TIPS - February 1987 [Vol.

81 57

provides the most direct measure

of Na+ channel function, it has not
been easily applicable to all expec-
mental preparations of pharma-
Cological interest. In particular, for
some of the work considered here,
Common modes of drug action measurement of the rate of rise of
the action potential (ir,, dV/dt) in
on Na+ channels: local cardiac preparations or mam-
malian neurons has been used as
anesthetics, antiarrhythmics an indirect measure of activation
and inactivation of Na+ channels.
and anticonvulsants In general, the dependence of ir,,
on Na+ permeability is curvilinear,
rising more sharply at low Na+
William A. Catterall permeability and approaching a
maximum value at high Na+
permeability. Measurements in
The development of agents useful in local anesthesia, therapy of cardiac most excitable cells span this
arrhythmias, and prevention of epileptic seizures has proceeded along nonlinear range. Therefore, meas-
independent pathways with use of appropriate pharmacological models for urements of ir,, must be
drug testing. William Catterall reviews recent studies of the mechanism of considered only a semiquantita-
action of these agents in peripheral nerve, cardiac muscle cells and central tive measure of Na+ channel
neurons. These have focused on a common array of effects on voltage-sensitive function.
Na+ channels which may underly much of their therapeutic utility. Nai In addition, some neurotoxins
channels are found to be inhibited at therapeutic concentrations of these agents have been useful in revealing new
in a voltage- and frequency-dependent manner that selectively blocks aspects of Na+ channel function
excitability of abnormally firing cells while leaving normally functioning cells and interaction with drugs. These
relatively unaffected. An allosteric or ‘modulated receptor’ model of drug action neurotoxins act at three separate
is sufficient to describe the major features of the voltage- and frequency- receptor sites (see review, Ref. 1).
dependent action of all three classes of drugs. Neurotoxin receptor site 1 binds
the water-soluble heterocyclic
guanidines tetrodotoxin and saxi-
Recent evidence has demonstrated first increases dramatically and toxin. These toxins inhibit Na+
that there may be a common then after 1 msec decreases to the channel ion transport by binding
molecular basis for the mechanism base-line level. The biphasic be- to a common receptor site that is
of action of local anesthetics, Class havior is described in terms of two thought to be located near the
I antiarrhythmics and some anti- voltage-dependent processes that extracellular opening of the ion-
epileptic agents that involves a control Na+ channel function: conducting pore of the Na+ chan-
voltage- and frequency-dependent activation, which controls the rate nel. 3H-Labeled derivatives of each
inhibition of Na+ channels. Multiple and voltage dependence of Na+ toxin are used in receptor binding
methods have been used to analyse permeability increase following studies. Neurotoxin receptor site 2
the action of drugs on Na+ chan- depolarization and inactivation, binds several lipid-soluble toxins
nels. Changes in ionic permeability which controls the rate- and including grayanotoxin and the
are accurately measured only if the voltage-dependence of the sub- alkaloids veratridine, aconitine,
membrane potential of the cell sequent return of Na+ permeability and batrachotoxin. These toxins
is controlled experimentally. The to the resting level during a cause persistent activation of Na+
voltage clamp method allows rapid maintained depolarization. The channels at the resting membrane
recording of ionic currents across Na+ channel can therefore exist in potential by blocking Na+ channel
cell membranes following changes three functionally distinct states or inactivation and shifting the volt-
in membrane potential so that groups of states: resting, active, age dependence of channel activa-
changes in Na+ permeability oc- and inactivated. Both resting and tion to more negative membrane
curring on the millisecond time inactivated states are noncon- potentials. [3H]Batrachotoxinin A
scale can be accurately estimated. ducting, but channels that have 20-o-benzoate has been used as a
This experimental approach show- been inactivated by prolonged labeled probe to measure toxin
ed in 1952 that the depolarizing depolarization are refractory un- interaction at this site. Neurotoxin
phase of the action potential in less the preparation is repolarized receptor site 3 binds polypeptide
squid giant axon results from a to allow them to return to the toxins purified from North African
voltage- and time-dependent in- resting state. These basic proper- scorpion venoms or sea anemone
crease in permeability to Na+. ties are now known to be character- nematocysts. These toxins Slow or
Following depolarization of the istic of Na+ channels in mam- block Na+ channel inactivation.
cell membrane, Na+ permeability malian myelinated nerve, neurons’ They also enhance persistent ac-
in cell culture, skeletal muscle tivation of Na+ channels by the
fibers, the fibers of the cardiac con- lipid-soluble toxins acting at
William Caiterull isProjessor and Chairman in neurotoxin receptor site 2 through
the Department of Pharmacology, S]-30,
duction system, and dissociated
Universityof Washington, Seattle, WA cardiac myocytes. allosteric interactions. ‘%Labeled
98195, USA. While the voltage clamp method derivatives of scorpion toxins and

