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INTRODUCTION |
Voltage-gated Na+ channels are responsible for the
initiation and propagation of action potentials in nerve, heart, and
skeletal muscle (1-5). The major structural component of voltage-gated Na+ channels is a 260-kDa
subunit, which forms the
voltage-gated, Na+-selective pore. In mammalian brain, the
subunit associates with auxiliary
subunits of 33-36 kDa (1, 6,
7). The
subunit contains four homologous domains (I-IV), each
containing six predicted transmembrane
-helices (S1-S6) and an
additional membrane-reentrant pore loop (1, 4, 5). The S6 segments of
each homologous domain are arranged in a square array surrounding the
inner pore, whereas the membrane-reentrant pore loops between the S5
and S6 segments line the narrower outer pore and form the ion
selectivity filter (1, 4, 5).
Clinically important drugs, including local anesthetics and some
anticonvulsants and antiarrhythmics, exert their therapeutic effects by
binding preferentially to the inactivated state and blocking
voltage-gated Na+ channels. Local anesthetics and the
related phenylalkylamine blockers of Ca2+ channels have
been characterized biophysically as pore blockers (8-10) and therefore
provide molecular probes for identification of pore-lining amino acid
residues. Photoaffinity labeling studies of phenylalkylamine binding to
L-type Ca2+ channels initially implicated the IVS6 segment
in forming the inner pore lining (11). Subsequent mutagenesis studies
of Na+ channels and Ca2+ channels (12, 13)
identified analogous sets of amino acid residues in the IVS6 segment
that form the receptors sites for these drugs and therefore line the
inner pores. Mutations F1764A and Y1771A in segment IVS6 reduced
affinity of inactivated Na+ channels for the local
anesthetic etidocaine by up to two orders of magnitude (12). Mutation
of these homologous residues also substantially reduced block of
inactivated Na+ channels by other local anesthetic,
antiarrhythmic, and anticonvulsant drugs in brain type IIA, brain type
III, and skeletal muscle Na+ channels (12, 14-20).
X-ray crystallographic analysis of a K+ channel from
Streptomyces lividans
(KcsA)1 shows directly that
the channel pore is formed by transmembrane segments analogous to the
S6 segments and S5-S6 pore loops of Na+ and
Ca2+ channels (21). A water-filled cavity on the
intracellular side of the ion selectivity filter includes the amino
acid residues analogous to Phe-1764 and Tyr-1771 of Na+
channels and likely corresponds to the binding site of pore-blocking drugs. In this structure, the S6-like segments cross to form an apparent barrier to ion movement at the intracellular end of the pore.
Therefore, in addition to their role in forming the inner pore lining,
the S6 segments may also serve as the activation gate and move during
activation (22, 23).
Because local anesthetics bind in a cavity in the pore formed by a
symmetrical array of all four S6 segments, it is likely that the S6
segments in domains I, II, and III of Na+ channels also
contribute to the receptor sites for local anesthetic, antiarrhythmic,
and anticonvulsant drugs. Consistent with this hypothesis,
site-directed mutagenesis of the skeletal muscle Na+
channel showed that replacement of Asn-434 and Leu-437 within transmembrane segment IS6 with lysine significantly reduced block by
etidocaine, suggesting that the positive charge of the inserted lysine
residue can disrupt normal drug binding (24). In this study, we have
used alanine-scanning mutagenesis to investigate the role of amino acid
residues in transmembrane segment IIIS6 in channel gating and block by
anticonvulsant and local anesthetic drugs. Our results further define
the pore-lining residues of the inner pore of Na+ channels
and identify new components of the receptor site for local anesthetics
and related pore-blocking drugs.
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EXPERIMENTAL PROCEDURES |
Mutagenesis of rIIA Channels--
Mutations were prepared by a
two-step polymerase chain reaction protocol using two mutagenic primers
and two restriction site primers. The mutagenic fragment and the
plasmid pCDM8rIIA were digested with BstEII and
BlpI restriction endonucleases. The mutagenic fragment was
then subcloned into the plasmid pCDM8rIIa via those restriction sites.
The mutations were confirmed by restriction mapping and DNA sequence analysis.
Na+ Channel Expression in Xenopus
Oocytes--
Plasmids encoding wild-type and mutant Na+
channel
subunits and wild-type
1 subunits were
linearized, and RNA was transcribed as described previously (25).
Xenopus laevis oocytes were harvested, maintained, and
injected with RNA by standard methods as described previously (25).
Two-microelectrode Voltage-clamp Recordings from
Oocytes--
Na+ channel recordings were obtained from
injected oocytes using a Dagan CA-1 voltage clamp (Dagan Corp.) as
described previously (25). The bath was continuously perfused with
Ringer solution containing (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 HEPES, pH 7.2, adjusted with NaOH. Recording
electrodes contained 3 M KCl and had resistances of <0.5
megohm. Stock solutions of lamotrigine and 619c89 (Glaxo Wellcome) were
prepared in 25 mM HCl, 227c89 (Glaxo Wellcome) was prepared
in water, and 4030w92 (Glaxo Wellcome) and etidocaine (Astra) were
dissolved in dimethyl sulfoxide. All drugs stocks were then diluted to
the desired concentration in the bath solution.
Analysis of Periodicity of Gating Perturbations--
To evaluate
the gating perturbations caused by IIIS6 mutants, we calculated the
difference in Gibb's free energy between closed and open states at 0 mV (
G0) according to
G0 =
RTV0.5/k, where R is the
gas constant, T is the absolute temperature in degrees K,
V0.5 is the half-maximal activation voltage, and k is a slope factor. V0.5 and
k values were determined from fits of activation curves to
single Boltzmann functions. The difference in
G0 between wild-type and mutant
channels was calculated according to
G0 =
Gmut0
Gwt0.
To evaluate the periodicity of gating perturbations produced by IIIS6
mutants, we used Fourier transform methods (26-29). The Fourier
transform power spectrum (P(
)) was calculated according to the equation,
|
(Eq. 1)
|
where X(
) =
j=1n [(Vj
V
)
sin(j
)]; Y(
) =
j=1n [(Vj
V
) cos (j
)],
and
is the angular frequency, n is the number of
residues in a segment, Vj is
|
G0| at a given position j,
and
V
is the average value of

