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1-subunit modifies the
kinetics and fatty acid block of hH1
Na+
channels
1 Charles A. Dana Research Institute and Harvard-Thorndike Laboratory, Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, 2 Department of Medicine, Massachusetts General Hospital, and 3 Department of Anesthesia, Brigham and Women's Hospital, 4 Harvard Medical School, Boston, Massachusetts 02215; and 5 Department of Biological Sciences, Murray State University, Murray, Kentucky 42071
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Voltage-gated cardiac Na+ channels are composed of
- and 
1-subunits. In this study
1-subunit was cotransfected with the -subunit of the
human cardiac Na+ channel (hH1
) in human
embryonic kidney (HEK293t) cells. The effects of this coexpression on
the kinetics and fatty acid-induced suppression of Na+
currents were assessed. Current density was significantly greater in
HEK293t cells coexpressing - and 1-subunits
(INa,
) than in HEK293t cells expressing
-subunit alone (INa,). Compared with
INa,, the voltage-dependent inactivation and activation of INa, were significantly
shifted in the depolarizing direction. In addition, coexpression with
1-subunit prolonged the duration of recovery from
inactivation. Eicosapentaenoic acid [EPA, C20:5(n-3)]
significantly reduced INa, in a
concentration-dependent manner and at 5 µM shifted the midpoint voltage of the steady-state inactivation by 
22 ± 1 mV. EPA also significantly accelerated channel transition from the resting state to
the inactivated state and prolonged the recovery time from
inactivation. Docosahexaenoic acid [C22:6(n-3)], -linolenic acid [C18:3(n-3)], and conjugated linoleic acid
[C18:2(n-6)] at 5 µM significantly inhibited both
INa, and INa,. In contrast, saturated and monounsaturated fatty acids had no effects
on INa,. This finding differs from the
results for INa,, which was significantly
inhibited by both saturated and unsaturated fatty acids. Our data
demonstrate that functional association of 1-subunit
with hH1 modifies the kinetics and fatty acid block of
the Na+ channel.
-subunit; cardiac sodium ion channel; polyunsaturated fatty
acids
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SEVERAL CLINICAL STUDIES suggest that diets containing long-chain n-3 fatty acids significantly reduce the incidence of sudden death from coronary heart disease (1, 13, 14, 34, 35). A diet high in fish oil, in contrast to saturated fat or monounsaturated olive oil, prevented ventricular fibrillation induced by coronary artery ligation in rats and increased the electrical ventricular fibrillation thresholds in marmosets (27, 28). Furthermore, an intravenous infusion of an emulsion, largely eicosapentaenoic acid (EPA) and docosahexaenoic acid [DHA, C22:6(n-3)], prevented ischemia-induced ventricular fibrillation in dogs (7, 8). Previous data (21) indicate that polyunsaturated long-chain fatty acids (PUFAs) reduced electrical excitability of rat cardiac myocytes by increasing the depolarizing current required to elicit an action potential and by markedly prolonging the relative refractory period. Activation of voltage-dependent Na+ channels leads to a rapid influx of Na+ and initiates an action potential in most cardiac myocytes. Extracellular application of free PUFAs significantly suppressed Na+ currents (INa,rat) and shifted the steady-state inactivation to more hyperpolarized potentials in cultured neonatal rat cardiomyocytes (41). PUFAs also inhibit Ca2+ (40) and K+ currents (9) in mammalian heart cells. These effects of PUFAs on ion channels may be critical for their antiarrhythmic action in vivo.
It is recognized that the human cardiac Na+ channel
consists of one large -subunit that alone creates a functional
membrane channel, which we have studied (43). In addition,
there is also a small 1-subunit (12). The
physiological consequences of 1-subunit modulation of
the voltage-dependent Na+ channel are controversial in the
literature (2, 19, 24, 25, 29, 31). Therefore, in this
study we assessed the effects of coexpressing the
1-subunit on the kinetics of the -subunit of the
voltage-dependent human cardiac Na+ channel
(hH1) transiently expressed in a mammalian cell line (HEK293t). We were surprised that the fatty acid specificity was
lost in the -subunit expressed in HEK293t cells in which monounsaturated and even saturated fatty acid had some suppressing effects on INa, (43), whereas
only PUFAs suppressed INa,rat in rat cardiac
myocytes (41). Two further aims of this study were
1) to learn how the complete human myocardial
Na+ channel (hH1) would be affected by
the antiarrhythmic PUFAs and 2) to learn whether the
addition of the 1-subunit to the hH1 -subunit would
reestablish the requirement that only PUFAs could modulate the fast
voltage-dependent Na+ current in the rat.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials and solutions.
Fatty acids obtained from Sigma (St. Louis, MO) were dissolved weekly
in ethanol at 10 mM and stored under a nitrogen atmosphere at 20°C
before use. The experimental concentration of fatty acids was obtained
by dilution of the stocks and contained negligible ethanol, which at
the dilution applied had no effect on Na+ currents. The
pipette solution for recording the inward Na+ current
contained (in mM) 100 CsCl, 40 CsOH, 1 MgCl2, 1 CaCl2, 11 EGTA, 10 HEPES, and 5 MgATP, pH 7.3. The bath
solution contained (in mM) 60 NaCl, 40 N-methyl-D-glucamine, 10 CsCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, and 10 glucose, pH
7.4. The Tyrode solution contained (in mM) 137 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, and 10 glucose, pH
7.4.