@ 1967. Elsevier Science Publis’ners B.V., Amsterdam 0165 - 6147/87/502.(30

58 77PS - February 2987 [Vol. 81

sea anemone toxins are used in
receptor binding studies. These
three groups of neurotoxins define
specific sites at which drugs may
Local anesthetic action on Na+ channels
Inhibition of Na+ channels was
first shown to be the mechanism of
local anesthetic action in blocking
nerve conduction in 1959. Earlier
observations had already shown
that the potency of local anesthetic
action is increased by increasing
the frequency of stimulation of
nerve fibers. These early results
(reviewed in Ref. 2) are now under-
stood to result from a fundamental
characteristic of local anesthetic
drugs: voitage-dependent and fre-
quency-dependent binding to
their receptor site(s) on Na+
channels. Quaternary, tertiary
amine and neutral local anesthetics
have differing profiles of activity
in this regard.
Frequency-dependent action of
local anesthetics was first charac-
terized for quaternary derivatives
which are permanently positively
charged. Relatively few Na+ chan-
nels are blocked in unstimulated
axons maintained at a negative
holding potential under voltage
clamp, but the fractional inhibi-
tion of Na+ currents is markedly
ina=eased by repetitive stimula-
tion’. The extent of inhibition is
increased by experimental mani-
potentially alter channel function
and provide biochemical assays of
aspects of Na+ channel function in
isolated excitable membranes.
pulations which increase the time
that Na+ channels spend in the
active state including increased
number and frequency of stimula-
ting pulses, more positive stimula-
ting pulses, or decreased Na+
channel inactivation. These find-
ings suggest that quatemary local
anesthetics act preferentially on
active channels.
Tertiary amine local anesthetics
which are used clinically also
inhibit Na+ channels in a fre-
quency- and voltage-dependent
manner, but with some character-
istic differences in mode of action.
Fig. 1 illustrates the frequency
dependence of lidocaine action.
Increased rates of stimulation in
the range of 1 to 8 Hz cause marked
enhancement of channel block.
Inhibition by amine anesthetics at
the resting membrane potential is
substantial and repetitive stimula-
tion causes an additional incre-
ment in drug effectS5. Moreover,
the voltage-dependence of Na+
channel inactivation is shifted
toward more negative membrane
potentials during exposure to the
drug at the resting membrane
potential without stimulation5
(Fig. 1B) and is increased upon
repetitive stimulation. Because
these drugs shift the voltage-
dependence of inactivation with-
out repetitive stimulation, their
inhibition of Na+ channels is
voltage-dependent with increas-
ing block at depolarized mem-
brane potentials within Ihe voltage
range of channel inactivationS5.
The neutral local anesthetic ben-
zocaine deviates further from the
behavior of the quatemary drugs.
Its inhibition is not increased by
repetitive stimulation with brief
pulses at less Wan 10 Hz, although
modest frequency dependence is
observed at high stimulus rates.
Like amine local anesthetics,
benzocaine shifts the voltage-
dependence of inactivation to
more negative membrane poten-
tial but this effect is not enhanced
by repetitive stimulation to acti-
vate Na+ channels5.
The modulated receptor
hypothesis
The unique characteristics pf
inhibition of Na+ channels by local
anesthetics described above led to
the development of the modulated
receptor hypothesis22*5, an exten-
sion of ideas developed for allo-
steric enzymes to the action of
drugs on ion channels. Local
anesthetics are proposed to inter-
act with the three main functional
states of Na+ channels, resting (R),

A B
Stimulation enhances block 1.0/c

1.0 ---E_+
-2
0.75
Q,
>.
.= m
z 4
.o a,
B .Z 0.5cI-
i 0.5 s
?i
Q cc

.-2 0.25, -
3
$
0 o-
b ’ i ’ i ’ i -150 -i20 -90 -60
Seconds of stimulation Membrane potential (mV)

