From the Department of Pharmacology, Box 357280, University of
Washington, Seattle, Washington 98195-7280
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INTRODUCTION |
In neurons and muscle cells, activation of voltage-gated
Na+ channels leads to inward Na+ current which
initiates the action potential (1). Within milliseconds after membrane
depolarization, Na+ channels pass through a series of
nonconducting closed states, enter an ion-conducting open state, and
finally convert into a nonconducting inactivated state. Inactivated
channels recover rapidly upon membrane repolarization and are thus
available for reactivation by subsequent depolarizing stimuli. As
inactivation exerts crucial control over Na+ channel
activity, understanding the molecular basis of this process is an
important step toward determining how Na+ channels
function. Three subunits comprise the brain Na+ channel:
of 260 kDa,
1 of 36 kDa, and
2 of 33 kDa (2). Expression of
the
-subunit alone produces functional Na+ channels in
Xenopus laevis oocytes (3, 4). Coexpression of the
1- and
2-subunits yields Na+ channels with kinetics and voltage
dependence of inactivation more closely resembling those found in
neurons (5, 6). The
-subunit has four homologous domains (I-IV),
each with six
-helical transmembrane domains (S1 through S6) (7, 8).
The S4 segments in each domain are thought to serve as voltage sensors,
and the S5 and S6 segments and the short SS1-SS2 segments between them are thought to form the walls of the transmembrane pore (reviewed in
Ref. 9).
Treatment of the intracellular surface of Na+ channels with
proteolytic enzymes prevents inactivation, implicating cytoplasmic components of the Na+ channel in the inactivation process
(10, 11). Site-directed antibodies against the intracellular loop
connecting domains III and IV (LIII-IV) block inactivation
(12, 13), and expression of the
-subunit as two proteins cleaved in
LIII-IV greatly slows inactivation (14). Mutations of a
cluster of three hydrophobic residues within this
loop-Ile1488, Phe1489, and Met1490
(IFM)-prevents fast inactivation (15) primarily by destabilizing the
inactivated state of the channel (16). Small peptides containing the
IFM sequence are sufficient to restore fast inactivation to Na+ channels with mutations in LIII-IV, leading
to the hypothesis that the IFM motif binds within the pore of the
Na+ channel and blocks it during inactivation (17). This
model is supported by recent results showing that a cysteine residue substituted in the IFM motif becomes inaccessible to reaction with
cysteine-specific reagents during the inactivation process (18).
This hypothesis for inactivation implies the presence of amino acid
residues in the intracellular mouth of the pore of the Na+
channel that are involved in conformational change(s) which couple activation to inactivation and bind the IFM motif in the inactivated state. For example, mutations of Phe1764 and
Val1774 at the intracellular end of segment IVS6 strongly
destabilize the inactivated state and increase the rate of recovery of
the channel from inactivation (19). Although these studies define a
region of the Na+ channel required for conformational
coupling and formation of a stable inactivation gate receptor, it is
unlikely that these residues are fully responsible for these
functions.
The S4-S5 intracellular loops in each Na+ channel domain
are good candidates for a role in the fast inactivation process. They are located near the intracellular mouth of the pore and are thus ideally situated to form part of the receptor for the pore-blocking inactivation gate. In addition, they are directly connected to the S4
segments which serve as the voltage sensors of Na+ channels
and move outward under the influence of the electric field to initiate
channel activation (14, 20, 21). The S4-S5 intracellular loop has been
implicated in inactivation of Shaker K+ channels (22, 23),
but its role in the inactivation of Na+ channels is
unknown. In these experiments, we have undertaken a systematic analysis
of the role of residues in the S4-S5 loop of domain IV (IVS4-S5) of the
rat brain type IIA Na+ channel using a combination of
deletion/insertion and scanning mutagenesis, expression in
Xenopus oocytes, and analysis by whole cell and
single-channel recording methods. Our results reveal several amino acid
residues in IVS4-S5 which are required for fast Na+ channel
inactivation. A preliminary report of these results has been published
in abstract form (24).
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EXPERIMENTAL PROCEDURES |
All experimental procedures were performed as described (19). In
brief, mutants were created using oligo-directed mutagenesis in an
M13mp18 construct containing a portion of the
-subunit of the rat
brain IIa Na+ channel that included the IVS4-S5
intracellular loop. The full-length, mutant Na+ channel was
produced by subcloning the mutant cassette into the full-length
-subunit in one of the following vectors: ZemRVSP6 (25), pVA2580
(8), or CDM8 (26). Each vector contained a bacteriophage RNA polymerase
promoter and the rest of the RIIa Na+ channel gene.
mRNA was produced in vitro from these mutant constructs
and then coinjected with
1-subunit mRNA into X. laevis oocytes for channel expression (19). 50 nl of a 2:1 (w/w)
mixture of rat brain Na+ channel
1 RNA and
-subunit
RNA containing 5-50 ng/µl
-subunit RNA were injected. Injected
oocytes were maintained for 2-5 days prior to electrophysiological
recording.
Two-microelectrode voltage clamp recording was used to record whole
cell currents. Macropatch and single channel data were collected from
cell-attached patches except for the excised macropatch experiments
with the KIFMK peptide. Pulses were applied and data acquired using a
personal computer-based data acquisition system (Basic-Fastlab, Indec
Systems, Sunnyvale, CA). Single channel openings were detected using
standard half-amplitude threshold analysis after filtering the data at
2 kHz and omitting overlapping events in multichannel patches (27).
