From the
Fast Na
Ionic current through voltage-dependent Na
The rat brain Na
Treatment of the intracellular surface of Na
The actions of local
anesthetics and functionally related drugs on Na
The cell-attached
configuration of the patch clamp technique (Hamill et al.,
1981) was used to obtain both macropatch and single channel data.
Oocytes were incubated in 200 mM potassium aspartate, 20
mM KCl, 1 mM MgCl
Single channel openings were detected
using standard half-amplitude threshold analysis after filtering the
data at 2 kHz (Colquhoun and Sigworth, 1993). Patches contained from
one to four channels. Channel number was estimated from the maximum
number of overlapping openings at potentials where the probability of
opening was high. Open times, closed times, and first latencies were
analyzed using PSTAT software (Axon Instruments, Foster City, CA).
Overlapping events were omitted from analysis.
The large sustained currents
caused by the mutations F1764A and V1774A suggested that these two
residues might play a particularly important role in Na
In addition to producing sustained currents,
IVS6 mutations also altered the voltage dependence of steady state
activation and inactivation (, Fig. 2). For example,
the voltage for half-maximal activation of mutants V1774A and
F1764A/V1774A was shifted by -8 to -10 mV compared with WT
(, Fig. 2A). In contrast, the voltage for
half-maximal activation of F1764A and several other mutants was
unaffected (, Fig. 2A). Apparent shifts in
activation toward more negative membrane potentials are expected for
mutations that strongly inhibit inactivation because of the kinetic
overlap of the activation and inactivation gating processes (Gonoi
et al., 1984; Gonoi and Hille, 1987). The lack of a strict
quantitative correlation between the extent of disruption of
inactivation and shifts in the voltage dependence of activation among
our different mutants suggests that IVS6 mutations may also have small
direct effects on the voltage dependence of activation.
Mutations
F1764A and V1774A also accelerated the rate of recovery from
inactivation at hyperpolarized potentials. We examined the time course
of recovery from inactivation by stepping the voltage to 0 mV for 15 ms
to inactivate channels and then repolarizing to negative potentials for
recovery intervals of varying duration. The time constants for recovery
derived from an exponential fit to the data are plotted as a function
of recovery voltage in Fig. 2C. For F1764A and V1774A,
the time constant for recovery was 2-3-fold faster than WT at all
potentials. This result gives further evidence that the inactivated
state is destabilized by these mutations.
To
examine this point more carefully, we measured tail currents recorded
after repolarization to -140 mV following these 11-ms-long
depolarizing pulses (Fig. 3D). These tail currents are
inward Na
Tail current amplitudes
for both F1764A and V1774A increased progressively and did not saturate
at positive potentials. Instead, the amplitudes of these tail currents
continued to increase at strongly positive membrane potentials
(Fig. 3D) where activation was essentially complete (see
Fig. 2A). This increase in tail current amplitude at
positive potentials reflects the progressive impairment of inactivation
at these potentials and the increased fraction of sustained
Na
In response to a 40-ms
depolarization to -20 mV, WT Na
With
strong depolarizations, the single channel open time gives an estimate
of the rate of channel entry into the inactivated state from the open
state, because the channel leaves the open state primarily by entry
into the inactivated state rather than by return to a closed state at
depolarized voltages (Armstrong, 1981; Aldrich et al., 1983;
Armstrong and Bezanilla, 1977). Mutations that slow the entry into the
inactivated state from the open state are expected to increase single
channel mean open time. Distributions of open times at -20 mV for
typical WT, F1764A, V1774A, and F1764A/V1774A single channel recording
experiments are illustrated in Fig. 5as histograms. WT, F1764A,
and V1774A single channel data were well fit with single exponentials,
suggesting that each of these channels had a single open state with a
short lifetime. The time constants (
The first latency is the time
before the first opening of a single Na
The first latency distribution for F1764A/V1774A
approached an open probability of 1.0 as for mutant V1774A; however,
there was no slow component to the distribution. Either this mutation
blocked transitions from the closed states to the inactivated state or
these channels leave the inactivated state so rapidly that the overall
first latency distribution is unaffected. The similar time course for
the rapid component of the cumulative first latency distribution for WT
and both mutant channels indicates that the activation process is not
strongly slowed by these mutations.
For the mutant V1774A, we
consistently observed an increase in single channel activity throughout
the duration of a stimulus pulse with increasingly strong
depolarizations (Fig. 6B). This behavior suggests that
the degree of destabilization of inactivation in this mutant was
voltage-dependent, as observed in recordings of sustained macroscopic
Na
First, we investigated
the role of hydrophobicity at the 1774 position. If this residue forms
part of a hydrophobic binding site for IFM, then substituting serine or
asparagine, two polar residues, for the native valine should disrupt
inactivation even more than substituting alanine. When analyzed as
described in Fig. 1, the fraction of sustained Na
As a second
strategy to investigate whether the residues Phe
For macroscopic Na
Inactivation of Na
Additional support for a role
of segment IVS6 in Na
Segment IVS6 is
considered to be an
Our
experimental results place limitations on the possible mechanisms by
which these amino acid residues participate in fast inactivation since
the rate of activation of the channel and the rate of entry into the
inactivated state are not substantially affected by the mutations in
segment IVS6. In addition, our results show that a receptor for the IFM
motif becomes available for binding and block of the open Na
For activation and
inactivation data, normalized conductance (see legend to Fig.
2A) and current (see legend to Fig. 2B) plots,
respectively, were fit with a Boltzmann equation (see
``Experimental Procedures'') to obtain V and k values.