Pig. 1. Inhibition of Na+ channels in myelinated nerve by 0.5 rnM lidocaine at pH 7.6. A. Frequencydependence.A frog node of Ranvier
underwltage clamp wasstimulatedat theindicahxl fkquencies wifh 7-msec restpulses to -20 mVfmma holdingporemialnear -80 mVat
10”Candtheresuiting:rla+cunents wererecorded. Increased frequencyofstimulaf resulfsinincreased block. 8. Shift of inactivationA
fw nodeof Ranver under voltage clamp was held at -90 mv, pulsed to the membrane potenlials indicated on the abscissa for 50 m&z
and lhen &fed wrth a single test puke to - 15 m V to elicit Na + currents. Progressive depolarization during the 50 msecpre-pulse produce;
inacfivabbn of the Na+ currenf 03 in the absence of drug. Lidocaine (1 mM) shiits fhe inactivationcurve to more negative membrane
PolentMs. (0): no drug; (*I: 1 mM lidocaine. Reproduced with permission from Ref. 5.
TlPS - February 1987 [Vol. 81
active or open (01, and inactivated
(I), according to the following
scheme:
R-Cl-1
kl Jt k-1 h Jt k-2 k3 Jt k-3
DR+DO-DI
Frequency-dependent inhibi-
tion is considered to arise from a
faster rate of drug binding to active
Na+ channels (k2 + kl, k3) which
are generated transiently during
each stimulation. Drug molecules
bind rapidly to activated channels
and are trapped in bound state as
the channel inactivates or returns
to the resting state. More drug
molecules are then bound during
the next stimulation. Drug inhibi-
tion increases with each succeed-
ing stimulus until the amount of
drug bound during the stimula-
tion pulse is equalled by the
amount of drug that dissociates
from resting and inactivated Na+
channels between pulses. As ex-
pected from this, drugs which can
dissociate rapidly from resting or
inactivated Naf channels require a
high frequency of stimulation to
observe enhanced inhibition, in
order to minimize drug dissocia-
tion between pulses.
Shifts in the voltage-depend-
ence of Na+ channel inactivation
are considered to result from high
affinity binding of local anesthe-
tics to the inactivated state of Na+
channels (k3/k-s % kllk-l, kz/k_z).
The high binding energy of local
anesthetics for inactivated states
stabilizes the channels in this state
relative to resting or active states
via the law of mass action, and
therebyincreasestheprobabilityof
channel inactivation at negative
membrane potentials.
The differences in behavior
among quatemary, tertiary amine,
and neutral local anesthetics are
thought to be controlled by their
rate of binding and unbinding at
their site of action in resting and
inactivated Na+ channels’. Qua-
ternary derivatives can bind and
unbind only to active Naf chan-
nels at an appreciable rate and
therefore have strong frequency-
dependence. Relatively hydro-
philic tertiary amines also show
substantial frequency-depen-
dence whiie hydrophobic tertiary
amines have less. The neutral local
anesthetic benzocaine can bind
and unbind rapidly at resting and
inactivated Na+ channels and its
action is therefore not strongly
frequency-dependent. Evidently,
access to the site(s) of local
anesthetic action in resting and
inactivated Na+ channels requires
transit through a hydrophobic
pathway which excludes charged
drugs. Activation of the Na+
channel creates a hydrophilic
pathway to the site(s) of action5.
Sitesoflocalanestheticactionon
Na+ channels
Devebpment of membrane im-
permeant, quaternary derivatives
of amine local anesthetics allowed
a decisive examination of which
side of the Na+ channel is sensitive
to local anesthetic action. In both
squid giant axons and frog mye-
linated nerves, permanently posi-
tively charged derivatives such as
the quaternary analogs of lido-
Caine, QX-314 and QX-572, block
Na+ currents effectively when
present inside the fiber, but have
little effect outside (see review,
Ref. 2). Tertiary amine local anes-
thetics are more difficult to study
because they exist in an equili-
brium mixture of neutral and
charged forms at physiological pH.
Examination of the effect of pH on
the rate of action of tertiary amine
local anesthetics applied from
outside the node of Ranvier shows
that tile cationic forms must pass a
substantial diffusion barrier be-
fore reaching their site of action,
whereas the freely membrane-
permeant neutral forms are not
delated in reaching the receptor
site . The results are consistent
with an internal receptor site for
both drug forms which is reached
by diffusion through the mem-
brane. Neutral forms have imme-
diate access while charged forms
diffise through the membrane
slowly.
Although local anesthetics act at
relatively high concentration (1 PM
to 10 mM) and their potency is most
strongly correlated with their lipid
solubility, suggesting that they
may act by perturbing the proper-
ties of the phospholipid phase of
biological membranes, recent ex-
periments on local anesthetic ac-
tion provide clear evidence that
these drugs block Na+ channels by
acting at specific receptor sites.
Charged forms of anesthetics act
on the Na+ channel from the
intracellular side. Since the lipid
bilayer membrane is expected to
59
be accessible from either the
intracellular or extracellular sides
of the membrane, these data
suggest an action on the Na+
channel protein itself. The modu-
lated receptor hypothesis, which
provides a clear model to explain
the prominent voltage- and fre-
quency-dependence of local anes-
thetic action, requires that local
anesthetics bind to a receptor site
whose accessibility and affinity
are different when Na+ channels
are in the resting, active and
inactivated states5. This receptor
site or sites must therefore be
located at least partially on the Na+
channel protein itself.
The action of some enantiomeric
local anesthetics is stereospecific.
The experimental anesthetic RAC
109-I (s-N-(apsaIon-diethylamino-
propyl)-1,2,3,4-tetranaphthalia-l-
spirosuccinimide) is nine times
more potent in frequency-depend-
ent inhibition of Na+ channels than
its enantiomer RAC 109-P. This
result also implies local anesthetic
action at a chiral site formed, at least
in part, by the Na+ channel protein,
since partition into the phospho-
lipid bilayer to cause general mem-
brane perturbation should not be
stereospecific.
Localanestheticandneurotoxin
receptorsites
Neurotoxins modify Na’ chan-
nel function by interaction with
several distinct receptor sites that
have been characterized exten-
sively in voltage clamp, ion flux,
and nelurotoxin binding studies.
Effects of local anesthetics on toxin
binding and action at some of
those receptor sites has been
examined in order to determine
whether local anesthetics may
exert their effects tbrrugh inter-
action with the same receptor sites.
Like local anesthetics, tetrodotoxin
and saxitoxin inhibit Na+ chan-
nels. However, local anesthetics
have no effect on binding of these
toxins to neurotoxin receptor site 1
on Na+ channels and therefore do
not act at that receptor site7,*.
Similarly, several local anesthetics
have no effects on binding of N-
scorpion toxins to neurotoxin re-
ceptor site 3 at concentrations that
effectively block Na+ channels,
ruling site 3 out as a potential site
of local anesthetic actions.
In contrast to these results, local
anesthetics have strong effects on
nemotoxin binding and action at
 TlPS - February 1987 fVo1. 81

1.0 -
B
-7 -6 -5 -4 -3 -2 0 5 10 15 20 25 G
Log [drugI, M Time (min)

Fg. 2. Mibition of batrachotoxinin A 2k-benzoate binding by local anesthetics. A. Concentration dependence of local anesthetic
binding. -es wenz incubated with 10 nM(3H]batrachotoxinin A 20-cu-benzoate and the indicated concentrations of tetracaine
(0). pmprano/ (0). or lidocaine (A) for 30 min and~~atrachototinin A 20-cu-benzoate specifically bound to neurotoxinreceptor site 2 on
Na+&annek was mBasursd. 0. Enhancement of toxin dlssoclation from receptor site 2. Dissociation of13H]batrachotoxinin A 20-a-
benzoatespeMkWybound to Na+channels in rat brain synaptosomes was measured in thepresence of the competing toxin aconitine at a
~tratkmof2iXl~(A)orin thepresenceof2OO~aconitineplus ldp~tetracaine (0). 470pMpdlocaine (@), 7.6mMocainide (A), 2.5
rn~ lidoceine (8). FL: n%zeptor-tigand complex. Reproduced withpermission from Ref. 13.

