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KPQ
Na+ channels
1 Department of Medicine, University of Wisconsin, Madison, Wisconsin 53792; and 2 Department of Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Na+
current (INa)
through wild-type human heart Na+
channels (hH1) is important for normal cardiac excitability and
conduction, and it participates in the control of repolarization and
refractoriness. INa kinetics
depend strongly on temperature, but
INa for hH1 has been studied previously only at room temperature. We characterized early INa (the
peak and initial decay) and late
INa of the
wild-type hH1 channel and a mutant channel (
KPQ) associated with
congenital long Q-T syndrome. Channels were stably transfected in
HEK-293 cells and studied at 23 and 33°C using whole cell patch
clamp. Activation and inactivation kinetics for early
INa were twofold faster at higher temperature for both channels and shifted activation and steady-state inactivation in the positive direction, especially for
KPQ. For early
INa (<24 ms),
KPQ decayed faster than the wild type for voltages negative to
20 mV but slower for more positive voltages, suggesting a
reduced voltage dependence of fast inactivation. Late
INa at 240 ms was
significantly greater for KPQ than for the wild type at both
temperatures. The majority of late
INa for KPQ
was not persistent; rather, it decayed slowly, and this late component
exhibited slower recovery from inactivation compared with peak
INa. Additional
kinetic changes for early and peak
INa for KPQ
compared with the wild type at both temperatures were
1) reduced voltage dependence of
steady-state inactivation with no difference in midpoint,
2) positive shift for activation kinetics, and 3) more rapid recovery
from inactivation. This study represents the first description of human
Na+ channel kinetics near
physiological temperature and also demonstrates complex gating changes
in the KPQ that are present at 33°C and that may underlie the
electrophysiological and clinical phenotype of congenital long Q-T
Na+ channel syndromes.
long Q-T syndrome; human heart; ion channels; sodium current
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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SODIUM ION CURRENT (INa) is important for cardiac excitability and conduction through effects on the action potential upstroke, and late INa is also involved in maintaining the action potential plateau. These effects in turn play important roles in the mechanisms of arrhythmia by reentry and triggered automaticity. INa has generally been studied at less than physiological temperatures for convenience and to reduce and slow the currents sufficiently for adequate voltage control. The few studies of nonhuman mammalian cardiac INa at higher temperature show a steep and sometimes complex temperature dependence that causes, for example, a shift in the inactivation relationship (2, 16, 17).
The congenital long Q-T syndrome (LQT) is a hereditary cardiac disorder
that causes syncope and sudden death from ventricular arrhythmias such
as torsades de pointes and ventricular fibrillation. LQT3 is one form
of the disease for which the defective gene has been identified as
SCN5A, which encodes the voltage-dependent cardiac
Na+ channel 
-subunit in humans
(hH1; see Refs. 8, 12, 23, 24). Deletion of three amino acids (KPQ:
lysine, proline, and glutamine) at positions 1505-1507 in the
intracellular linker between domain III and domain IV is one of the
mutants in LQT3 (23, 24). Altered current decay rates and a small
"persistent" current at depolarized potentials have been reported
for the KPQ (1, 3, 6, 22). These studies have reported diverse results for the voltage dependence of current decay rates and the
magnitude of late currents, both of which are presumed to be important
in the arrhythmogenic mechanism that results in the clinical phenotype.
No studies have reported the recovery characteristics of the late
current. Moreover, just as with the wild-type human Na+ channel, all previous reports
of KPQ kinetics were obtained at room temperature. It is not known
whether or not the kinetic effects of KPQ are the same at more
physiological temperatures.
We characterized gating kinetics of the wild-type channel and KPQ
hH1 INa at room
temperature and at 33°C, comparing
1) kinetics of early macroscopic
INa decay,
2) amplitude and decay of late INa,
3) steady-state inactivation and
activation of peak
INa, and
4) recovery from inactivation of
peak and late
INa. Some of these data have been reported previously in abstract form (18).