We thank R. MacKinnon for suggesting the analysis of
suppressor mutations using a thermodynamic cycle and S. Love for
helpful discussions.
channel inactivation is thought to
occur by the binding of an intracellular inactivation gate to regions
around or within the Na
channel pore through
hydrophobic interactions. Previous studies indicate that the
intracellular loop between domains III and IV of the Na
channel
subunit (L
) forms the inactivation
gate. A three-residue hydrophobic motif (IFM) is an essential
structural feature of the gate and may serve as an inactivation
particle that binds within the pore. In this study, we used
alanine-scanning mutagenesis to examine the functional role of amino
acid residues in transmembrane segment IVS6 of the Na
channel
subunit in fast inactivation. Mutant F1764A, in the
center of IVS6, and mutant V1774A, near its intracellular end,
exhibited substantial sustained Na
currents at the end
of 30-ms depolarizations. The double mutation F1764A/V1774A almost
completely abolished fast inactivation, demonstrating a critical role
for these amino acid residues in the process of inactivation. Single
channel analysis of these three mutants revealed continued reopenings
late in 40-ms depolarizing pulses, indicating that the stability of the
inactivated state was substantially impaired compared with wild type.
In addition, the cumulative first latency distribution for the V1774A
mutation contained a new component arising from opening transitions
from the destabilized inactivated state. Substitution of multiple amino
acid residues showed that the disruption of inactivation was not
correlated with the hydrophobicity of the substitution at position
1774, in contrast to the expectation if this residue interacts directly
with the IFM motif. Thermodynamic cycle analysis of simultaneous
mutations in the IFM motif and in IVS6 suggested that mutations in
these two regions independently disrupt inactivation, consistent with
the conclusion that they do not interact directly. Furthermore, a
peptide containing the IFM motif (acetyl-KIFMK-amide) restored
inactivation to the F1764A/V1774A IVS6 mutant, indicating that the
binding site for the IFM motif remains intact in these mutants. These
results suggest that the amino acid residues 1764 and 1774 in IVS6 do
not directly interact with the IFM motif of the inactivation gate but
instead play a novel role in fast inactivation of the Na
channel.
channels initiates the action potential in neurons and other
excitable cells. Upon membrane depolarization, Na
channels undergo conformational changes through a series of
nonconducting closed states to a conducting open state and then to a
nonconducting, inactivated state within 1 or 2 ms. Upon membrane
repolarization, Na
channels recover from fast
inactivation within a few milliseconds and are again available for
activation. Understanding the molecular basis of fast inactivation
gating is an important step in defining the mechanisms of Na
channel function.
channel is
a heterotrimeric protein consisting of
(260-kDa),
1
(36-kDa), and
2 (33-kDa) subunits (reviewed in Catterall(1992)).
The
subunit, a 2005-amino acid glycoprotein, contains four
homologous domains, each with six probable
-helical transmembrane
segments (S1-S6; Noda et al., 1986b; Auld et
al., 1988). The
1 subunit, a 218-amino acid glycoprotein, has
a single transmembrane segment (Isom et al., 1992). Only the
cloned
subunit is required for the expression of functional
Na
channels in Xenopus laevis oocytes (Noda
et al., 1986a; Goldin et al., 1986). Co-expression of
the
and
1 subunits, however, is required for the expression
of Na
channels displaying normal time course and
voltage dependence of inactivation (Isom et al., 1992).
and
K
channels with proteolytic enzymes specifically
disrupts fast inactivation, implicating peptide segment(s) on the
cytoplasmic surface of these channels in the inactivation process
(Armstrong et al., 1973; Armstrong, 1981; Hoshi et
al., 1991). One current hypothesis for the mechanism of fast
inactivation is that a tethered inactivation gate, composed of these
cytoplasmic peptide segments, binds to a region within the pore during
fast inactivation, blocking ion conductance and immobilizing gating
charge (Armstrong and Bezanilla, 1977; Armstrong, 1981; Hoshi et
al., 1991; Zagotta et al., 1990). Studies using
site-directed antibodies and site-directed mutagenesis indicate that
the intracellular loop between homologous domains III and IV of the
subunit (L
) is involved in inactivation
(Vassilev et al., 1988; 1989; Stühmer et al.,
1989). Mutation of three hydrophobic amino acids, Ile
,
Phe
, and Met
(IFM), within this loop to
glutamine disables fast inactivation (West et al., 1992a). In
contrast, mutations of charged amino acid residues elsewhere in this
loop do not disrupt inactivation (Moorman et al., 1990; Patton
et al., 1992). Small peptides containing the IFM sequence are
sufficient to restore fast inactivation to Na
channels
with mutations in L
, demonstrating that the IFM motif
has affinity for a region within the Na
channel pore
(Eaholtz et al., 1994). These results have led to the proposal
that Na
channel inactivation occurs when
L
, serving as an inactivation gate, closes over the
intracellular mouth of the pore and binds to other regions of the
Na
channel. The IFM residues are thought to act as a
hydrophobic molecular latch that holds the gate shut by forming a
hydrophobic interaction with residues within or near the intracellular
mouth of the pore (West et al., 1992a; Scheuer et
al., 1993; Eaholtz et al., 1994).
channels are similar to the actions of the fast inactivation gate
in that they act from the cytoplasmic side of the Na
channel to inhibit ionic current and immobilize gating charge
(Cahalan and Almers, 1979; Hille, 1992). Some local anesthetics, like
lidocaine, stabilize the inactivated state (Hille, 1977; Bean et
al., 1983), whereas other compounds, like
N-methylstrychnine, prevent inactivation (Cahalan and Almers,
1979). These observations suggest that local anesthetics and related
drugs interact with regions of the Na
channel that are
important for inactivation. We have recently identified amino acid
residues in the S6 transmembrane segment of domain IV of the
Na
channel
subunit (segment IVS6) that are
critical determinants of local anesthetic action (Ragsdale et
al., 1994), and we have shown that mutation of a cluster of three
residues near the cytoplasmic end of IVS6 strongly disrupts fast
inactivation (McPhee et al., 1994). These results indicate
that segment IVS6 is important for both drug action and fast
Na
channel inactivation. In the present work, we have
undertaken a systematic analysis of the role of segment IVS6 in
inactivation by alanine-scanning site-directed mutagenesis and
identified individual amino acid residues that are important for
inactivation.