neurotoxin receptor site 2 on Na+
channels. Like local anesthetics,
batrachotoxin, veratridine, and
aconitine are lipophilic amines
and their binding and action is
enhanced by repetitive stimula-
tion to activate Na+ channels (see
review, Ref. 1). Local anesthetics
inhibit batrachotoxin action com-
petitivel~il. The binding of
batrachotoxinin A 20-or-benzoate
to neurctoxin receptor site 2 on
Na+ channels in synaptosomes is
also competitively inhibited bv a
wide range of local anesthehcs
(Fig. 2A) at concentrations which
block Na+ channels in myelinated
nerve and inhibit batrachotoxin-
induced depolarization of synap-
toneurosomes12~13. The action of
the enantiomeric local anesthetics
RAC 109-I and RAC 189-11 is
stereospecific with RAC 189-I
having g-fold higher affinity in
occupying neurotoxin receptor site
2 than it has in blocking Na+
channelsr3. All these studies are
consistent with binding and action
of the drugs at neurotoxin recep-
tor site 2.
However, two other aspects of
the interaction of local anesthetics
and neurotoxins at receptor site 2
rule out that site as the local
anesthetic receptor. Although local
anesthetics are competitive inhibi-
tors of activation of Na+ channels
by the full agonist batrachotoxin,
they are mixed competitivelnon-
competitive inhibitors of persis-
tent activation by the partial
agonist veratridine which also acts
at neurotoxin receptor site 2 (Ref.
8). This behavior is expected of
allosteric inhibitors of toxin action
but not of simple competitive
inhibitors that act by stericall
interfering with toxin binding’ r .
In addition, although the local
anesthetics increase the & for
binding of [3H]batrachotoxinin A
20-ar-benzoate without altering
B as expected for a competitive
i;bitor, they also accelerate the
dissociation of the preformed
toxin/receptor complex (Fig. 28,
Ref. 13). This action cannot be
mediated by binding at neuro-
toxin receptor site 2 since it is
already occupied by toxin. There-
fore, the local anesthetics must act
at a distinct site or sites on the Na+
channel protein and alter neuro-
toxin binding and action at recep-
tor site 2 by indirect allosteric
interactions mediated by con-
formational changes in the Na+
channel protein.
The allosteric antagonism be-
tween local anesthetics and neuro-
toxins that activate Na+ channels
by binding at site 2 is easily
understood in terms of an allo-
steric mod.el 4 drug and toxin
actiorP. These neurotoxins cause
persistent activation by binding
with high affinity to the active
states of Na+ channels and thereby
stabilizing them. In contrast, local
anesthetics bind with high affinity
to inactivated states of Na+ chan-
nels and stabilize them. The local
anesthetics and activating neuro-
toxins therefore interact competi-
tively by shifting the Na+ channels
to states with lowest binding
affinity for each other.
Local anestheticsand the
transmembrane pore of theNa+
channel
Since access to the local anes-
thetic receptor site is most rapid
when the channel is in the open
state and dissociation of bound
local anesthetic is also most rapid
from the open state of the channel,
it has been proposed that this site
may be located within the lumen of
the transmembrane pore such that
it is accessible from the intracel-
lular side of the Na+ channel only
when it is open*,‘.‘. While this is an
attractive interpretation of the
results, it is not a necessary one.
The conformational changes that
lead to Na+ channel activation and
inactivation involve many differ-
ent regions of the Na+ channel
protein. For example, binding of
toxins at neurotoxin receptor site 2
on Na+ channels is accelerated by
repetitive stimulation to activate
Na+ channels in the same way as
TIPS - February 1987 [Vol. 81 61
local anesthetic action is (reviewed dence on this point is examination ment through the channel. Thus,
in Ref. 1). However, neurotoxin of the effects of extracellular per- this effect of permeant cations is
receptor site 2 cannot be located in meant cations on ivthibition of ha+ most easily interpreted in terms of
the lumen of the channel since large channels in squid giant axon. competition with the charged form
neurotoxins bind there and acti- Inward mcwcment of p-meant of local anesthetics for an anionic
vate the channel without blocking cations through the activated binding site in the Na+ channel.
it. Thus, access to receptor sites on channel appears to oppose the Additional structural information
the Na+ channels can be restricted block of open channels by quater- seems necessary, however, before
in closed and inactivated states, nary local anesthetic derivatives”. a definite conclusion on the loca-
even if the site is not located in the This effect is blocked by tetrodo- tion of local anesthetic receptor
lumen of the transmembrane pore. toxin since it prevents ion move- sites can be made.
Is there any direct evidence to
place the local anesthetic receptor Antiarrhythmic drug action on Na’ channels
site in the intracellular end of the The Class I or q&idine-like In addition to quinidine, this class
transmembrane pore of the Na+ antiarrhythmic drugs inhibit vol- of drugs includes the local anesthe-
channel? Probably the experiment tage-sensitive Na+ channels in the tics lidocaine and procaine
that comes closest to direct evi- heart as their major mode of action. and their numerous derivatives,
phenytoin, a drug known mainly
as an anticonvulsant (see below),
and a host of newer agents such as
aprindine and amiodarone. In
*Or addition, cardiac drugs with other
primary actions also share Class I
antiarrhythmic potency. Notable
60 among these is the B-adrenergic
antagonist propranolol which
*.-._r_,,-r -- A blocks Na+ channels at high
A
concentrations.
40
Because many drugs are effez-
tive both as local anesthetics ant’,as
antiarrhythmic drugs, it is ex-
pected that these two drug cJ;lsses
will share many common proper-
ties. Early electrophysiological
,I- studies showed that reduction
0 5 10 of ir,, of the cardiac action
time(s) potential by quinidine and pro-
cainamide is enhanced by depolar-
B ization or repetitive stimulation of
the preparation (reviewed in Refs
15, 16). Subsequent studies have
confirmed bothvoltage-dependent
and frequency-dependent inhibi-
tion of cardiac Na+ channels by a
wide range of antiarrhythmic
drugs acting at therapeutic con-
centrations’ *16.In each case, inhi-
bition of Na+ channels is increased
by depolarization of the cardiac
muscle cell and by increased rates
of stimulation of the cell. Although
much of this work . employed
measurements of V-, the
strong similarity to observations of
local anesthetic action in myelin-
Lidocaine. pi ated nerve under voltage clamp led
to the proposal that the same
mechanisms described by the