Cell culture and transient transfection of Na+ channels. The method for the culture of HEK293t cells was as described previously (11). Briefly, cells were grown to 50% confluence in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin solution, 3 mM taurine, and 25 mM HEPES. Cells were split twice per week.
HEK293t cells were transfected with cloned hH1
Na+ channels by a calcium phosphate precipitation method in
a TI-25 flask. A reporter plasmid CD8-pih3m (1 µg, cell surface
antigen) and hH1 cDNA clone (10 µg) in the pcDNA1/amp
vector (Invitrogen, San Diego, CA) were prepared in 250 mM
CaCl2, added to a test tube containing 0.36 ml of Hanks'
balanced salt solution (2×) (in mM: 274 NaCl, 40 HEPES, 12 dextrose,
10 KCl, and 1.4 Na2HPO4, pH 7.05), and
incubated at 22°C for 20 min. The DNA solution was then
dripped over a cell culture (30-50% confluence) containing 7 ml
of DMEM. The transfection was satisfactory under these conditions (38). For coexpression of the rat brain
1-subunit with hH1 (hH1, coexpression of the channel), saturating levels (>10-fold molar excess) of 1-subunit cDNA were used to
ensure that the currents recorded were from channels composed of both - and 1-subunits. The transfected cells were
trypsinized and replated 15 h later to an appropriate density in
35-mm tissue culture dishes (which also served as recording chambers)
containing 2 ml of fresh DMEM. Transfected cells were incubated at
37°C in air with 5% CO2 added and 98% relative humidity
and were used within 3 days. Transfection-positive cells, which were
identified by binding immunobeads (CD8-Dynabeads M-450, Dynal, Oslo,
Norway) coated with a monoclonal antibody (ITI-5C2) specific for CD8
antigen, were selected for patch-clamp experiments.
Electrophysiological recordings.
During an experiment HEK293t cells plated in a culture dish were
continuously superfused (1-2 ml/min) with the Tyrode solution. Recording glass electrodes had a resistance of 1-3 M
when
filled with the pipette solution and were connected via Ag-AgCl wire to
an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). A cell
coated with CD8 beads in HEK293t cells was chosen for patch-clamp study. After a conventional gigaseal was formed, the capacitance of an
electrode was compensated. Additional suction was used to form the
whole cell configuration. Whole cell membrane capacitance was measured
by using the method described previously (42). The average
membrane capacitance was 37 ± 0.8 pF (n = 179)
for HEK293t cells. Correction of cell capacitance and series resistance was then performed before application of experimental voltage-clamp protocols. After the whole cell configuration was formed, the cells
were dialyzed for 5-10 min before data were acquired. With our
internal and external recording solutions, we found that maintaining a
tight seal for a relatively long period became difficult at holding
potentials more negative than 90 mV. Therefore, we held the membrane
potential at 90 mV in most experiments to ensure that cells would
remain stable long enough for us to examine
INa, or INa,
from the same cell before, during, and after exposure to fatty acids.
In addition, the amplitude and current-voltage (I-V)
relationship curve were not altered when
INa, or INa, was
elicited by pulses from a holding potential of 150 mV or from 90 mV
with a 400-ms hyperpolarizing prepulse to 160 mV. Our experiments
show that the 400-ms hyperpolarizing prepulse to 160 mV was
sufficient to remove fast and slow inactivation. Na+
currents were activated by 10- or 20-ms test pulses. Bath solutions with or without fatty acids were rapidly exchanged by using a modified
puffer-pipette system (41). Experiments were conducted at
22-23°C.
Statistics.
Data from two groups were analyzed by the unpaired Student's
t-test. Variance analysis (ANOVA) was used to compare the
difference derived from three or more group experiments. The level for
statistical significance was set at P < 0.05. Data are
presented as means ± SE. Depending on the experiment, some data
were fit with a logistical equation, (A1 A2)/[1 + (x/x0)p + A2], where x0 is the
center, p is power, A1 is the initial y value, and A2 is the final
y value. Other data were fit by either a Boltzmann equation
{1/[1 + exp(V1/2 V)/k], where V1/2 is the half-inactivation potential, V is the voltage potential, and
k is the slope factor (in mV/e-fold change in
current} or least-squares fitting (y = A0 + A1
expt/
1 + A2 expt/2,
where t is time and 
1 and 2
are the time constants of the fast and slow components of inactivation,
respectively) (Origin 4.1, Microcal Software, Northampton, MA) with a
single or double exponential function.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Voltage-dependent Na+ currents in HEK293t cells
transfected with hH1 (INa,) or
hH1 (INa,).
Voltage-gated Na+ currents with fast activation and fast
inactivation kinetics were evoked by depolarizing pulses in HEK293t cells transiently transfected with hH1 or cotransfected
with hH1 and 1-subunit (Fig.