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Clones and construction of KPQ
mutation. The human heart
Na+ channel clone that we used was
kindly provided by Dr. H. Hartmann (Baylor College of Medicine,
Houston, TX). This channel is designated hH1a because it differs in
nine amino acids with one deletion from the channel reported as hH1
(9): V120I, A180G, R552A, T559A, H987Q, Q1027R, W1085G, R1087E, G1088A,
deletion Q1077. These differences are not known to cause functional
effects; therefore, for simplicity we refer to the channel as hH1. The
nucleotide and amino acid numbering follow Hartmann et al. (9).
The KPQ mutation was made by polymerase chain reaction (PCR)
techniques according to Higuchi et al. (10). The primers used generated
PCR products through the Kpn I (base
pair 4253) and BstE II (base pair
4657) sites of hH1a. The mutant primers deleted bases 4539 through
4547, which code for amino acids lysine (K1504), proline (P1505), and
glutamine (Q1506). In addition, these primers introduced a silent
restriction site by a C-to-A mutation at base 4532, which adds a
BamH I site without altering the amino
acid sequence. The PCR products were subcloned into hH1a in the pGEM3 construct at Kpn I and
BstE II, and expression was first
tested in Xenopus oocytes. The mutant
region was then shuttled to hH1a in a mammalian expression vector
Rc/CMV (Invitrogen) using unique Age I
(base pair 1045) and BstE II (base
pair 4657) sites. The entire PCR generated region was completely
sequenced and confirmed the deletion and also confirmed that no other
unwanted changes were made in the channel.
Cell preparation and transfection. Approximately 5 × 105 cells from a transformed human embryo kidney cell line (HEK-293) were seeded on a 60-mm-diameter plate (Falcon 3001) with 3 ml of culture medium a day before the transfection. Culture medium was MEM complete medium containing MEM (Eagle's salts and L-glutamine), 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid solution, 1 mM MEM pyruvate solution, 10,000 units penicillin, and 10,000 g streptomycin.
Transfection was carried out by using a cationic liposome method. The cationic lipid used was N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) at a concentration of 1 µg/µl. DOTAP was obtained from the Medical School Vector Core Laboratory, University of Wisconsin-Madison. DOTAP (20 µg) and plasmid DNA (5 µg) were diluted with Opti-MEM (GIBCO-BRL), and final volume for transfection was 200 µl. The DOTAP-DNA mixture was incubated with cells for 5 h in the 2 ml of Opti-MEM medium. After incubation, cells with the DOTAP-DNA mixture were replaced with 3 ml of normal culture medium. To select stably transfected cells, geneticin (G418 sulfate; GIBCO-BRL) at a total concentration of 800 µg/ml was added for ~15 days, at which time surviving single colonies were isolated and cultured with 400 µg/ml geneticin for 1-3 wk. Cells were then treated with a trypsin-EDTA solution (0.25% trypsin, 1 mM EDTA; GIBCO-BRL), centrifuged at 2,000 rpm for 2 min, and frozen in FBS (GIBCO-BRL) with 7% DMSO for storage up to 6 mo. Cells for study were thawed, transferred to normal culture media for 1-6 h, and then transferred directly to the experimental chamber.
Electrophysiological recordings.
Macroscopic INa
was recorded using the whole cell patch-clamp technique. The bath
(extracellular) solution contained (in mM) 140 NaCl, 4 KCl, 1.8 CaCl2, 0.75 MgCl2, and 5 HEPES (pH 7.4 with
NaOH). The pipette solution contained (in mM) 120 CsF, 20 CsCl, 5 EGTA,
and 5 HEPES (pH 7.4 set with CsOH). The electrodes were pulled (P-87;
Sutter Instrument) from borosilicate glass and heat polished with a
microforge (MF-83) to a final resistance of <1.2 M
when filled with the electrode solution. Cells for study were placed in
a Plexiglas chamber with continuous-flowing bath solution mounted on an
inverted microscope (Nikon) in a Faraday cage. The temperature was
controlled via heat exchange through a water jacket surrounding the
bath solution, and the bath temperature was monitored.