G0 for the segment.
 |
RESULTS |
Effects of Mutations in Segment IIIS6 on
Voltage-dependent Activation of Na+
Channels--
Alanine-scanning mutagenesis of amino acid residues in
transmembrane segment IIIS6 of the
subunit of the rat brain type IIA Na+ channel (rIIA) was used to investigate their
functional role in activation, inactivation, and binding of
pore-blocking drugs. Conversion of hydrophobic amino acid residues to
alanine has only small effects on protein secondary structure but
changes the size and chemical properties of the residue significantly
(30, 31). To identify functionally important residues in transmembrane
segment IIIS6, we substituted alanine for individual native amino acid residues from Val-1454 to Ile-1473. We coexpressed the wild-type and
mutant Na+ channel
subunits and wild-type
1 subunits in Xenopus oocytes and measured
Na+ current using a two-microelectrode voltage clamp.
Mutations throughout the IIIS6 segment caused significant negative and
positive shifts in the voltage dependence of activation compared with
wild-type. For example, the voltage for half-maximal activation of
mutants L1467A, F1468A, and I1469A was shifted by
5,
14, and +8 mV,
respectively (Fig. 1A).
Mutations V1454A, F1456A, I1458A, F1459A, T1464A, V1471A, and I1472A
also caused significant negative shifts in voltage dependence of
activation (Fig. 1B). In contrast, mutations G1460A, F1462A,
L1465A, N1466A, G1470A, and I1473A caused significant positive shifts
(Fig. 1B). The steepness of the voltage dependence of the
activation process (k = 5.4 ± 0.1 mV for
wild-type) was significantly decreased by mutations L1465A
(k = 6.2 ± 0.2 mV), N1466A (k = 6.5 ± 0.2 mV), and L1467A (k = 7.6 ± 0.2 mV).

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Fig. 1.
Effects of point mutations on Na+
channel activation. A, conductance-voltage curves
for wild-type, L1467A, F1468A, and I1469A. Peak current
versus voltage relationships were measured using 30-ms test
pulses to potentials from 50 mV to +70 mV from a holding potential of
90 mV. Conductance was determined as
IP/(VR V), where IP is peak inward
current, VR is the reversal potential, and
V is the test pulse voltage. Normalized conductance was fit
with a single Boltzmann relationship of the form:
G(V) = 1/(1 + exp[(V V0.5)/k ]), where V0.5
is the half-maximal activation voltage and k is a slope
factor. Mean V0.5 and k values were
determined for each mutant. The curves shown are plots of the Boltzmann
relationship using these mean values. B,
V0.5 for activation of IIIS6 mutants compared
with the wild-type Na+ channels. The histogram shows the
differences between voltage for the half-maximal activation of the
wild-type and mutant Na+ channels. Mean
V0.5 values were obtained from Boltzmann fits of
normalized conductance versus voltage plots as described
above. The asterisks indicate significant differences from
wild-type as determined by t test (p < 0.01).
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Periodicity of Effects of Mutations on
Voltage-dependent Activation--
By analogy with the
bacterial K+ channel KcsA, whose structure is known (21),
S6 transmembrane segments are assumed to be
-helical in structure
and to line the inner aspect of the ion-conducting pore. Therefore, one
surface of each S6 helix is expected to face the lumen of the pore,
whereas the other sides interact with adjacent transmembrane segments.
S6 transmembrane segments are proposed to change conformation via
rotation during channel activation (22, 23, 32, 33), leading to changes
in side-chain interactions with neighboring
-helices. Power spectral
analysis of periodicity in the effects of mutations in successive
positions along the
-helix can reveal position-dependent
interactions with neighboring structural elements during activation
gating (34-36).
Changes in free energy of activation
(
G0) for each mutation were
calculated from the results of Fig. 1 and plotted in Fig. 2A. The