Open times, closed times, and first latencies were analyzed using PSTAT
software (Axon Instruments, Foster City, CA).
Conductance (g) was calculated from peak current
(I) during depolarizations as
I/(V-Vrev), where V was
the test pulse potential and Vrev was the
reversal potential. Normalized conductance-voltage and inactivation
curves were fit with a Boltzmann relationship, A/[1 + exp[(V-V1/2)/k)]] + (1
A), where V1/2 is the voltage of
half-maximal conductance, g1/2, or the voltage of
half-maximal inactivation, h1/2, and A is
the fraction of g or h that varies with
voltage.
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RESULTS |
Deletion and Insertion Mutations in Segment IVS4-S5 Affect Fast
Inactivation--
To determine whether the IVS4-S5 region of the
-subunit has a role in the fast inactivation of rat brain type IIA
Na+ channels, we examined the effects of one deletion and
one insertion mutation in this region (Fig.
1).
WT1 or mutant
-subunit RNA
was co-expressed with
1-subunit RNA in X.
laevis oocytes, and Na+ currents were analyzed
via two-microelectrode voltage clamp recording as described under
"Experimental Procedures." Deletion
4Na45, which deletes the
Leu, Met, Met, and Ser residues in positions 1653-1656, and insertion
I4Na45, which inserts four alanine residues between Met1654
and Met1655, both disrupt fast inactivation. Upon
depolarization, the WT Na+ channels rapidly activate and
then inactivate within a few milliseconds (Fig. 1). In contrast, I4Na45
channels inactivate very slowly, and both these and
4Na45 channels
inactivate incompletely during the pulse, leaving a sustained current
at the end of the 30-ms depolarization. These results implicate IVS4-S5
in the inactivation process.

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Fig. 1.
Effects of deletion, insertion, and heptad
repeat mutations in IVS4-S5 on fast inactivation.
Two-microelectrode voltage clamp recordings of currents due to
expression of WT, 4Na45, L1639A/I1646A, I1653A/L1660A, and I4Na45
Na+ channel -subunits in combination with 1-subunits
in Xenopus oocytes. Currents were evoked by 30 ms pulses to
10 mV from a holding potential of 90 mV. Normalized currents are
shown. Oocytes were injected with RNA encoding the appropriate
-subunit construct together with a 2-fold excess of RNA encoding the
rat brain 1-subunit (5).
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Mutations of a Heptad Leucine Repeat Slow Inactivation--
The
S4-S5 loop contains four Leu and Ile residues spaced at seven-residue
intervals (8) as shown in Scheme 1.
Such heptad repeats are often involved in protein-protein
interactions including leucine zippers (28, 29). A similar repeat in
the Shaker K+ channel S4-S5 loop plays a role in
voltage-dependent gating (30). The double mutations
L1639A/I1646A and L1653A/L1660A each disrupt half of this heptad
leucine repeat. As observed for the deletion mutation
4Na45, both
L1639A/I1646A and L1653A/L1660A channels inactivate incompletely during
the pulse, and a substantial sustained current remains at the end of
the 30-ms depolarization (Fig. 1). Evidently, the integrity of the
leucine heptad motif is essential for normal fast inactivation.
Scanning Mutagenesis of Segment IVS4-S5--
To examine the role
of individual amino acid residues in the IVS4-S5 segment in fast
inactivation, we substituted each amino acid in this region (Scheme 1)
with alanine except for the charged residues in this region which were
mutated to glutamine. Substitution of alanine residues is expected to
alter the chemical characteristics of each residue without causing
substantial conformational change because alanine is found with high
frequency in both
-helices and
-sheets and has limited
hydrophobic and hydrophilic character (31-34). While most of these
mutations had no effect on Na+ channel function, four of
them caused slow and/or incomplete inactivation during depolarizing
test pulses to
10 mV in two-microelectrode voltage clamp. L1639A had
slowed inactivation, L1660A had incomplete inactivation, and F1651A had
both dramatically slowed and incomplete inactivation (Fig.
2A). No inactivation could be
observed during 30-ms test pulses with the N1662A mutant (Fig.
2A). The double mutant combination of F1651A and L1660A also
completely prevented inactivation (Fig. 2A). These results
indicate that the transition between the open state and the inactivated
state and/or the stability of the inactivated state is impaired by
these mutations.

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Fig. 2.
Effects of point mutations in IVS4-S5 on fast
inactivation. A, two-microelectrode voltage clamp recordings
of currents due to expression of WT, L1639A, F1651A, L1660A, N1662A,
and F1651A/L1660A Na+ channels. Experimental details are
the same as described in legend to Fig. 1. B, steady state
inactivation curves for WT (filled circles), L1639A
(inverted triangles), F1651A (open squares), and
L1660A (open circles). Currents were elicited by test pulses to 0 mV following 100-ms conditioning pulses to various potentials from
a holding potential of 90 mV. Peak test pulse current from each
experiment was plotted as a function of prepulse potential, normalized
and fit with a Boltzmann equation as described under "Experimental
Procedures." Mean V1/2 and k were
determined for each mutant construct. The curves shown are plots of the
Boltzmann equation using these mean values. C, recovery from
inactivation at 90 mV. Recovery from inactivation was studied using
two 30-ms long depolarizations separated by a recovery interval of
variable duration. Fractional recovery for each recovery time was
determined as (peak current during the second test pulse)/(peak current
during the first test pulse). Mean recovery time courses for WT
(filled circles), F1651A (open squares), L1660A
(open circles), I1663A (open triangles), L1664A
(diamonds), and L1666A (inverted triangles) are
plotted as mean fractional recovery versus recovery time.
