Local Anesthetics" Hydrophilic

and Hydrophobic Pathways

for the Drug-Receptor Reaction
BERTIL HILLE
From the Department of Physiology and Biophysics, University of Washington School of
Medicine, Seattle, Washington 98195

AB S T R AC T The properties of Na channels of the node of Ranvier are altered by

neutral, amine, and quaternary local anesthetic compounds. The kinetics of the Na
currents are governed by a composite of voltage- and time-dependent gating
processes with voltage- and time-dependent block of channels by drug. Conven-
tional measurements of steady-state sodium inactivation by use of 50-ms prepulses
show a large negative voltage shift of the inactivation curve with neutral benzocaine
and with some ionizable amines like lidocaine and tetracaine, but no shift is seen
with quaternary QX-572. However, when the experiment is done with repetitive
application of a prepulse-testpulse waveform, a shift with the quaternary cations
(applied internally) is seen as well. 1-min hyperpolarizations of lidocaine- or tetra-
caine-treated fibers restore two to four times as many channels to the conducting
pool as 50-ms hyperpolarizations. Raising the external Ca ++ concentration also has
a strong unblocking effect. These manipulations do not relieve block in fibers
treated with internal quaternary drugs. The results are interpreted in terms of a
single receptor in Na channels for the different drug types. Lipid-soluble drug
forms are thought to come and go from the receptor via a hydrophobic region of
the membrane, while charged and less lipid-soluble forms pass via a hydrophilic
region (the inner channel mouth). The hydrophilic pathway is open only when the
gates of the channel are open. Any drug form in the channel increases the
probability of closing the inactivation gate which, in effect, is equivalent to a
negative shift of the voltage dependence of inactivation.

INTRODUCTION

This p a p e r continues the analysis o f two major questions c o n c e r n i n g local

anesthetic action: where on the Na channel is the receptor, and how do the
anesthetic molecules get there? In this work the anesthetic solution is applied
long e n o u g h for the distribution o f d r u g to a p p r o a c h an equilibrium. N o n e t h e -
less the degree o f block is shown to be strongly d e p e n d e n t on the rate a n d
waveform o f the testing voltage clamp pulses, on the holding potential, a n d on
the external Ca ++ concentration. T h e persistence o f after effects o f recent
stimulation is quite analogous to the r a t e - d e p e n d e n t block o f Na channels seen
in myocardial fibers treated with a n t i a r r h y t h m i c d r u g s (see references in Gettes
and Reuter, 1974). As has already been f o u n d in previous work on myelinated
nerve (Strichartz, 1973; K h o d o r o v et al., 1974; C o u r t n e y , 1975; K h o d o r o v et al.,

THE JOURNAL OF G E N E R A L PHYSIOLOGY " VOLUME 69, 1977 • pages 497-515 497
498 TI~E JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 69 " 1977

1976), the reactivity o f the local anesthetic r e c e p t o r is a sensitive function o f the

state o f the v o l t a g e - d e p e n d e n t gates o f the Na channel. Factors that affect gating
affect the susceptibility to block with local anesthetics, a n d block by anesthetics
affects gating. As in the p r e c e d i n g p a p e r (HiUe, 1977), m e a s u r e m e n t s are m a d e
by using neutral, ionizable, and p e r m a n e n t l y cationic d r u g molecules with a
r a n g e o f lipid solubilities in o r d e r to e x a m i n e the effect o f c h a r g e a n d h y d r o p h o -
bicity on the d r u g - r e c e p t o r interaction.

MATERIALS AND METHODS

Single myelinated nerve fibers were studied under voltage clamp as described in the
preceding paper. All experiments were done at 15°C with an external solution containing
NaCI 100-115 mM, CaC12 2 mM, and tris(hydroxymethyl) aminomethane-HC1 buffer 1-6
mM. The pH was always 7.4 in these experiments. In most cases 1-7 mM tetraethylammo-
nium ion (TEA) was added to block currents in K channels. The ends of the fibers were
cut in unbuffered 120 mM KCI, and drugs were added to the "internal" or external
solutions as before. The structures of" the drugs are given in Fig. 1 of the preceding
paper.