Fig. 3. Frequency- and voltage-dependent inhibition of Na+ channels in cardiac muscle modulated receptor hypothesis of
by lidocaine. A. Frequency dependence. Rabbit Purkinje fibers under voltage damp
local anesthetic action are opera-
were maintained at a holding potenlial of - 105 mV and were stimulafed with 500 msec
pulses fo -35 mVat a frequency of 1.OHz. Membrane currents in response to each pulse tive*5J6. Voltage-dependence of
are plotted as a funcfion of puke number. B. Voltage-dependent blndlng. Rabbit channel inhibition near the resting
Purkinje fibers under voltage clamp were maintained at a holding potenrial (VH) of membrane potential arises primar-
-65 #W(A) or - 120 mV(@). The fibers wemexpcsed to fheindicatedconcenlretMnsof ily from preferential binding of the
lidocaine in pennuted order and Na + currenfs were measured in test pulses to -40 mV.
Depoiarizafion markedly increases block by lidocaine. The smcwlh curves r’epreSMl
drugs to the inactivated state of
hyperbolic adsorbtion isotherms for 16 values of 10~ at -65mV and 353 ph4 al Na+ channels. Frequency-depend-
- 120 mV. Reproduced with permission from Ref. 17. ence arises from rapid binding to
active states.
62
Recent improvements in f&C-
trophysiological methods now al-
low effective voltage clamping of
Na+ channel currents in rabbit
Purkinje fiber preparations using
traditional procedures” and in
enzymatically dissociated myo-
cytes using whole cell patch vol-
tage clamp procedureslK1q. The
pdctions of the modulated re-
ceptor model of ant&rhythmic
drug action on cardiac Na+ than-
nels have now been rigorously
tested using these preparations.
For lidocaine, both frequency-de-
pendent and voltage-dependent
blocks have been directly mea-
sured (Fig. 3). Inhibition at thera-
peutic concentrations (20 w) is
small on the first stimulus from a
negative holding potential but
increases during repeated stimuli
(Fig. 3A). High concentrations of
lidocaine are required to block Na+
channels at a negative holding
potential where few Na+ channels
can inactivate, but concentrations
lower by a factor of 20 are required
to block channels at -65 mV where
99% of Na+ channels are inacti-
vated (Fig. 38). From these data, Kd
values for lidocaine binding to the
resting and inactivated states of
Na+ channels were estimated as
440 v and low, respectively”.
These and other data show that
lidocaine binds rapidly to the
active state of Na+ channels and
has highest affinity for the in-
activated state of the Channel.
These state-dependent differences
in the rate and affinity of drug
binding result in voltage- and
frequency-dependent inhibition
of Na+ channels.
Many additional antiarrhythmic
drugs have now been studied
using vnl, or voltage-clamp
measurement$j. Many, like lido-
Caine, show a strong preferential
binding to the inactivated state of
Na+ channels. Others, like quini-
dine, show enhanced affinity for
both the active and inactivated
states of Na+ channels compared to
the resting state. Detailed models
are now under development to
correlate the characteristics of
preferential drug binding to dis-
tinct states of Na+ channels with
their action on particular classes of
cardiac arrhythmias.
The voltage- and frequency-
dependence of antiarrhythmic
drug action is likely to be particu-
larly important for the ability of
these drugs to prevent impulse
initiation and propagation in car-
diac arrhythmias. Normally, car-
diac rhythm is controlled by the
firing rate of the sino-atrial node.
Arrhythmias are considered to
arise from damaged cardiac tissue
which has been depolarized to
cause ectopic pacemakers which
compete with the sinoatrial node
for control of cardiac rhythm or to
provide slow conduction path-
ways which allow re-entry of
cardiac impulses at abnormal
phases of the cardiac Eycle. Ir.
optimal cases, effective antiar-
rhythmic drug therapy can block
abnormal initiation and propaga-
tion of impulses without signifi-
cantly compromising normal car-
diac function. Since damaged car-
Anticonvulsant action on Na’ channels
A broad range of drugs of
multiple structural and pharma-
cological classes are effective in
treating one or more of the major
classes of epileptic seizures. Their
cellular and molecular mechan-
isms of action have only recently
begun to come into focus. Pheny-
toin, which is particularly effective
in management of grand ma1 and
partial seizure disorders, blocks
Phenobarbital
Diazepam
Sodium valproale
Trimethadione
Ethosuximlde
-2. .A
0 10 20 30 40 50
% InhIbition of brain Nat channels
at therapeubc drug levels
Fig. 4. Inhibition of Na+ channels at
therapeutic concentrations of anttcon-
v&ants. Apparent K,, values were
detemrined from inhibition curves for
block of neurotoxin-activated =Na+
influx detemrined as in willow. et al.?
Tnie I& vatues were also measured
fmm block of specific f3Hlbatracho-
toxinin A 20-&en&at6 by the
various anticonvulsants*5. levels of
anticonvulsants present in rat brain
durim effective vrevention of exoeri-
menta seizures.were taken froh the
literature references given in Willow &
Cafterall*?Percent inhibition at the
meantherapeutic brain /eve/ of each
anticonvulsant was then calculated
from the relationship % I = 100 ftDMcI
+ /D]). Only ca;bamazepi&i and
phenytoin cause significant inhibition
of Na+ channels at therapeutic con-
centrations.
TIPS - February 1987 Wol. 81
disc tissue is usually depolarized
with respect to normally function-
ing cells, inhibition of Na+ chan-
nels in damaged cells is enhanced
by the increased binding of anti-
arrhythmic drugs to inactivated
states ol Na+ channels. The
frequency-dependence of anti-
arrhythmic drug action is particu-
larly important in limiting propa-
gation of rapid, arrhythmogenic
cardiac impulses beyond the dam-
aged tissue in which they origin-
ate. Selective limitation of rapidly
fired impulses and preferential
block of Na+ channels in depolar-
ized cells act together to allow
reversal and prevention of arrhy-
thmias without general inhibition
of cardiac function.
voltage-sensitive Na+ channels in
squid giant axonzo and frog mye-
linated nerve’l under voltage
clamp. Carbamazepine, which has
a similar therapeutic profile to
phenytoin also inhibits Na+ chan-
nels in squid giant axon=. Thus,
the Na+ channel is a potential site
for pharmacological action of anti-
convulsants.
Phenytoin also blocks vera-
tridine-induced depolarization of
squid giant axon= and both
phenytoins and carbamazepinez4
block veratridine- and batracho-
toxin-dependent activation of Na+
I channels in mouse neuroblastoma
cells and rat brain synaptosomes.
As observed for local anesthetics,
/ the inhibition of batrachotoxin-
activation is competitive while
inhibition of veratridine-activa-
tion at the same receptor site is
mixed competitive/noncompeti-
tive, consistent with designation
of phenytoin and carbamazepine