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For the single mutants which retained significant inactivation, we also
measured the voltage dependence of inactivation during 100-ms prepulses
to a range of negative membrane potentials (Fig. 2B). This
protocol measures primarily inactivation from closed states which are
predominant at potentials more negative than
20 mV. For WT channels,
100-ms prepulses to potentials more positive than
30 mV cause nearly
complete inactivation, leaving only 3 ± 0.3% noninactivating
current. The L1639A mutant also is nearly completely inactivated at
positive voltages, although its voltage dependence of inactivation is
shifted to more positive potentials (Fig. 2B). In contrast,
the steady state inactivation curves for F1651A and L1660A approach
non-zero asymptotes of 18 ± 1% and 29 ± 4%, respectively
(Fig. 2B), indicating that inactivation is incomplete at
positive potentials for these mutants as expected from their large
sustained currents at the end of depolarizing test pulses (Fig.
2A). These inactivation curves also reveal that the voltage
dependence of inactivation is shifted to more positive membrane
potentials for F1651A and L1660A (Fig. 2B). Consistent with
the positively shifted inactivation curves, recovery from inactivation
at negative membrane potentials was accelerated in these mutants (Fig.
2C). Thus, these mutations have important effects on
inactivation from both open and closed states.
Although these selected mutations had large effects on Na+
channel inactivation, most of the mutations of amino acid residues in
IVS4-S5 had little or no effect. The relative effects of all of the
scanning mutations on the fraction of noninactivating Na+
current at the end of the test pulse are illustrated in Fig. 3A. Of 26 single residue
mutations analyzed, only 3 caused a significant increase in the
fraction of noninactivating current. Evidently, the effects of the
mutations at positions 1651, 1660, and 1662 on the fraction of
noninactivating current are highly specific, suggesting that these
residues have an important role in determining the stability of the
inactivated state.

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Fig. 3.
Summary of effects of IVS4-S5 Na+
channel point mutations on the steady state fraction of noninactivating
current and the voltage dependence of activation and
inactivation. A, fraction of current that fails to
inactivate for each mutant construct. Currents were elicited by pulses
to 5 mV as described in the legend to Fig. 1. The fraction of
noninactivating current was determined as the current 15 ms after the
beginning of the pulse divided by the peak inward current. Mean ± S.E. is plotted for each IVS4-S5 mutant. Data are from three to eight
experiments for each mutant. B, shift of activation curves
for mutants relative to WT. Currents were elicited by 30-ms long,
two-microelectrode voltage clamp pulses to a range of test potentials
from a holding potential of 90 mV. Peak inward current was measured,
converted to conductance and fit with a Boltzmann relationship as
described under "Experimental Procedures." The mean WT
g1/2 value ( 18.6 ± 4.6 mV) was subtracted
from the mean g1/2 value for each mutant, and the
difference ± S.E. was plotted. C, shift of steady
state inactivation curves for mutants relative to WT. Inactivation
curves were recorded and fit with a Boltzmann relationship as described for Fig. 2B. The average WT h1/2 value
( 47.4 ± 4.2 mV) was subtracted from the mean
h1/2 value for each mutant, and the difference ± S.E. was plotted.
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Although the mutations in IVS4-S5 caused large and specific effects on
the rate and extent of inactivation, their effects on the voltage
dependence of activation and inactivation were smaller and less
specific (Fig. 3, B and C). Of the IVS4-S5
mutations discussed above, only N1662A caused a large (+12 mV) shift in the potential for half-maximal activation. As illustrated in Fig. 2B, the L1639A, F1651A, and L1660A mutants each had small
positive shifts in the half-maximal steady state inactivation curves of 8, 9, and 5 mV, respectively. Other mutations in the IVS4-S5 loop affected the voltage dependence of steady state activation or inactivation without producing sustained or slowly decaying
Na+ currents (Fig. 3, B and C). The
mutant with the most dramatic effect (S1656A) had a large negative
shift of 14 mV in both the steady state activation and inactivation
curves, but no effect on the rate or extent of inactivation during test
pulses. The results show that the effects on the kinetics and extent of
fast inactivation observed for L1639A, F1651A, L1660A, and N1662A are not secondary to changes in the voltage dependence of channel gating.
In contrast to the mutants that disrupted inactivation, mutation of 3 residues at the COOH terminus of this region enhanced closed state
inactivation as revealed by large negative shifts in steady state
inactivation curves (I1663A, G1664A, and L1666A; Fig. 3C).
Mutant G1664A differed from the other two mutants in that activation
(Fig. 3B) and inactivation (Fig. 3C) are shifted to similar extents compared with WT and recovery from inactivation (Fig. 2C) was indistinguishable from WT. Because
inactivation is coupled to activation, the negative shifts in
h1/2 for G1664A and S1656A are probably secondary to
negative shifts in activation. In contrast, the shifts of inactivation
curves for mutants I1663A and L1666A occurred without concomitant
shifts in the voltage dependence of activation (Fig. 3B),
suggesting a specific effect on inactivation. In addition, recovery
from inactivation was greatly slowed in these two mutants (Fig.
2C). Thus, inactivation of closed Na+ channels
is stabilized in mutants I1663A and L1666A in the carboxyl-terminal portion of the IVS4-S5 loop.