RESULTS

Fig. 1 shows a family o f voltage c l a m p c u r r e n t s f r o m a fiber b e f o r e a n d d u r i n g

e x p o s u r e to 1 m M lidocaine at p H 7.4. In the u n t r e a t e d fiber (Fig. 1 A), the test
pulses elicit the usual transient increase o f sodium permeability PNa that reaches
a p e a k in the 1st ms and then a slower rise o f potassium permeability P~ that
takes m o r e than 10 ms to d e v e l o p fully (not shown in its entirety). With a 50-ms
prepulse to - 1 4 3 m V (Epp) to r e m o v e resting s o d i u m inactivation, the p e a k
inward sodium c u r r e n t INa reaches - 3 3 . 2 nA in the step to - 2 3 m V , a n d the
o u t w a r d potassium c u r r e n t rises to 19.1 nA at 67 m V (at 10 ms but not d r a w n in
the figure). W h e n the same fiber is treated with 1.0 m M lidocaine and tested with
the same pulses (Fig. 1 B), peak Psa is r e d u c e d to 15% o f the control a n d p e a k PK
is r e d u c e d to 80%. This observation c o n f i r m s the general finding with myeli-
nated fibers that local anesthetics block Na channels m o r e than K channels
(Hille, 1966; A r h e m and F r a n k e n h a e u s e r , 1974). F r o m now on Results focus on
c o m p a r i n g the block o f Na channels with amine, neutral, a n d q u a t e r n a r y
anesthetics, and in most e x p e r i m e n t s PK is deliberately d e p r e s s e d by a d d i n g
t e t r a e t h y l a m m o n i u m ion to the solutions. T h e results are divided into two
sections. T h e first concerns the effect o f prepulses, holding potential, and Ca ++
on steady-state block with low rates o f stimulation. T h e s e factors are m o d u l a t o r s
o f the resting state o f channels. T h e second section concerns the cumulative
effect o f higher rates o f stimulation on block. T h e depolarizing stimulus pulses
activate Na channels and modify their probability o f b e c o m i n g blocked.

Prepulse, Holding Potential, and Ca ++

SODIUM INACTIVATION SEEN WITH SHORT PREPULSES. I n n o r m a l myeli-
nated fibers roughly 30-50% o f the Na channels are inactivated at the resting
potential, and their resting inactivation may be r e m o v e d by a 30-45-mV hyper-
polarizing p r e p u l s e lasting tens o f milliseconds ( F r a n k e n h a e u s e r , 1960). T h e s e
p h e n o m e n a are well described in n o r m a l fibers by the conventional inactivation
HXLL£ ModulatedReceptorfor Local Anesthetics 499

variable h o f H o d g k i n and H u x l e y (1952). After local anesthetic t r e a t m e n t ,

however, the extent o f resting sodium inactivation is increased in a m a n n e r
which may involve the d e v e l o p m e n t o f new inactivated states. ( K h o d o r o v et al.,
1974, 1976; C o u r t n e y , 1974, 1975).
For some but not all local anesthetics changes o f inactivation may be seen in
e x p e r i m e n t s with 50-ms prepulses p r e c e d i n g the test pulse. An increase o f
resting inactivation with 1 mM lidocaine is shown in Fig. 1. In the u n t r e a t e d fiber
(Fig. 1 A and C), r e m o v i n g the 50-ms prepulse (Epp) decreases peak PNa by 35%,
while in the treated fiber (Fig. 1 B and D) r e m o v i n g the prepulse decreases PNa
by 90%. T h e voltage d e p e n d e n c e o f sodium inactivation m e a s u r e d with 50-ms
prepulses f r o m a holding potential o f - 8 3 mV is shown in m o r e detail in Fig. 2.

t(ms)
0 1 2
'A' ' ' B C

. lmM ~R in,~e~ .1 M.
-20 nger Lidocaine

oL
. VV/EH =-83mY
Epp=-143rnV Epp= -83rnV
FIGURE 1. Families of voltage clamp currents before and during treatment with 1
mM lidocaine. Solutions contain no TEA. The node is held at a holding potential
EH = -83 mV and depolarized by test pulses ranging in 15 mV steps from -68 to
+67 mV. In (A) and (B) the test pulse is preceded by a 50-ms conditioning
prepuise to -143 mV. In (C) and (D) there is no prepulse. Test pulses applied at
intervals of 1.0 s. Same node as in Figs. 3 and 5.