as allosteric inhibitors of neuro-
toxin binding and action at recep-
tor site 2 on Na+ channels. Both
of these drugs specifically block
binding of batrachotoxinin A 20-
ar-benzoate to Na+ channels, in
part by increasing the rate of
dissociation of the toxin-receptor
complexZ5. These results confirm
that these anticonvulsants, like the
local anesthetics and antiarrhy-
thmic drugs, are allosteric inhibi-
tors of neurotoxin binding and
action at receptor site 2 on Na+
channels.
The several classes of clinically
effective rnticonvulsants are all
lipophilic compounds which are
W’S - Februal-j 1987 [Vol. 81
expected to have multiple actions
on excitable membranes. It is
important therefore to correlate
quantitatively the concentration of
these drugs that act on Na+ chan-
nels with the concentrations that
are effective in preventing sei-
zures. Comparison of therapeutic
brain concentrations of anticon-
v&ants with those that block
batrachotoxinin A 20-cr-benzoate
binding and batrachotoxin-activa-
tion of Na+ channels in rat brain
synaptosomes allows an estimate
of the fraction of Na+ channels that
would be blocked at therapeutic
brain levels of drr1g2*,~~(Fig. 4).
Phenytoin and carbamazepine are
predicted to inhibit 30% and 37%
of Na+ channels at a mean thera-
peutic concentration, respectively.
In contrast, the barbiturate pheno-
barbital, the benzodiazepine dia-
zepam, Na+ valproate, ethosux-
imide, and trimethadione all are
predicted by this assay method to
inhibit less than 3% of Na+ chan-
nels at therapeutic concentrations.
Based on these findings, it was
proposed that phenytoin and car-
bamazepine are Na+ channel-
selective anticonvulsants which
block Na+ channels as their major
mode of action, while the several
other classes of agents tested exert
their antiepileptic action on other
physiological processes**-.
These neurochemical assays of
drug action allow a direct compari-
son of therapeutic concentrations
with pharmacologically effective
concentrations in adult rat brain,
but do not directly measure the
physiological activity of Na+ chan-
nels. However, several electro-
physiological studies on mam-
malian neurons in cell culture have
confirmed the major aspects of the
profile of drug action (Fig. 4). In
mammalian dorsal root ganglion
or spinal cord neurons maintained
in cell culture, phenytoin blocks
action potentials and repetitive
firing at therapeutically relevant
concentrations, but alters GABA-
mediated inhibitory synaptic res-
ponses and calcium-dependent
action potentials only at higher
concentrationP,*‘. In mouse
neuroblastoma cells, Na+ currents
are blocked by therapeutic concen-
trations of henytoin and car-
bamazepine* 2:lz9, but other classes
of anticonvulsants do not have
effects at therapeutic concentra-
tions. Thus, both physiological
- .
and neurochemical assays of drug
action support the conclusion that
phenytoin and carbamazepinr
block Na+ channels preferentially
at therapeutic concentrations.
Voltage- andfrequency-dependent
drugbindinginanticonvulsanf
action
At therapeutic doses, anticon-
v&ants prevent seizures without
causing sedation or diminishing
normal electrical activity in the
brain. General inhibition of Na+
channels in central neurons would
be expected to cause nonspecific
sedation. Therefore, the sugges-
tion that a subclass of anticonvul-
sant drugs may act primarily by
s 0-
-135 -105 -75 -45
Holding potential (mV)
Fig. 5. Voltage-depandentinhibition of
Na+ channels by phenyiotn and
carbamazaptne. Families of inward
Na+ currents were generated in
response fo test pulses (10 msec)
from a range of holding potentials
(-135 to -30 mv). A 90 mssc
prep&e at - 105 mVp:eceded each
test pulse. A period of 1 min was
allowed between chanoes in holdina
potential The e&t ofkhenytoin (6)
and carbamazepine (O), bW .Y?23
phf, on maximum mw~.;. ‘3;;.i.2n.
(measured at +15 mv) at differed
holding potentials is shown. Each
ordinate value is compared to a
oontro/at the same ho/ding potential.
The results show increased inhibition
of Na+ channels at depolanked
mombrane potentials. Reproduced
with permission from Ref. 29.
inhibition of Na+ channels re-
quires consideration of possible
mechanisms whereby their action
might be selective for neurons
responsible for generation of seiz-
ure discharges. Voltage- and fre-
quency-dependent binding of
these drugs to their receptor site on
Na+ channels provides a possible
mechanism for their antiepileptic
specificity.
Several early studies of the
action of phenytoin established
that it inhibits repetitive firing at
lower concentrations than it blocks
action notential generation. Mam-
63
mahan spinal cord neurons in
cell culture respond similarly
with half-maximal bjo:k of action
pOtential trains at 5 PM and
half-maximal reduction of action
potential rate of rise (Pm=) at
18 uMz. In voltage clamp studies of
mouse neuroblastoma cells, inhi-
bition of Na+ channels by both
phenytoin*s,” and carbamaze-
pine29 is increased by repetitive
stimulation of cells.
The voltage-dependence of ac-
tion of phenytoin and carbamaze-
pine on Na+ channels in mouse
neuroblastoma cells is even more
prominent than their frequency-
dependenceBZ9. Long depolariz-
ing pulses enhance inhibition of
Na+ channels by phenytoin and
long hyperpolarizing pulses re-
verse it. Typical results are illus-
trated in Fig. 5 which shows that
the percent inhibition of Na+
currents by a fixed concentration
(20 PM) of phenytoin or carbama-
zepine increases from approxi-
mately 5% at a holding potential of
-120 mV to 60-70% at -45 mV.
The voltage-dependence is steep-
est between -90 mV and -45 mV
where inactivation of Na+ chan-
nels normally occurs and the
voltage for half-maximal inactiva-
tion of Na+ currents (-70 mV, Ref.
29) is similar to the mid-point for
the voltage-dependence of drug
binding (Fig. 5). These results
indicate that phenytoin and car-
bamazepine bind preferentially to
the inactivated state of NaC chan-
nels that is generated by prolonged
depolarization.
How might frequency- and vol-
tage-dependent binding of anti-
convulsants allow selective action
of these drugs on neurons under-
going epileptic discharges? Neur-
ons in experimental epileptic foci
undergo an unusual electrical ac-
tivity, termed the paroxysmal de-
polarization shift, which consists
of prolonged depolarizations of 15
to 20 mV lasting for hundreds of
milliseconds with high frequency
trams of superimposed action
potentials (reviewed in Ref. 30).
These electrical transients are of
the appropriate voltage and fre-
quency to cause enhanced inhibi-
tion of the Na+ channels present in
neurons undergoing epileptic dis-
charge. Stimulation of neuroblas-
toma cells under voltage clamp
with similar voltage transients
results in additive voltage-depen-
dent and frequency-dependent in-