1A). Functional
association of 1-subunit with hH1
significantly enhanced the peak current densities (Fig. 1B).
The current densities (elicited by voltage pulses from 150 to 30
mV) were 74 ± 8 pA/pF for INa,
(n = 27) and 106 ± 9 pA/pF for
INa, (n = 39, P < 0.05; Fig. 1B), respectively. Compared
with the current densities of 68 ± 7, 17 ± 3, and
0 ± 0 pA/pF for INa, (n = 27) elicited by the corresponding voltage commands from 120, 90,
and 70 mV to 30 mV, the corresponding values of
INa, (n = 39) were
105 ± 9, 85 ± 9, and 25 ± 5 pA/pF (Fig.
1B). Whereas >80% of peak INa, was elicited by pulses from 90 to
30 mV, the same voltage step evoked only 18% of peak
INa,. In addition, a considerable amount of
INa, (23%) was activated by voltage
pulses from 70 to 30 mV, but no INa, was
evoked with the same voltage protocol (Fig. 1A). These
results indicate that coexpression of 1-subunit modifies
the voltage-dependent availability of Na+ channels.
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Modification of channel activation and inactivation by the
1-subunit.
Figure 2A shows the original
current traces, and Fig. 2B shows the I-V
relationship of the voltage-dependent activation of hH1 and hH1. Na+ currents
were activated at around 60 mV and reached the maximal amplitude at
30 mV for both INa, (n = 7) and INa, (n = 6). Although
the general shape of the I-V relationship did not change,
the midpoint of the normalized I-V curve (from 60 to 30
mV) for INa, had an ~10-mV shift in the
positive direction. This result suggests that the
1-subunit may modify the activation process of
hH1 Na+ channels. Normalized whole cell
activation conductance from peak Na+ currents confirmed the
modulatory effect of the 1-subunit on INa, activation, which caused an 8-mV
positive shift at V1/2 (P < 0.05, Fig. 2C). The average V1/2 and
k (slope) values for the fitted functions were 42.2 ± 0.81 and 5.3 ± 0.17 mV, respectively, for
INa, and 50.1 ± 0.74 and 5.5 ± 0.56 mV, respectively, for INa,.
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1-subunit on the fast steady-state
inactivation were examined by measuring the amplitude of peak currents evoked by a two-pulse protocol. From a holding potential of 90 mV, we
delivered 500-ms prepulses ranging from 160 to 10 mV (in 5-mV
increments) and then measured the available current elicited by a 10-ms
pulse to 30 mV. The average V1/2 of the fast
steady-state inactivation curve of INa, was
97 ± 2.3 mV with a k value of 6.7 ± 0.8 mV
(n = 6). In contrast, coexpression of
hH1 with 1-subunit caused a 22 ± 0.7 mV shift of the V1/2 of
INa,, which was 75 ± 2.7 mV with a
k value of 5.5 ± 0.5 mV (n = 9, P < 0.001; Fig. 2D). In another series of
experiments (data not shown), we used a protocol similar to that of
Bendahhou and colleagues (4) to examine the effects of
1-subunit coexpression on the slow steady-state
inactivation. We used 45-s conditioning pulses ranging from 140 to 10 mV (in 10-mV increments) followed by a 100-ms recovery pulse to 120
mV and a subsequent 10-ms test pulse to 30 mV. We found that the
V1/2 values for the slow inactivation curves of
INa, (62.7 ± 1.9 mV,
n = 7) and INa, (62.0 ± 2.1 mV, n = 6) were not significantly
different (P > 0.05). These results suggest that
functional association of 1-subunit with
hH1 causes a significant shift of the fast steady-state inactivation but does not markedly affect the slow steady-state inactivation.
Coexpression of 1-subunit with hH1
slows the recovery from inactivation.
Voltage-activated cardiac Na+ channels may directly transit
from the resting state to the inactivated state without opening of the
channel. This process of inactivation is referred to as resting
inactivation (6, 15, 18,
22, 33). To assess the effects of
1-subunit coexpression on the development of resting inactivation of hH1 Na+ channels, a
conditioning pulse to 65 mV with variable durations was followed by a
10-ms test pulse to 30 mV (Fig.
3A). We selected 65 mV as
the conditioning voltage because the depolarization was large enough to
ensure that the inactivation of both INa, and INa, neared completion but small enough
to ensure that the channels did not open. Figure 3B shows
that the amplitudes of INa, and
INa, dramatically decreased as the duration
(
t) of conditioning pulses was prolonged, indicating that
an increasing proportion of channels entered the inactivated state. The
decay time constant of inactivation development was 26.2 ± 3.6 ms
for INa, (n = 6) and
32.2 ± 4.2 ms for INa, (n = 7) (Fig. 3B). Our results
indicate that coexpression of the 1-subunit with
hH1 does not significantly (P > 0.05)
alter the development of inactivation.