In the control these m e a s u r e m e n t s define a conventional h~ curve, but with

anesthetic the situation is m o r e complicated and the curve will be called simply
the inactivation curve. T h e changes with 1 mM lidocaine may be described as a
30-mV shift to more negative potentials o f the midpoint o f the curve accompa-
nied by a reduction o f slope. Inactivation curves f r o m the same fiber are also
shown d u r i n g partial block with the u n c h a r g e d molecule benzocaine and the
q u a t e r n a r y cation QX-572. A 23-mV shift to m o r e negative potentials is f o u n d
with 1 mM benzocaine, but no change is f o u n d after external application o f 0.6
mM QX-572. In o t h e r e x p e r i m e n t s with internal application o f q u a t e r n a r y QX-
572 or RAD 250 I, there was at most a 5-mV shift o f the inactivation curve at low
rates o f stimulation, although P•a was r e d u c e d by the drugs to 25-40% o f the
control value. Some amine anesthetics shift m u c h less than lidocaine does. For
instance, in two e x p e r i m e n t s I f o u n d that 2/~M tetracaine reduces P~a to 8.5% o f
control and shifts the inactivation curve by only 7 mV, and C o u r t n e y (1975)
500 THE .JOURNAL OF GENERAL PHYSIOLOGY • VOLUME ~9 " 1977

showed that GEA 968 gives no shift. It is i m p o r t a n t to note that the inactivation
r e f e r r e d to here is defined by the effects o f 50-ms prepulses on currents
m e a s u r e d at stimulation rates too slow to give the kind o f cumulative block
described later.
MODULATIONBY HOLDINGPOTENTIAL. In addition to the rapid effects f r o m
short conditioning prepulses, there are slower inactivation-like processes m o d u -
lating the available Psa in myelinated nerve (Peganov et al., 1973; Fox, 1976;
N e u m c k e et al., 1976). A local anesthetic-dependent inactivation with 200-900-
ms time constants at r o o m t e m p e r a t u r e was discovered and analyzed extensively
by K h o d o r o v et al. (1974, 1976). T h e s e authors used the name slow sodium
inactivation and designated the time constant ~'s and the fraction o f available
channels s=. In the experiments to be described, removal o f such slow c o m p o -

1.0 1.0

OY5 OY5

~ 0.5c
0 NO drug ~ \ 0 25 o No drug
o.25 • 1raM Lidocoine ~'~ ~ • olmM Benzocoim
After Q,6_mM ~b,.~
-, 9xs7,~ ' " " ~ o i i A

-~8o -~5o -~2o -9o -6o -180 -150 -120 - 9 0 -60

Prepulse potential (mY)
FIGURE 2. Effect of 50-ms conditioning prepulses on peak Na currents with
neutral, amine, and quaternary anesthetics. All observations from the same node
held at En = -90 mV and tested with test pulses to -15 inV. For each treatment,
the current at the most negative prepulse potential shown is normalized to 1.0.
Curves are drawn according to the formula 1/(1 + exp ((E - Eo)/k)) where from left
to right in the figure E0 = - 109, -79, - 102, -79 mV, and k = 14.4, 7.5, 9, 7.5 inV.

nents o f inhibition was accomplished by m a k i n g the holding potential m o r e

negative in steps o f 15 mV and holding at the new level for about 1 min before
m e a s u r e m e n t s were made.
Fig. 3 shows families o f currents measured on the same fiber as was used for
Fig. 1. T h e external solution contains 1 mM lidocaine and 3.6 mM T E A . In A the
peak INa is only - 0 . 5 nA at the holding potential E~ of - 8 3 mV without a
prepulse. In B a 60-mV h y p e r p o l a r i z i n g prepulse lasting 50 ms increases peak
Psa 10-fold. According to the inactivation curve o f Fig. 2, PNa should now be
within 15% o f its m a x i m u m value. Nevertheless, as is shown in Fig. 3 C and D,
peak PNa can be increased a f u r t h e r 70% or 250% over that in Fig. 3 B by stepping
the holding potential to - 9 8 or - 1 4 3 mV for 1 rain. L o n g hyperpolarization
relieves anesthetic block. T h e voltage d e p e n d e n c e o f this slow effect o f holding
potential is shown by triangles in Fig. 4 for the lidocaine experiments o f Fig. 3
and for a different e x p e r i m e n t with 2 /~M tetracaine. For c o m p a r i s o n the
c u r r e n t amplitudes (circles) f o u n d in inactivation m e a s u r e m e n t s with 50 ms
prepulses starting f r o m En = - 8 3 mV are shown as well. With 1 mM lidocaine
HILL~: Modulated Receptorfor Local Anesthetics 50I

less t h a n 5% o f the N a c h a n n e l s a r e available w h e n h o l d i n g at - 8 3 m V w i t h o u t