Analysis of Mutants by Macropatch Recording--
To examine the
inactivation properties of selected IVS4-S5 mutants in more detail, we
recorded macroscopic currents from cell-attached macropatches.
Recordings from macropatches give better time resolution and voltage
control than two-microelectrode voltage clamp recordings. Fig.
4A shows typical records
obtained during depolarizations to +20 mV. At this voltage, activation
is fast relative to inactivation, so the decay of currents reflects
mainly the rate of channel inactivation from the open state (35, 36).
The mutants L1639A, F1651A, and L1660A all inactivate more slowly than
WT, and all cause a detectable level of noninactivating Na+
current at the end of the 11-ms test pulse (Fig. 4A). The
alterations of the Na+ currents conducted by mutant and WT
channels are similar in the macropatch and two-microelectrode voltage
clamp measurements, except that a small noninactivating current is
observed in the macropatch recordings for L1639A. We could detect no
N1662A current in macropatches, consistent with the relatively poor
expression of this mutant observed in two-microelectrode voltage clamp
recordings.

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Fig. 4.
Voltage dependence of inactivated state
stability. A, normalized currents evoked by depolarizations
to +20 mV from a holding potential of 140 mV in cell-attached
macropatches on X. laevis oocytes expressing WT,
L1639A, F1651A, and L1660A mutants. Each trace is an average of 10-15
sweeps. B-E, normalized currents from cell-attached
macropatches on oocytes elicited by 11-ms pulses to 40, 20, 0, 20, and 40 mV from cells expressing WT, L1639A, F1651A, and L1660A,
respectively. F, the fraction of noninactivating current in
cell-attached macropatches on oocytes expressing F1651A and L1660A was
determined at each potential and plotted as a function of test pulse
voltage. This fraction was defined as the ratio of sustained current at
10 ms after the beginning of the depolarizing pulse to peak current.
The data are from three experiments each.
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Comparison of the Na+ current records during test pulses to
potentials from
40 to +40 mV showed substantial impairment of inactivation of L1639A, F1651A, and L1660A at a wide range of potentials (Fig. 4, C-E) compared with WT (Fig.
4B). At potentials more positive than 0 mV at which most
Na+ channels are fully activated, the impairment of
inactivation increased with depolarization, as illustrated by the
increased fraction of noninactivating current at the end of the test
pulse (Fig. 4F). These results imply that the stability of
the inactivated state is progressively impaired with increasing
depolarization in these mutant channels.
Alterations in Gating of Single Mutant Na+
Channels--
Each of the IVS4-S5 mutants having impaired inactivation
was analyzed by single-channel recording methods as outlined under "Experimental Procedures." Na+ channel gating can be
described simply by the reaction pathway illustrated in Scheme
2 (16, 35, 37, 38). Upon depolarization, Na+ channels undergo voltage-dependent
transitions through multiple closed states (states Cn through
C0), open (state O), and then inactivate (state I). The
inactivated state can be reached from the open state and also from one
or more closed states. For WT Na+ channels, inactivation is
essentially irreversible at depolarized potentials, indicating that the
O-I transition strongly favors inactivation at those potentials.
In response to 40-ms depolarizations to
20 mV, WT Na+
channels generally opened once or twice at the beginning of the
depolarization and then inactivated and did not reopen (Fig.
5A). Most depolarizations of
mutant L1639A channels showed similar behavior to WT (Fig. 5B,
top trace). However, occasionally one channel in a multichannel patch generated sustained bursts of openings and had delayed
inactivation (Fig. 5B, bottom trace). In contrast, nearly
all F1651A channels had long openings and an increased frequency of
reopenings relative to WT (Fig. 5C). L1660A channels had
short openings like WT, but had a high frequency of reopenings
throughout the depolarizing pulse (Fig. 5D). The ensemble
currents for these mutants recreated the slow decay and significant
level of non-inactivating current observed in macropatch recordings
(Fig. 5, B, C, and D, bottom traces).

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Fig. 5.
Single channel records and ensemble
averages. A-E, examples of single channel traces
(upper traces) and ensemble averages (lower
trace) from cell-attached patches for each of the indicated constructs. Single channel activity was elicited by 40 ms pulses to
20 mV from a holding voltage of 140 mV. The number of channels in
the patches were 2, 4, 1, 2, and 1 for WT, L1639A, F1651A, L1660A, and
N1662A, respectively. Sweeps with exceptionally long bursts of openings
suggesting a change of gating mode (47-49) and series of null sweeps
representing slow inactivation (37) were omitted from ensemble
averages.
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Patches containing the N1662A mutant channel were rare due to its low
expression at the whole cell level; therefore, we used large patches
suitable for macropatch recording to obtain single channel data. The
openings of the N1662A mutant channels are long, and reopenings occur
at a high frequency for this mutant relative to WT (Fig.
5E). The ensemble average currents show no appreciable inactivation during the test pulses, as observed in two-microelectrode voltage clamp recordings of this mutant channel.
Total channel activity can be estimated as the integrated probability
(Po) of opening for the duration of the 40-ms depolarizing sweeps. The value of Po for the WT
channels is low, <0.5%. For each of the three IVS4-S5 mutants with
substantial noninactivating current, the Po was
higher than WT: F1651A, 11.2%; L1660A, 8.4%; and N1662A, 57.0%.
Thus, this quantitative measure of increased channel activity also
shows the substantial impairment of inactivation in these mutants.
