From the University of Washington, Seattle, Washington 98195-7280
A relatively large group of compounds, which includes
local anesthetics, antiarrhythmics, and anticonvulsants
induce voltage- and/or frequency-dependent block of
sodium and other ion channels. Current block increases as the stimulus voltage and frequency of depolarization is increased. These voltage- and frequency-dependent blocking properties have been recognized
for over 40 yr (Weidmann, 1955 A characteristic set of features describes local anesthetic block, but the prevalence (or presence) of any
one of these characteristics varies with drug structure
and physical properties (Butterworth and Strichartz,
1990 Although much of this basic phenomenology was established over time, it was during the 1970's that many
of the key features of block by local anesthetics were
outlined and their mechanisms of action explored intensively. Studies of tertiary amine and quaternary local
anesthetics led Hille (1977) Inactivated channels do appear to be stabilized by local anesthetics, as steady state inactivation curves are
shifted toward negative potentials. In addition, recovery from drug block after repolarization resembles a
slowed recovery from channel inactivation, as if local
anesthetic-bound channels have difficulty in recovering from inactivation (and deactivating). Thus, local anesthetic-like molecules were proposed to bind to depolarized channels and perhaps stabilize the inactivated
state (Hille, 1977 In the 20 yr that have passed, many local anesthetics
and related compounds have been studied in many different tissues on many different sodium channel molecules. Each compound and tissue displays its own idiosyncrasies of block. Nevertheless, perhaps the most often repeated statement in these various studies is that
these compounds cause use- or voltage-dependent
block by virtue of their ability to stabilize the inactivated state.
The article from Vedantham and Cannon (1999) Vedantham and Cannon (1999) Despite the surprising nature of their basic result,
and its intrinsic contradiction of the idea that local anesthetics stabilize the fast-inactivated state, Vedantham
and Cannon (1999) Vedantham and Cannon's findings also indicate that
anesthetic-dependent block can occur without movement of the inactivation gate. As the lidocaine concentration is increased, up to 30% of the channels become
blocked even though there has not been any movement of the inactivation gate as judged by the SH reactivity. There must be a component of the lidocaine-
dependent block that does not involve closure of the
inactivation gate. Also, the rate of modification does not
drop as steeply as the current magnitude as lidocaine
concentration is increased. Both results are expected if
channels can be blocked by binding drug molecules
with no closure of the inactivation gate. The modulated
receptor hypothesis provides that fully resting channels
bind local anesthetics, although the binding affinity is
lower than that for binding to depolarized channels.
Even increased lidocaine block in response to depolarization can occur without movement of the inactivation gate. In the absence of lidocaine, channel availability for modification tracks the inactivation curve for
the channel closely. The modification rate thus reports
the position of the inactivation gate. In the presence of
1 mM lidocaine, inactivation gate closure is not detected until the channel is depolarized beyond This dissociation between depolarization and inactivation gate closure may be relatively subtle, but the
events occurring on repolarization to the holding potential after a depolarization are not. Whereas the inactivation gate becomes fully accessible to modification (reopens) at the normal rate by, at most, 30 ms after repolarization, the channels take ~1 s to recover
completely from the voltage-dependent block induced
by lidocaine during a 20-ms conditioning depolarization. That is, lidocaine blocks the current for at least
100× longer than it takes for the inactivation gate to
reopen. Something else must be stabilizing the lidocaine molecule in its receptor besides the closed inactivation gate.
The findings of Vedantham and Cannon (1999) What do these results mean for the modulated receptor model? The basic model remains intact. What
changes is the role of the inactivation gate. Whereas
the inactivation gate previously was considered to be
central for local anesthetic block, the results of Vedantham and Cannon (1999) The literature that examines block by local anesthetics and related compounds is large, and many members
of the ion channel biophysics community have contributed to the current knowledge. This rich literature contains many findings (or conclusions) that are difficult
to reconcile completely with the present findings and
bear reexamination and reinterpretation. One finding
that is particularly perplexing in view of the present results is the loss of depolarization and use-dependent
block when the inactivation gate is disabled by proteases (Cahalan, 1978
ARTICLE
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References
), and the voltage- and
use-dependent properties of a wide range of molecules
have been described in detail.