p r e p u l s e s , a n d m o r e t h a n 50% o f the c h a n n e l s a r e r e s t o r e d to the c o n d u c t i n g
pool by l o n g h y p e r p o l a r i z a t i o n s . T h e situation with 2 t~M tetracaine is similar. By
contrast in e x p e r i m e n t s with the q u a t e r n a r y molecules QX-572 a n d R A D 250 1 (N-
t(ms)
0 I 2
5
A B C D

o _ _

-I0
EH= -83rnV EH:-83mV EH=-98mV EH=-143rnV
-15 Epp=-83mV Epp:-14 3rnV Epp=-158mV Epp=-143rnV
FIGURE 3. Relief of lidocaine block by more negative holding potentials. Families
o f voltage clamp currents in the presence o f 1 mM lidocaine and 3.6 mM TEA.
Same node and same test pulse potentials as in Figs. 1 and 5. Voltage of holding
potential En and of the 50 ms prepulse Epp are indicated below.

] mM Lidocaine 2/~M Tetracaine

I.OC I00 0 ControI,EH=-83
E~,pvaried
• Drug,E~ =-143
. 0.75 0.75 EHvaried
• Drug,EH=-83
, ~ ~ , ~ q' Eppvaried
0.5C 0.50

0.25
4
I -150 -125 -I00 -75-
Prepulse Or holding potential(rnV)
FIGURE 4. Slow and fast inactivation in fibers treated with lidocaine and tetra-
caine. T h e relative peak INn is shown (filled circles) as a function of the prepulse
potential with a constant holding potential (Eu = - 8 3 mV) or (filled triangles) as a
function o f the holding potential with a constant prepulse potential (Epp = -143
mV). Unlike in Fig. 2, all currents are normalized with respect to the largest current
in the control measurement before drug treatment. Smooth curves for controls are
like those in Fig. 2 with Eo = - 8 0 mV for the left curve and Eo = - 8 2 mV for the
right, and k = 7.5 mV. Curve through filled circles for lidocaine hasEo = - 110 mV,
k = 14.4 mV, and an asymptote o f 0.2. Other curves arbitrary.

m e t h y l m e p i v a c a i n e ) a n d with n e u t r a l b e n z o c a i n e , c h a n g i n g the h o l d i n g p o t e n -
tial f o r 1 min h a d n o a p p a r e n t effect on the available Psa. T h e h o l d i n g potential
also h a d n o significant effect o n fibers in the d r u g - f r e e c o n t r o l solution.
T h e time c o u r s e o f the effects o f h o l d i n g potential was m e a s u r e d by s t e p p i n g
~02 THE JOURNAL OF GENERAL ]PHYSIOLOGY • VOLUME 6 9 " 1977

EH for varying periods o f time b e f o r e testing with a standard prepulse to ca.

-125 mV followed by a test pulse to - 5 mV. With 1 m M lidocaine, changes o f EH
f r o m --80 to --125 mV or back again gave nearly full effects in less than 200 ms.
In this case it was difficult to separate the "slow" process f r o m removal or
d e v e l o p m e n t o f normal inactivation since their time courses o v e r l a p p e d consid-
erably. With 2 /.tM tetracaine the separation was clear. Unblock on stepping EH
f r o m --80 to -- 125 mV developed with a time constant ~'s o f 10.2 s, and block on
stepping back to - 8 0 mV developed with a ~'s o f 2.8 s. T h e s e time constants at
13°C may be c o m p a r e d with values o f 10 s for 0.6 mM GEA 968 at 10°C and - 7 4
mV (Courtney, 1975) and 220-370 ms for 0.18 mM trimecaine and 640-700 ms
for 0.9 mM procaine both at 20°C and - 1 0 0 mV ( K h o d o r o v et al., 1976).
MODULATION BY E X T E R N A L CALCIUM. T h e effects o f hyperpolarizing
holding potentials may be imitated by raising the external Ca ++ concentration.
Fig. 5 shows m e a s u r e m e n t s on the same fiber as was used for Figs. 1 and 3. T h e
top line shows currents r e c o r d e d in the standard 2 mM Ca ++ Ringer with 1 mM
lidocaine as before. T h e lower line shows currents in 12.8 mM Ca ++ with 1 mM
lidocaine. C o m p a r i n g f r a m e A with f r a m e D shows that elevating Ca ++ dramati-
cally increases the available Psa when the fiber is held near the resting potential.
C o m p a r i n g B with D and C with E shows that hyperpolarizing the fiber in 2 mM
Ca by 15 mV for 1 min restores the peak sodium c u r r e n t in a similar m a n n e r to
raising Ca ++ to 12.8 mM. According to earlier studies on the node o f Ranvier
(Hille, 1968; Vogel, 1974; Hille et al., 1975), increasing Ca ++ f r o m 2 mM to 12.8
mM would be equivalent to a d d i n g a 16.l-mV bias to the voltage d e p e n d e n c e o f
activation o f Na channels. I n d e e d , in Fig. 5 the m a x i m u m inward c u r r e n t occurs
at - 8 mV in 12.8 mM Ca ++ and at - 2 3 mV in 2 mM, and the entire PNa-E relation
is shifted about 15 mV. H e n c e , at least to a first a p p r o x i m a t i o n , steady h y p e r p o -
larization and elevated Ca ++ probably modulate lidocaine effects t h r o u g h a
c o m m o n mechanism involving the electric field i n the m e m b r a n e . In parallel
with the lack o f effect o f holding potential on block with q u a t e r n a r y molecules
inside, raising the external Ca ++ concentration did not increase peak PNa with
QX-572 or RAG 250 I inside. I n d e e d , in two experiments with RAG 250 I,
elevating Ca ++ actually decreased peak PNa m e a s u r e d at a stimulation rate o f 1
$-1.