G0 values parallel the changes
in the voltage for half-maximal activation from which they were derived
(Fig. 1B). Inspection of the

G0 values reveals that they are
consistently negative for the six most extracellular residues of the
helix, indicating that these mutations favor activation. This pattern
is interrupted at G1460A, mutation of which produces a strongly
positive 
G0 value. Progressing
inward through the IIIS6 segment, the

G0 values assume a periodic
pattern of positive and negative changes. These results suggest that
the mutated residues make structurally or functionally important
contacts with other transmembrane segments that differ depending on
their location around the IIIS6 helix. If interactions of IIIS6 with
different surrounding transmembrane segments have different effects on

G0 for activation, a pattern of

G0 values with
-helical
periodicity of approximately 100° is expected. Power spectral
analysis of the effects of mutations of the residues intracellular to
G1460 that caused positive

G0 values yields a peak at
approximately 97° (Fig. 2B), consistent with an
-helical structure in which the native residues on one face of the
helix make interactions that favor activation. Residues intracellular
to G1460 with negative 
G0
values yielded a less well-defined peak of angular periodicity at
101°, suggesting that a different, broader face of the
-helix makes interactions that favor the resting state of the channel and
oppose activation (data not shown). All mutations except I1458A and
F1468A make relatively small (<1 kcal) perturbations in

G0, indicating that the change
in energy of interaction for any single residue is relatively
small.

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Fig. 2.
Power spectral analysis of changes in the
free energy of activation associated with mutations in transmembrane
segment IIIS6. A, free energy changes
( G0) were calculated from fits
to activation curves reported in Fig. 1B as described
previously (34, 35). B, angular frequency of positive shifts
in  G0 determined for mutations
of residues in the intracellular portion of IIIS6 between F1462A and
I1473A (26, 36). Periodicity was evaluated using the Fourier transform
power spectrum P( ) and plotted versus angular
frequency.
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Effects of Mutations in the IIIS6 Segment on
Inactivation--
Most mutations of amino acid residues in the
cytoplasmic half of the IIIS6 segment also caused strong shifts in the
voltage dependence of inactivation during 100-ms prepulses to the
indicated membrane potentials (Fig.
3A). For example, the voltages
for half-maximal inactivation of mutations L1465A and L1467A were
shifted by
11 and
14 mV, respectively (Fig. 3A).
Significant negative shifts were also caused by mutations F1463A,
N1466A, F1468A, G1470A, V1471A, and I1472A (Fig. 3B). In
contrast, mutations I1473A and I1469A caused significant positive
shifts in the voltage dependence of inactivation (Fig. 3, A
and B). Inactivation gating was unaffected for most of the
mutations in the extracellular half of IIIS6. Only I1458A caused a
significant negative shift (Fig. 3B). The two
mutations giving positive shifts (I1469A and I1473A) are four residues
apart, and mutations F1463A, L1467A, and V1471A, which define local
peaks in the negative shifts of inactivation, are also four residues
apart in the sequence. Therefore, the changes in voltage
dependence of inactivation for mutations at the intracellular end of
IIIS6 also conform approximately to an
-helical periodicity of 3.6 residues per 360° turn.

