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Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Several aspects of the effect of the KPQ
deletion mutation on Na+ channel
gating remain unresolved. We have analyzed the kinetics of the early
and late currents by recording whole cell and single-channel currents
in a human embryonic kidney (HEK) cell line (HEK293) expressing
wild-type and KPQ deletion mutation in cardiac
Na+ channels. The rate of
inactivation increased three- to fivefold between
40 and
80 mV in the mutant channel. The rate of recovery from
inactivation was increased twofold. Two modes of gating accounted for
the late current: 1) isolated brief
openings with open times that were weakly voltage dependent and the
same as the initial transient and 2)
bursts of opening with highly voltage-dependent prolonged open times.
Latency to first opening was accelerated, suggesting an acceleration of
the rate of activation. The
KPQ mutation has multiple effects on
activation and inactivation. The aggregate effects may account for the
increased susceptibility to arrhythmias.
long Q-T syndrome; sodium channel; embryonic kidney cells; patch clamp
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INTRODUCTION |
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RECENT STUDIES HAVE SHOWN that the genetic defect in a
subgroup of patients with the autosomal dominant form of the long Q-T syndrome is linked to mutations in the cardiac
Na+ channel
-subunit gene on
the short arm of chromosome 3 (37, 38). The most severe defect is a
deletion mutation of nine bases that code for Lys-1505, Pro-1506,
Gln-1507 in the linker between domains III and IV of the
Na+ channel
-subunit. Prior
studies have shown that this linker is a highly conserved region of the
Na+ channel and plays a central
role in inactivation (15, 30, 36, 41).
Initial reports using heterologous expression of the mutant
Na+ channel
-subunit in frog
oocytes by Bennett et al. (6) and Dumaine et al. (10) demonstrated a
small component of Na+ current
that persisted after termination of the initial transient. This late
component of Na+ current could
prolong the duration of the cardiac action potential and the Q-T
interval of the surface electrocardiogram (ECG). There is some
controversy as to the nature of the persistent current and of the
effect of the KPQ deletion mutation on other aspects of
Na+ channel gating (6, 10). In
their initial report, Bennett et al. reported an acceleration of the
macroscopic inactivation of the current, whereas Dumaine et al.
reported no change. Bennett et al. described a single mode of gating,
with bursts accounting for the late current. Dumaine et al. suggested
two modes of gating accounting for the late current: isolated openings
and bursts of openings. Neither study provided data on the time
dependence of late events. When the
Na+ channel
-subunit of the
brain and skeletal muscle is expressed in frog oocytes, the
inactivation kinetics are slower than those of the native channel (20,
22). This change in gating is corrected by coexpression of the
- and
-subunits (18). In the case of the cardiac
Na+ channel, the data are
controversial, with studies showing no effect or an acceleration of
inactivation with
-subunit coexpression (24, 26). The large size of
the oocyte and the geometric complexity of its surface membrane limit
its utility in the analysis of Na+
channel kinetics (21, 25). Bennett et al. reexamined the gating
kinetics of the long Q-T interval-associated mutant channels expressed
in a mammalian cell line using whole cell recordings. In contrast to
their earlier studies in the frog oocytes, they showed an acceleration
of recovery from inactivation. Their data also suggested that overlap
in the activation and inactivation curves also contributes to the late
current, a conclusion that differs from that of Dumaine et al. (10).
We have examined the kinetics of gating of the wild-type and
KPQ
mutant Na+ channel
-subunit
stably expressed in a human embryonic kidney (HEK) cell line (HEK293).
The small size and spherical shape of the HEK293 cells permit rapid
voltage clamping. The Na+ channel
kinetics more closely resemble those in the native cells (33). We have
combined whole cell and single-channel recordings to determine the
basis of the persistent current and the other effects of the KPQ
deletion on channel gating. By combining whole cell and single-channel
recordings, it was possible to relate changes in the whole cell current
waveform to the underlying changes in gating. Our results show that the
KPQ deletion has a wide range of effects on both the activation and
inactivation of the Na+ channel.
Robust persistent currents were observed at potentials of
20 and
10 mV, well outside the range of activation and inactivation overlap but sufficiently depolarized to contribute to prolongation of
the plateau of the action potential. The composite effects of the
mutation on gating may be important in the increased susceptibility to
arrhythmias in patients with this mutation.