Electrophysiological recordings were carried out at room temperature of
23°C and at 33°C. Experiments were attempted at 37°C, but
the patch-clamp seal did not remain stable for a time sufficient to
complete the protocols. Membrane currents were recorded with an
Axopatch 200 amplifier (Axon Instruments). Data were acquired using
pCLAMP v6.03. Data were digitized at 100 kHz for early current or 10 kHz for late current and were low pass filtered at 10 kHz. Holding
potential was 150 mV except where otherwise noted as in, for
example, recovery from inactivation protocols.
We observed a time-dependent shift of the steady-state inactivation relationship even though the preparation was otherwise stable. To minimize effects of the time-dependent change of availability, we recorded over a 5-min period beginning 2 min after the whole cell configuration was achieved. Steady-state availability was assessed initially and at the end of this period, and the shift was usually <3 mV. In those cases in which the shift was >3 mV the data were discarded.
Data analysis. Passive leak
subtraction of peak and late currents was performed by subtracting a
linear leak extrapolated from holding currents at subthreshold
potentials (less than 100 mV) to the potential of interest (15).
Saxitoxin (STX)-sensitive currents were measured by subtracting a trace
obtained in the presence of 5 µM STX, a concentration that completely
blocked INa, from
a trace obtained earlier in the absence of STX (referred to as STX
subtraction). Data were fit to model equations using nonlinear
regression using pCLAMP v6.03 or SigmaPlot 3.0. Goodness of fit was
judged both visually and by the sum of squared errors. In choosing
models with a greater number of parameters to fit the data, as in a
two-exponential fit over a single exponential fit, an
F-ratio test was used
(P < 0.05) to account for the
increased number of free parameters. Mean data are expressed with their SE. All determinations of statistical significance of mean data were
performed by using a Student's t-test
for comparisons of two means. A P
value of <0.05 was considered statistically significant.
Technical considerations. Mammalian
cell lines such as HEK-293 cells are tools widely used in studying
structure-function relationships of ion channels, but this use is based
on the supposition that endogenous currents are absent or minimal.
Endogenous currents were measured in nontransfected HEK-293 cells and
also in cells transfected with hH1 and the KPQ where
INa was blocked
by STX. Under our study conditions (CsF in pipette solution) a
noninactivating outwardly rectifying current was measured at voltages
>0 mV. This current was between 100 and 500 pA and activated over
several hundred milliseconds during a depolarizing step. This contrasts with a previous report in these cells of a rapidly activating and
inactivating outward endogenous current under other conditions (KCl in
pipette solution; see Ref. 27) and emphasizes the need to consider
endogenous currents under the specific study conditions. The endogenous
current also gradually increased over the data collection period;
therefore, STX subtraction did not perfectly correct for the endogenous
current. At voltages <0 mV, the range of primary interest, the
endogenous current was negligible (<1% of late
INa at 240 ms)
but tended to increase with length of depolarization.
Adequate voltage control was achieved by using low-resistance pipettes, high conductive solutions, and series resistance compensation. To further minimize possible errors arising from loss of voltage control, we selected clonal lines expressing lower INa density. The small size and low capacitance (<10 pF) of these cells allowed for very rapid charging of the membrane capacitance. Two indexes of voltage control that we required to be present were a graded slope of the activation curve (Boltzmann slope factor >5.0) and scaling of currents of different amplitudes to the same test potential as in steady-state inactivation and recovery protocols. These indirect measures are standard, and they have been validated in other preparations (15). The data at higher temperatures where the currents are larger and faster showed no differences in slope of the activation relationship (Table 1), suggesting that voltage control was maintained.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Characterization of macroscopic
currents. The activation relationship for
INa was obtained
from peak INa
resulting from a 24-ms step to various test potentials from a holding
potential of 150 mV. Figure
1A shows
representative current tracings for the wild-type current and the
KPQ at 23 and 33°C from different cells. At the higher
temperatures, macroscopic currents peaked earlier and decayed faster,
and peak INa was
greater. The KPQ showed faster initial decay compared with the
wild-type current, and the peak of the current-voltage relationship was
shifted in the positive direction (Fig.