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1-subunit
affected the recovery from inactivation, a double-pulse protocol was
used to test the recovery from resting inactivation at 65 mV. A 10-s depolarizing conditioning pulse to 65 mV was followed by a variable recovery interval at 140 mV and then a subsequent test pulse to 30
mV (Fig. 3C). The 10-s conditional pulse to 65 mV failed to elicit channel opening but ensured that Na+ channels
entered the inactivated state. The time course of recovery from
inactivation of both INa, and
INa, was well fit by a double exponential
function (Fig. 3D). For INa, (n = 6), 1 was 2.1 ± 0.3 ms
(A1 = 0.73) and 2 was
232 ± 78 ms (A2 = 0.27). For
INa, (n = 8),
1 was 10.6 ± 2.1 ms
(A1 = 0.69) and 2 was
698 ± 275 ms (A2 = 0.31),
respectively. The values of 1 and
2 between INa, and
INa, were significantly different
(P < 0.05, Fig. 3D).
To more carefully examine the recovery time course of the fast
component, we used a pulse protocol similar to that shown in Fig.
3C. We delivered a 400-ms conditioning pulse to 65 mV and a recovery pulse to 140 mV with 1-ms increments (t) to
30 ms, as well as a 10-ms test pulse to 30 mV. The data fit well with a single, but not a double, exponential function for both
INa, and INa,.
The time constants were 3.56 ± 0.14 and 5.11 ± 0.22 ms for
INa, (n = 14) and
INa, (n = 8, P < 0.05), respectively. We tested four recovery
potentials, 100, 120, 140, and 160 mV. The recovery rate for
both channels became more rapid when more negative potentials were
used, but the difference in recovery rate between
INa, and INa,
remained significant. These data suggest that 1-subunit
modifies the inactivation kinetics of hH1 channels by
slowing the recovery from both fast and slow inactivation.
Voltage- and concentration-dependent suppression of
INa, by EPA.
It has been reported that coexpression with the
1-subunit reduced the affinity of resting channels to
lidocaine by a factor of two in oocytes (24). In a recent
study (39), we showed that coexpression of
hH1 with the 1-subunit elicited a
positive shift in state-dependent cocaine block of the Na+
channel. In another study (41, 43), we
demonstrated that INa, was more sensitive to
EPA than was INa,rat. To determine whether
coexpression with the 1-subunit had an effect on EPA block of hH1 channels similar to that observed in local anesthetic experiments and to determine whether the different sensitivity to PUFAs
between INa, and
INa,rat might result from a lack of the
1-subunit, experiments were designed to look at the
effects of EPA on INa,. The inhibition of
INa, initiated within 20 s and
reached the maximal effect within 3 min after application of 5 µM
EPA. INa, returned toward the pretreatment level after washout of EPA with 0.2% fatty acid-free BSA solution. Figure 4 shows a voltage-dependent
inhibition. The original current traces of
INa, were evoked by single-step pulses
from 150, 120, 90, and 70 mV to 30 mV in the absence
(control) and presence (EPA) of 5 µM EPA (Fig. 4A). The
reduction of INa, caused by 5 µM EPA at
all of the tested voltage steps is statistically significant
(n = 15, P < 0.001; Fig.
4B). In hH1, EPA (5 µM) inhibited
INa,, evoked by a single-step voltage command from 150, 120, or 90 mV to 30 mV, by 67 ± 6, 82 ± 5, or 97 ± 1% (n = 15), respectively.
Coexpression with the 1-subunit decreased the degree of
block by 5 µM EPA, because with the same voltage steps
INa, was inhibited by 50 ± 5, 61 ± 5, or 90 ± 4% (n = 15), respectively.
The inhibition is more profound at pulses from 90 to 30 mV. This
result suggests that the EPA-induced suppression of
INa, is voltage dependent.
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by EPA was
concentration dependent. Figure 5 shows
the inhibitory effects of EPA on INa, and
INa,. Currents were elicited by single-step
pulses from 120 to 30 mV every 5 s. The IC50
values of EPA were 3.9 ± 0.3 and 0.51 ± 0.06 µM for
INa, and INa,,
respectively. These data indicate that functional association of the
1-subunit with hH1 reduces the apparent
affinity of the channel for EPA 7.6-fold compared with expression of
the -subunit alone. The data in Figs. 4 and 5 suggest that
coexpression of the 1-subunit with hH1
reduces the channel sensitivity to EPA and that the change in the
effectiveness of EPA may be related to the
1-subunit-induced shift of the steady-state
inactivation.
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Effects of EPA on activation and inactivation of
INa,.
EPA (5 µM) block of INa, did not alter
the I-V relationship, but the decrease in amplitude of
INa, in the presence of 5 µM EPA was
significantly different at the voltages from 40 to 30 mV (Fig.
6A, n = 7).
The Na+ current was activated at 60 mV and achieved its
maximal amplitude at 30 mV in either the absence or presence of 5 µM EPA. The inhibition was reversible after washout of EPA with the
bath solution containing 0.2% BSA (data not shown). The activation
curves calculated from normalized conductance were superimposed in the
absence and presence of 5 µM EPA (n = 7, Fig.
6B). The 50% channel activation was at 42.2 ± 0.81 mV with a k value of 5.3 ± 0.17 mV for control and at
41.5 ± 0.21 mV with a k value of 6.0 ± 0.15 mV
for 5 µM EPA.