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Fig. 3.
Steady-state inactivation of the wild-type
and mutant Na+ channels. A, steady-state
inactivation curves for wild-type, L1465A, L1467A, and I1469A.
Inactivation curves were measured using 100-ms prepulses to the
indicated potentials followed by a test pulse to 0 mV. Peak test pulse
current was plotted as a function of prepulse potential, normalized,
and fit with a Boltzmann function, I = 1/(1 + exp[(V V0.5)/k]), where
V0.5 is the membrane potential at the
half-maximal current and k is a slope factor. Mean
V0.5 and k values were determined for
each mutant. Curves shown are plots of the Boltzmann function using
these mean values. B, V0.5 for
inactivation of IIIS6 mutants compared with the wild-type
Na+ channels. The histogram shows the differences between
voltage for the half-maximal inactivation of the wild-type and mutant
Na+ channels. V0.5 values were
obtained from Boltzmann fits of normalized current versus
voltage plots. The asterisks indicate significant
differences from wild-type as determined by t test
(p < 0.01).
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Effect of Mutations in Transmembrane Segment IIIS6 on Affinity of
Inactivated Na+ Channels for Anticonvulsants and Local
Anesthetics--
The anticonvulsant lamotrigine, which is used for
treatment of epilepsy and bipolar disorder, acts by blocking brain
Na+ channels in a voltage- and
frequency-dependent manner (37-39). Etidocaine, an
effective local anesthetic that is used for regional anesthesia, also
blocks brain Na+ channels with strong voltage and frequency
dependence (40). The efficacy of these drugs as anticonvulsants and
antiarrhythmics stems from their ability to selectively block
Na+ channels during abnormal membrane depolarizations and
rapid bursts of action potentials that characterize neuronal and
cardiac pathologies (8, 9, 41). The selectivity of these drugs for
Na+ channels in depolarized cells results from the
preferential binding to the open and inactivated states that
predominate at depolarized membrane potentials. This
state-dependent drug action can be explained by an
allosteric model in which a modulated drug receptor is in a low
affinity conformation when the channel is in the resting state and
converts to a high affinity conformation when the channel is
inactivated by depolarization (8, 9).
Etidocaine, lamotrigine, and related compounds have been previously
used to identify amino acid residues involved in binding of
pore-blocking drugs in transmembrane segment IVS6 (12, 42). For primary
screening of mutants in segment IIIS6 for their effect on drug binding
to the inactivated state of the Na+ channel, block of
inactivated Na+ channels by lamotrigine or compound 619c89,
a tricyclic lamotrigine congener (43), was determined during a test
pulse to 0 mV following a 15-s depolarization to a holding potential at
which 70-80% of the Na+ current was inactivated (Fig.
4A, control trace
c). At such a depolarized holding potential (
50 mV), addition of
100 µM lamotrigine (Fig. 4A, trace
d) reduced the Na+ current by 75%. At the same
concentration, no significant block of Na+ current was
observed when the holding potential was
120 mV (Fig. 4A,
traces a and b). From experiments like the one
illustrated in Fig. 4A, we determined the dissociation
constant for the inactivated state (KI) for
wild-type and mutant Na+ channels according to Kuo and Bean
(44). Apparent affinities for lamotrigine are presented in Fig.
4B and for 619c89 in Fig. 4C. Wild-type
Na+ channels were inhibited by lamotrigine and 619c89 with
dissociation constants of 32 and 10 µM, respectively, in
agreement with previously reported results (42). L1465A and I1469A
decreased the affinity of lamotrigine for the inactivated state of the
channel, resulting in 8- and 3-fold increases in
KI, respectively (Fig. 4B). The same
mutations increase KI of 619c89 for the
inactivated state of the channel approximately 3-fold (Fig.
4C). Both L1465A and I1469A caused positive shifts in
voltage dependence of activation (Fig. 1B). To test whether
these shifts in activation could account for the observed decrease in
drug affinity by preventing full activation of the Na+
channels, we also examined inactivated-state affinity using strong depolarizations for which both wild-type and mutant channels were fully
activated. We applied pulses to +20 mV for 15 s followed by a
short interpulse interval at
120 mV to allow recovery from fast
inactivation but not from drug block, and then by 15-ms test pulse to
+20 mV. This protocol yielded dissociation constants for the drugs in
agreement with those reported in Fig. 4B (data not
shown).

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Fig. 4.
Affinity of inactivated wild-type and mutant
Na+ channels for lamotrigine and 619c89. A,
representative current traces for wild-type Na+ channels in
the absence (traces a and c) or in the presence
of 100 µM lamotrigine (traces b and
d). Traces a and b were measured
during a test pulse to 0 mV following a 15-s step to 120 mV. Traces
c and d were measured at 0 mV following a 15-s
step to 50 mV. B and C, apparent dissociation
constants for block of inactivated channels by lamotrigine
(B) and 619c89 (C). For mutant Na+
channels, the depolarized holding potential was varied so that 70-80%
of the channels were inactivated after the 15-s conditioning prepulse.
The dissociation constant for the inactivated state
(KI) for each mutant was calculated according to
Kuo and Bean (44) as KI = (1 h)(Emax/E 1)[D], where h is the fraction of inactivated
channels, Emax is the maximal block that is
assumed to be the complete block of the current, and E is
the amount of block at drug concentration of [D].
Error bars indicate S.E. The asterisks indicate
significant differences from wild-type as determined by t
test (p < 0.01).
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Because mutations L1465A and I1469A had effects on the affinity of
lamotrigine, we examined the affinities of these mutants for the
structurally related compounds 4030w92 and 227c89 as well as the
structurally unrelated local anesthetic etidocaine. L1465A, N1466A, and
I1469A decreased the affinity of 4030w92 5-, 3-, and 5-fold,
respectively (Fig. 5A). Block
by compound 227c89 was disrupted in L1465A and I1469A mutants by about
3-fold (Fig. 5B). Larger disruptions in block by etidocaine
were observed with mutations L1465A, N1466A, and I1469A resulting in
6-, 8-, and 7-fold increases in KI, respectively
(Fig. 5C). Thus, mutations L1465A and I1469A decreased the
affinity of all compounds tested substantially. In contrast, mutation
N1466A caused the largest disruption of etidocaine binding but had no
effect on binding of compound 227c89 or of lamotrigine and 619c89.