Site-directed Mutagenesis
Mutants were produced
in a 1719-base pair DNA fragment excised from the rat brain type IIA
Na channel
subunit cDNA (Auld et al.,
1988; Auld et al., 1990) and inserted into M13 mp18. A
single-stranded, uridine-containing form of the template was isolated
from dut
ung
E.coli cells.
Oligonucleotides containing the mutation to be introduced were annealed
to the template and used to direct the transcription of the
complementary strand containing thymidine in vitro. After
transforming normal bacteria with this heterodimeric, double-stranded
template, only the mutant, thymidine-containing strand was replicated,
whereas the wild type (WT),
(
)
uridine-containing
strand was not. The double-stranded mutant DNA was isolated and
sequenced to confirm the identity of the mutation. The full-length,
mutant Na
channel was produced by subcloning the
mutant cassette into either vector ZEMRVSP6-2580 (West et
al., 1992b) or vector pVA2580 (Auld et al., 1988)
containing the remainder of the Na
channel and a
bacteriophage RNA polymerase promoter (Kunkel, 1985; West et
al., 1991; West et al., 1992b).
Na
RNA
encoding WT and mutant sodium channel Channel Expression
subunits and WT
1
subunits was synthesized in vitro using the Ambion mMessage mMachine
kit containing either SP6 or T7 bacteriophage RNA polymerase for the
ZemRVSP6 or pVA2580 cDNAs, respectively. In preparation for expression
of WT and mutant Na
channels, pieces of ovary were
surgically removed from anesthetized X. laevis frogs. Oocytes
were separated and defolliculated by shaking gently for 2 h in 1.5
mg/ml collagenase in OR-2 (82.5 mM NaCl, 2 mM KCl, 1
mM MgCl
, and 5 mM HEPES, pH 7.5). After
an overnight incubation at 18 °C in Barth's medium (88
mM NaCl, 1 mM KCl, 0.82 mM MgSO
,
0.33 mM Ca(NO
)
, 0.41 mM
CaCl
, 2.4 mM NaHCO
, and 10 mM
HEPES, pH 7.4) supplemented with 5% fetal bovine serum and 40 µg/ml
gentamicin, healthy stage V and VI oocytes were pressure-injected with
50 nl of a 10:1 mixture of rat brain Na
channel
1
and
subunit mRNA with
concentrations ranging from 5 to 50
ng/µl of injected solution. For injection, RNA was diluted in a
solution of 1 mM Tris-HCl, pH 7.5, and 0.01 mM EDTA.
Injected oocytes were maintained for 2-3 days as described above
before electrophysiological recording.
Electrophysiological Recording
Two-microelectrode
voltage clamp recordings were obtained from injected oocytes using a
Dagan CA-1 voltage clamp (Dagan Corp., Minneapolis, MN). The amplitude
of expressed Na currents was typically 1-5
µA. The bath was continuously perfused with Frog Ringer (115
mM NaCl, 2.5 mM KCl, 1.8 mM
CaCl
, 10 mM HEPES, pH 7.2). Recording electrodes
contained 3 M KCl and had resistances of <0.5 megaohms.
Pulses were applied, and data were acquired using a personal
computer-based data acquisition system (Basic-Fastlab, Indec Systems,
Sunnyvale, CA). Maximum possible series resistance compensation was
used to avoid errors due to relatively large Na
currents and to maximize resolution of their rapid kinetics.
Capacity transients were partially canceled using the internal clamp
circuitry. The remaining transients and leak were subtracted using the
P/4 procedure (Armstrong and Bezanilla, 1974).
, 10 mM EGTA,
and 10 mM HEPES for 5-10 min prior to removing the
vitelline layer with fine forceps (Methfessel et al., 1986).
Oocytes were then transferred to a solution containing 110 mM
KCl, 10 mM NaCl, 10 mM EGTA, 1 mM
MgCl
, 10 mM HEPES, pH 7.2, to depolarize the
membrane potential. Microelectrode measurement of oocyte membrane
potential after incubation in this solution gave a value of -3.25
± 0.25 mV (n = 4). For experiments with the
peptide blocker, macropatches were excised and placed beneath a stream
of bath solution containing 50 µM KIFMK peptide. Pipettes
were pulled from Corning 7052 (Garner Glass Co., Claremont, CA), coated
with Sylgard, and fire-polished before filling with Frog Ringer. Single
channel currents from the patch clamp amplifier (List EPC-7) were
sampled at 50 kHz and filtered at 5 kHz. Macropatch records were
filtered at 7 kHz.
Data Analysis
Normalized conductance-voltage
curves and inactivation curves were fit with the expression
A/{1 + exp[(V -
V)/k]} + (1 - A), where
V is the test pulse voltage (for activation) or prepulse
voltage (for inactivation), V is the midpoint of the curve,
k is a slope factor, and A is the amplitude. Least
squares fitting was done with the Sigma-Plot program (Jandel
Scientific, San Rafael, CA).
RESULTS
Point Mutations in Segment IVS6 Affect Fast
Inactivation
Fig. 1A illustrates the amino acid
sequence of segment IVS6, which contains both hydrophobic and neutral
polar amino acids. In order to identify functionally important
residues, we substituted alanine at each position by
oligonucleotide-directed mutagenesis. Alanine is small and minimally
hydrophobic, has a small effect on protein secondary structure, and is
frequently located in -helical transmembrane regions (Richardson,
1981; Zhang et al., 1992; Eriksson et al., 1992;
Blaber et al., 1993). Accordingly, alanine substitutions
should change the size and hydrophobicity of each residue in the helix
without disrupting overall protein structure. We also mutated
methionine 1770 to valine to recreate a naturally occurring mutation
associated with hyperkalemic periodic paralysis in the skeletal muscle
Na
channel (Rojas et al., 1991).