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in
HEK293t cells. The membrane potential of the cells was held at 90 mV.
Currents were elicited with a double-pulse protocol, which consisted of
a 500-ms prepulse and a 10-ms test pulse to 30 mV. The prepulses
varied from 160 to 50 mV in 5-mV increments with a stimulatory rate
of 0.1 Hz. Bath perfusion of 5 µM EPA solution significantly
suppressed INa,. The current was almost
completely inhibited when the prepulses were more positive than 70 mV
in the presence of EPA. The V1/2 of the
normalized steady-state inactivation curve of
INa, was significantly shifted to the
negative direction, from 74.8 ± 0.3 mV (k = 5.7 ± 0.23 mV) to 97.5 ± 0.3 mV (k = 9.0 ± 0.32 mV, n = 7, P < 0.001)
in the absence and the presence of 5 µM EPA, respectively. After
washout of EPA with 0.2% fatty acid-free BSA, the steady-state
inactivation curve was shifted back to 81.9 ± 0.4 mV at the
V1/2 point with a k value of 6.9 ± 0.34 mV. The values of the V1/2 of the
steady-state inactivation curves between control and washout were not
significantly different (P > 0.05), but the difference
between EPA and washout was statistically significant (P < 0.01).
EPA-induced acceleration of inactivation development.
To assess the effects of EPA on the development of resting inactivation
of INa,, conditioning pulses to 65 mV
with increasing durations were followed by a test pulse to 30 mV
(Fig. 7A). Because the
conditioning pulses to 65 mV did not evoke any current (Fig. 2), the
development of inactivation proceeded directly from the resting or
preactivated states. Figure 7B shows that increases in
duration of the conditioning pulse gradually increased the population
of hH1 channels into the inactivated state. EPA at 5 µM significantly accelerated the process of this transition. The data
were well fit by a single exponential decay with a time constant of
32.2 ± 0.8 and 8.3 ± 0.8 ms for control and EPA
(P < 0.01), respectively. It is interesting that
compared with hH1 alone, coexpression of the
1-subunit with hH1 reduced the rate of
inactivation development in the presence of EPA (Fig. 7C).
The time constant was significantly different between INa, (3.6 ± 0.2 ms, n = 6) and INa, (8.3 ± 0.8 ms,
n = 7, P < 0.05; Fig. 7C).
The data suggest that functional association of the
1-subunit with hH1 reduces the effects of
EPA on the development of resting inactivation of cardiac
Na+ channels.
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EPA slows the recovery of INa, from inactivation.
We examined the kinetics of recovery of
INa, from inactivation in the absence and
presence of EPA using a protocol similar to that shown in Fig.
3C. In these experiments we used 100 mV as the holding and
recovery potential. Note from Fig. 7 that 10 s at 65 mV would
more than suffice to convert all Na+ channels to the
inactivated state. We determined the recovery from inactivation by
increasing the duration (t) of recovery pulses and
measuring the available current elicited by a test pulse to 30 mV. In
control saline, the time course of recovery from inactivation of
INa, was fit by a double exponential function, and most of the recovery was in the fast component (Fig. 8). The time constants for the recovery
from inactivation of INa, in control
saline were 9 ± 7 ms (1;
A1 = 0.78) and 1,149 ± 256 ms
(2; A2 = 0.22). In the
presence of 5 µM EPA, the recovery from inactivation of
INa, was also fit with a double exponential function (Fig. 8) with the time constants of 33 ± 7 ms (1; A1 = 0.65) and
5,363 ± 845 ms (2; A2 = 0.35). Both 1 and 2 are significantly
slower in the presence of 5 µM EPA (P < 0.05). To
obtain a better view of the fast component, the x-axis was
plotted to 1 s (Fig. 8, inset). At the recovery
potential of 100 mV, the time required for 50% channel recovery from
the fast component of inactivation was significantly delayed, from 13 ± 1 ms for control to 63 ± 7 ms for 5 µM EPA
(n = 9, P < 0.01), respectively. The
time required for 50% recovery from the fast component of inactivation
was also significantly prolonged when the recovery potential was 150
mV, from 5.7 ± 0.6 ms for control to 21.8 ± 1.6 ms for 5 µM EPA (n = 8, P < 0.01). These
results indicate that EPA slows recovery of
INa, from both the fast and slow
components of inactivation.
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A higher efficacy of EPA on inactivated Na+ channels.
Only closed resting channels are able to open in response to a
depolarizing pulse. During a cycle of depolarization and
repolarization, channels are in dynamic equilibrium between the resting
and inactivated states. Therefore, current amplitude is proportional to
the number of channels in the resting state before a depolarizing
pulse. The EPA-induced suppression of INa,
was voltage dependent (Fig. 4), and the midpoint of the steady-state
inactivation of INa, was significantly
shifted to the negative direction in the presence of EPA (Fig. 6). The
voltage-dependent block and the negative shift in channel availability
could result from preferential EPA action on inactivated channels
compared with closed resting channels (3). Therefore, we
tested the effects of EPA on the resting and inactivated states of
Na+ channels. Figure 9,
A and B, shows that 5 µM EPA suppressed
superimposed Na+ currents evoked by depolarization pulses
to 30 mV from the holding potentials of 150 and 70 mV; from the
latter holding potential INa, was
completely inhibited.