1B).
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To compare the differences in the time course of current activation and
decay, representative currents for test potentials of 40,
20, 0, and 30 mV were normalized to their peak values, and the
traces for wild-type current and the KPQ were superimposed (Fig.
2). Current decays were more rapid for the
KPQ than the wild-type current at 40 and 20 mV, but,
at 30 mV, current decays were more rapid for the wild-type current.
Currents recorded at the higher temperature (Fig. 2,
bottom) showed more rapid kinetics, but the differences between
INa decay rates
for KPQ and the wild type remained.
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Figure 3 shows summary data for the voltage
dependence of time to peak amplitude of
INa, time
constants of INa
decay, and fractional amplitude of
INa decay at 23 and 33°C. Time to peak INa, an index of
activation rate, was reduced at higher temperatures by about one-half
(compare Fig. 3, A and
B).
INa for the
KPQ peaked earlier than the wild type at 50 mV but tended to
peak later at more depolarized potentials, and the crossover point was
temperature dependent.
INa decay, an
indicator of inactivation rate, at most potentials was best fit to two
exponentials over 24 ms, and the time constant for the fast component
is shown in Fig. 3, C and
D. Like time to peak, decay time
constants were faster at the higher temperature. The first component of
the time constants for macroscopic current decay was significantly
(P < 0.05) more rapid for the KPQ
than for the wild-type current from 40 to 10 mV but
crossed over at potentials more positive than 0 mV (Fig.
3C) at 23°C and at 15 mV
(Fig. 3D) at 33°C. The
difference in INa
decay for the KPQ compared with the wild-type current at 33°C,
however, was less than that at 23°C and did not reach statistical
significance. This finding, therefore, may be of biophysical importance
but less interesting for physiological or pathophysiological mechanisms.
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Late INa.
Late INa was
investigated using prolonged (240-ms) depolarizing steps to various
test potentials from a holding potential of 150 mV. The KPQ
showed a readily apparent late inward
INa (called
persistent, "plateau," or "pedestal"
INa previously)
at both 23 and 33°C (Fig.
4A). We
measured INa at
240 ms and normalized it by dividing by the maximum peak amplitude of
INa from the
current-voltage relationship. The mean values of normalized late
INa at 240 ms for
the wild-type current were 0.21 ± 0.02% at 23°C
(n = 15 experiments) and 0.19 ± 0.03% at 33°C (n = 8 experiments), and values of normalized late
INa for the
KPQ were 0.76 ± 0.06% at 23°C
(n = 22 experiments) and 0.60 ± 0.10% at 33°C (n = 13 experiments). The KPQ exhibited significantly more current at 240 ms
than the wild type at both 23 and 33°C
(P < 0.01), but there was no
significant difference between 23 and 33°C.
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KPQ was not persistent but decayed in a multiexponential manner
(Fig. 4A). This decay was faster at
33°C compared with 23°C (Fig.
4A). To verify that these late
currents were
INa, we used STX,
a relatively specific blocker of
Na+ channels. Representative
INa traces for
the KPQ in response to a depolarization to 20 mV without STX
(Fig. 4B,
left) and with STX subtraction (Fig.
4B,
right) confirm the late currents as
INa. Data for the
current-voltage relationship of leak-subtracted late currents measured
at 20, 40, 120, 240, 480, and 720 ms are shown without STX (Fig.
4C,
left) and with STX subtraction (Fig. 4C,
right), indicating that late
INa was STX
sensitive and decayed at all potentials.
To further characterize the decay of late
INa, we fit
INa traces over
240 ms with a three-component exponential function for the KPQ, and
the results of the fitting are shown in Fig. 3, E and
F, for time constants of decay and in
Fig. 3, G and
H, for the relative amplitudes of the
corresponding components as well as the "baseline" or persistent
current. Only two component results are shown for wild-type late
INa because three
exponential fits were not consistently found, perhaps because of the
smaller size of the late
INa in the wild
type. At 23°C both KPQ and the wild-type current showed a second
slow decay time constant between 3 and 8 ms with a modest voltage
dependence (Fig. 3E), and this time constant was decreased by about one-half at 33°C (Fig.