Figure 1:
Effects of point mutations in
transmembrane segment IVS6 on Na channel macroscopic
inactivation. A, single letter code for the amino acid
sequence of transmembrane segment IVS6 of the WT rat brain type IIA
Na
channel
subunit. Sequence numbers of the
amino and carboxyl termini are given (Auld et al., 1988). Each
native residue was individually mutated to alanine. Methionine 1770 was
also mutated to valine. B, two-microelectrode voltage clamp
recordings of currents due to expression of WT, F1764A, V1774A, and
F1764A/V1774A 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 as well as a 10-fold
excess of RNA encoding the rat brain
1 subunit (Isom et
al., 1992). C, fraction of current that fails to
inactivate for each mutant construct. Currents were elicited by pulses
to -5 mV as described above. The fraction of non-inactivating
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 IVS6 mutant. The asterisks indicate significant
differences from WT as determined by t test (p <
0.01). Data are from 3-10 experiments for each mutant. The
histogram data shown at position 1770 is for mutant M1770A. The data
for the mutant M1770V (not shown) were also not significantly different
from WT.
We
co-expressed WT and mutant Na channel type IIA
subunit RNAs with
1 subunit RNA in X. laevis oocytes and
analyzed Na
currents due to expression of WT and
mutant Na
channels by two-microelectrode voltage clamp
recording. Coexpression of WT
and
1 subunits yielded
Na
currents that closely resemble native rat brain
Na
currents in that they inactivated rapidly and
almost completely (Fig. 1B) (Isom et al.,
1992). A number of mutations in segment IVS6 caused incomplete
inactivation, resulting in sustained currents at the end of a
30-ms-long depolarization (Fig. 1, B and C).
The mutants F1764A and V1774A exhibited the largest sustained
Na
currents (Fig. 1C), whereas mutants
S1763A, Y1771A, I1775A, and L1776A had significantly increased but
smaller sustained Na
currents. Thirteen of the
remaining mutants (including M1770V) did not significantly affect
inactivation, while inactivation of Y1759A and V1768A was significantly
more complete than inactivation of WT.
channel inactivation. This suggestion is further supported by the
finding that simultaneous mutation of both residues (mutant
F1764A/V1774A) almost completely eliminated fast inactivation
(Fig. 1, B and C). These data indicate that
segment IVS6 plays an essential role in Na
channel
inactivation and that residues Phe
and Val
within this segment may be particularly important in the
inactivation process.
Figure 2:
Effects of point mutations on
Na current activation and inactivation. A,
activation curves. 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
(I
) was measured. Conductance was determined as
I
/(55 mV - V), where V was
the test pulse voltage and +55 mV was the approximate reversal
potential in our recording conditions. Normalized conductance was fit
with a Boltzmann relationship (see ``Experimental
Procedures''), and mean V and k values were
determined for each mutant (Table I). The curves shown are plots of the
Boltzmann relationship using these mean values. B, steady
state inactivation curves. 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 was plotted
as a function of prepulse potential, normalized and fit with the
Boltzmann equation. Mean V and k were determined for
each mutant construct (Table I). Curves shown are plots of the
Boltzmann equation using these mean values. C, recovery from
inactivation. Na
channels were inactivated by
15-ms-long pulses to 0 mV. The membrane was then repolarized to a
recovery potential for an interval of variable duration that was then
followed by a test pulse to 0 mV. Peak current during the test pulse
was divided by peak current during the inactivating pulse and plotted
as a function of time between the pulses. Such plots were fit with
single exponential functions to determine the recovery time constant at
each recovery potential. The time constant of the fit is plotted as a
function of recovery potential.
Steady state
inactivation was determined using a test pulse applied after a 100-ms
voltage step to various prepulse potentials. Mutations F1764A and
F1764A/V1774A shifted the voltage dependence of steady state
inactivation by +6 mV and +15 mV, respectively, whereas
V1774A was similar to WT (, Fig. 2B).
Prepulses more depolarized than -30 mV resulted in almost
complete inactivation of WT channels. In contrast, the steady state
inactivation curves for F1764A, V1774A, and F1764A/V1774A approached
nonzero asymptotes with strong depolarization (Fig. 2B).
This residual, non-inactivating component of the Na current was 31 ± 1% for V1774A, 12 ± 1% for F1764A,
and 79 ± 4% for F1764A/V1774A but only 3 ± 0.3% for WT.
Thus, the non-inactivating currents of mutant Na
channels, detected during 30-ms-long test depolarizations
(Fig. 1), showed little additional decay at 100 ms.
Inactivation of Mutant Na
To examine the
inactivation properties of WT and the F1764A, V1774A, and F1764A/V1774A
mutants in more detail, we recorded macroscopic currents from
cell-attached patches (macropatches), which gives better time
resolution and voltage control than two-microelectrode voltage clamp.
Fig. 3A shows typical records obtained by depolarization
to +20 mV. The overall characteristics of the records in the
macropatches were similar to those obtained in two-microelectrode
recordings although the fraction of sustained NaChannels
Is Less Complete at Positive Voltages
current was smaller. This difference between the two recording
techniques was probably due to incomplete resolution of the peak
current in two-microelectrode recording. With strong depolarizations as
depicted in Fig. 3A, activation is fast compared with
inactivation (Aldrich et al., 1983; Aldrich and Stevens,
1987), and the time constant (
) of current decay reflects mainly
the rate of channel inactivation. The
of WT current decay was
0.21 ± 0.008 ms. For F1764A, the
of the current decay was
similar to WT, 0.27 ± 0.003 ms, indicating that this mutation
did not substantially disrupt the rate of channel entry into the
inactivated state. In contrast, the time constant for V1774A (0.49
± 0.03 ms) was slower than WT, suggesting that for these
channels, entry into the inactivated state was slowed. Thus, F1764A and
V1774A mutations greatly destabilized the inactivated state, but entry
into the inactivated state was unaffected or slightly slowed.