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Fig. 5.
Affinity for block of inactivated
Na+ channels by 4030w92, 227c89, and etidocaine.
Dissociation constants for the inactivated state
(KI) of the indicated mutant channels was
determined for 4030w92 (A), 227c89 (B), and
etidocaine (C) as described in the legend to Fig. 4.
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Effect of Mutations L1465A, N1466A, and I1469A on Block of Resting
Na+ Channels by Lamotrigine and Etidocaine--
To
determine whether IIIS6 mutants affected block of resting
Na+ channels at negative membrane potentials, we applied
15-ms test pulses from holding potentials of
80 to
120 mV in the
absence and the presence of 500 µM lamotrigine. Mutations
L1465A, N1466A, and I1469A increased affinity of lamotrigine for
resting channels approximately 2-fold (Fig.
6A). L1465A and N1466A also
increased affinity of etidocaine for resting channels 3-fold (Fig.
6B). In contrast, I1469A did not affect the affinity of
etidocaine for resting channels (Fig. 6B). No IIIS6 mutants
caused significant decreases in resting affinity of lamotrigine or
etidocaine (data not shown). Thus, the loss of drug binding affinity of
these mutant channels is specific for the high affinity inactivated
state.

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Fig. 6.
Affinity for block of resting Na+
channels by lamotrigine and etidocaine. Voltage dependence of the
affinity (Kr) for block of resting wild-type and
mutant Na+ channels by lamotrigine (A) and
etidocaine (B). Test pulses to 0 mV were applied after
stepping to the indicated holding potentials
(Vh) for 60 s. Kr
was calculated according to a single-site binding isotherm:
Kr = [D][(1/E) 1], where E represents the fraction of current remaining at
drug concentration [D].
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Effect of Mutations F1462A, L1465A, N1466A, and I1469A on
Frequency-dependent Block of Na+
Channels--
Frequency-dependent block of Na+
channels is observed for drugs that bind to the channel rapidly in the
open state and then bind with high affinity to the inactivated state
(8, 9). Compounds 619c89 and etidocaine produce strong
frequency-dependent block of the rat brain type IIA
Na+ channels (Fig. 7) (12,
42). To determine whether IIIS6 mutants alter
frequency-dependent block by compound 619c89, we applied 10-Hz trains of 20-ms pulses to 0 mV from a holding potential of
90
mV and recorded Na+ currents. Only I1469A significantly
reduced frequency-dependent block by 619c89 compared with the
wild-type (Fig. 7A). In contrast, mutations F1462A and
N1466A substantially increased frequency-dependent block.
Surprisingly, given the reduced affinity of these inactivated channels
for block by 619c89, L1465A had no significant effect on
frequency-dependent block. Mutations L1465A and I1469A
significantly reduced use-dependent block by etidocaine
during 2-Hz trains (Fig. 7B). In contrast, mutations N1466A
and F1462A had little effect on frequency-dependent block
by etidocaine (Fig. 7B).

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Fig. 7.
Frequency-dependent block of
wild-type and mutant Na+ channels by 619c89 and
etidocaine. Frequency-dependent block of wild-type and
mutant Na+ channels by 50 µM 619c89
(A) and 100 µM etidocaine (B).
Cells were held at 90 mV and stimulated by 20-ms test pulses to 0 mV
in 10-Hz (A) or 2-Hz (B) pulse trains. The peak
current amplitude of each pulse in the presence of the drug was
measured, normalized with respect to control currents elicited before
drug application, and plotted versus the pulse number.
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Effects of Mutations F1462A, L1465A, N1466A, and I1469A on Recovery
of Na+ Channels from Inactivated-state Block--
Recovery
of drug-blocked inactivated Na+ channels to the resting
state was studied for the IIIS6 mutants that affected voltage- and/or
frequency-dependent block of Na+ channels by
619c89 and etidocaine. We measured the rate of recovery by applying a
500-ms conditioning prepulse to 0 mV to produce drug block of
inactivated channels followed by a recovery interval of variable
duration and a test pulse to 0 mV. In control conditions, recovery
after depolarization follows a double-exponential time course (Fig.
8A). In the presence of 50 µM 619c89, the fast time constant reflects recovery from
inactivation of the small fraction of channels that was not blocked
during the conditioning prepulse. The slow time constant reflects slow
dissociation of the drug from the channels that were blocked during the
conditioning prepulse (
drug). For drug concentrations in
which a large fraction of channels was blocked during the
depolarization, only a single drug-induced recovery component was
observed despite the two exponential components observed in control.
Slow time constants (
drug) induced by 619c89 are
presented in Fig. 8C. For wild-type channels
drug was 384 ± 15 ms.
drug for
F1462A was not significantly different (379 ± 27 ms) (Fig. 8,
A and C). In contrast, I1469A recovered with
drug of 187 ± 6 ms (Fig. 8, B and
C), 2-fold faster than the wild-type recovery rate. Recovery
from block of both L1465A and N1466A was about 2-fold slower than for
wild-type channels with
drug values of 710 ± 61 ms
and 820 ± 30 ms, respectively. The slower dissociation of 619c89
from L1465A would counteract the effect of reduction in binding
affinity by that mutation, and the slower dissociation from N1466A
would enhance frequency-dependent block.