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evoked by depolarization pulses from
a holding potential of 150 to 30 mV (Fig. 9A). At this
holding potential, virtually all channels were in the closed resting
state. The concentration-dependent curve gave an estimated
Kr, the equilibrium constant for drug binding or
interaction with resting channels (3), of EPA at a
concentration of 5.29 ± 0.56 µM (Fig. 9C).
Because inactivated channels do not open during depolarization, the
effects of drug binding to the inactivated state must depend on a small
portion of channels in the resting state. We therefore set the membrane
holding potential at 70 mV and assessed the concentration-dependent
relationship of EPA, Ki (Fig. 9, B and C). Here, Ki is the equilibrium
constant for block of inactivated channels at a 70-mV holding
potential at which channel inactivation was 80% (Fig. 2B).
Ki measured at this holding potential was
0.02 ± 0.01 µM (Fig. 9C). Thus Na+
currents in HEK293t cells coexpressing 1-subunit and
hH1 displayed a 265-fold greater sensitivity to EPA in
the inactivated state than in the resting state.
EPA block of resting hH1 channels.
Several Na+ channel blockers show a tonic and
frequency-dependent inhibition of voltage-gated Na+
channels (32). Figure
10A shows EPA-induced
inhibition of INa, without and with
predepolarizing pulses. The current was evoked by a voltage command
from a holding potential of 140 mV to 30 mV every 5 s. After
10 control current traces were collected, 5 µM EPA solution was
washed into the bath. Na+ currents were then recorded after
3 min of EPA perfusion. The amplitude of peak currents were elicited by
a train of 30 pulses in the presence of 5 µM EPA. The amplitude of
peak INa, elicited by the first pulse of
the train was markedly reduced. This inhibition is referred to as tonic
block, because the block developed at a holding potential of 140 mV
and without opening channels. During subsequent pulses in the train, no
additional block developed. The amplitude of peak
INa, evoked by the 30th pulse was similar
to the value evoked by the 1st pulse. Therefore, EPA-induced
suppression of INa, was mainly due to
fatty acid block of closed resting channels and did not require that
the channels enter the open state to gain access to a "binding"
site. The inhibition was removed after washout of EPA. In addition, the
time course of the EPA-induced suppression of
INa, in another group of experiments was
not altered when the stimulating rate of pulses was altered from 0.1 to
1 Hz (data not shown). These results are consistent with our previous findings (40, 41, 43) that EPA-induced inhibition of
cardiac Na+ and Ca2+ channels are time
dependent, but not use dependent, in rat cardiac myocytes and in
HEK293t cells transfected with hH1.
|
channels, we
used the same experimental protocol as in Fig. 10A and
examined the concentration dependence of tonic block of closed,
resting-state channels (Kr) and that of
state-dependent block of open channels (Ko, an
estimate) (32). Figure 10B shows that EPA has
very similar effects on the resting state and the open state of
hH1. The IC50 of EPA was 4.72 ± 1.26 and 4.82 ± 1.36 µM for the resting-state channel block and
the open-state channel block, respectively (n = 9, P > 0.05). The data further support the suggestion
that EPA-induced inhibition of cardiac Na+ currents is time
dependent, not use dependent, which is consistent with its high lipid
solubility (16).
Fatty acid effects on Na+ currents in HEK293t cells
transfected with hH1 or hH1 plus
1-subunit.
Table 1 summarizes the inhibitory effects
of several fatty acids on INa, and
INa,. EPA, DHA, -linolenic acid, conjugated linoleic acid (all 5 µM), and retinoic acid (10 µM) are
significant inhibitors of these Na+ currents. EPA ethyl
ester at 5 µM had no effect on either
INa, or INa,.
Monounsaturated and saturated fatty acids (5 µM) had no significant
inhibitory effects on INa, but
significantly inhibited INa,. This loss of
the characteristic fatty acid specificity on
INa, was recovered when the
1-subunit was coexpressed in HEK293t cells.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Modulation of hH1 channels by the
1-subunit.
In our present study, coexpression of the rat brain
1-subunit with the hH1 Na+
channel in HEK293t cells significantly increased the current density by
43% of INa, elicited by pulses from a
holding potential of 150 mV to 30 mV (Fig. 1B). The data
are consistent with other reports that described the enhancement of
Na+ current amplitude in oocytes coexpressing
1-subunit with either hH1, rat
H1, or rat brain -subunits of Na+
channels (19, 29, 31). In this
study coexpression of the 1-subunit with
hH1 not only markedly shifts the voltage-dependent activation to a more positive membrane potential but also significantly shifts the steady-state inactivation to the positive (depolarizing) direction. In addition, coexpression of the 1-subunit
with hH1 prolongs the duration of recovery from
inactivation. Our results demonstrate that the 1-subunit
enhances hH1 Na+ currents in HEK293t cells
and modifies some of the kinetic properties of hH1 channels.
