§
From the * Program in Neuroscience, Division of Medical Sciences and Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts 02115; and § Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02214
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ABSTRACT |
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Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through
preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has
been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that
reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted
at phenylalanine 1304 in the rat adult skeletal muscle sodium channel subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during
lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the
fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of
site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More
significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a
slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.
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INTRODUCTION |
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The gating of voltage-sensitive Na+ channels determines the time course of the rising phase of the action
potential and the length of the refractory period in
nerve, skeletal muscle, and heart. As a result, Na+ channels are the targets of several classes of drugs that modulate electrical excitability, including antiarrhythmics,
local anesthetics, antimyotonics, and anticonvulsants.
Among these, lidocaine and related local anesthetics
have received a great deal of experimental attention
because of their striking effects on Na+ channels: they
induce a voltage-dependent inhibition of the peak current upon infrequent stimulation (tonic block), and
they dramatically slow repriming of sodium channels
after depolarizations (use-dependent block), thereby
preventing the repetitive discharges that occur in cardiac arrhythmia, epilepsy, and myotonia (Butterworth
and Strichartz, 1990).
Several experimental findings implicate a role for the
Na+ channel fast inactivation mechanism in generating
these effects: depolarization favors local anesthetic
binding, many local anesthetics shift the steady state
availability (h) curve in the hyperpolarizing direction
(Bean et al., 1983
; Hille, 1977
), and fast-inactivation- defective Na+ channels are more resistant to some of
the effects of local anesthetics than are normal channels (Cahalan, 1978
; Wang et al., 1987
; Yeh and Tanguy, 1985
).
However, a number of questions remain. Is there cooperativity, negative or positive, between lidocaine and the fast-inactivation gate? Does use-dependent block involve an accumulation of fast-inactivated sodium channels? Is there a direct, mutually stabilizing interaction between lidocaine binding and closure of the fast-inactivation gate? Answers to these questions have been difficult to obtain, primarily because gating transitions that occur in drug-bound channels do not involve changes in ionic current, and are thus electrophysiologically silent. Indeed, neither the ionic current nor the gating current provides direct information about the position of the fast-inactivation gate during local anesthetic block.
To circumvent this difficulty, we have employed a conformational marker for the position of the fast-inactivation gate, the reactivity of a cysteine substituted for phenylalanine 1304 in the rat adult skeletal muscle sodium
channel subunit with the thiol-modifying reagent
[2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET).1 Site 1304 lies in the sodium channel III-IV
interdomain and plays a crucial role in fast inactivation
(West et al., 1992
). In a previous study, we have demonstrated that the reaction rate of the substituted cysteine
with MTS-ET follows closely the voltage dependence of
steady state fast inactivation (Vedantham and Cannon, 1998
). This has enabled us to use this reaction rate as a
measure of the fraction of channels whose fast-inactivation gates are shut under conditions of particular interest.
In this study, we have determined the position of the fast-inactivation gate in channels that are bound to the local anesthetic drug lidocaine under several experimental conditions. We found that lidocaine does not compete with closure of the fast-inactivation gate; on the contrary, the fraction of blocked channels that are fast inactivated increases with depolarization and with drug concentration. More surprisingly, our data show that recovery from fast inactivation precedes recovery of the ionic current in drug-bound channels and is just as fast as recovery in the absence of drug, demonstrating that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these findings, we propose a model in which lidocaine binding affinity is modulated by gating transitions along the activation pathway, without a direct interaction between lidocaine binding and fast inactivation.
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MATERIALS AND METHODS |
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Expression of Na+ Channels
The construction of cDNAs encoding F1304C and human Na+
channel 1 subunit in pGEMHE is described in Vedantham and
Cannon (1998)
. RNA for F1304C and
1 subunit were all generated by in vitro translation of linearized plasmids (Message
Machine kit; Ambion Inc.). Xenopus oocytes were harvested and
coinjected with F1304C + human
1 RNA as described in
Chen and Cannon (1995)
. Before electrophysiological recording, oocytes were incubated for 2-3 d at 18°C in ND-96 (96 mM
NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES,
pH 7.6) supplemented with pyruvate (2.5 mM) and gentamicin
(50 µg/ml).