3F). A third decay constant between
70 and 100 ms was detected for KPQ, again increasing modestly with
stronger depolarizations (Fig. 3E).
The time constant for this third component of decay, however, was not
as strongly temperature dependent (Fig.
3F). Figure 3, G and
H, shows the relative amplitude of
each component of decay and the baseline or persistent current from the
fit. For both wild-type and KPQ the relative amplitude of the
fastest decay component was comparable at 23°C (Fig.
3G) and 33°C (Fig.
3H). The second or intermediate slow
component amplitude was greater for the wild-type current than for
KPQ at both 23°C (Fig. 3G) and 33°C (Fig. 3H), perhaps
because the third component and baseline were too small to be
distinguished in the wild-type current and were included in this
component for the wild-type but not for KPQ. At 23°C the
relative amplitude of the third component of decay for KPQ and the
baseline component was comparable at or near 1% of peak
INa (Fig.
3G), but at 33°C the baseline
current was smaller, and the third component was larger (Fig.
3H).
Activation and steady-state
inactivation. The voltage dependence of steady-state
inactivation was assessed by a two-pulse protocol as indicated in the
protocol diagrams in Fig. 5, insets. Peak INa was
measured at a test potential of 20 mV after 1-s-long conditioning potentials between 150 and 30 mV. Peak
currents were normalized to the largest peak current obtained, and data were fitted with a Boltzmann function to yield a midpoint and slope
factor. Table 1 shows the parameters of Boltzmann fits at 23 and
33°C. The midpoint for availability was not significantly different
between the wild-type current and the KPQ, although the slope factor
was significantly different for KPQ at 23°C. There were no
significant differences in either the slope factor or the midpoint
between the wild-type current and the KPQ at 33°C.
The voltage dependence of activation was evaluated as a normalized peak
conductance-voltage relationship (Fig. 5)
and was fitted with a Boltzmann function. The midpoint of the
activation relationship for the KPQ was shifted in the positive
direction at 23°C (<0.05) without a significant difference in
slope factor. At 33°C, both the slope factor and midpoint between
the wild-type current and the KPQ were significantly different
(Table 1). As for the effects of temperature on the activation and
inactivation kinetics, at 33°C both the inactivation and the
activation curves for both channels tended to shift in the positive
direction compared with 23°C although a significant difference was
observed only for the KPQ (Table
1).
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Recovery from inactivation of peak and late
INa.
Recovery from inactivation at 120 mV was studied with a
two-pulse protocol (see Fig. 6,
inset). Cells were stepped to
20 mV for 1 s during which time
INa was
inactivated almost completely, and test pulses to 20 mV were
delivered after recovery steps to 120 mV. Recovery from
inactivation was best fit to a double exponential function, and the
fast time constant was significantly faster in the KPQ at 23°C.
At 33°C, recovery from inactivation was more rapid than that at
23°C, and both the fast and the slow time constants were
significantly faster in the KPQ (Table
2). Interestingly, although the time
constants themselves indicated that recovery was faster at higher
temperature, the relative contribution of the slow component of the
recovery process was increased at the expense of the fast component in
both the wild-type current and the KPQ at the higher temperature
(see Table 2).
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20 mV, see Fig. 6,
inset), but a prolonged test
potential was used, and the
INa at 240 ms was
measured and plotted for the recovery time (Fig. 6). Approximately 40%
of late INa at
240 ms did not inactivate with a 1-s conditioning pulse, corresponding
to the proportion of baseline current noted previously (Fig. 3,
G and
F). Late
INa for KPQ
recovered more slowly from inactivation compared with peak
INa, and the
recovery process was more rapid at 33 than at 23°C.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Temperature dependence of human heart
INa.