No significant effects on the single channel conductance were observed
for any of the IVS4-S5 mutants. The WT single channel current at
20
mV was 1.05 ± 0.005 pA. The single channel currents for the
F1651A, L1660A, and N1662A mutants were: 1.1 ± 0.06 pA, 1.03 ± 0.04 pA, and 1.06 ± 0.06 pA, respectively.
Decreased Inactivation from Closed States in Single Channel
Recordings--
Many depolarizations of single Na+
channels produce "null" sweeps containing no single channel
activity (35-37). This behavior results from direct inactivation of
the single channel from a closed state (Scheme 2) (19, 35). The number
of nulls decreased for each of the three IVS4-S5 mutants which caused
substantial noninactivating current. For WT channels, approximately
35% of the recorded sweeps were nulls. The number of nulls observed
for the F1651A, L1660A, and N1662A mutants were 3.6, 1.0, and less than
1.0%, respectively. The decrease in the number of nulls for these
mutants correlates with their increased frequency of reopenings during
a depolarizing pulse (see below). Thus, the loss of nulls is caused by
a combination of slow entry into the inactivated state from closed
states (decreased rate constant e in Scheme 2) and frequent return from
the destabilized inactivated state (increased rate constants f and h in
Scheme 2).
Latency to First Activation--
The latency before the first
single channel opening following depolarization reflects the dwell time
in the multiple closed states in the activation pathway prior to
opening (Cn through C0, Scheme 2) (35). Fig.
6 shows the cumulative first latency
distributions for WT, F1651A, L1660A, and N1662A channels at a test
potential of
20 mV, corrected for the number of channels in the patch
(39). We were unable to obtain patches with small numbers of L1639A
channels and, thus, could not obtain an adequate estimate of first
latency or closed times for this mutant. For WT, single channels open
rapidly, but the maximum probability of opening is only about 0.65 because 35% of the depolarizations fail to elicit channel opening,
resulting in null sweeps. As noted above, these nulls represent
transitions directly from the closed states in the activation pathway
to the inactivated state during depolarization (35, 36) (Scheme 2).

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Fig. 6.
First latency for single channel
activation. Cumulative first latency distributions for WT, F1561A,
L1660A, and N1662A. Distributions at 20 mV are compared after
correcting the WT data for the presence of 2 channels (39). The
ordinate corresponds to the cumulative probability that a
channel has opened at the indicated time after depolarization.
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Most F1651A channels open rapidly like WT, but the open probability
reached after this early rapid phase is nearly 0.85 (Fig. 6). The high
open probability suggests that these channels may have slowed
inactivation from closed states resulting in a higher proportion of the
channels being available for opening. A small slow phase of activation
is also apparent and is consistent with a low frequency of channel
opening after first inactivating from the closed state in agreement
with the observation of increased channel reopenings for this
mutant.
The cumulative first latency distribution for mutant L1660A rises
rapidly to an open probability of 0.6 similar to WT channels and then
continues to increase slowly to an open probability approaching 1.0 (Fig. 6). The fast component represents channels that open normally,
indicating that channel activation is not affected by this mutation.
The second, delayed component represents channels that inactivate
normally from closed states, but unlike WT, leave the destabilized
inactivated state and then open.
For mutant N1662A, the first latency distribution rises rapidly to 1.0, but at a slightly slower rate than for WT. Thus, N1662A channels open
rapidly like WT but rarely inactivate irreversibly from the closed
state. As was suggested for F1651A, these characteristics are likely to
result from a combination of slow entry into the inactivated state and
frequent reopening if they do enter the destabilized inactivated state.
The slightly slower opening rate agrees well with the observed positive
shift of the voltage dependence of N1662A macroscopic activation. The
first latency curves for F1651A, L1660A, and N1662A all contain a rapid
opening rate similar to WT channels and open to an equal or greater
open probability than WT channels, suggesting that these mutations have
little effect on the activation process and predominantly affect the inactivation process.
Mean Open Times--
At depolarized potentials, the mean open time
depends primarily on the rate of entry of single Na+
channels into the inactivated state from the open state, because the inactivation rate is much faster than the closing rate
(g
d in Scheme 2) and the channel
leaves the open state primarily via entry into the inactivated state
rather than via return to the closed state (11, 16, 35). Consequently,
if mutant channels enter the inactivated state more slowly from the
open state (reduced g in Scheme 2), they will have an
increased mean open time. Open time data for WT, L1639A, and L1660A
could be fit with a single exponential (Fig.
7). The mean time constants for these
Na+ channels were short, 0.33 ± 0.27 ms
(n = 5), 0.39 ± 0.09 ms (n = 3),
and 0.35 ± 0.03 ms (n = 4) (Fig. 7, A
and C). For each of these channels, therefore, the rate of
entry into the inactivated state from the open state is similar to WT.
In contrast, the open time histogram for F1651A is fit with a single,
longer exponential time constant. The mean time constant was 0.90 ± 0.23 ms (n = 3), significantly longer than that for
the WT channel (Fig. 7B). Thus, this mutant has a decreased
rate of entry into the inactivated state relative to WT. The
distribution of open times of N1662A could be best fit with 2 exponentials. The data in Fig. 7D were fit by a mean open
time of 0.54 ms for 80% of the openings and a mean open time of 1.8 ms
for the remaining 20%. For some patches, the fraction of openings with
the more rapid mean open time approached 100%, but the mean open time
was always longer than WT. The overall average mean open time for
N1662A is 0.94 ± 0.15 ms (n = 3). Therefore, this
mutant, like F1651A, has slowed inactivation from the open state.