Electrophysiology
Recording conditions and solution exchange were as described
in Vedantham and Cannon (1998). The pipette solution contained (mM): 100 NaCl, 10 HEPES, 2 CaCl2, 1 MgCl2, pH 7.6. The bath contained 100 KCl, 10 HEPES, 5 EGTA, 1 MgCl2, pH
7.6. Lidocaine powder (hydrochloride salt; Sigma Chemical Co.)
was added to the bath solution in appropriate amounts to obtain
a final concentration of 0.5, 1, 2, 4, or 8 mM lidocaine. Stock solutions (2 mM) of MTS-ET (Toronto Research Chemicals Inc.)
were prepared from the solid in 1 ml of distilled, deionized H2O
and placed on ice at the beginning of each recording day. Appropriate amounts were diluted into 10 ml of bath solution (to a final concentration of 8 µM) after suitable patches were obtained
and immediately before use. MTS-ET solutions were never used
for >10 min after dilution from the stock. Our method for determining the fidelity of solution exchange is described in Vedantham and Cannon (1998)
, with one change: patches in which the
seal formed without suction were included in the data set even if
they did not last long enough for a switching test (such patches
invariably exhibited rapid exchange kinetics).
Data Analysis
Curve fitting was performed off line using a custom AxoBasic
analysis program (Axon Instruments, Inc.) or SigmaPlot (Jandel Scientific Co.). Steady state fast inactivation, h, and the voltage
dependence of the modification rate were fitted to Boltzmann
curves with maximum values, Imax, and nonzero pedestals, c, calculated as I/Ipeak = [Imax
c]/{1 + exp[(V
V1/2)/k]} + c,
where V1/2 is the voltage at half maximum, and k is the slope factor. Error bars indicate means ± SEM.
For modification experiments, the fraction modified after a
given pulse of MTS-ET was calculated by averaging the value of the Na+ current between 3 and 3.5 ms after depolarization to
20 mV. For each experiment, the fraction modified (F) versus
cumulative exposure time (texp) were fit to a monoexponential:
F = (Imax
Fo)[1
exp(
texp/
mod)] + Fo, where
mod is the reciprocal of the reaction rate, Fo is the mean value of the current
between 3 and 3.5 ms before any exposure has occurred, and
Imax, the maximum value of the mean current between 3 and 3.5 ms, was a free parameter in the fit.
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RESULTS |
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Modification of F1304C by MTS-ET in the Presence of Lidocaine
All experiments were conducted in excised inside-out
patches pulled from Xenopus oocytes coinjected with
rat adult skeletal muscle sodium channel subunit
F1304C and human
1 subunit RNA. Fig. 1 A shows
macroscopic current traces elicited by depolarization from
120 to
20 mV before and after a 5-s application of 8 µM MTS-ET to the intracellular side. As reported previously for this channel (Vedantham and
Cannon, 1998
) and for the rat brain IIA homologue
(Kellenberger et al., 1996
), MTS-ET modification
causes an increase in the peak current and a dramatic
disruption of fast inactivation, consistent with the importance of F1304 (F1489 in the brain IIA channel) for
fast inactivation (West et al., 1992
).
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As in the absence of lidocaine, MTS-ET increases the
peak current and disrupts fast inactivation in the presence of 1.0 mM lidocaine (Fig 1 B). However, block by
lidocaine reduces the apparent fraction of channels
that fail to inactivate and attenuates the increase in
peak current associated with MTS-ET modification. These effects of lidocaine on F1304C-ET are similar to
its effects on fast inactivation-defective Na+ channels
studied in other contexts (Balser et al., 1996; Bennett et al., 1995
; Wang et al., 1987
). When lidocaine is subsequently washed out, the current traces were indistinguishable from the case when no lidocaine was present
during the modification (data not shown), demonstrating that lidocaine does not prevent the modification reaction from reaching completion.
Measurement of the Modification Rate of in the Presence of Lidocaine
For accurate measurement of the rate of modification
of F1304C by MTS-ET, we used a rapid perfusion system (Vedantham and Cannon, 1998) to apply controlled, brief exposures of MTS-ET to the intracellular
face of inside-out patches. Fig. 2 A shows an example of
such an experimental protocol: a series of 20-ms exposures to 8 µM MTS-ET at
120 mV, with a test pulse
used to assay the macroscopic current between each exposure. The rate of modification was determined by averaging the value of the macroscopic current between
3.0 and 3.5 ms after depolarization (Fig. 2 B), plotting the averages from successive traces against cumulative
exposure time, and fitting the resulting curve with a
monoexponential (Fig. 2 C). For the experiment
shown in Fig. 2, the time constant of this curve was 0.16 s,
giving a rate of 0.781 µmol
1 s
1.