This study is the first report of human cardiac
INa at near
physiological temperatures (technical considerations limited the study
to 33°C rather than 37°C). In general, the temperature
dependence of the gating kinetics for the human heart
Na+ channel is much like that
reported for nonhuman cardiac channels (13, 14, 17) with a temperature
coefficient for indexes of activation and inactivation of about two for
both the wild type and the KPQ and a shift in activation. An
interesting finding noted only briefly previously (14) is that the
relative amplitude of the slow component of recovery is increased at
higher temperatures for both the wild-type current and the KPQ. This
implies that entry into the kinetic state(s) from which recovery is
slow has a higher temperature dependence than entry into the more rapid recovery state(s). Both steady-state inactivation and activation relationships were more positive with increasing temperature in both
the wild-type current and the KPQ. This observation has also been
reported by Murray et al. (17) for guinea pig ventricular cells; the
mechanism for this positive shift is unknown but is probably related to
a different temperature dependence of the individual gating
transitions. Repolarization of the action potential depends on a
delicate balance of relatively small inward and outward currents during
the cardiac action potential plateau. As computer modeling is
increasingly used to explore the underlying arrhythmogenic mechanism
(13, 26), including details of these kinetic differences with different
isoforms, mutants, and temperature will be important in understanding
the relative contributions of each current to the arrhythmia.
KPQ was maintained at each temperature.
With more rapid decay at higher temperature, late current might be
expected to be smaller at higher temperature. The third or slowest time
constant for KPQ
INa decay,
however, was not significantly decreased at higher temperatures in
contrast to the first and the second time constant (Fig. 3,
C-F) . Slower recovery of late
INa compared with
peak INa was
found at both temperatures (Fig. 6). These results suggest that the
late INa kinetics
(slow inactivation decay and slow recovery) are different from peak
INa and also
correspond with the reported mechanisms that the late
INa in the KPQ
may result from mode switching to the slow gating kinetics (3, 4, 6).
Comparison of the wild type and KPQ
kinetics. The KPQ mutation lies in the III-IV linker
thought to be the inactivation gate, and defective inactivation and
persistent current have been postulated as the cause of Q-T
prolongation (7, 9, 25). In the present study, however, we found
complex effects of the KPQ mutation on kinetics:
1) initial current decay was
actually more rapid and less voltage dependent, that is, faster for
negative test potentials and slower for positive test potentials;
2) late currents for KPQ were
larger than for the wild-type current but smaller than reported
previously by other investigators;
3) late currents were not time
independent but rather inactivated slowly with a multiexponential time
course and recovered from inactivation with slower time course compared
with peak INa;
4) activation was shifted to more
positive voltages; 5) the midpoint
of steady-state inactivation was not different, but slope factor was
changed at 23°C; and 6) recovery
from inactivation was accelerated.
The present study showed that currents for KPQ actually have a more
rapid initial decay at most negative voltages, but a lower voltage
dependence results in a crossover (Figs. 2 and 3) such that at positive
voltages decay is faster for the wild type. Wang et al.
(22) reported that the fast time constant of the KPQ
was larger than the wild type at potentials greater than 20 mV
for a holding potential of 120 mV, but their data also clearly
indicate a lesser voltage dependence of the fast time constant of the
KPQ. An et al. (1) reported that the onset of inactivation, using a
holding potential of 90 mV, was faster for the KPQ, but
current decay was fitted by a single exponential function. A recent
report by Chandra et al. (4) for channels expressed in mammalian cells
showed that early decays fit by a single exponential were more rapid
for KPQ at negative voltages, results very similar to Fig.
3C. Other reports (3, 6) using the
oocyte expression system showed faster current decay for KPQ at test
potentials of 20 or 10 mV. Taken together, these
observations suggest that macroscopic current decay for the KPQ has
lower voltage dependence compared with the wild-type current and decay is faster at negative voltages. A positive shift of the activation curve for KPQ compared with the wild-type current agrees with the
data by Wang et al. (22). However, this kinetic change should not
necessarily be attributed to a mutation-induced change in activation
kinetics, because the faster initial rate decay might account for some
of this change.