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METHODS |
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Construction of hH1 and deletion mutant KPQ/hH1. The 5' segment of the human cardiac Na+ channel gene hH1 from the starting Met at a position 150 bp to the Xho I site at 683 bp was synthesized using polymerase chain reaction (PCR). A Hind III site was introduced preceding the ATG codon to facilitate cloning of the 5' end. The PCR product was restricted with Hind III and Xho I and cloned into the corresponding sites in pREP4 plasmid (Invitrogen, San Diego, CA). hH1 in pSP64 parent plasmid (clone generously provided by Dr. A. George) was restricted with Xho I and Sfi I. The ~5.5-kb band was excised and cloned into the corresponding sites in pREP4. The Sfi I site had to be repaired in both the plasmid and insert because the sequence of the site was different.
To perform the
KPQ mutation, hH1 was first cloned into pcDNA3
(Invitrogen) by using the above strategy, except the
Sfi I repaired end of hH1 was ligated
to the Xba I repaired end of pcDNA3. The deletion of KPQ was done by recombinant PCR (16). The PCR product
carrying the mutation was restricted with
Kpn I and
BstE II and used to replace the
wild-type segment in hH1. All PCR products were sequenced in their
entirety by using the chain termination method of Sanger et al. (32).
Expression of hH1 and KPQ deletion mutant in mammalian cells. DNA (10 µg) was transfected into 293-EBNA cells (pREP4 plasmid) or HEK293 cells (pcDNA3 plasmid) by using Lipofectamine (GIBCO-BRL, Gaithersburg, MD) in DMEM without serum or antibiotics. After 4 h of incubation at 37°C, an equal amount of DMEM containing 20% FBS was added to the medium over transfected cells. After 24 h of transfection the cells were trypsinized, counted, and aliquoted into a 96-well plate at a density of 10,000 cells/well. Individual colonies were picked, grown, and tested for Na+ channel expression by whole cell voltage clamping.
Cells transfected with hH1 cloned in pREP4 were grown and selected in DMEM containing 10% FBS, nonessential amino acids, penicillin (100 U/ml), streptomycin (100 µg/ml), and hygromycin (300-500 µg/ml; Boehringer Mannheim, Indianapolis, IN). Cells transfected with KPQ deletion mutant cloned in pcDNA3 were grown and selected in DMEM containing 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and G-418 (500 µg/ml; GIBCO-BRL).Experimental setup.
A coverslip containing cultured HEK293 cells was superfused in a tissue
bath for at least 10 min before recordings were initiated. For whole
cell recordings, cells were perfused with an
Na+ external solution.
Micropipettes were filled with a
Cs+ internal solution. The
internal Cs+ block the endogenous
K+ current in these cells. The
only ionic current measured under these conditions is
Na+ current. For single-channel
recordings, cells were perfused with a
high-K+ solution. This solution
reduced the membrane potential to 0 mV. Membrane potential is reported
as absolute values. Na+ and
K+ were the major cations in the
microelectrode solution. Occasionally (<1% of trials), we observed
long single-channel openings that apparently did not result from the
opening of Na+ channels; the
current was outward between
30 and 0 mV. Single Na+ channel currents were inward
in this potential range. The outward channel openings are probably the
endogenous K+ currents resolved at
the single-channel level. Depolarizing trials showing outward currents
were excluded from analysis.
Recording techniques.
Whole cell and single-channel currents were recorded with an
integrating patch-clamp amplifier (model 3900 A, Dagan, Minneapolis, MN) with a whole cell expander module (model 3911 A, Dagan). Whole cell
currents were recorded with 0.5- to 1.5-M
microelectrodes. An
Ag-AgCl electrode wire coupled each microelectrode to the input of the
amplifier. A similar wire embedded in agar-micropipette solution formed
the bath reference. Series resistance compensation was achieved using
conventional and supercharging techniques (4). Each whole cell
voltage-clamp experiment started with an assessment of adequacy of
voltage control, as previously described (11). The holding potential
was set at
100 mV. Stable whole cell recordings could not be
obtained in these cells for prolonged periods at hyperpolarized
potentials. The current-voltage relationship was determined with 20-ms
pulses of increasing amplitude applied at 1,000-ms intervals. The pulse
amplitude was incremented in 5-mV steps from a potential of
60
to +60 mV. The steady-state inactivation curve was determined by the
application of 200-ms prepulses from
130 to +55 mV. A 20-ms test
pulse to
20 mV followed the prepulse. The prepulse potential was
incremented in 5-mV steps. The development of inactivation was
determined at
40,
60, and
80 mV by application of
prepulses of increasing duration followed by a test pulse to
20
mV. Recovery from inactivation was determined with a 50-ms prepulse to
20 mV, a variable recovery interval at a conditioning potential
(Vc), then a
test pulse to
20 mV. Whole cell currents were filtered at 10 kHz, digitized at 40 kHz, and stored on the fixed drive of a
microcomputer (Compaq 386/20). Voltage command pulses were also
provided with a microcomputer equipped with a digital-to-analog
interface (TL1 interface with Labmaster boards, Axon Instruments,
Burlingame, CA).