1-subunit coexpression with
Na+ channel -subunits vary depending on the expression
system used. A summary of these differences was discussed in a previous
report (39). In general, coexpression of the
1-subunit with Na+ channel -subunits in
the oocyte or Chinese hamster cell expression systems elicits
hyperpolarizing shifts in channel activation and/or inactivation
(5, 10, 19), whereas in the
HEK293 (2) or HEK293t (Ref. 39 and this study) expression
systems there is a marked depolarizing shift in channel kinetics. To
our knowledge, a precise mechanism has not been identified that could
explain these differences among the expression systems currently in
use. The possibilities may include, but are not limited to, differences in constitutive second messenger systems, differences in
phosphorylation levels, and differences in posttranslational
modification of protein products.
We note that the depolarizing shifts in channel activation and
inactivation reported here are larger than the shifts reported in a
previous study (39). There are several differences between these two studies. First, the internal and external solutions in the
two studies were different. Second, when a reversed Na+
gradient was used (39), the cell was dialyzed for 30 min
to ensure that the outward Na+ currents were stable. In
contrast, when a more traditional Na+ gradient is used,
such as in the present study, the inward Na+ currents
become stable within 10 min. Therefore, the time-dependent shifts in
channel kinetics (37) for the two studies were different. Third, the holding potential in the previous study was always 140 mV,
whereas the holding potential in the present study was typically 90
mV. The holding potential likely influences the rate and degree of the
time-dependent shifts in channel inactivation. Fourth, the internal
solution in the present study contained ATP, which would most likely
elevate the degree of channel phosphorylation. Thus the numerous
technical differences between these two studies make it impossible to
determine with any certainty a direct cause or set of causes that could
explain the differences in the magnitude of the
1-subunit-induced depolarizing shift.
Kinetic resemblance between INa, and
INa,rat.
The voltage-gated Na+ channel isolated from rat brain is a
heterotrimeric protein, including an -subunit (260 kDa), a
noncovalently associated 1-subunit (36 kDa), and a
disulfide-linked 2-subunit (33 kDa) (12).
Na+ channels in the skeletal muscle and heart are complexes
of a large -subunit and only one smaller 1-subunit
(25, 36). We have shown that the
half-inactivation potential of hH1 is 97 mV when
expressed alone in HEK293t cells (43). A comparison of the
kinetics of INa, and
INa, with the functional properties of
Na+ currents in native heart cells indicates that
coexpressed cardiac Na+ channels composed of
hH1 and 1-subunits behave similarly to
native Na+ channels. In rat (41) and rabbit
(23) cardiac myocytes, the half-inactivation potentials
are 73 mV and 80 mV, respectively, which are very close to the
half-inactivation potential of 75 mV for
INa, in HEK293t cells. Our results
indicate that coexpression with 1-subunit modulates the
kinetics of INa, so as to resemble the
behavior of native mammalian cardiac Na+ channels. This is
consistent with the results in oocytes that coexpressed hH1
Na+ channels composed of - and 1-subunits
behave as Na+ channels of cardiac myocytes
(29).
Suppression of INa, by EPA.
To increase our understanding of EPA-induced inhibition of
hH1 currents (43), in this study we report
that human cardiac Na+ channels in HEK293t cells
coexpressing hH1 are sensitive to extracellular
application of PUFAs in a concentration- and voltage-dependent manner.
The activation of INa, was not altered in
the presence of 5 µM EPA. However, EPA significantly shifted the
steady-state inactivation of INa, to the hyperpolarizing direction. A similar shift of the steady-state inactivation of Na+ currents in HEK293 cells expressing
hH1 was observed previously in the presence of
arachidonic acid (4).
by PUFAs
was state dependent, EPA suppressed INa,
with a 265-fold lower concentration of IC50 for the
inactivated state than for the resting state of hH1 Na+
channels. EPA markedly sped up the transition of Na+
channels from the resting state to the inactivated state and significantly prolonged the time for recovery from inactivation. These
effects of EPA are consistent with the previous findings, which showed
a prolongation of the recovery from inactivation of
INa, in the presence of PUFAs
(4, 41, 43).
Modulatory effects of 1-subunit on fatty
acid-induced inhibition of Na+ currents.
In cultured neonatal rat cardiac myocytes the IC50 of EPA
is 4.8 µM for INa,rat (41), which
is significantly higher than the IC50 of 0.51 µM for
INa, in HEK293t cells (43).
Coexpression of the 1-subunit with hH1 in
HEK293t cells reduced the percentage of block of Na+
channels by EPA, and the IC50 of EPA was 3.9 µM. A
similar phenomenon for lidocaine block of cardiac Na+
currents has been observed in oocytes coinjected with
hH1 and 1-subunits (24). The
affinity of resting channels to lidocaine was twofold lower in oocytes
coexpressing hH1 and 1-subunit than in
oocytes expressing hH1 alone. In the present study EPA-induced inhibition of cardiac Na+ currents was very
much voltage dependent and had a higher affinity to inactivated
channels. The significant positive shift of the steady-state
inactivation in HEK293t cells coexpressing the 1-subunit increases the channel availability, which reduces the inhibition of
INa, mainly due to state-dependent block
by EPA. This hypothesis is supported by the evidence that the
IC50 was similar between INa,
and INa, when the holding potential was set
at 150 mV. The data are consistent with the findings that
coexpression of the 1-subunit with hH1
shifts the state-dependent cocaine block of hH1
Na+ channels to the positive direction (39).