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Concentration Dependence of Site 1304 Accessibility in Lidocaine-bound Channels
We estimated tonic block in F1304C by measuring the
peak current elicited from excised inside-out patches
by infrequent depolarization from 120 to
20 mV
under control conditions, in the presence of a fixed
concentration of lidocaine applied to the intracellular face (0.1, 0.5, 1.0, 2.0, 4.0, or 8.0 mM), and then back
in control solution. The fraction of tonic block was obtained by dividing the peak current measured in the
presence of lidocaine by the average of the peak current measured before exposure to lidocaine and after
washout. These data (Fig. 3,
) were fit to a binding curve with a hill coefficient of 1.0, yielding a Kd of 1.9 mM.
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We compared the relative accessibility of site 1304 to
the degree of tonic block at specific lidocaine concentrations by dividing the modification rate in the presence of 0.5, 1.0, 2.0, or 4.0 mM lidocaine by the rate
measured with no lidocaine present (Fig. 3, ). We
found that the accessibility of site 1304 was less sensitive to lidocaine than the peak current (Ka = 6.6, as compared with 1.9), indicating that, at
120 mV and
lidocaine concentrations below the Kd, the majority of
tonic block is caused by lidocaine binding to noninactivated Na+ channels. However, as drug concentration is
increased, the fraction of inactivated channels appears
to increase as well, although we cannot rule out the
possibility that nonspecific effects cause the reduction
of reaction rate at such high drug concentrations.
The Voltage Dependence of Site 1304 Accessibility Is Shifted in the Presence of Lidocaine
We confirmed that lidocaine causes a hyperpolarizing shift in the steady state availability curve for F1304C using 1.0 mM lidocaine. In five experiments, the steady state availability curve was measured with 200-ms prepulses, first in control solution, and then in 1.0 mM lidocaine. Each curve was fit independently to a Boltzmann (Fig. 4). We observed a hyperpolarizing shift in the half-maximal voltage (10.8 ± 2.6 mV), and a reduction in the maximum value of 22 ± 6%.
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Although such a shift strongly suggests a stabilizing
effect of lidocaine on fast-inactivated channels, it is in
principle possible that the decrease in available channels due to lidocaine does not involve closure of the
fast-inactivation gate, but rather to the intrinsic properties of drug-block. Indeed, a hyperpolarizing shift in
the apparent h curve does not preclude the possibility of competition between lidocaine binding and closure
of the fast-inactivation gate (for example, if lidocaine
and the fast-inactivation gate compete for a single binding site). In the case of a stabilizing interaction, the
voltage dependence of site 1304 accessibility should
shift in the same direction as the steady state availability curve, while in the case of competition it would not.
We therefore measured the modification rate at a variety of conditioning voltages using the protocol shown
in Fig. 5 A in the absence of lidocaine (Fig 5 B, ), and
in the presence of 1.0 mM lidocaine (Fig. 5 B,
). The
data were fit with Boltzmanns containing maximum
rates (Rmax), minimum rates (Rmin), slope (k), and half-maximal voltage (V1/2) as free parameters. With no
lidocaine present, Rmax = 0.71 µmol
1 s
1, Rmin = 0.12 µmol
1 s
1, k = 3.3 mV, and V1/2 =
79.7 mV; while in
1.0 mM lidocaine, Rmax = 0.68 µmol
1 s
1, Rmin = 0.08 µmol
1 s
1, k = 6.6 mV, and V1/2 =
89.9 mV.
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The voltage at half-maximal modification is shifted
by 10.2 mV in the hyperpolarizing direction, similar to
the 10.8-mV shift seen in the steady state availability
curves (Fig. 2 A). Thus, depolarization increases the
fraction of blocked channels that are inactivated: at
120 mV, very few of the blocked channels are inactivated, while at
90 mV, nearly all of the blocked channels (~40-50% of the total) are inactivated. Both the
direction of the voltage shift and the fact that Rmin is
unchanged demonstrate that lidocaine does not compete with the fast-inactivation gate. Rather, it tends to
favor closure of the fast gate in a voltage-dependent manner. Also, the Rmax was not significantly different
for control and 1.0 mM lidocaine. To confirm this convergence of Rmax values, further experiments were performed at conditioning voltages of
160 and
200
mV. We found no significant difference between control and 1.0 mM lidocaine conditions (data not shown).