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Fig. 7.
Single channel open times. Open time
histograms are displayed for L1639A, F1651A, L1660A, and N1662A
mutants. Data were collected during pulses to 20 mV. The solid
lines are exponential fits to the binned data at times 100 µs.
Time constants are 0.21, 1.09, 0.33, and 0.54/1.80 (80%/20%
approximate weight) ms, respectively. The dotted line in
each mutant panel is a single exponential fit with a representative WT
time constant of 0.33 ms. These histograms are representative plots
from three, three, four, and three experiments for L1639A, F1651A,
L1660A, and N1662A, respectively.
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Closed Times--
Analysis of closed time distributions for single
channels provides information about the closed states which they enter
between openings. For WT channels the inactivation rate is rapid, and return to closed states (Cn through C0, Scheme 2)
after the first opening is rare. For mutants with slowed entry into the
inactivated state, return to the last closed state becomes more
probable and these closures are expected to add additional components
to the distribution of closed times. In addition, return from the
inactivated state of mutant channels adds another component to the
distribution of closed times. The closed time distributions for the
F1651A, L1660A, and N1662A mutants all are best fit by two exponentials
with a short closed time of about 0.2 ms and a second closed time about
an order of magnitude longer (10 ms for F1651A, 3.3 ms for L1660A, and
1.4 ms for N1662A) (Fig. 8, A-C). In each case, this longer closed time is longer than
the first latency, and therefore cannot represent return to a closed state in the activation pathway (40). Thus, the long closed times must
represent the dwell times of these channels in the destabilized
inactivated state. These closed times provide a direct measure of the
increased rate of return from the inactivated state for these mutants.
The long closed times due to closure to the inactivated state for these
three mutants are voltage-dependent and become shorter at
more depolarized voltages (Fig. 8D). Evidently, the
inactivated state is less stable at more depolarized voltages, as was
also suggested by the increased noninactivating Na+
currents at more depolarized potentials (Fig. 4F).

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Fig. 8.
Single channel closed times. A-C,
closed time histograms are displayed for F1651A, L1660A, and N1662A
mutants. Data were collected during pulses to 20 mV. The solid
lines are exponential fits to the binned data at times 100 µs.
In each case the data are fit with two time constants 0.10/10.57
(99%/1%), 0.16/3.31 (90%/10%), and 0.26/1.40 (about 85%/15%) ms,
respectively. The dotted line in each panel is a single
exponential fit with a representative WT time constant of 0.21 ms.
These histograms are representative plots from three, four, and three
experiments for F1651A, L1660A, and N1662A, respectively. D,
mean values of the slower time constant (long closed times) for F1651A,
L1660A, and N1662A. Data from single channel patches are displayed as a
semilog plot.
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Effects of the Inactivation Gate Peptide KIFMK on Decay of
Na+ Currents Through the F1651A/L1660A Double
Mutant--
The intracellular loop between domains III and IV forms
the Na+ channel inactivation gate (12-14). Mutagenesis
studies have identified four residues that are critical components of
the gate, Ile1488, Phe1489, and
Met1490 (IFM), and Thr1491. Mutation of
Phe1489 to Gln is sufficient to nearly completely block
fast inactivation (15). The IFM residues are thought to bind to a
hydrophobic site in or near the mouth of the Na+ channel
pore (16, 17). One possible interpretation of the effects of the F1651A
and L1660A mutations is that the native residues at these positions
form the hydrophobic binding site for the IFM motif so that mutations
of these amino acids disrupt the binding site.
To test this hypothesis, we examined the effects of application of a
peptide, acetyl-KIFMK-amide (KIFMK), to the intracellular surface of
excised membrane patches from oocytes expressing mutant Na+
channels. Mutations of an inactivation gate receptor would be expected
to affect block by this peptide. Application of this peptide to the
intracellular surface of patches containing Na+ channels
with a mutation in the critical Phe of the intrinsic IFM motif (F1489Q)
reduces the peak Na+ current and produces current decay
during the pulse that resembles fast inactivation from closed and open
states, respectively (19, 41) (Fig.
9A). The time course of the
Na+ current in the presence of KIFMK can be fit to a model
in which inactivation by binding KIFMK occurs in parallel with
inactivation by the intrinsic inactivation gate as illustrated in
Scheme 3.

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Fig. 9.
Effect of KIFMK peptide on Na+
currents in excised, inside-out macropatches from oocytes expressing
the F1489Q and F1651A/L1660A mutants. Currents elicited by
depolarizing pulses to 20 mV from a holding potential of 140 mV in
the absence and presence of 50 µM KIFMK peptide. The
traces shown are representative of 2 patches for each mutant construct.
The smooth lines are fits of Scheme 3 to the data as
described (16). For peptide block of F1489Q, the channel makes the C to
I-KIFMK transition (Scheme 3) 28% of the time that it arrives in state
C0, whereas for F1651A/L1660A it makes that transition a
maximum of 5% of the time. The rate constants for the fit to F1489Q
alone were: a, 12,000 s 1; b, 100;
c, 10,000; d, 500; e, 2,000;
f, 200; g, 80; and h, 160. In the
presence of KIFMK additional rate constants were: i, 4,600 s 1; j, 45; k, 346; and
l, 68. The rate constants for the fit to F1651A/L1660A alone
were: a, 9,000 s 1; b, 100;
c, 16,000; d, 500; e, 430;
f, 9; g, 425; and h, 285. In the
presence of KIFMK a was increased to 16,000 s 1 and rate
constants for KIFMK block were: i, 800 s 1;
j, 5; k, 485; and l, 92.