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Fig. 8.
Recovery of the wild-type and mutant
Na+ channels from drug block of inactivated channels.
A and B, representative time courses of
Na+ channel recovery from inactivation in control
(filled symbols) and in the presence of 50 µM
619c89 (open symbols) for wild-type (A) and
I1469A (B) channels. Recovery was measured using a 500-ms
conditioning pulse to 0 mV followed by a recovery interval of the
indicated duration (1.5-4000 ms) at 90 mV followed by a test pulse
to 0 mV. The peak test pulse current was divided by the peak
conditioning pulse current and plotted against the recovery interval.
The curves are least-squares fits of a two-exponential function to the
data. C and D, recovery time courses in the
presence of 50 µM 619c89 (C) or 100 µM etidocaine (D) were fit with two
exponentials. Mean values of the slow time constant
( drug) are plotted for the indicated mutant channels.
Note the different units of the ordinates in C and
D. Error bars indicate S.E. The
asterisks indicate significant differences from wild-type as
determined by t test (p < 0.01).
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Wild-type Na+ channels recovered from block by etidocaine
with
drug of 2.3 ± 0.2 s, 6-fold slower than
for compound 619c89. F1462A and I1469A recovered with
drug values of 1.7 ± 0.3 s and 2.0 ± 0.3 s, respectively, which were not significantly different from
the wild-type channels. L1465A and N1466A recovered with
drug values of 0.76 ± 0.05 s and 1.05 ± 0.05 s, respectively (Fig. 8D), which were
approximately 3- and 2-fold faster than the recovery of wild-type channels.
We also examined the rate of onset of block by 619c89 and etidocaine of
F1462A, L1465A, N1466A, and I1469A during depolarizations. Only F1462A
affected onset block by 619c89 significantly. Onset of block was
2.7-fold faster than with the wild-type channel (data not shown). This
is consistent with the faster and greater development of
use-dependent block during a train by 619c89 in this mutant channel (Fig. 7A). Only L1465A had a significant effect on
the rate of onset of block by etidocaine, which was 3-fold slower compared with the wild-type channel (data not shown).
 |
DISCUSSION |
Position-dependent Shifts in the Voltage Dependence of
Activation Suggest Rotational Gating Movement of the IIIS6
Segment--
Point mutations throughout the IIIS6 segment of the
Na+ channel
subunit affected the voltage dependence of
activation gating. In the cytoplasmic two-thirds of IIIS6 the reduction
of size, hydrophobicity, and/or polarity of residues produced by
alanine substitutions stabilized or destabilized the open state of the channel, depending on the position of the mutated residue on the circumference of the
-helix. Mutations F1462A, L1465A, N1466A, I1469A, and I1473A induced positive shifts of the voltage for half-maximal activation and positive

G0 values (Figs. 1B
and 2A). The positive shift of the activation curve of these
mutants indicates that the native residues at these positions make
interactions that stabilize the open channel or destabilize closed
channels. These amino acid residues spread across a 100° section of
the circumference of the IIIS6
-helix. They are proposed to face the
lumen of the pore in the activated and inactivated states of the
channel, because Leu-1465 and Ile-1469 are required for high affinity
binding of the pore-blockers lamotrigine and etidocaine (Fig.
9).