Lidocaine Does Not Impede Recovery of Site 1304 Accessibility
Use-dependent block results from the slowing of the recovery of Na+ channel availability, and explains the
ability of lidocaine to prevent rapid, high-frequency discharges in excitable tissues (Bean et al., 1983). We measured this effect in F1304C inside-out macropatches using a two-pulse recovery protocol with a 20-ms conditioning pulse to 0 mV, a variable recovery period, and a test pulse to 0 mV. The peak current from the test pulse
divided by the peak current from the conditioning
pulse is a measure of the amount of repriming that
takes place during the recovery period. Experiments
were performed in control solution (Fig. 6,
), and in
1 mM lidocaine (Fig. 6,
). Consistent with previous
studies, lidocaine dramatically slowed recovery of Na+
channel availability
by ~100-fold under our experimental conditions.
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If use-dependent block involves accumulation of fast-inactivated channels, then the accessibility of site 1304 should be reduced for hundreds of milliseconds after a brief depolarization, in accordance with the reduction in Na+ channel availability. However, if recovery from fast inactivation is unaffected by lidocaine, then the accessibility of site 1304 after a short depolarization should not change in the presence of lidocaine, even though Na+ channel availability is reduced.
The protocol we used to resolve this issue (Fig. 7 A)
consisted of a series of 20-ms conditioning pulses to 0 mV, each followed by a brief, experimentally measured
7.5-ms delay at 120 mV, and then a 20-ms exposure to
8 µM MTS-ET, also at
120 mV. Relative to the time
course of Na+ channel repriming, the duration of MTS-ET exposure corresponds to the shaded area of Fig. 6
B. After each depolarization and MTS-ET exposure,
sufficient time was allowed for complete recovery before assaying the macroscopic current. Experiments
were performed in control solution and in 1.0 mM
lidocaine. Averages of modification time courses, analogous to the individual time course in Fig. 2 C, are
shown in Fig. 7 B (
, control;
, 1.0 mM lidocaine). The reaction rates were not significantly different for
the two conditions: 0.63 ± 0.07 µmol
1 s
1 for control,
and 0.60 ± 0.07 µmol
1 s
1 for 1.0 mM lidocaine, and
are within 10-14% of Rmax, the maximum rate of modification estimated from the data of Fig. 5. The dashed line shows the modification time course that would be
expected if site 1304 accessibility mirrored the availability of Na+ current (~0.13 µmol
1 s
1; see Fig. 7 legend
for calculation). These data demonstrate that recovery
from fast inactivation is not significantly affected by lidocaine, and thus that use-dependent block does not involve the accumulation of fast-inactivated Na+ channels.
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DISCUSSION |
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Accessibility of Site 1304 During Lidocaine Block
The major findings of this study are that (a) lidocaine
does not compete with the fast-inactivation gate, (b)
lidocaine potentiates the degree to which depolarization favors closure of the fast-inactivation gate, and (c)
lidocaine does not measurably affect the rate of recovery of the fast-inactivation gate. These observations
were made possible by our ability to follow the position
of the fast-inactivation gate with a conformational
marker, the reactivity of site 1304 with MTS-ET, characterized in detail in a previous study (Vedantham and
Cannon, 1998).
In the first set of experiments, we determined the
position of the fast-inactivation gate as a function of
lidocaine concentration during tonic block, the inhibition of peak sodium current that occurs with infrequent depolarization. Our results indicate that at 120
mV, for lidocaine concentrations below the Kd for
block, the majority of blocked channels are not fast-
inactivated. Above the Kd for block, the data suggest
that lidocaine favors closure of the fast-inactivation
gate, although the certainty of this conclusion is undermined by the possibility of nonspecific effects interfering with the reaction between MTS-ET and site 1304 at such high drug concentrations. (Our data on the voltage dependence of the reaction rate show that nonspecific reduction of the reaction rate is not occurring at
1.0 mM lidocaine: the reaction rates in 1.0 mM
lidocaine and control conditions are equal at very hyperpolarized voltages.)