Figure 3:
Voltage dependence of macroscopic
inactivation. 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,
F1764A, V1774A, and F1764A/V1774A mutants. Each trace is an
average of 10-15 sweeps. B, normalized currents from a
cell-attached macropatch on an oocyte expressing mutant V1774A elicited
by 15-ms pulses to -20 and +20 mV. C, the fraction
of non-inactivating current in cell-attached macropatches on oocytes
expressing WT (), F1764A (
), and V1774A (
) 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, three, and five experiments, respectively.
D, normalized tail current amplitudes in cell-attached
macropatches expressing F1764A, V1774A, and F1764A/V1774A (
)
mutants as a function of depolarizing pulse voltage. Tail currents were
measured at -140 mV following 11-ms depolarizing pulses to the
indicated potentials. Peak amplitudes were normalized to the tail
obtained after the pulse to +130 mV for each mutant. Data are from
representative experiments displayed as a semilog
plot.
The
inactivation of mutant F1764A and V1774A becomes progressively impaired
with increasingly positive depolarizing pulses. For example, the
records illustrated in Fig. 3B show that mutant V1774A
inactivated more completely at -20 mV than at +20 mV. The
progressively larger fraction of sustained current observed at more
positive voltages reveals this voltage-dependent impairment of
inactivation (Fig. 3, B and C). In contrast,
the fraction of sustained WT current was not voltage-dependent.
currents flowing through Na
channels that were open at the end of the depolarizing pulse and
then closed progressively following repolarization. Their amplitudes
are proportional to the fraction of Na
channels that
were open at the end of the depolarizing pulse. They are large and
easily measured because of the greatly increased driving force for
Na
influx at -140 mV.
current at the end of more positive depolarizing
pulses (Fig. 3, B and C). In contrast, for
mutant F1764A/V1774A, the amplitude of tail currents saturates at
positive voltages. Inactivation of this mutant is minimal at all
potentials, and the probability of channel opening is already high (see
Fig. 4
); therefore, further destabilization of inactivation at
positive potentials can have little additional effect. The
destabilization of inactivation with increased depolarization may be a
general property of Na
channels. Similar behavior is
seen in naturally occurring Na
channels with
incomplete inactivation such as those of the squid giant axon (Chandler
and Meves, 1970; Correa and Bezanilla, 1994a). This voltage-dependent
impairment of inactivation is normally masked in WT Na
channels with virtually irreversible inactivation, such as those
studied here, but is revealed by our mutations which destabilize the
inactivated state.
Figure 4:
Single
channel records and ensemble averages. Examples of single channel
traces (uppertrace) and ensemble averages (lowertrace) from cell-attached patches for each of the
indicated constructs. All depolarizations were to -20 mV.
Arrows indicate the beginning of 40-ms depolarizations from a
holding potential of -140 mV. The number of channels in the
patches was two, two, two, and one for WT, F1764A, V1774A, and
F1764A/V1774A, respectively. Single channel current magnitudes for each
mutant at -20 mV were similar to WT (WT, i = 1.26
± 0.02 pA (n = 5 patches); F1764A, i = 1.35 ± 0.07 (n = 2); V1774A, i = 1.32 ± 0.06 (n = 4);
F1764A/V1774A, i = 1.36 ± 0.06 (n = 3)). Sweeps of hyperactive moding behavior (Patlak and
Ortiz, 1986; Nilius, 1988; Zhou et al., 1991) or series of
null sweeps representing slow inactivation were omitted from ensemble
averages (Horn et al., 1984).
Gating of Single Mutant Na
To determine the mechanisms underlying the different
inactivation gating of the WT and mutant NaChannels
Is Altered
channels
observed in measurements of macroscopic Na
currents,
we examined the behavior of single Na
channels
(Fig. 4). Gating of single sodium channels is described in terms
of the general kinetic model shown in Fig. S1(Armstrong and
Bezanilla, 1977; Aldrich et al., 1983; Horn and Vandenberg,
1984; Scanley et al., 1990).
Figure S1:
Scheme 1.
Upon depolarization, sodium
channels undergo voltage-dependent transitions through multiple closed
states (states C through C
), open (state O),
and then inactivate (state I). The inactivated state can also be
reached from one or more closed states as illustrated in Fig. S1.
At depolarized potentials, inactivation of WT sodium channels is
irreversible on the millisecond time scale.
channels opened
once or twice at the beginning of the depolarization but then
inactivated and did not reopen until the membrane patch was repolarized
(Fig. 4). Ensemble averages of WT single channel current records
had a time course like WT currents in whole cell and macropatch
recordings. F1764A, V1774A, and F1764A/V1774A, on the other hand, all
exhibited repeated openings during 40-ms depolarizations
(Fig. 4), suggesting that inactivation was rapidly reversible for
these mutations. Ensemble averages showed sustained Na
currents for V1774A and F1764A/V1774A similar to those observed
in macroscopic current recordings. For F1764A, no clear sustained
current was detected in ensemble averages despite the frequent channel
openings observed late in the pulse. This finding is consistent with
the relatively small sustained currents seen with this mutant at the
macroscopic level (Fig. 3, A and C). These
results indicate that the behavior of single WT and mutant channels
observed in patch recordings accounts for the properties of
Na
currents observed at the macroscopic level.
) of these fits correspond to
the mean open time. The values for WT, F1764A, and V1774A were 0.24
± 0.03 ms (n = 5), 0.21 ± 0.04 ms (n = 2), and 0.33 ± 0.01 ms (n = 3),
respectively. The values for WT and F1764A were similar, indicating
that the F1764A mutation did not alter the rate of entry into the
inactivated state. The V1774A mean open time was slightly longer than
that for WT, suggesting that the rate of entry into the inactivated
state is slightly slower for this mutant. The mean open times obtained
from single channel recordings were similar to the
values for
inactivation determined from macropatches. The open times were not
obviously voltage-dependent, consistent with the idea that the rate of
entry into the inactivated state from the open state is
voltage-independent (Armstrong, 1981; Aldrich et al., 1983;
Armstrong and Bezanilla, 1977; Zagotta and Aldrich, 1990).