microelectrodes coated with Sylgard (Dow
Corning, Midland, MI) up to the tip. The holding potential was usually
set at
100 mV, and 200-ms test pulses were applied to various
potentials. In a majority of experiments, test pulses were applied to
20 and
50 mV. Steps to other potentials were applied if
the recording condition remained stable. The time dependence of the
persistent current was obtained with 200-s depolarizing pulses to
potentials of
50 to 0 mV. Currents were filtered at a corner
frequency of 2-2.5 kHz by using an eight-pole Bessel filter (model
902 LPF, Frequency Devices, Haverhill, MA). Filtered currents were
digitized at 20 kHz.
Data analysis. The procedures for analyzing whole cell and single-channel data are similar to those reported in previous studies from this laboratory (11, 13). Peak currents were measured with custom software written in C programming language. Activation and inactivation were fit with a Boltzmann function (Eq. 1) using a Marquardt routine
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(1) |
rec) was obtained from the
following equation
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(2) |
is the
steady-state current at the test potential. The recovery from
inactivation was fit by a single exponential at a recovery potential of
90 mV. The rate of development of inactivation was also fit with
a single exponential function.
The residual Na+ current at late
times was determined from leakage-subtracted currents. Whole cell
currents were recorded at high gain using five voltage steps, each of
10-120 mV (in 10-mV increments). The currents for the 10-mV step
were averaged, scaled, and subtracted from the current obtained at
other test potentials. Single-channel currents were also leakage and
capacity transient subtracted before analysis. Currents during each
depolarizing trial were scanned, and those trials without events
(nulls) were collected. The nulls were averaged and subtracted from
each trial to remove residual leakage and capacity current. In some
multichannel patches, no nulls were observed. In those experiments,
20-30 trials with depolarizations of 10 or 20 mV were performed.
These trials were scanned, and the nulls were averaged, scaled, and
used for leakage and capacity transient subtraction. An automatic
detection scheme with the threshold set at 0.5 times the single-channel amplitude was used to identify channel openings. Closed times were
determined during segments of current records that had no overlapping
events. Less than 12% of trials were excluded because of overlapping
events.
Open and closed time histograms were fit with exponentials using a
least-squares procedure. The bin width was set at an integer multiple
of the sampling interval. Using simulated data from a gating model with
known rate constants, we have shown that our fitting technique provides
an accurate measure of event distribution (17). Most open times of
single Na+ channels span a single
logarithmic unit. Under these circumstances, logarithmic binning as
proposed by Sigworth and Sine (34) provides no advantage over linear
binning. Values are means ± SE. Comparisons were made by unpaired
t-test or ANOVA. Channel openness
during 2-s segments of 200-s depolarizing trials was compared by
2 analysis.
P < 0.05 was considered significant.
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RESULTS |
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Whole cell currents.
For the wild-type and
KPQ mutant
Na+ channel, we identified two
clones from each construct that expressed robust
Na+ currents of several
nanoamperes. In <5% of untransfected cells we observed endogenous
whole cell Na+ currents of
10-20 pA. These were not of sufficient amplitude to affect the
results reported in this study. Figure 1
illustrates the current-voltage relationship and conductance-voltage
and steady-state inactivation curves in cells expressing the wild-type
and
KPQ mutant Na+ channels.
Measurable current was observed at
60 mV. There was a gradual
rise in current amplitude with increasing depolarization for both
channel types; peak inward current was observed at
20 mV.