; Hille, 1992
), which in turn affects the qualitative
characteristics of current block. In quiescent cells with
a very negative resting potential, even relatively high local anesthetic concentrations have little effect on electrical activity. However, when depolarizing pulses arrive at high frequency, the peak sodium current elicited by each subsequent depolarization becomes
smaller until the current reaches a new steady level. The degree of this use dependent or phasic block will
increase with increasing depolarization frequency. Local anesthetics also respond to steady depolarizations.
When the membrane is depolarized before the test depolarization that elicits the sodium current, this will increase the fraction of the current that is blocked by a
local anesthetic. When this effect of earlier depolarization on local anesthetic block is measured as a function
of voltage using a voltage-clamp protocol designed to
measure the voltage dependence of channel inactivation or availability, local anesthetics shift the voltage
dependence of channel availability to more hyperpolarized potentials. At any particular potential, a smaller
fraction of the channels is available for activation by
depolarization. Finally, when local anesthetic block is
increased by depolarization, the recovery from block
upon repolarization is slow and may not be complete
before the next depolarization. Local anesthetics that cause slow recovery from block after depolarization
produce use-dependent block. When recovery from
block on repolarization occurs rapidly, use dependence is not observed. Whether or not a given local anesthetic produces use-dependent block thus will vary
with the binding kinetics of the local anesthetic and
the stimulus frequency.
and Hondeghem and Katzung (1977)
to propose a model, termed the modulated receptor hypothesis, that explained many features
of block by local anesthetic-like compounds. This
model built on explorations of state-dependent block
by quaternary ammonium blockers of potassium channels by Armstrong (1969)
. This work provided insights
into the key feature of block by local anesthetics,
namely that steady state inactivation is enhanced and
recovery from inactivation is slowed. The modulated receptor hypothesis, as originally proposed, suggested
that local anesthetics bind with different affinities to
different conformational states of the channel. In particular, the drug affinity for depolarized conformations
of the channel is higher than for hyperpolarized conformations. Allosteric coupling, in turn, causes the
high affinity drug binding to depolarized channels to
stabilize these conformations relative to the conformations having the low drug affinity. Finally, if the high-
affinity, depolarized conformation were the inactivated state, this would further enhance the effect of a blocking drug.
; Hondeghem and Katzung, 1977
). A
key experimental test of the model was provided by
testing local anesthetics on channels in which inactivation had been removed by the protease pronase. In
such channels, use-dependent block by local anesthetics was lost (Cahalan, 1978
). Another test made use of
the fact that sodium channel inactivation immobilizes a fraction of the charge associated with channel gating
(Armstrong and Bezanilla, 1977
). Local anesthetics
also immobilize a fraction of gating charge, and this
charge immobilization seemed to occlude charge immobilization by inactivation (Cahalan and Almers,
1979
). This finding suggested that the charge immobilized by local anesthetics was the same component of
charge that was immobilized by channel inactivation
(Cahalan and Almers, 1979
). These results were expected if local anesthetics acted by stabilizing the inactivated state.
in
this issue of The Journal of General Physiology provides a
new experimental test of this statement
with a surprising result. Vedantham and Cannon made use of the observation that the loop connecting the homologous domains III and IV of the sodium channel is critical for inactivation and is proposed to be the inactivation gate
(Vassilev et al., 1988
; Stühmer et al., 1989
). A phenylalanine residue in a hydrophobic triplet approximately
one third of the distance from the NH2 terminus of
that loop is critical for inactivation (West et al., 1992
).
When this phenylalanine is replaced with a cysteine
and the intracellular surface of the channel is exposed to the water-soluble methane thiosulfonate reagent,
methane thiosulfonate ethyltrimethylammonium (MTSET), the cysteine is modified and inactivation is
blocked. By examining its ability to react with MTS derivatives, this loop was found to change conformation
when the channel inactivates (Kellenberger et al., 1996
;
Vedantham and Cannon, 1998
). In the hyperpolarized,
noninactivated channel, the cysteine at this position is
highly reactive, but if the channel is depolarized, the
same cysteine becomes unreactive. Furthermore, the
voltage dependence of the reaction rate tracks the voltage dependence of inactivation. Thus, the reaction rate
of this substituted cysteine is a measure of the number
of sodium channels with open inactivation gates.