Figure 5:
Properties of single channel open times.
Open time histograms are displayed for WT, and the F1764A, V1774A, and
F1764A/V1774A mutants. Data were collected during pulses to -20
mV. For WT, F1764A, and V1774A, the solidlines are
fits of single exponentials to the binned data at times 100
µs. Time constants are 0.23, 0.18, and 0.33 ms, respectively. For
F1764/V1774, the data were fit with the sum of two exponentials of
approximately equal weight with time constants of 0.46 and 1.20 ms. The
dottedlines in each mutant panel are single
exponentials with the WT time constant of 0.23 ms. These histograms are
representative plots from five, two, three, and three experiments for
WT, F1764A, V1774A, and F1764A/V1774A,
respectively.
Fits of
the open times of F1764A/V1774A required two exponentials of nearly
equal weight, suggesting that the extensive disruption of inactivation
seen with this mutant reveals a second open state (Fig. 5). The
mean open times from three experiments were 0.37 ± 0.06 ms and
1.01 ± 0.16 ms. Two open states have been previously suggested
for Na channels from squid giant axon and mouse
neuroblastoma cells but are not normally observed for the WT brain
channel (Nagy et al., 1983; Nagy, 1987; Chandler and Meves,
1970; Correa and Bezanilla, 1994b).
channel
following depolarization and indicates the rate of transition of
Na
channels through the multiple closed states in the
activation pathway and subsequent channel opening (Fig. S1)
(Armstrong and Bezanilla, 1977; Armstrong, 1981; Aldrich et
al., 1983; Zagotta and Aldrich, 1990). Fig. 6A shows first latency distributions for WT, V1774A, and
F1764A/V1774A, corrected for the number of channels in the patch
(Patlak and Horn, 1982) and plotted as a cumulative probability density
function versus time. These plots illustrate the rate and
final extent of opening of single Na
channels as a
function of time after depolarization. For WT at -20 mV, single
channels opened rapidly, but the cumulative first latency curve reached
a maximum probability of only 0.65 because 35% of the depolarizations
failed to elicit channel openings and therefore resulted in null
sweeps. At the holding potential of -140 mV, it is unlikely that
any of the channels were inactivated. Thus, null sweeps represent the
irreversible transitions of WT Na
channels directly
from closed states in the activation pathway to the inactivated state
during depolarization (Fig. S1) (Aldrich et al., 1983;
Aldrich and Stevens, 1987).
Figure 6:
Properties of single channel closed times.
A, cumulative first latency distributions for WT, V1774A, and
F1764A/V1774A. Distributions at -20 mV are compared after
correcting for channel number (Patlak and Horn, 1982). For V1774A, two
distributions are shown, one obtained from a single channel patch and
the other obtained from a two-channel patch. The similarity of the two
distributions is an empirical verification of the correction procedure.
B, representative 40-ms-long traces of single V1774A channel
activity during depolarizations to the indicated voltages are shown.
This patch contained one channel. C, mean closed times for
V1774A. Data are from a single channel patch. Mean closed times were
determined with first latencies excluded and are displayed as a semilog
plot. The solidline is a least squares fit with a
slope corresponding to an e-fold change in closed time per 35 mV.
Qualitatively similar behavior was seen in three other patches
containing from two to five channels.
The cumulative first latency
distribution for V1774A also had a rapid component with a maximum open
probability of about 0.6, but virtually no null sweeps were observed
for this mutant. Instead, the first latency distribution for V1774A had
an additional slow component, and the total distribution increased to a
probability of opening of 1.0 by 20 ms (Fig. 6A). The
fast component of this distribution reflects channels that opened
normally, whereas the slow component likely represents channels that
inactivated from closed states and then opened. This component appears
as delayed openings in the V1774A mutant because the inactivated state
is rapidly reversible, but it appears as nulls in WT recordings because
WT inactivation is irreversible at -20 mV on the time scale of
our recordings.
currents and tail currents (Fig. 3). We
examined this phenomenon in more detail by measuring closed times in a
V1774A patch that contained only a single active channel. The voltage
dependence of these closed times is illustrated in
Fig. 6C, where mean closed times are plotted as a
function of membrane potential. The shortest mean closed time was
greater than 2 ms. This long duration is much longer than the mean
first latency for channel activation from closed states
(Fig. 6A), indicating that the mean closed times are
dominated by openings of single sodium channels from the destabilized
inactivated state. The mean closed times were shorter with stronger
depolarizations. This voltage dependence of the closed times in V1774A
indicates that the rate for exiting the inactivated state is increased
with depolarization. This increased frequency of reopening at more
positive membrane potentials may underlie the increase in sustained
macroscopic currents and tail currents observed at more positive
depolarizing potentials with this mutant (Fig. 3).
Mutational Tests for Hydrophobic Interactions of Residues
Phe
Studies using site-directed antibodies
(Vassilev et al., 1988, 1989) and mutagenesis (Stühmer
et al., 1989) indicate that the intracellular loop between
domains III and IV of the and Val
in Segment
IVS6 with the IFM Motif
subunit forms the Na
channel inactivation gate. Three hydrophobic residues,
Ile
, Phe
, and Met
(IFM),
are crucial components of this inactivation gate, and mutations of
residue Phe
to hydrophilic residues are sufficient to
nearly completely prevent fast inactivation (West et al.,
1992a). The IFM motif is thought to bind to a hydrophobic site in or
near the inner mouth of the channel pore (Scheuer et al.,
1993; Eaholtz et al., 1994). One possible interpretation of
the results of mutations F1764A and/or V1774A is that the native
residues at these positions form the binding site for the IFM motif and
interact directly with it. We investigated this possibility by
analyzing two additional types of mutants.