Potential for half-activation (
37.4 and
37.9 mV) and
slope factor (5.8 and 7) were similar for the two channel types. The inactivation curves show a progressive decline in current amplitude as
the conditioning potential was reduced. There was no crossover of the
current waveform as current amplitude declined. Potentials for
half-inactivation were
83.2 and
91.5 mV; slope factors
were 6.5 and 5.3 for wild-type and
KPQ mutant channels,
respectively. Unlike our observations and those of others in native
cells, these parameters were stable under the present recording
conditions (14). At 15 and 30 min, these parameters were
84.5 mV
and 6.4 and
84.9 mV and 6.4, respectively, for the cell
expressing the wild-type channel. Similar stability was noted during
long recordings from another cell.
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KPQ mutant
Na+ channel are compared in Table
1. The potential for half-activation is
similar to that reported for Na+
current recorded from rabbit atrial cells with the perforated-patch technique (40). It is positive to that observed with
F
-containing micropipette
solution in the conventional whole cell configuration (11). The
potential for steady-state half-inactivation was similar in wild-type
and mutant Na+ channels. It was
intermediate between that observed with the perforated and conventional
whole cell techniques (11, 40). There were clear differences in the
kinetics of development of and recovery from inactivation between the
wild-type and
KPQ mutant Na+
channels. At a potential where there was no significant inward current
(
80 mV) and at potentials at which there was measurable inward
Na+ current (
60 and
40 mV), inactivation developed much more rapidly in the
KPQ
mutant channel. Similarly, the rate of recovery from inactivation at
90 mV was almost twice as fast in the
KPQ mutant Na+ channel.
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100 to
40 mV. For cells expressing each
channel type, the relaxation of the current was well fit by single
exponentials. The time constants
(
c) for wild-type and
KPQ
mutant currents were 3.1 and 1.6 ms, respectively. Summary data are
presented in Fig. 2C. Between
50 and +40 mV,
c
decreased sevenfold for the wild-type channel. In contrast,
c for the
KPQ mutant channel was weakly voltage dependent, decreasing only 1.7-fold over the same
voltage range.
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KPQ mutant. Figure
3 shows leakage-subtracted
Na+ currents recorded at high gain
(A) and
Na+ current recorded at a lower
gain (B) from the same cell. Holding and test potentials were
100 and
20 mV, respectively. The
initial transient of inward current saturated the amplifier when
recorded at high gain. At the end of the 200-ms pulse, inward current
of amplitude 1% that of the peak current persisted. In the last 50 ms
of the pulse, there was no perceptible time-dependent change of the
current. The initial transient and persistent current were normalized
to the current at
20 mV and plotted against test voltage in Fig.
3C. The amplitude of the persistent
current paralleled that of the peak current, suggesting that the
persistent current results from a transient change in gating of a
fraction of the channels. This result contrasts with that observed for
the transient and persistent Na+
current in rat ventricular myocytes in which the current-voltage relationship of the persistent current is shifted to more negative potentials by ~20 mV (31).
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Single-channel recordings.
We examined the bases for some of these changes observed at the whole
cell level by recording single-channel
Na+ current in the cell-attached
configuration. Depending on the choice of microelectrode tip size and
the density of channels in the cell, we were able to record from
patches that contained a few or many
Na+ channels. For detailed kinetic
analysis during step depolarizations, we used patches that contained a
few Na+ channels. Figure
4 compares membrane currents recorded
during 10 consecutive voltage steps from
100 to
40 mV in
cells expressing the wild-type and
KPQ mutant
Na+ channels. The maximum number
of overlapping events suggests that there were at least three
functioning channels in the patch for the wild-type channel. Channel
openings occurred after a brief latency and consisted predominantly of
single or overlapping openings having a mean open time of 1 ms. At
later times, e.g., in the first trial, occasional openings were
observed. There was evidence for three functioning channels in the
patch containing the
KPQ mutant. The initial channel openings of
0.9-ms mean duration also occurred after a brief latency. However, late
openings were observed in many trials. As was observed in frog oocytes,
these took the form of isolated brief background openings and bursts of
openings. The fifth trial shows an isolated brief opening late in the
trial without a preceding early opening. This suggests that a closed channel may have passed directly to the inactivated state but visited
the open state by a return from inactivation. The trials containing
bursts tended to cluster, suggesting that the channels functioned in a
distinct mode for a period of time well beyond the interpulse interval
(1,000 ms). The probability that a channel would fail to open at late
times
(Pfl)
was estimated from the nth root of the
fraction of trials with no openings after 20 ms, where
n is the number of channels in the
patch and is estimated from the maximum number of overlapping openings
during the initial transient. The complement of
Pfl
is the probability that a channel will open after 20 ms. These
probabilities were 0.01 and 0.22 for the wild-type and mutant channels
in Fig. 4. The estimates should be regarded as the upper limit of the
probabilities, because the number of channels in the patch is likely to
have been underestimated. The averaged current in the lowest trace of
Fig. 4 shows that those late openings summed to produce a persistent component of inward current. The persistent current amounted to 4% of
the peak current. The two forms of late openings are similar to those
originally described in cardiac and skeletal muscle by Patlak and Ortiz
(28, 29).