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Fig. 9.
Lamotrigine binding to transmembrane segments
IIIS6 and IVS6 of the rat brain type IIA Na+ channel.
A, side view of the proposed location of the lamotrigine
binding site within the pore. B, -helical representation
showing the axial position of mutations causing reduction in affinity
of lamotrigine (LTG) for inactivated Na+
channels.
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Thr-1464, Leu-1467, Phe-1468, Gly-1470, Val-1471, and Ile-1472 are
spread across 200° of the opposite side of the circumference of IIIS6
segment (Fig. 9). Point mutations of Thr-1464, Leu-1467, Phe-1468,
Val-1471, and Ile-1472 to alanine induced negative shifts of the
voltage for half-maximal activation (Figs. 1B and
2A). These residues are proposed to face away from the pore.
The negative shifts caused by these mutations indicate that the native
residues at these positions contact other protein components of the
channel and stabilize the closed state and/or destabilize the open state.
This pattern of shifts in the voltage dependence of activation is
consistent with an
-helical conformation of the IIIS6 segment and
with a rotational motion of this helix during activation. Rotational
movement of these mostly hydrophobic residues (i.e., Phe-1462, Leu-1465, Asn-1466, Ile-1469, and Ile-1473) to face the lumen
of the pore during activation would break interactions with surrounding
transmembrane segments in domain III and allow new interactions of
these amino acid residues with the adjacent S6 segments from domains II
and IV. These new interactions would be destabilized by alanine
substitutions, resulting in impaired channel activation. Rotational
motion of an S6-like segment has been detected with EPR probes in the
KcsA channel (22), and movements of the S6 segment of K+
channels have been proposed to open the activation gate (22, 23, 32,
33, 35, 45, 46). Our results provide support for rotational movement of
the IIIS6 segment of Na+ channels during activation.
This helical pattern of effects on activation for the inner two-thirds
of the IIIS6 segment contrasts with our results for the extracellular
one-third of the IIIS6 segment located on the N-terminal side of
Gly-1460. In this outer part of the segment, reduction of size and
hydrophobicity of residues by substitution of alanine caused negative
shifts of activation at all positions except Ile-1457, indicating that
interactions of the native residues at all these positions favor the
closed state of the channel. By analogy with the structure of the KcsA
channel (21), it is thought that this outer part of IIIS6 interacts
with the pore loop that forms the narrow part of the conduction pathway
rather than with the water-filled lumen of the pore. Therefore, this outer part of the transmembrane segment has all of its residues in
contact with neighboring peptide segments rather than water. The
different effects of mutations in the intracellular and extracellular parts of IIIS6 on the activation process may result from their different interactions; i.e. peptide segments on all
sides for the extracellular part compared with the interaction of one
face of the inner segment with water in the lumen of the pore.
It is of interest that Gly-1460 defines the boundary between these two
parts of the IIIS6 segment. Glycine residues have no side chain and
therefore provide points of flexibility where
-helical segments can
kink or unwind. Gly-1460 may provide flexibility to allow partially or
completely independent movements of the inner and outer parts of the S6
segment during activation gating. For example, the inner part of the
helix may rotate to open the activation gate, while the outer part
remains comparatively immobile and retains its association with the
pore loop region.
Effects of Mutations in the Inner Part of the IIIS6 Segment
on the Voltage Dependence of Closed-state Inactivation--
Mutations
of amino acid residues in the cytoplasmic half of the IIIS6 segment
also strongly affected inactivation gating. Most residues in this
region caused negative shifts in the voltage dependence of inactivation
when mutated to alanine (e.g. Phe-1463, Leu-1465, Asn-1466,
Leu-1467, Phe-1468, Gly-1470, Val-1471, and Ile-1472). In contrast,
alanine substitutions of residues Ile-1469 and Ile-1473, which are
proposed to face the pore lumen (Fig. 9), induced strong positive
shifts in the voltage for half-maximal inactivation. The voltage
dependence of inactivation during long prepulses to membrane potentials
more negative than
40 mV measures primarily the voltage dependence of
inactivation from closed states, because channel openings are very rare
at such negative potentials. In contrast to several mutations in the
IVS6 segment (25, 47), no alanine substitution mutations in IIIS6
disrupted inactivation from open states, because none slowed the rate
of decay of the Na+ current. Thus, residues in the
cytoplasmic half of IIIS6 appear to play an important role in
conformational changes leading to channel inactivation from closed
states but not from open states.
Mutations L1465A, N1466A, and G1470A produced opposite shifts of the
voltage dependence of activation and inactivation, shifting activation
to more positive potentials and inactivation to more negative
potentials. These mutations therefore impair the coupling of channel
activation to channel opening, allowing channels to activate and
undergo closed-state inactivation, but not to open. Evidently, these
amino acid residues participate both in closed-state inactivation and
in the coupling of activation to channel opening.
A Receptor Site for Local Anesthetic and Anticonvulsant Drugs in
Transmembrane Segments IIIS6 and IVS6 of the Na+
Channel--