current for mutant V1774S was 0.28 ± 0.02 at 15 ms after
the beginning of a pulse to -5 mV, which is smaller than that
found for the mutant V1774A (0.38 ± 0.01). The fraction of
sustained current for mutant V1774N was 0.07 ± 0.003, which is
only slightly larger than WT (0.04 ± 0.005) (see
Fig. 1B for V1774A and WT). These data suggest that the
effects of mutations at position 1774 are not closely correlated with
the hydrophobicity of the substituted amino acid. In contrast, a series
of substitutions for Phe
in the IFM motif did show a
close correlation of inactivation with hydrophobicity (Scheuer et
al., 1993). Thus, it is unlikely that Phe
in the
IFM motif forms a required hydrophobic interaction with residue
Val
during the inactivation process.
and
Val
of transmembrane segment IVS6 and residue
Phe
of the IFM motif interact, we examined whether the
mutation F1489W, which increases the size and hydrophobicity of the
critical residue of the IFM motif, could compensate for the reduced
size and hydrophobicity of the F1764A and V1774A mutants and restore
inactivation when combined with these IVS6 mutations. This approach is
analogous to the analysis of suppressor mutants isolated by selection
in genetic screens. Fig. 7compares Na
currents
elicited with depolarizing pulses to +20 mV in macropatches
expressing WT, the single mutants F1489W, F1764A, and V1774A, or the
double mutants F1489W/F1764A and F1489W/V1774A. F1489W caused a small
sustained current, similar in size to that observed for F1764A.
Combining F1489W with F1764A or V1774A resulted in substantially
greater disruption of inactivation than observed for either of the
single mutants. Thus, Trp
is not as effective as
Phe
in mediating inactivation of Na
channels having WT or mutant IVS6 segments, and mutation F1489W
does not compensate for the effects of F1764A or V1774A.
Figure 7:
Analysis of single and double mutants
of the III-IV loop residue F1489W and the IVS6 residues F1764A or
V1774A. Normalized currents from cell-attached macropatches on X.
laevis oocytes expressing WT, F1489W, F1764A, V1774A,
F1489W/F1764A, and F1489W/V1774A mutants at +20 mV are shown. Each
trace is an average of 10-15 sweeps. Data for the
thermodynamic analysis described in the text is from four, three, two,
four, four, and two patches, respectively. The mean values for the
fraction of sustained current for WT, F1489W, F1764A, V1774A,
F1489W/F1764A, and F1489W/V1774A were 0.01 ± 0.005, 0.04
± 0.003, 0.08 ± 0.03, 0.28 ± 0.02, 0.15 ±
0.02, and 0.80 ± 0.002.
The results
of these double mutation experiments argue against a direct hydrophobic
interaction between the critical Phe residue of the IFM motif and
either the Phe or Val
residues. To test
this hypothesis more quantitatively, we examined the independence of
the changes in the free energy (
G) of the inactivation
process caused by these mutations using a thermodynamic cycle analysis
originally developed by Carter et al.(1984) to examine
structural changes in the active site of tyrosyl-tRNA synthetase.
According to this analysis, if two residues do not interact, then the
changes in the free energy of the inactivation process caused by their
mutation should be independent of each other and therefore additive if
the two mutations are combined. Conversely, if the residues interact,
then the changes in free energy of the inactivation process caused by
their mutation may be interdependent and therefore substantially
greater than or less than additive.
currents recorded during strong depolarizations, the fraction of
sustained current (S) is approximately equal to
k
/(k
+
k
), where k
is the rate
constant governing the transition from the open to inactivated state,
and k
is the rate constant governing the reverse
transition from the inactivated state to the open state
(Fig. S1). This equation can be rearranged to give the
equilibrium constant K
=
k
/k
=
(1/S) - 1. The free energy of inactivation is
G = -RTlnK
, where R and T are the universal gas constant and temperature,
respectively. The values for
G of inactivation at
+20 mV and their standard errors (Taylor, 1982) for WT and each
mutant were calculated from the mean values for the fraction of
sustained Na
current for each channel given in the
legend to Fig. 7. The changes in these values,
(
G) that were caused by each mutation are shown in
Fig. S2
.
Figure S2:
Scheme 2.
In these thermodynamic cycles, the sum of
(
G) values in the transitions from WT to each of the
individual mutations (e.g. WT
F1764A + WT
F1489W) should equal the predicted
(
G) value for the
double mutant (e.g. WT
F1764A + F1764A
F1489W/F1764A) if the two individual mutations act independently. The
sum of the free energy changes from WT to the double mutant in either
direction around the cycle gives an identical value for
(
G), as required for a thermodynamic cycle. For the
double mutant F1489W/F1764A,
(
G)was 1.60 ±
0.29 kcal/mol, a value indistinguishable from the value of 1.92
± 0.29 kcal/mol expected for independent effects. For the double
mutant F1489W/V1774A,
(
G) was 3.41 ± 0.27
kcal/mol, a value similar to the value of 2.84 ± 0.39 kcal/mol
expected for independent effects. This thermodynamic analysis,
therefore, supports the conclusion that the mutations at positions 1764
and 1774 act independently of the mutation at position 1489 to disrupt
inactivation (Carter et al., 1984).