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KPQ mutant (Fig.
5). Each trial was arbitrarily divided into an early segment (0-20 ms) and a late segment (20-200 ms).
Twenty milliseconds should be sufficient for the initial transient to reach steady state, inasmuch as the time constant of macroscopic inactivation was <3 ms at all test potentials. At each potential the
distribution of open times was well fit with a single exponential. For
the isolated brief openings, mean open times were 0.58 and 0.62 ms at
test potentials of
50 and
20 mV, respectively. In contrast, the mean open time during the burst increased 2.7-fold over
the same range of membrane potential. Summary data over a range of
membrane potential are presented in Fig. 6.
Whereas mean open times for the isolated opening showed little voltage
dependence, those during the bursts increased progressively with
membrane depolarization.
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KPQ mutant. The holding and test potentials were
100 and
50 mV, respectively. The analysis of consecutive bursts provides a large number of events for kinetic analysis, yet the heterogeneity associated with bursting in different patches should be reduced. The
records imply a complex gating mechanism during bursts. Channel openings are interrupted by brief closures, many of which are not well
resolved. Longer closures separate groups of openings. The histogram of
the distribution of closed times is well fit by two exponentials, with
time constants of 0.22 and 1 ms. For illustrative purposes, the
histogram was truncated at 7.5 ms. Occasional events exceed this limit.
However, there were insufficient long-lasting events to attempt a
higher-order (i.e., 3) exponential fit. When data from other patches
with few bursts were combined at each test potential, multiple
exponentials were usually required to fit the histogram of closed
times.
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KPQ mutant.
For patches with one or two active channels, a majority of late
openings were single isolated events occurring during the 200-ms
depolarizing trials (Fig. 4). Closely spaced openings during 200-ms
trials or constant depolarization (see Fig. 10,
insets) are likely to be the result
of opening of separate channels. The closed times between such openings
are not informative.
We examined the time dependence of the persistent current by applying a
steady holding potential for 200 s. Data acquisition was
interrupted every 200 ms for a period of 1 ms to reset the buffers.
Data from an illustrative experiment are presented in Fig.
9. The holding potential was
20 mV.
Channel "openness" was quite variable over the 200-s interval. A
2 analysis suggested that the
200-ms segments were not homogenous. However, there was no tendency for
the total current to decrease at late times. Occasional long openings
were observed during steady depolarization. However, they were isolated
(e.g., segment B) rather than
occurring in bursts. When the distribution of open times of all events
observed during steady depolarization was compared, the open times
followed a single distribution. As illustrated in Fig.
10, their mean open time and lack of
voltage dependence suggest that the openings during steady
depolarization reflect the isolated openings.
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KPQ mutant channels. At
potentials close to threshold, the macroscopic inactivation is
dominated by the first latency distribution. At these potentials, opening probability is low. Therefore, a large number of trials is
required to estimate the first latency distribution. Figure 11 shows cumulative first latencies in
single patches, each expressing the wild-type and
KPQ mutant
Na+ channels. The cumulative
probabilities are conditional that a channel opened (nulls were
omitted). Data are based on 550 trials of the wild-type channel and 996 trials of the
KPQ mutant channels during step depolarizations from
100 to
50 mV. Mean open times of the wild-type and
KPQ
mutant channels were 0.6 ± 0.6 and 0.8 ± 1 ms, respectively.
The first latency distribution of the wild-type channel took a simple
form. The first 10 ms was fit by a single exponential with a time
constant of 3.1 ms. The cumulative distribution of the
KPQ mutant
took a more complex form. After an initial rapid rise, there was a slow
progressive tail, reflecting first openings occurring at late times.
The initial 10-ms period was fit by a double exponential with time
constants of 0.6 and 25 ms. These data suggest that early openings
occur faster in the
KPQ mutant channel.
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DISCUSSION |
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Initial transient current.