A major difference of our findings from previous reports was that
persistent current was much less than that reported previously. An et
al. (1) and Wang et al. (22) found that persistent currents were 4.0 and 2.6% of the peak amplitude, respectively, using the same
expression system (HEK-293), whereas our value of persistent current
was 0.76% at 23°C. Late
INa, however,
slowly decayed in a time-dependent manner, a finding not previously
recognized. The magnitude of late
INa, therefore,
will depend on the time of measurement. We estimated the ratio of
persistent current at 240 ms, whereas previous reports used earlier
time points. The lower value for late current that we report results,
in part, from the time-dependent decay of late
INa. Late
INa measured in our preparation at the same time points as those reported previously, however, still yielded a smaller relative late
INa amplitude.
Although no significant difference in midpoints of steady-state
inactivation between the wild type and KPQ were detected in the
present study, the slope factors at 23°C were significantly different. Depending on the extent to which macroscopic current decay
represents inactivation, this could be consistent with the observed
decrease in the voltage dependence of decay rates for KPQ (Fig.
3C). Macroscopic decay, however, is
a convolution of the kinetics of activation and inactivation, and the
changes in steepness of the inactivation curve and the decay curves may
also indicate an alteration of coupling between the activation and inactivation process.
Implication for the clinical
phenotype. The rapid onset of fast inactivation and
faster recovery from inactivation for the KPQ are generally reported
findings (1, 22). Recently, fast recovery from inactivation was
reported as a possible mechanism for arrhythmogenesis in the SCN5A
mutation that caused idiopathic ventricular fibrillation (5). Although
the persistent current of
INa contributing
to prolongation of the Q-T interval is likely to be an important
mechanism for arrhythmogenesis, the complex gating changes in the
present study suggest that the arrhythmogenic effects may not be
limited to Q-T prolongation in the KPQ but may also be exacerbated
by altered excitability of the channel and effects on conduction and
refractoriness because of these altered kinetics.
A new finding in this paper was that late
INa recovered
from inactivation more slowly than peak
INa. The time
constants are such that late
INa would be
decreased with high-frequency depolarization because of accumulation of
inactivation. This mechanism may explain the shortening of the
prolonged Q-T interval with increased heart rate reported in the LQT3
patients (20) and the shortening of action potential duration by rapid
pacing in the experimental model for LQT3 (19).
Human heart INa
demonstrates more rapid gating kinetics with increased temperature
similar in magnitude to that in previous reports in nonhuman channels.
A mutant channel, KPQ, associated with the congenital LQT exhibits
complex gating changes, including a relative increase in late currents
that may account for the clinical phenotype. This late current shows a
gradual decay and slow recovery from inactivation, indicating that it
is subject to a slow inactivation process.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-56441 (J. C. Makielski) and HL-20592 (J. W. Kyle), by grants from the University of Wisconsin Cardiovascular Research Center and the Oscar Rennebohm Foundation, and by a travel grant to T. Nagatomo from the Fukuda Memorial Foundation.
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FOOTNOTES |
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Address for reprint requests: J. C. Makielski, Univ. of Wisconsin Clinics and Hospitals, 600 Highland Ave H6/349, Madison, WI 53792.
Received 24 November 1997; accepted in final form 6 August 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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1.
An, R.-H.,
R. Bangalore,
S. Z. Rosero,
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 J. A. Towbin, Z |
) and 
) for the same cells in A. Peak of
the current-voltage relationship was more positive for 
f;
C and
D), slow time constant
(
; third: 
) at 23°C
(n = 8 experiments) and at 33°C
(n = 4 experiments). The portion of
the trace between 90% of peak
INa and 240 ms
was fit with three exponential functions:
INa(t) = 1 
) and 
) are plotted. For 23°C,
n = 5 (WT) and
n = 8 (
]
1;
normalized GNa = [1 + exp(V1/2 