. The first was
Figure 4. Histograms of the open-channel lifetime
in the different gating modes. The data could be
fitted monoexponentially in the F-mode, M1-mode,
M2-mode, and P-mode, and biexponentially in the
S-mode. Mean open times (␶o) are indicated (ventricular cell from a nonfailing human heart, prepulse potential ⫺120 mV, pulses to ⫺40 mV, filter
10 kHz, baseline method, same patch and sweeps
as in Figure 3).
Böhle et al
Human Naⴙ-Channel Gating Modes
425
Figure 5. First-latency distributions in the
F-mode, M1-mode, M2-mode, S-mode, and
P-mode, respectively. The time before which
half of the first channel openings occurred
(t0.5) is indicated in each plot (ventricular cell
from a nonfailing human heart, prepulse
potential ⫺120 mV, pulses to ⫺40 mV, filter
10 kHz, same patch and sweeps as in
Figure 3).
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long (␶o1⫽1.25 ms) and the second was short (␶o2⫽0.12 ms).
Because we were not able to separate these two time
constants by selecting different traces, we conclude that only
in this case switching between two different gating modes
happened within the same trace. We assume that the long
open time represents the S-mode, and the short open time
represents the P-mode.
Cumulative first-latency distributions of different gating
modes allowed determination of the time before which half of
the first channel openings occurred (t0.5). This was fast and
nearly identical in the F-mode (t0.5⫽0.26 ms), the M1-mode
(t0.5⫽0.31 ms), the M2-mode (t0.5⫽0.36 ms), and the S-mode
(t0.5⫽0.30 ms). In contrast, it was slow in the P-mode (t0.5⫽0.92 ms).
From the Hodgkin-Huxley fits of the ensemble-averaged
currents, activation and inactivation kinetics of the different
gating modes were calculated. The activation-time constant
(␶m) was fast and similar in the F-mode (␶m⫽0.14 ms), the
M1-mode (␶m⫽0.17 ms), the M2-mode (␶m⫽0.17 ms), and
the S-mode (␶m⫽0.14 ms), and it was slow in the P-mode
(␶m⫽0.29 ms). The inactivation-time constant (␶h) was fast in
the F-mode (␶h⫽0.35 ms) and successively slower in the
M1-mode (␶h⫽0.62 ms), the M2-mode (␶h⫽1.56 ms), and the
S-mode (␶h⫽4.57 ms). In the P-mode (␶h⫽4.50 ms), it was
identical to the S-mode.
The mean single Na⫹-channel current amplitude was identical in atrial and ventricular myocytes, in normal and
diseased tissue, and in different gating modes, ie, it was not
altered by mode switching.
When the five gating modes of native human cardiac Na⫹
channels are compared with those of adult white mice (see
Table 1 in Böhle and Benndorf13; see Figure 3 in Böhle et
al15), only slight differences can be found. The mean open
times (␶o), the activation-time constants (␶m), and the
inactivation-time constants (␶h) are almost identical. Whereas
in human cardiac Na⫹ channels the time before which half of
the first channel openings occurred (t0.5) was slightly slower
in the F-mode, M1-mode, M2-mode, and S-mode, it was
slightly faster in the P-mode. In tissue from adult white mice,
fast back and forth switching was observed only between the
S-mode and the P-mode. In contrast in human tissue, we
found fast back and forth switching between all five gating
modes. Thus, the main difference in modal gating of myo-
cardial cells from adult white mice compared with human
tissue is the respective lifetime of the five gating modes. In
human tissue, in all five modes it was found to be only short.
In tissue from adult white mice, a long lifetime could be
shown in the F-mode, M1-mode, M2-mode, and the P-mode,
but in the S-mode it was found to be only short. In the
P-mode, it could be short, too.
All gating modes were found in both normal and diseased
human atrium and ventricle, but no regulation site is yet
detected. Modal gating may be regulated by (1) phosphorylation through protein kinase C22 (brain Na⫹ channels),23–24
(cardiac Na⫹ channels), and/or by protein kinase A24 –26
(cardiac Na⫹ channels),27–30 (cardiac L-type Ca2⫹ channels),
and (2) mechanisms involving intracellular Ca2⫹,31–33 or (3)
mechanical stress on the plasma membrane during contraction of the heart muscle cell. In this context, it is important
that the contraction-relaxation cycle of single heart-muscle
cells is altered in terminal heart failure. Compared with
nonfailing cells, the intracellular Ca2⫹ concentration during
relaxation, ie, during the diastolic phase, is increased, and
during contraction systole, it is depressed. Additionally, the
diastolic decay of the intracellular Ca2⫹ concentration is
markedly slowed.17 When regulation sites (2) or (3) are
discussed, it might be of further importance that different
types of Na⫹ current, presumably reflecting different gating
modes, have been identified in single beating cells of chick
embryo heart and have been correlated to different phases of
the action potential by direct measurement.34 –36 From these
results, we suspect that synchronous appearance of gating
modes may be triggered by contraction of the muscle cell.
Acknowledgments
This research was supported by grants from the Bundesministerium
für Bildung, Forschung und Technologie (BMBF 01 KS 9502,
ZMMK, Projekt 4), and the M. and W. Boll-Stiftung. We thank
Nadine Henn and Iris Berg for their excellent technical assistance,
Harald Metzner and Jürgen Staszewski (Center of Physiology and
Pathophysiology, University of Cologne) and their colleagues for
their continual technical advice and support, and Prof DeVivie
(Department of Cardiac Surgery, University of Cologne) and his
colleagues for providing the myocardial tissue.
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Identification of Gating Modes in Single Native Na+ Channels From Human Atrium and
Ventricle
Thomas Böhle, Mathias C. Brandt, Michael Lindner and Dirk J. Beuckelmann
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Circ Res. 2002;91:421-426; originally published online August 15, 2002;
doi: 10.1161/01.RES.0000033521.38733.EF
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