have cleverly made
use of the accessibility of this cysteine residue as an indicator of the position of the inactivation gate. Using
this readout, they followed the position of the inactivation gate during depolarization-dependent block by
the local anesthetic, lidocaine. They got the unexpected result that the inactivation gate reopens with virtually unchanged kinetics after a depolarizing pulse
whether or not lidocaine is present. Current through
the channels, however, still recovers extremely slowly
after depolarizations in the presence of lidocaine. This
leads to the conclusion that drug binding is linked only tenuously to the molecular machinery that causes fast
inactivation. The drug molecule remains tightly bound
in the channel but the inactivation gate reopens. Because recovery of the inactivation gate and recovery
from depolarization-induced lidocaine block have drastically different time courses, the slowly recovering lidocaine block does not appear to depend on the stability of the inactivated state.
find that many key predictions of
the modulated receptor model concerning the stabilization of the inactivated state are verified. At a potential of
100 mV, where the inactivation gate was open
(and readily modifiable) in the absence of lidocaine,
lidocaine (binding) causes the gate to close and become unavailable for modification. Lidocaine thus
seems to stabilize the inactivation gate in the closed position. Likewise, lidocaine shifts the voltage dependence of inactivation gate closure toward more negative potentials. These results are in agreement with
both basic tenets of the modulated receptor hypothesis.
100 mV.
At this membrane potential, however, 1 mM lidocaine
has blocked substantial current that was available for
activation at
140 mV. That is, although there had been enough of a conformational change in the channel molecule (in response to depolarization) to increase the lidocaine block and reduce current, this
conformational change was not due to, or accompanied by, closing of the inactivation gate. This change in
conformation in response to depolarization without
movement of the inactivation gate separates movement
of the inactivation gate from the primary effect of depolarization on the lidocaine-induced block.
distinguish multiple voltage-dependent processes that occur in response to repolarization. Immediately upon repolarization, the channels close. Presumably, channel
closure occurs with approximately normal kinetics.
This is the first voltage-dependent conformational
change. The reopening of the inactivation gate occurs
with a slower time course and, in the absence of drug,
would be expected to be associated with reversal of the
charge immobilization that accompanies channel inactivation (Armstrong and Bezanilla, 1977
). This process
is highly voltage dependent (e.g., Kuo and Bean,
1994
). It is assumed that the voltage dependence of
these conformational changes remains invariant in the
presence of lidocaine. However, this remains to be
tested. A third voltage-dependent time process is the
reversal of block by the local anesthetic, in this case
lidocaine. Recovery of channel availability at different
membrane potentials in the presence of local anesthetics also is highly voltage dependent (Bean et al., 1983
;
Kuo and Bean, 1994
). This suggests that part of the
voltage-sensing apparatus of the channel is stabilized by
the drug and undergoes an additional voltage-dependent conformational change in response to repolarization long after the inactivation gate has reopened.
Taken together, these results lead to the conclusion that there are at least three distinct voltage-dependent
events that occur in succession upon repolarization in
the presence of local anesthetic block: channel closure,
recovery from inactivation, and deactivation of local anesthetic blocked channels.
suggest that it may play a
more peripheral role. Nevertheless, local anesthetics
still bind with higher affinity to depolarized conformations of the channel. The closed configuration of the
inactivation gate is still stabilized in the presence of local anesthetics, but its stabilization seems to be an allosteric one, secondary to stabilization of other depolarized configurations of the channel. Vedantham and
Cannon (1999)
explain these findings by proposing
that local anesthetics bind more avidly to, and stabilize,
activated (depolarized) conformations of the channel.
These same depolarized conformations favor inactivation. Stabilizing the channel in an activated configuration by virtue of local anesthetic binding will, in turn,
favor a configuration with a higher affinity for the inactivation gate, as was measured in their study. The rapid
opening of the inactivation gate on repolarization,
however, means that this reciprocal stabilization somehow breaks down upon repolarization. Additional quantitative measurements of charge and protein movement
under a broader array of experimental paradigms will
be necessary to further refine this aspect of the model.
) or by generation of the IFM
QQQ mutation, which also prevents inactivation (Bennett et al., 1995
; Balser et al., 1996
). Vedantham and
Cannon (1999)
explain this by proposing a loss of inactivation gate stabilization by the depolarized channel
an idea that needs to be further developed. Whatever
the final interpretation of the results of Vedantham and Cannon (1999)
, they remind us once again of the
power of measures of conformational change, such as
changes in cysteine or fluorescence accessibility to challenge, and test and refine biophysical hypotheses that
propose changes in channel protein conformation.
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