Assuming that the modification rate faithfully reports
the position of the fast-inactivation gate even above the Kd
for tonic block, our observations on concentration dependence are consistent with state-dependent binding of
lidocaine to channel conformations that are populated
significantly only at depolarized potentials in the absence
of drug. As the lidocaine concentration is increased, the population of channels that are in these "depolarized" conformations will increase by mass action, even
at 120 mV. Because depolarized states favor closure
of the fast-inactivation gate, increasing lidocaine concentration should also favor closure of the fast gate.
That the modification rate is reduced by 40-50% in
the presence of 4.0 mM lidocaine at 120 mV predicts
a dramatically altered h
curve: at
120 mV, a significant fraction of channels must be unavailable. We
found, consistent with our data, that in 4.0 mM
lidocaine, availability at
120 mV is somewhere on the steep portion of the h
curve, although we could not
accurately estimate the relative availability at
120 mV
because patches do not survive the strong hyperpolarizations (less than
140 mV) that would be required to
determine the maximum availability (data not shown).
Our next set of experiments on the voltage dependence of site 1304 accessibility in the presence of
lidocaine showed a 10.2-mV hyperpolarizing shift of
the half-maximal modification rate, similar to the 10.8-mV hyperpolarizing shift of the V1/2 of the h curve. However, Rmax and Rmin were not significantly changed,
even though the maximum value of the h
curve was reduced by 22% in 1.0 mM lidocaine.
Most state-dependent models predict that block at very hyperpolarized voltages reflects binding of drug to noninactivated channels. Rmax, the limiting modification rate at such hyperpolarized voltages, reflects the position of the fully accessible fast-inactivation gate and should not, according to a state-dependent model, be reduced in the presence of 1.0 mM lidocaine, even if 22% of the channels are blocked. As the channels are depolarized, however, a state-dependent mechanism favors binding to channels further along in the activation pathway and predicts that the fraction of blocked channels that are fast-inactivated will increase. This explains the observed left shift of the voltage dependence of the modification rate. Rmin, which reflects the maximal degree of gate closure in F1304C, is not significantly changed in the presence of lidocaine, a finding that is also predicted by a state-dependent mechanism favoring inactivation.
The final set of experiments was directed at the effect
of lidocaine on the recovery of site 1304 accessibility after brief depolarizing pulses. We first confirmed that
1.0 mM lidocaine dramatically slows the recovery of
F1304C availability at 120 mV after a 20-ms depolarization to 0 mV. In the absence of lidocaine, the time
constant of recovery is on the order of 1-2 ms, while in
1.0 mM lidocaine, it is ~100-200 ms. This effect produces use-dependent block, a frequency-dependent,
cumulative inhibition of sodium current with repetitive
depolarizations. Between 7.5 and 37.5 ms, only ~20-
30% of channels recover in the presence of lidocaine, whereas >90% recover with no lidocaine present. By
contrast, the modification rate was not changed at all in
the presence of 1.0 mM lidocaine, demonstrating that
lidocaine does not significantly alter the kinetics of recovery from fast inactivation.
A Possible Mechanism of Lidocaine Action
At first glance, the results of these experiments seem to
be in conflict: on the one hand, lidocaine shifts the h
curve in a way that favors fast inactivation, suggesting a
stabilizing interaction between lidocaine block and fast-inactivated channels. On the other hand, lidocaine has
no measurable effect on the off rate of the fast-inactivation particle, suggesting that it does not preferentially
stabilize fast inactivation.
One model that reconciles our results is shown in
Fig. 8. Following Kuo and Bean (1994), we employ a
model for sodium channel gating consisting of several
closed, noninactivated states, each in equilibrium with
a fast-inactivated state (Fig. 8 A). For convenience, only
a few such equilibria are depicted. Horizontal equilibria represent the voltage-dependent transitions along
the activation pathway, with depolarization favoring a
rightward shift in the distribution of populated states.
The vertical transitions, by contrast, are voltage independent, and the rightmost equilibria favor inactivated,
rather than noninactivated, channels. According to this
model, depolarization moves the distribution of channels to the right and down, while hyperpolarization
tends to shift the distribution to the left and up.