Our present results for transmembrane segment IIIS6 and
similar data for transmembrane segment IVS6 (42) define components of
the receptor site for the anticonvulsant lamotrigine and related drugs
and for the local anesthetic etidocaine. In the IIIS6 segment, mutation
of amino acid residues Leu-1465 and Ile-1469, which are located on the
same side of the
-helix (Fig. 9), decreased the inactivated-state
block by lamotrigine and related compounds and by etidocaine. Mutation
N1466A also reduced inactivated-state block by compound 4070w92 and had
a particularly strong effect on block by etidocaine. Local anesthetics
like etidocaine are thought to bind in the channel pore based on
biophysical studies (8). Thus, we propose that residues Leu-1465,
Asn-1466, and Ile-1469 define a pore-facing surface of the IIIS6
-helix and form part of receptor sites for anticonvulsant and local
anesthetic drugs. Similarly, previous studies indicate that residues
Phe-1764, Tyr-1771, and Ile-1760 of segment IVS6 face the pore and form part of the receptor site for anticonvulsants and local anesthetics (12, 14, 42). Evidently, this site for binding of pore-blocking drugs
involves one face of at least two of the four symmetrically located S6
segments that form the inner pore. Further work will be required to
assess the possible roles of amino acid residues in the S6 segments of
domains I and II in the receptor site for anticonvulsant and local
anesthetic drugs.
In previous work (42), we found that the pattern of effects of
mutations of Ile-1760, Phe-1764, and Tyr-1771 in transmembrane segment
IVS6 differed for lamotrigine and its three congeners, suggesting
specific interactions of the different chemical moieties of these drugs
with individual amino acid residues in the receptor site. In contrast,
in the present experiments we found a more similar pattern of effects
of the mutations in transmembrane segment IIIS6 on affinity for
lamotrigine, its three congeners, and etidocaine. The affinity of
inactivated Na+ channels for all of these drugs was
substantially reduced by mutation of Leu-1465 and Ile-1469. Only
mutation N1466A revealed drug-specific differences. The affinity for
etidocaine was decreased 8-fold, the affinity for 4030w92 was reduced
3-fold, and the affinity for the other drugs was unaffected. Therefore,
differential interactions with amino acid residues in transmembrane
segment IVS6 may be primarily responsible for mediating drug-specific
effects on Na+ channels, but the interactions of amino acid
residues in transmembrane segment IIIS6 with these drugs are less
likely to contribute to drug-specific effects within this family of compounds.
State-dependent Effects of Mutations in the IIIS6
Segment--
In contrast to their inhibitory effects on
inactivated-state block, mutations L1465A, N1466A, and I1469A increased
resting-state block by lamotrigine. L1465A and N1466A also increased
resting-state block by etidocaine, but I1469A had no effect. We suggest
that, in the resting conformation of the channel, each of these
mutations that substitute alanine for a larger hydrophobic residue
creates additional space for the drug molecule to reach its binding
site in the resting state of the channel and thereby enhances
resting-state block.
Frequency-dependent block involves drug entry and binding
to the open state of the channel followed by stabilization of the bound
drug during inactivation. Mutations L1465A and I1469A reduced frequency-dependent block by etidocaine, and I1469A reduced
frequency-dependent block by 619c89. Measurement of on- and
off-rates of 619c89 and etidocaine for L1465A, N1466A, and I1469A
revealed different effects of these mutations on the kinetics of
interaction of these drugs with their receptor site. None of these
mutations had any effect on the rate of onset of channel block by
619c89. In contrast, L1465A decreased the onset rate of etidocaine
block, whereas N1466A and I1469A had no effect. The rate of recovery
from 619c89-bound channels at the resting membrane potential was faster
for I1469A and slower for L1465A. In contrast, L1465A induced faster
recovery of etidocaine-bound channels and I1469A had no significant
effect. Thus, both faster recovery from block and reduced
inactivated-state affinity contribute to the reduction in
frequency-dependent block by I1469A, whereas reduced
inactivated-state affinity is the primary effect for L1465A. Increased
use-dependent block of N1466A by 619c89 is due to slower
recovery rate from block by this compound. Reduced affinity for
inactivated channels, slower onset rate, and faster recovery rate all
contribute to the large decrease in frequency-dependent
block of L1465A by etidocaine, whereas the smaller effect of I1469A on
frequency-dependent block is due entirely to reduced
affinity for inactivated-state block. The complex effects of these
mutations on the kinetics of drug binding and dissociation suggest that
interactions of these amino acid residues with different chemical
moieties of the drug molecules can specifically affect the kinetics of
these processes.
Overall, our results show that the effects of these mutations are
greatest for high affinity drug binding to the inactivated state of
Na+ channels and are smaller and variable for low affinity
drug binding to resting and open states. We conclude that the high
affinities of local anesthetic and anticonvulsant drugs for inactivated
Na+ channels depend on interactions with Leu-1465 and
Ile-1469 in the IIIS6 segment and Ile-1760, Phe-1764, and Tyr-1771 in
the IVS6 segments. Gating movements of the IIIS6 and IVS6 segments may
allow access of these drugs to their receptor site, which becomes
available as the channel opens and increases in affinity as it inactivates.