We have used whole cell and single-channel recordings to determine the
changes in kinetics of the Na+
channel that result from the KPQ deletion. Experiments were performed in HEK cells. These cells have diameters of
10 µm and readily formed gigaohm seals with microelectrodes of ~1 M
resistance. In
130 mM external Na+, the
Na+ current was usually well
controlled, as judged by the slope factor of the activation curve and
the lack of overlap of the current waveform as the current amplitude
was reduced with the inactivation voltage-clamp protocol (11). Voltage
dependence of steady-state activation and inactivation were stable
during prolonged recordings from these cells.
KPQ mutant. The rate of recovery from
inactivation was unchanged. Presumably,
h, the rate constant for the
onset of inactivation, must have increased. However, this parameter was
not reported. An et al. (3) examined the kinetics of inactivation of
KPQ mutant Na+ channels
expressed in HEK cells. As reported in the present study, they also
observed an acceleration in the rate of recovery from inactivation. The
difference in the kinetics of recovery in frog oocytes and HEK cells
may reflect the presence of regulatory subunits in the latter cell
type.
As has been reported in two earlier studies, we observed an
acceleration of the rate of macroscopic inactivation (6, 10). The
effect was most marked around the threshold for measurable inward
current at about
50 mV. The faster relaxation of the current could have resulted from a decrease in mean open time or of latency to
first opening. We did not observe a significant change in mean open
time at
50 mV. In fact, in the experiment illustrated in Fig.
11, mean open time was longer in the
KPQ mutant channel (0.8 vs. 0.6 ms). The studies of Aldrich et al. (1, 2) emphasize the importance of
the first latency in determining the relaxation of the macroscopic
current at a neuronal Na+ channel.
This factor is also important in cardiac muscle for small
depolarization. Latency to first opening was changed by the mutation.
An initial early component was accelerated, and a later component
reflected the occurrence of isolated brief openings without earlier
openings. Such opening may reflect direct passage of closed channels to
the inactivated state and a later brief visit to the open state. The
acceleration of the rate of development of inactivation at
80 mV
supports an increased rate of inactivation of closed channels. The
acceleration of the initial component of the first latency suggests an
acceleration of the rate of activation by the
KPQ mutation. Inasmuch
as this mutation affects a segment of the
Na+ channel usually associated
with inactivation, the two processes may be coupled. These results are
consistent with those of O'Leary et al. (27). They showed that when
vicinal Tyr-1494 and -1495, located between the IFM and
KPQ
residues, were mutated to Gln (YY/QQ), macroscopic inactivation is
accelerated for small depolarizations and latency to first opening is
shortened.
Late components of
Na+ current.
The nature of the late component of
Na+ current in the
KPQ mutant
is controversial. Bennett et al. (6) observed that the late openings
result from a modal shift to a single bursting state. On the other
hand, Dumaine et al. (10) observed two modes of gating in cell-attached
patches in frog oocytes. We examined the voltage and time dependence of
the late Na+ channel current at
the single-channel level in cell-attached recordings. Our results are
consistent with two modes of gating at late times: isolated brief
openings and bursts of opening. The open times of the isolated openings
were weakly voltage dependent. They were of the same duration as the
open times of the events that make up the early transient current. The
simplest explanation for these openings is that they result from an
increase in the reversibility of fast inactivation (10). Wild-type
Na+ channels open once at most
depolarized potentials; i.e., the fast inactivated state is absorbing.
The
KPQ mutant Na+ channels
return from the fast inactivated state at a slow but significant rate.
Inasmuch as the closure mechanisms are postulated to be without memory,
the late openings should close at the same rate as those during the
early transient.
30 to
10 mV and a decline around threshold and at strongly depolarized
potentials (42). However, special recording conditions, e.g., high
external Na+ concentration, are
required to obtain accurate kinetics at the extremes of voltages. Over
the limited range of voltage studied, our results are consistent with
the earlier studies of voltage dependence of open times.
The persistence of the isolated brief openings when depolarization is
maintained for as much as 200 s suggests that the channels are not
undergoing slow inactivation. However, there are a number of other
possibilities. At the voltages tested, slow inactivation may not be
complete; i.e., at equilibrium, there may be significant occupancy in
the fast inactivated state. Clarkson et al. (8) reported steady-state
values of slow inactivation of ~0.5 s in the 0- to
60-mV range
of membrane potentials. An alternative possibility is that the
KPQ
mutation may have also modified the kinetics of slow inactivation (10).