(43). The present data
indicate that coexpression of 1-subunit with
hH1 restores the selective effect of free long-chain
PUFAs on hH1 channels. The mechanism underlying the 1-subunit-induced restoration of selectivity to fatty
acids is unknown, but the shifted steady-state inactivation of
INa, may account for this restoration, at
least partially. Our data show that coexpression of
1-subunit and hH1 modifies the kinetics
of activation and inactivation of the channel, which is consistent with
our previous results with local anesthetics. These data support the
findings from another study (2), which suggests that
1-subunit functionally interacts with the regulatory segments for activation and inactivation of the Na+
channel. Our data also show that coexpression of
1-subunit and hH1 modifies the effects of
PUFAs on cardiac Na+ currents. Although the underlying
mechanism of the 1-subunit-induced changes in the
kinetics of activation and inactivation of
INa, is unclear, we speculate that
1-subunit increases the stability of Na+
channel structure conformation and reduces the sensitivity to voltage
alteration and chemical stimulation. Thus our results have implications
for the reduction of cardiac arrhythmias in vivo.
Mechanism of the prevention of ischemia-induced arrhythmias by
PUFAs: a hypothesis.
It has been reported that PUFAs affect the voltage-dependent
inactivation of cardiac Na+ channels in native cardiac
myocytes (41) and in HEK293t cells expressing
hH1 (4, 43). In the present
study we report that EPA shifts the steady-state inactivation in the
hyperpolarizing direction and has no effects on the activation of
INa,, INa,, and
INa,rat. The inhibitory effects of PUFAs on
human cardiac Na+ channels, which result from the
voltage-dependent shift of the steady-state inactivation potential to
more hyperpolarized values, as found in this as well as our previous
(43) report, may be critical for the potent antiarrhythmic
actions of these PUFAs. Ischemia-induced fatal ventricular arrhythmias
have been prevented in rats by fish oil feeding (17,
26, 27) and in dogs by intravenous
administration (7, 8). During a
myocardial infarction, there occurs a spectrum of depolarization of
myocytes in the ischemic tissue. Cells in the central core of the
ischemic tissue rapidly depolarize and die. Depolarization results from
the dysfunctional state of Na+-K+-ATPase and
the rise of interstitial K+ concentrations in the
ischemic tissue. However, at the periphery of the ischemic
zone, myocytes may be only partially depolarized. They become
hyperexcitable because their resting membrane potentials become more
positive, approaching the threshold for the gating of the fast
Na+ channel. Thus any further small depolarizing stimulus
may elicit an action potential, which, if it occurs out of phase with
the electrical cycle of the heart, may initiate an arrhythmia. In the
presence of the n-3 PUFAs, however, a voltage-dependent shift of
the steady-state inactivation to more hyperpolarized potentials occurs.
The consequence of this hyperpolarizing shift is that the negative
potential necessary to return these Na+ channels to a
closed resting but activatable state requires a physiologically
unobtainable hyperpolarized resting membrane potential. Also, these
partially depolarized cells have Na+ channels, which
quickly can slip into "resting inactivation" from the closed
resting state without eliciting an action potential in the presence of
EPA (see Fig. 7). The result of these two effects of the n-3 PUFAs
is that these partially depolarized myocytes are quickly eliminated
from function, and their potential arrhythmic mischief is aborted (see
Fig. 4, noting especially the absence of
INa, at a depolarizing stimulus from 70
to 30 mV in the presence of EPA). Myocytes in the nonischemic
myocardium with normal resting membrane potentials will not be so
drastically eliminated from function by PUFAs and continue to function
normally. This effect of the n-3 PUFAs on Na+
channels, together with their effect to inhibit the cardiac L-type Ca2+ channels and prevent triggered arrhythmic
afterpotential discharges due to excessive systolic Ca2+
fluctuations (20, 40), we currently think are
the major mechanisms for the antiarrhythmic effects of these PUFAs.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. R. G. Kallen for the hH1 clone,
Drs. L. L. Isom and W. A. Catterall for the rat brain
1-subunit clone, and Dr. S. C. Cannon for the CD8
clone and the HEK293t cell line.
| |
FOOTNOTES |
|---|
This study was supported in part by American Heart Association Grant 9930254N (to Y. F. Xiao) and National Institute of Health Grants DK-38165 and HL-62284 (to A. Leaf), DA-11762 (to J. P. Morgan), and GM-35401 (to G. K. Wang).
Address for reprint requests and other correspondence: Y.-F. Xiao, Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215 (E-mail: yxiao{at}caregroup.harvard.edu).
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. §1734 solely to indicate this fact.
Received 9 August 1999; accepted in final form 29 December 1999.
| |
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RESULTS
DISCUSSION
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) and
presence (EPA, 
) of 5 µM EPA. The testing currents were
normalized to their maximal values of INa,