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As for F1489Q, Na+ currents in excised patches
containing the double mutant F1651A/L1660A have impaired inactivation
in the absence of peptide, and KIFMK increases both the rate and extent of inactivation (Fig. 9B). However, the same concentration
of peptide had different effects on this mutant compared with F1489Q. There was little reduction of the peak current (Fig. 9B).
Because the reduction in peak current reflects inactivation of closed Na+ channels, this effect of the F1651A/L1660A mutation
suggests that binding of KIFMK to the closed states of this mutant
channel is substantially impaired. Fit of the data to a model based on Scheme 3 revealed a 5.7-fold reduction in the maximum value of rate
constant i for KIFMK-mediated inactivation from the closed state. This impairment of binding of the IFM motif may contribute to
the loss of closed state inactivation of the F1651A and L1660A mutants
revealed by the increase in their maximum probability of opening (Fig.
6). In contrast to the difference in the reduction of peak
Na+ currents between the inactivation gate mutant F1489Q
and mutant F1651A/L1660A, there is not a marked difference in the
effects of KIFMK on the decay of the Na+ current during the
test pulse (Fig. 9). For both mutants, the peptide increases the rate
of current decay markedly and decreases the level of current remaining
at the end of the test pulse. Fits of these results to a model based on
Scheme 3 showed that the rate constants k and l,
governing the transition from the open state to the inactivated state,
were similar for both mutants. Therefore, the double mutation
F1651A/L1660A has a specific effect on KIFMK binding to closed channels
and little or no effect on KIFMK binding to open Na+
channels.
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DISCUSSION |
Mutations of F1651, L1660, and N1662 in the IVS4-S5 Loop Impair
Fast Inactivation of Open Na+ Channels--
The mutations
F1651A, L1660A, and N1662A all slow the rate of decay of
Na+ currents. Analysis of single channel currents shows
that all three mutations impair the stability of the inactivated state, equivalent to increasing rate constant, h, for exit from the
inactivated state in Scheme 2. In addition, F1651A and N1662A also slow
entry into the inactivated state from the open state, equivalent to decreasing rate constant g in Scheme 2. Thus, these amino
acid residues are necessary for rapid transition from open to
inactivated Na+ channels and for the irreversibility of
that transition at depolarized membrane potentials.
Mutations of F1651, L1660, and N1662 in the IVS4-S5 Loop Impair
Fast Inactivation of Closed Na+ Channels--
Two lines of
evidence support the conclusion that these mutations in the IVS4-S5
loop also impair fast inactivation from closed states. First, these
mutations all reduce the frequency of null channel traces in single
channel analysis, consistent with slowing the entry of channels from
the closed state into the inactivated state (rate constant
e, Scheme 2) and increasing the frequency of opening from
the inactivated state (rate constants f and h). Second, F1651A and L1660A both shift the voltage dependence of steady
state inactivation to more positive membrane potentials, consistent
with an increased energy requirement for the transition from the closed
states to the inactivated state. Evidently, these amino acid residues
in the IVS4-S5 loop are involved in both of the dual pathways to the
inactivated state from the closed and open states as illustrated in
Scheme 2.
Molecular Basis for Impairment of Fast Inactivation by Mutations in
the IVS4-S5 Loop--
Our results with the KIFMK peptide give
additional insight into the molecular basis for the effects of these
mutations on inactivation from open and closed states. Previous results
have shown that the IFM motif enters into a hydrophobic interaction with a putative receptor in or near the intracellular mouth of the pore
during the inactivation process (16). Disruption of this interaction by
substitution of hydrophilic amino acid residues for Phe1489
in the inactivation gate greatly impairs the stability of the inactivated state and slows entry into the inactivated state. Therefore, it is expected that mutation of hydrophobic amino acid residues in the inactivation gate receptor to less hydrophobic ones
would also slow entry into and destabilize the inactivated state by
impairing the hydrophobic interaction with the IFM motif. The
phenotypes of mutants F1651A, L1660A, and N1662A are consistent with
this type of mechanism. They each slow entry into and/or reduce the
stability of the inactivated state.
This mechanism can be tested specifically with the KIFMK peptide since
it is thought to bind to the inactivation gate receptor but it is not
coupled physically to the gating processes of the channel protein.
Surprisingly, our results show that amino acid residues
Phe1651 and/or Leu1660 are essential for rapid
interaction of the KIFMK peptide with closed channels but not with open
channels. We cannot assess the effects of these two mutations
individually because they do not prevent inactivation completely enough
when present singly to allow clear effects of the KIFMK peptide to be
observed. Nevertheless, the finding that the region of the IVS4-S5 loop
defined by these two residues may interact with the IFM motif during
inactivation of closed Na+ channels is an important
advance. In our previous studies of candidate amino acid residues for
interaction with the IFM motif, we showed that those mutants were
blocked normally by KIFMK, implying that those residues do not interact
directly with the IFM motif (19). Thus, Phe1651 and
Leu1660 are the first residues implicated in the receptor
for the IFM motif using this criterion.