In contrast to the isolated openings, the mean open times of the bursts
were strongly voltage dependent. They increased 2.7-fold over a 40-mV
range of membrane potential. In the wild-type channels, Patlak and
Ortiz (29) proposed that bursts result from a transient failure of fast
inactivation. The Na+ channel
kinetics are then reduced to the simple form
|
(3) |
m is the activation rate
constant, and
m is the
deactivation rate constant. Channels closed by deactivation with a mean
open time
o are given as
follows
|
(4) |
KPQ mutant is not
possible, inasmuch as the analysis is critically dependent on the
assumption that fast inactivation is absorbing (1).
The kinetic scheme outlined in Eq. 3
indicates that the distribution of closed times should reflect the
kinetics of activation. A fit to the distribution of closed times
required at least two exponentials. The rate constants of these
exponentials do not reflect the elemental rate constant
m and
2
m, but the eigenvalues of the
submatrix describing the transitions to the open state. The short time
constant is of the same order of magnitude as the short time constant
of the first latency distribution. At room temperature the rising phase
of the macroscopic current was not sufficiently resolved to fit
multiple exponentials to it. Therefore, the closed time constant could
not be compared with the time constants of the rising phase of the
macroscopic current.
The two modes of gating observed at late times with the KPQ deletion
mutant are qualitatively similar to that observed in native cardiac
cells. The reduction in the action potential duration by tetrodotoxin
and Na+ channel-blocking
antiarrhythmic drugs supports the presence of a slow component of
Na+ current in cardiac cells (9,
39). Single-channel recordings have identified both forms of late
gating in ventricular and Purkinje cells (12, 19, 28). The KPQ deletion
increases the likelihood of the slow gating mechanism.
Implications of the changes in Na+ channel gating. This study extends the earlier analysis of Bennett et al. (6), Dumaine et al. (10), and An et al. (3) in defining the voltage and time dependence of gating of the KPQ deletion mutant Na+ channel. These studies provide a basis for the prolongation of the Q-T interval on the surface ECG. We explored the impact of the changes in kinetics of the Na+ current on membrane excitability. The model of the cardiac action potential recently developed by Luo and Rudy (23) does not include the late component exhibited by wild-type and mutant Na+ channels. We varied the noninactivating component of Na+ current in the model of Beeler and Reuter (5) and examined its impact on the action potential. There was a very narrow range of conductance in which the noninactivating Na+ current resulted in marked increases in action potential duration and early afterdepolarizations. For example, with a peak conductance of 4 mS and zero noninactivating current, the resulting action potential duration was 290 ms. For a noninactivating Na+ current of 0.038 mS (<1% of peak Na+ conductance), the action potential duration increased to 670 ms with early afterdepolarizations. Further increases in the noninactivating current resulted in a sustained plateau. Acceleration of the recovery of the Na+ channel from inactivation may also increase the window of vulnerability during phase 3 of the action potential. Smith et al. (35) suggested that the large current tail observed in cells expressing the human ether-á-go-go (HERG) cardiac K+ channel would decrease excitability during phase 3. By diminishing the magnitude of this current, the HERG-associated mutations may enhance excitability during phase 3. The occurrence of persistent inward Na+ current at hyperpolarized potentials may also lower the threshold for excitability.
Prolongation of the Q-T interval on the surface ECG is a relatively common observation. However, its association with torsade de pointes is uncommon. Enhanced excitability during phase 3 is a potential mechanism by which the long Q-T interval-associated Na+ and K+ channel mutations lead to torsade de pointes. The acceleration of the onset of activation observed in the present study further supports coupling between the activation and inactivation mechanisms. The S4 segment of each domain of the Na+ channel is thought to play a critical role in activation. A change in activation after a mutation in the linker between domains III and IV suggests that significant change in the relative positions of the neighboring S4 segments and the III-IV linker may occur during channel gating. The region of the KPQ deletion has multiple Pro residues. Their relatively rigid structure may form a pivot around which some intramolecular movement may occur. The examination of other mutations in this region of the Na+ channel may provide further insight into the basis for the coupling of activation and inactivation.| |
ACKNOWLEDGEMENTS |
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Our preliminary results have been published in abstract form (7).
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FOOTNOTES |
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Address for reprint requests: A. O. Grant, Duke University Medical Center, Box 3504, Durham, NC 27710-3504.
Received 2 September 1997; accepted in final form 4 February 1998.
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