64
TAME 1.Gla&fi&ion of anticonvulsantsaccording to effects on seizures, Na+ channels, and
GABAreceptors
Class Anticonvulsant
Partial Grand
seizures mal
I Phenytofn
Carbamazepine
II Phenobarbital
Genzodiazepines
Valcroate
Ill Ethbsuximide
TdmethadMe
ll’hii abbreviated presentationof pharmawlcgical pmfile is derived from Glaser, G. H. (1989)
A& bheuml.27,593-575.
t Datataken from MacDonald, R. L. and Barker, J. L. (1979) Neuro/ogy29,432-447 and (1978)
Nat~ri3271.583-584; Baldino, F. and Getler, f-l. (1981) J. Phannaccl. Exp. Ther. 217,445-459;
and Nicoll, R. A., and Wojtowiu, J. M. (1980) Brain Res. 191. 225232.
NA, data not available.
h!bitior?. These two effects are
sufficient to allow inhibition
of repetitive action potential gen-
eration in neurons of epileptic
foci without inhibition of action
potential generation by normal
central neurons.
Classification of anticonvulsant
drugs
The different classes of anticon-
v&ants have different profiles of
clinical effectiveness against speci-
fic classes of seizures. The results
reviewed here show that two
major anticonvulsants, phenytoin
and carbamazepine, block Na+
channels at therapeutic concentra-
tions in a manner that selects for
inhibition of repetitive action
potential generation by neurons in
epileptic foci. Other anticonvul-
sant drugs do not share this
activity at therapeutic concentra-
tions (Fig.’ 4). Results in the
literature show that barbiturates,
benzodiazepines and Na+ val-
proate enhance inhibitory res-
ponses to GABA at concentrations
that are achieved therapeutically.
It is likely that these three anticon-
v&ants prevent seizures by en-
hancing inhibitory transmission at
GABAergic synapses. Surcinim-
ides and oxazolidinediones, which
are specifically effective against
petit mal seizures, have not been
reported to have effects on either
voltage-sensitive Na+ channels or
GABA receptors. In view of the
differential profile of clinical effec-
tiveness of the anticonvulsants
against specific classes of seizures,
the differential effects on Na+
channels noted in the studies
reviewed here and the differential
effects of these drugs on GABA
Drug actions at
therapeutic
Pharmacological profile* concsntratlons
Myo- Peti! Na+ GABA
clonic mal than- recep-
nets torst
+ + - - + side of the Na+ channel. This site is
+ + - - + NA
+ + + - - +
+
f f + + -
+ + + + - +
+ - NA
+ - NA
receptors reported elsewhere in
the literature, we previously pro-
posedz5 that the anticonvulsant
drugs can be classified into three
groups (Table I). The Class I
anticonvulsants, phenytoin and
carbamazepine, have a similar
spectrum of clinical potency with
efficacy primarily against partial
seizures and tonic-clonic (grand
mal) seizures. We propose that
these agents are Na+ channel-
selective anticonvulsants which
modulate Na+ channel properties
as their principal mechanism of
action. The Class II anticonvul-
sants (phenobarbital, the benzo-
diazepines, and possibly sodium
valproate) have a broad spectrum
of clinical potency with significant
efficacy against partial seizures,
grand mal seizures, myoclonic
seizures, and petit mal seizures.
On the basis of work in the
literature, we proposed that these
agents are GABA receptor-selec-
tive anticonvulsants which en-
hance inhibitory synaptic trans-
mission as their principal mechan-
ism of action. The Class III
anticonvulsants (ethosuximide,
trimethadione, and related suc-
cinimides and oxazolidinediones)
have a narrow spectrum of clinical
effectiveness which is comple-
mentary to that of the Class I
agents. Their effectiveness is re-
stricted primarily to treatment of
petit mal seizures. At present, no
satisfactory pharmacological mech-
anism for their action has been
proposed.
cl cl 0 Catterall, W. A. (1978) Biophys. J. 23,
Inhibition of Na+ channels in
peripheral nerve, cardiac muscle,
TlPS - February 1987 [Vol. 81
and central neurons is the mol-
ecular basis of action of local
anesthetics, Class I antiarrhythmic
drugs, and a subclass (Class I) of
anticonvulsants, respectively. The
action of the drugs is best ex-
plained by binding at a site or sites
available from the intracellular
not one of the well-defined neuro-
toxin receptor sites on the channel,
but occupancy of it allosterically
inhibits interaction of activating
neurotoxins with receptor site 2. In
each case, the interaction of the
drugs with their receptor site(s)
on Na+ channels is frequency-
and voltage-dependent. The fre-
quency- and voltage-dependence
arises from rapid access of the
drugs to their receptor site in the
active state of the channel and
selective high affinity binding to
the inactivated state of the channel
as postulated by the modulated
receptor hypothesis of drug action.
Voltage- and frequency-depen-
dent binding is probably respon-
sible for the selectivity of action of
the antianhythmic drugs to block
rapid cardiac impulses generated
by damaged, depolarized cells and
of the anticonvulsants to block
seizures without preventing nor-
mal neural activity. This property
of drugs may have broad implica-
tions for their actions in excitable
tissues.
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foot shock) for performing a Mgh-