VOLTAGE- AND FREQUENCY-DEPENDENT INHIBITION. Strichartz (1973) f i r s t

described what he called voltage-sensitive inhibition with q u a t e r n a r y derivatives
o f lidocaine (especially QX-314) inside myelinated fibers. T h e d e g r e e and rate o f
block o f Na channels d e p e n d e d on the amplitudes and durations o f prepulses
and test pulses, and the blocking or unblocking effects accumulated with succes-
sive pulses given at rates down to 0.1 s -1. He suggested that the gates o f Na
channels have to be open for the cationic d r u g molecules to e n t e r or leave the
internal r e c e p t o r site and that in addition the externally applied electric field
acting on the cationic d r u g alters the rates o f entering and leaving open chan-
nels. C o u r m e y (1974, 1975) described a related p h e n o m e n o n that he called
frequency- or u s e - d e p e n d e n t inhibition with amine local anesthetics including
lidocaine, procaine, procaine amide, and most especially the lidocaine derivative
GEA 968. T h e faster the rate o f stimulation, the m o r e the block d e p e n d e d on
HILLE Modulated Receptorfor Local Anesthetics 503

pulse amplitudes and durations. Courtney added to Strichartz's hypothesis the

suggestion that channels blocked by amine anesthetics also have their 1~ curve
shifted strongly to the left. A test for voltage- and f r e q u e n c y - d e p e n d e n t inhibi-
tion with the three charge classes of anesthetic molecules is now described.
Fig. 6A shows four pulse waveforms which were applied repetitively to
observe voltage- and frequency-dependent inhibition in my experiments. In
waveform 1 the m e m b r a n e is hyperpolarized by 45 mV for 50 ms and then
depolarized by 60 mV for 1 ms to measure the peak sodium current INa. In
waveform 2 the hyperpolarization is deleted. In waveforms 3 and 4 an extra 5 ms
depolarization is a d d e d after the test pulse. T h e amplitude is 150 mV in no. 3

t(ms)
0 1 2
i i i
4 A O C
2

~-2
-4
-6 EH= - 8 3 rnV EH = - 9 8 mV EH= -98mV
-8 Epp=-83mV Epp =-98mY Epp =- 158mY
D E

EH=-83mV EH=-83mV
Epp = -83mY Epp=-143rnV

FIGURE 5. Parallelism between relief of block by elevated external [Ca ++] and
relief by hyperpolarizing holding potentials with 1 mM lidocaine. Same fiber and
test pulses as in Figs. 1 and 3. For (A), (B), and (C) [Ca ++] = 2 mM and for (D) and
(E) [Ca ++] = 12.8 mM. Holding potential (EH) and prepulse (Epp) given below.