The gating diagrams of Schemes 2 and 3 include only a single
inactivated state, suggesting that the IFM motif should have the same
molecular interactions in inactivation of closed or open Na+ channels. However, this gating scheme is a
simplification, and it is clear that a complete scheme for inactivation
must include formation of multiple closed/inactivated states as well as
an open/inactivated state (42). Most likely, there is an inactivated state associated with each of the Cn through C0
closed states, as presented in previous complete gating models for
Na+ channels (42, 43). In the context of these more
complete gating models, our results indicate that rapid entry into the inactivated state C0I is likely to require hydrophobic
interactions of the IFM motif of the inactivation gate with
Phe1651 or Leu1660 in IVS4-S5. In contrast,
these interactions are less important for rapid entry into the
inactivated state I from the open state O. Thus, the IFM motif may form
a series of interactions with different amino acid residues in a
complex inactivation gate receptor region, depending on the activation
gating status of the intracellular end of the pore of the
Na+ channel.
Additional Effects of Mutations at the Ends of the IVS4-S5
Loop--
The effects of the L1639A mutation, located at the
NH2-terminal boundary of IVS4-S5, are different from the
other mutations located in the central segment of this intracellular
loop. At the macroscopic level, the decay of the Na+
current through the L1639A mutant channel is slowed, suggesting an
effect on inactivation. Although the overall behavior of single L1639A
channels is not qualitatively different from WT, the channels undergo
occasional transient failures of inactivation that are observed as
prolonged openings or bursts of openings. These bursts of activity are
responsible for the sustained current observed in macropatch
recordings. These high open probability events ended before the end of
the test pulses, explaining the minimal increase in the noninactivating
current at the end of depolarizations. The L1639A mutation may cause a
conformational change in IVS4-S5 that slows the development of
inactivation, perhaps by slowing formation of the inactivation gate
receptor. The position of this residue at the inner end of segment IVS4
suggests that it may participate in coupling of voltage sensor
movements to the inactivation process and that this coupling may
occasionally fail in the mutant channel.
Two mutations at the COOH-terminal end of IVS4-S5, I1663A and L1666A,
increase the stability of the inactivated state. This result suggests
that the natural Ile and Leu residues at these positions actually
destabilize the inactivated state. Thus, the WT residues in IVS4-S5
contribute both stabilizing and destabilizing interactions within the
overall energetics of inactivation. The sum of these interactions will
set the voltage dependence of inactivation, the rate of inactivation
during depolarizations, and the rate of recovery from inactivation
between depolarizations.
Comparison with Results of IVS4-S5 Mutants from Other
Na+ Channels--
Recent studies of mutations made in the
IVS4-S5 loops of human heart (hH1), rat skeletal muscle (rSkM1), and
human skeletal muscle (hSkM1) Na+ channels have yielded
some similar and some contrasting results to those presented here for
the rat brain Na+ channel (rIIA). Similar to the data
presented for the rat brain F1651A mutant, mutations in the
corresponding residue in human (F1473S) and rat (F1466C) SkM1 also
produce substantial slowing of Na+ current decay for these
channels (44, 45). Apparently, a role for this Phe residue in
inactivation is conserved in different Na+ channels. In
contrast, mutation of Leu1660 to Ala in type IIA channels
produced dramatic effects on inactivation, but the similar mutations in
human SkM1 (L1482A) or rat SkM1 (L1475C) produced little or no change
in steady state inactivation or recovery from inactivation (44, 45). A
double mutation of M1651/M1652 of the human heart Na+
channel to either QQ or AA caused substantial slowing of
Na+ current decay, a positive shift of steady state
inactivation, and an increased rate of recovery from inactivation. In
contrast, the single mutations of each Met in rIIA (M1654A and M1655A)
or the rSkm1 (M1469C, M1470C) had little or no effect on inactivation (44-46). Apparently, species or tissue differences in the role of
specific amino acid residues in inactivation exist, but the importance
of Phe1651 and the IVS4-S5 loop in the inactivation process
is conserved across species and tissue boundaries.
Possible Function of IVS4-S5 in Inactivation--
The
characteristics of the F1651A, L1660A, and N1662A mutants show that the
IVS4-S5 region is important for the inactivation of brain
Na+ channels. What is the exact role of these residues?
This region of the Shaker K+ channel is also important in
inactivation and has been suggested to form the receptor for the
NH2-terminal inactivation particle of K+
channels (22, 23). In agreement with this suggestion, the IVS4-S5
region is strategically located near the intracellular mouth of the
pore and is proximal to a voltage sensor of the channel. The phenotype
of the F1651A, L1660A, and N1662A mutants makes them potentially good
candidates to form part of the receptor for the Na+ channel
inactivation gate, since each mutation decreases the stability of the
inactivated state by increasing the rate of reversal of inactivation.
The effect of the L1639, F1651A, and N1662A mutations to slow entry
into the inactivated state may indicate that these residues participate
in a conformational change which is required for formation of an
effective inactivation gate receptor. Results with the KIFMK peptide
implicate Phe1651 or Leu1660 in interaction
with the IFM motif of the inactivation gate during inactivation of
closed Na+ channels but these residues are not as important
for fast inactivation of open Na+ channels. Thus, these
residues may form part of a multifaceted inactivation gate receptor
region which interacts with the inactivation gate differently depending
on the functional state of the channel gating machinery. Further
definition of these state-dependent events may elucidate
molecular interactions important for gating transitions among closed,
open, and inactivated states of Na+ channels.