Facilitation of memory and probability response such as step-
ping off a platform or entering a
cognition by drugs dark compartmeot. The animal’s
‘memory’ for the punished re-
sponse is measured by its latency
George A. Moise to perform that response when it is
returned to the punishment site,
usually 24 hours later; the longer
George Heise surveys the expe~mental research with un~maZson three groups the latency, the better the ‘mem-
of compounds - cholinergics, nootropics, and neuromodulators - that facilitate ory’. The popularity of this pro-
memory and cognition in animals, with emphasis on the adequacy and logic of cedurc is based on the mtnimal
‘animal models’ of normal or deficient human performance that underhe the amount of training required, on
research. Although there are compuunds that drumatica~ly improve the sensitivity of the procedure
performance in animals, the beneficial effects of these compounds on human and on the experimenter’s ability
memory or cognitive performance, while sometimes statistically significant, to specify precisely the time of
have been smali and of ~~es~ona&leclinical relevance. No clearly effective presentation of the to-be-remem-
‘reference’ compound has as yet been discovered. He makes suggestions for bered information and the dur-
redirecting the animal research towards more effective models. ation of the retention (memory)
interval. However, variability be-
Spurred by a concern for treating pharmacological candidates and tween animals tends to be high in
the normal and pathological defi- by more extensive clinical data. passive avoidance and changes in
cits in memory and cognitive ‘Memory’ in animals refers to arousal or response bias can under
performance that accompany old the behavior observed in experi- some circumstances alter retest
age, the use of drugs to facilitate ments in which a delay (the latency independently of any spe-
cognition, learning and memory retention interval) intervenes be- cific drug effects on memory. In
is a lively area of current re- tween the presentation of infor- contrast, such procedures as de-
search. This review examines the mational stimuli and the occasion layed response, delayed matching
methods, strategies, assumptions, for responding, The terms ‘cog- and the radial maze make it
anA accomplishments of the animal nition’ or ‘cognitive processes’ are possible to define more precisely
research on memory and cognition applied to situations where inte- the behavior affected by the drug,
facilitators. gration or reorganization of the to separate specific effects on
For many years there has been stimulus input is required for an memory from nonspecific effects
the need for more rigorous experi- appropriate response. There is no that do not depend critically on
mental definitions of phenomena, generally accepted, invariant cor- delay and to obtain repeated
while there has been overempha- respondence between ‘memory’ or observations upon the same
sis on passive avoidance in testing ‘cognition’ as defined behaviorally animal.
effects of compounds on memory. and any specific biochemical
The search for new drugs has been or neurophysiological process; Cholinergic drugs
handicapped by the lack of a hence, ‘animal models’ of memory Arguably, the most comprehen-
‘comparison standard’ - a refer- and cognition are of necessity sive and integrated approach to
ence compound that reliably facili- behavioral and evidence for the the discovery of memory and
tated learning or memory per- benefical effects of drugs on cognition facilitating drugs has
formance in humans (see Ref. 1). memory or cognitive dysfunction been that based on functions of the
Nevertheless, there has been sub- in animals must ultimately depend central cholinergic system. Much
stantial progress recently, as indi- on behavioral measurement. of the research has been guii’.ed by
cated by an enlarged range of An understanding of the be- the ‘cholinergic hypothesis of
experimental methods and animal havioral methods used to measure geriatric dysfunction’, which as-
models, by an abundance of drug effects on memory or cogni- serts, in essence, that the deFcits
tion will aid in the evaluation of in cognitive and memor7 a~~8~~-
the expe~ment~ findings. In the ante observed in aged human- and
George fieise is Professor of Psychology at
ubiquitous passive avoidance pro in Alzheimer’s disease p&tientsare
lndiana University, Department of Psy due at least in Dart to deficient
chology, Psychology Building, Bloomington, cedure, the animal - typically a rat
Indiana, 47605, USA. or mouse - is punished (usually by cholinergic functionine3.
@ 1987, Ekvier seipnm PublIshem B.V.. Amsterdam Olbs- 6147/87/$ot.O0