Effects of Mutations in Segment IVS6 on Interaction with
a Free Peptide Containing the IFM Motif
Intracellular
application of the peptide acetyl-KIFMK-amide (KIFMK) containing the
IFM motif restores fast inactivation to Na channels
with mutations in their intrinsic IFM motif (Eaholtz et al.,
1994). Application of KIFMK to the intracellular surface of
Na
channels in excised membrane patches also restores
fast inactivation and binds to open Na
channels with a
K
of 21 µM (Eaholtz et
al., 1995). As an additional test of the hypothesis that
Phe
and Val
interact with the IFM motif
during inactivation, we examined the effect of application of the
peptide KIFMK to the intracellular surface of excised membrane patches
from Xenopus oocytes expressing these mutant Na
channels. Application of 50 µM KIFMK to excised
macropatches containing Na
channels with the F1489Q
mutation, which disables the intrinsic inactivation gate, caused
strong, time-dependent block of the current elicited by a depolarizing
pulse to -20 mV (Fig. 8A). These results are
consistent with restoration of fast inactivation by the peptide
(Eaholtz et al., 1994). The same concentration of peptide
similarly blocked the Na
current conducted by the
F1764A/V1774A mutant (Fig. 8B). The restoration of
inactivation of the IVS6 double mutant by KIFMK was comparable with
that observed with mutant F1489Q. In contrast, the inactive peptide
acetyl-KIQMK-amide (Eaholtz et al., 1994) at 300 µM did not restore inactivation of F1764A/V1774A (data not shown).
These results indicate that the mutations at positions 1764 and 1774 do
not alter the binding site for the free peptide containing the IFM
motif.
Figure 8:
Effect of KIFMK peptide on excised,
inside-out macropatches from oocytes expressing the F1489Q (A)
and F1764A/V1774A (B) 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 are shown. The traces shown were obtained without
averaging and are representative of three patch experiments for each
mutant construct.
DISCUSSION
We have identified amino acid residues in transmembrane
segment IVS6 of the Na channel
subunit that are
critical for fast inactivation. Point mutations near the middle
(F1764A) and toward the cytoplasmic end (V1774A) of this transmembrane
segment caused sustained Na
currents, indicating that
these mutations destabilized the inactivated state. The double mutation
F1764A/V1774A prevented fast inactivation nearly completely. Single
channel analysis revealed that the sustained currents caused by
mutation of these residues were due to a large increase in the
frequency of reopenings from the inactivated state without a major
alteration in the rate of activation or the rate of entry into the
inactivated state. These reopenings account for the sustained currents
seen at the macroscopic level. Apparently, the Phe
and
Val
residues play a particularly important role in fast
inactivation.
channels is
hypothesized to involve a cytoplasmic inactivation gate folding into
and occluding the ion conducting pore. Experimental evidence with
site-directed antibodies (Vassilev et al., 1988, 1989) and
mutations (Stühmer et al., 1989) indicates that the
intracellular loop connecting domains III and IV acts as the
inactivation gate. The hydrophobic motif IFM is a critical component of
the inactivation gate, and these residues are thought to interact with
a receptor site that becomes available in the activated Na
channel (West et al., 1992a; Eaholtz et al.,
1994). This interaction is due primarily to hydrophobic forces (Scheuer
et al., 1993). Although mutations of amino acid residues in
segment IVS6 disrupt the stability of the inactivated state, our
results indicate that these residues probably do not form this
hydrophobic receptor for the IFM motif. Thus, the destabilization of
inactivation caused by substitution at position 1774 was not correlated
with amino acid hydrophobicity; thermodynamic analysis of double
mutations at positions 1764 and 1774 in IVS6 and at position 1489 in
the IFM motif suggest that the critical residues in these two regions
act independently in the process of inactivation; and the KIFMK peptide
restored inactivation to the F1764A/V1774A double mutant as effectively
as to a mutation in the IFM motif itself. The results of each of these
tests contradict the predictions of models in which these IVS6 residues
interact directly with the IFM motif. Therefore, we have identified
amino acid residues in segment IVS6 of the Na
channel
that are critical for fast inactivation and for the stability of the
inactivated state but do not appear to interact directly with the IFM
motif in the fast inactivation gate.
channel inactivation comes from
studies of mutations in the skeletal muscle Na
channel
that cause hyperkalemic periodic paralysis. One of these mutations is
analogous to M1770V within segment IVS6 (Rojas et al., 1991).
Expression of skeletal muscle Na
channels containing
this mutation in mammalian cells causes small sustained Na
currents (Cannon and Strittmatter, 1993). In our work, expression
of rat brain Na
channels with this mutation in
Xenopus oocytes did not produce major effects on inactivation,
indicating that the role of this amino acid residue may be less
critical than others identified in this study. Mutations causing
substantial inhibition of Na
channel inactivation as
observed in our experiments would probably be lethal and therefore are
not expected to be found in human genetic studies.
helix (Noda et al., 1986b; Durell
and Guy, 1992). Helical wheel analysis (Schiffer and Edmundson, 1967)
indicates that Phe
and Val
fall on the
same face of the helix separated by an angle of approximately 80°
and are 3 helical turns (15 Å) distant from each other.
Interestingly, two other mutations that disrupted inactivation, Y1771A
and I1775A, also fall on the same helix face. The mutations F1764A and
Y1771A also reduce the affinity of Na
channels for
local anesthetic drugs by up to 2 orders of magnitude (Ragsdale et
al., 1994). These drugs are thought to bind to a receptor site
within the pore of the Na
channel and to interact with
the closed inactivation gate (reviewed in Hille(1992)). Thus, an
intriguing possibility is that these residues are oriented toward the
inner lumen of the ion conducting pore, where they play a role in
stabilizing both drug binding and channel inactivation.
channel by the peptide KIFMK at approximately the same rate in WT
and mutant Na
channels. These results appear to rule
out major effects of the IVS6 mutations on activation, coupling of
activation to inactivation, closure of the inactivation gate, and
formation of the hypothetical receptor for the IFM motif. Possibly,
these critical amino acid residues in segment IVS6 bind to an
unidentified motif in the inactivation gate whose interaction with the
pore is also critical for stable fast inactivation of the Na
channel. Alternatively, binding of the free KIFMK peptide might
not be destabilized by these mutations, whereas binding of the IFM
motif in its naturally occurring, tethered form is. Experiments that
test these alternatives will further define the mechanisms of
Na
channel inactivation and the role of segment IVS6
in this process.
Table: Voltage dependence of activation and
inactivation for selected mutants
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.