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Fig. 8 B presents a qualitative model for how
lidocaine affects the states depicted in Fig. 8 A. We assume that each state can bind lidocaine, since our data
suggest that both inactivated and noninactivated channels may experience block. We incorporate state dependence by postulating that lidocaine binds more favorably to channels that are further along in the activation pathway (towards the right), regardless of whether
they are noninactivated or inactivated. In other words,
lidocaine is sensitive to position along the horizontal, voltage-dependent axis of the state diagram, but not
the vertical, voltage-independent axis. In this model,
lidocaine does not directly affect the equilibrium constants between inactivated and noninactivated channels
(the equilibrium distributions for Cn In and CnL
InL are equal). Consequently, lidocaine binding does
not affect the rate of recovery from fast inactivation by
very much, in agreement with our findings on the recovery of accessibility of site 1304. However, the voltage-dependent equilibria in the activation pathway are
altered in lidocaine-bound channels, shifting the overall distribution of channels to the right in Fig. 8 B.
The model also explains why lidocaine causes a hyperpolarizing shift in the h curve. By mass action, addition of lidocaine at any given voltage will tend to shift
the distribution of channels towards the right in the
state diagram of Fig. 8 B. Since the vertical equilibria
will favor fast-inactivated states as the distribution of
channels moves sufficiently rightward along the activation pathway, the addition of lidocaine will indirectly promote fast inactivation. This phenomenon also explains our tonic block measurements: the greater the
lidocaine concentration, the greater the rightward shift
along the activation pathway, and hence the greater the
fraction of inactivated channels.
The model also predicts a reciprocal effect of fast inactivation on lidocaine action: the presence of the fast-inactivation gate promotes block, because (like lidocaine) the fast-inactivation particle binds more tightly
to the rightmost states on the activation pathway. This
would partly explain why channels with disrupted fast
inactivation show a reduction in sensitivity to lidocaine effects (Cahalan, 1978; Yeh, 1978
; Bennett et al., 1995
;
Balser et al., 1996
). We need not attribute this reduction in sensitivity to an essential role played by inactivation in the mechanism of lidocaine action.
Use-dependent block, in our model, is a consequence of a slow off rate of drug from the drug-bound, non-fast-inactivated states. Recall that at depolarized potentials, our data show that both lidocaine and the fast-inactivation particle are bound (i.e., the back, lower row in Fig 8 B is populated), and that on repolarization the fast-inactivation particle dissociates rapidly, populating the back, upper row of Fig. 8 B. The transitions from the back, upper row to the front, upper row, along with full leftward movement along the activation pathway, is rate limiting and slow (100-fold slower than recovery from fast inactivation), and generates use- dependent block when further depolarization occurs before full recovery.
A remaining question concerns the kinetics of leftward movement along the activation pathway upon repolarization. Because inactivation is not intrinsically voltage dependent, but derives its voltage dependence from activation, some leftward movement along the activation pathway must precede recovery from inactivation. In other words, some inward charge movement must occur if recovery from inactivation is to occur. Unfortunately, whether and to what extent lidocaine impedes inward charge movement upon repolarization has not been examined carefully. Our results predict that some component of the gating charge must remain relatively free to move even in lidocaine-bound channels, and that inward movement of this fraction must be sufficient for complete recovery of the fast- inactivation gate. Further experiments will be required to elucidate the details of the coupling between inactivation and gating charge movement in the presence of lidocaine, and thereby to determine how far the distribution of channels must move to the left on repolarization for full recovery from inactivation to occur.
Relation to Previous Work on Lidocaine
Our model is a version of the modulated receptor hypothesis (Hille, 1977; Hondeghem and Katzung, 1977
),
in which the affinity of a single receptor site for
lidocaine is altered by the conformational state of the
channel. Our model differs from Hille's original presentation and from that of Bean et al. (1983)
by not
treating the inactivated state as the high-affinity state.
Instead, we propose that transitions along the activation pathway (outward movement of S4 segments and/
or opening of the activation gate) affect the affinity of
lidocaine for its receptor, following the proposals of
Wang et al. (1987)
, Strichartz and Wang (1986)
, and Yeh and Tanguy, 1985
. Several lines of evidence support our hypothesis.
First, numerous studies have shown a reduction in
the potency of local anesthetics in fast-inactivation defective sodium channels (Cahalan, 1978; Yeh, 1978
;
Bennett et al., 1995
; Balser et al., 1996
). However, despite the loss of potency, local anesthetics do retain
their ability to generate tonic and use-dependent block in these channels (Shepley et al., 1983
; Strichartz and
Wang, 1986
; Wang et al., 1987
). As noted above, this is
consistent with the predictions of our model: the inactivation gate potentiates the effects of local anesthetics,
but is not necessary to generate those effects. There is
also evidence that at least some local anesthetic molecules can be trapped by closure of the activation gate,
suggesting a possible mechanism for use-dependent
block that does not involve the fast-inactivation gate
(Strichartz, 1973
; Yeh and Tanguy, 1985
).
Gating-current studies have revealed that lidocaine
can produce a hyperpolarizing shift in the Q/V curve
(Hanck et al., 1994; Josephson and Chi, 1994
) along
with a reduction in the total amount of on-gating current. A possible interpretation of this finding is that
some of the voltage sensors of drug-bound channels
move outward at less depolarized potentials than normal. This would entail, at any given voltage, a drug-
induced rightward shift in the distribution of channels
along the activation pathway diagrammed in Fig. 8, as
our model predicts.
Finally, site-directed mutagenesis has placed the receptor for lidocaine roughly in the middle of the S6
transmembrane segment (Ragsdale et al., 1994). Extrapolation to Na+ channels of a recent substituted cysteine accessibility study in segment S6 of Shaker K+
channels (Liu et al., 1997
) suggests that the position of
the activation gate is likely to be very close to the local
anesthetic binding site. Thus, it would not be surprising if the primary action of lidocaine is to interact with
activation gating, perhaps by stabilizing the channel in
the open conformation.
We wish to emphasize that our results are not sufficient to determine uniquely our particular model of
lidocaine action. Although our results do suggest a very
limited role for fast inactivation in generating use-
dependent block, it is still possible that the affinity of
lidocaine for its receptor is increased by closure of the
fast-inactivation gate in the intact channel (i.e., with a
phenylalanine at site 1304). Another possibility is that
the Na+ channel slow inactivation mechanism plays a
role in lidocaine action. Our finding that recovery from
fast inactivation precedes recovery of the ionic current
in the presence of lidocaine parallels an earlier finding
that recovery from fast inactivation precedes recovery
from slow inactivation (Vedantham and Cannon, 1998), and raises the possibility that the two slowly recovering
states are related in some way. For example, lidocaine
might accelerate the rate of entry into slow-inactivated
states.
Also, the mechanism of lidocaine action might vary
among sodium channel isoforms. Our experiments
were conducted in skeletal muscle sodium channels,
which have a lower apparent lidocaine affinity that cardiac channels (Hille, 1978; Nuss et al., 1995
). However,
most of this difference is attributable to relative shifts in
voltage-dependent gating between the two isoforms,
rather than to differences in the putative binding site
(Wright et al., 1997
), suggesting that our results with
skeletal muscle channels will probably hold for cardiac
channels as well. We should also emphasize that our results may not hold for all local anesthetics, which exhibit considerable variation at the chemical level as well
as in their effects on sodium channels (Hille, 1977
).
These uncertainties aside, our data do enable us to place important new constraints on the possible forms that models for lidocaine action can take. Any such model must involve cooperativity between lidocaine binding and fast inactivation, and must incorporate a state that is slowly recovering, but not fast inactivated, to explain use-dependent block.
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FOOTNOTES |
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Address correspondence to Dr. Stephen Cannon, EDR413A, Massachusetts General Hospital, Boston, MA 02214. Fax: 617-726-3926; E-mail: cannon{at}helix.mgh.harvard.edu
Original version received 30 July 1998 and accepted version received 19 October 1998.
We thank Adriana Pechanova for assistance with mRNA preparation. We are also grateful to Bruce Bean, Jim Morrill, and Masanori Takahashi for comments on the manuscript.
This work was supported by the National Institutes of Health (AR-42703), the Harvard-Mahoney Neuroscience Institute (V. Vedantham) and the Esther A. and Joseph Klingenstein Fund, Inc. (S.C. Cannon).
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Abbreviation used in this paper |
---|
MTS-ET, [2-(trimethylammonium)ethyl]methanethiosulfonate.
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