and 60 mV in no. 4. Frames B-F show the relative peaklNa measured d u r i n g the
test pulse when the pulse waveforms are applied repetitively at rates o f 3.3-5 s-1.
Where a waveform different from no. 1 is used, the n u m b e r of the waveform is
given above the data points. In each case the fiber was rested for more than 1
min before the test sequence began.
In the control m e a s u r e m e n t without d r u g treatment (Fig. 6 B) the relative INa
does not vary from the first to the last stimulus o f waveform 1, and it drops to
0.67 when the prepulse is removed, as expected from conventional sodium
inactivation. A d d i n g a 150 mV pulse after the m e a s u r e m e n t has no effect on
subsequent measurements o f l s a . With 1 mM benzocaine (Fig. 6 C) the situation
is nearly the same except that the resting sodium inactivation is far larger than
without drug. By contrast after external application of lidocaine or QX-572 or
internal application o f RAD 250 I (Fig. 6D, E, F), there is clear evidence of
504 T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y - V O L U M E 6 9 - 1977

voltage- a n d f r e q u e n c y - d e p e n d e n t inhibition. O n starting the pulse p r o g r a m

a n d at each c h a n g e o f w a v e f o r m , the relative INa drifts t o w a r d a new steady level
in a m a n n e r d e m o n s t r a t i n g the cumulative effect o f closely spaced stimuli.
Additional depolarization (waveform 3 or 4) a f t e r the test pulse significantly
decreases the available PNa in the next test pulse. In s u m m a r y , a m i n e a n d
q u a t e r n a r y molecules give strong voltage- a n d f r e q u e n c y - d e p e n d e n t inhibition
while the neutral molecule benzocaine gives n o n e . Surprisingly, w h e n the same
series o f stimuli was applied at 5 s -1 in the p r e s e n c e o f 2 /zM tetracaine (not
shown), t h e r e was little drift a f t e r each change o f w a v e f o r m a n d no e x t r a block
on switching to w a v e f o r m s 3 and 4.

Pulse sequences [.~ 1[ ~ ' ,,,,-J" _ F~

, n - - -
--1 . . . . 1 - - -:-L.2"J .... ~os~
lmM
2 j'[ 4 ~---~ / No drug ~ ,~tp Benzocoine
L ~ -~ 0 ~, , i ~ "tJ
0 5 I0 15 0 5 I'0 15
t(s) t(s)

D , 2, ~. 4 E , 2 , ,3. 4 F ~ .3.

° . . - -

•~ 0 5
m °o | ,._ After 0.6mM
•~ 0.5raM Lidocoine | QX-572 ut in 0.2

% ~ ,'o ','5 #o '6 6 2'o 3'0 4'o io o 20 40 ~o so ,oo

tls) t(s) t(s)

FIGURE 6. Modulation of anesthetic block by repetitive stimulation with four

different voltage clamp waveforms. Holding potential - 9 0 mV. Stimulus interval
of 200 ms except in (F) where the interval was 300 ms. (A) shows the four pulse
waveforms composed of a 50-ms prepulse to - 9 0 or -135 mV, a 1-ms test pulse to
- 3 0 mV, and a 5-ms post pulse to -90, -30, or +60 mV. The fibers were resting
until 0 s and then waveform 1 was started. Other waveforms were applied during
the periods indicated by a bar. (B), (D), and (E) are done with the same fiber. (E)
was measured after a solution containing 0.6 mM QX-572 had been applied
externally for 3 min and then briefly washed off. (F) was measured 40 min after
cutting the current-pool internode in 0.2 mM RAD 250.

T h e drifts o f p e a k INa for each pulse w a v e f o r m are qualitatively similar with

lidocaine, QX-572, a n d RAD 250 I, but the rates o f r e a c h i n g the final value are
obviously different. For e x a m p l e , in Fig. 6, on switching to w a v e f o r m 2 f o u r
pulses suffice with lidocaine to get to the new level o f INa, while the 75 pulses
shown for QX-572 only just suffice a n d the 66 pulses for RAD 250 I are far f r o m
e n o u g h . T h e rate d i f f e r e n c e shows u p a n o t h e r way. At stimulation rates below 1
s -~ lidocaine- or p r o c a i n e - t r e a t e d fibers show no cumulative effect o f repetitive
stimulation ( C o u r t n e y , 1974, 1975; Hille et al., 1975) while fibers t r e a t e d with the
q u a t e r n a r y c o m p o u n d s QX-314 (Strichartz, 1973), QX-572, a n d RAD 250 1 show
cumulative effects even at 0.2 s -~.
Fig. 6 reveals a n o t h e r aspect o f s o d i u m inactivation with q u a t e r n a r y a n d
a m i n e molecules. As was already discussed, switching f r o m w a v e f o r m 1 to
HILLE ModulatedReceptorfor Local Anesthetics 505

waveform 2 reduces INa at once by permitting resting inactivation to remain and

then depresses INa f ur t he r by some cumulative effect o f repetitive stimulation.
Courtney (1974, 1975) found a similar effect with the amine anesthetic GEA 968.
He noted that the inactivation curve with GEA 968 is unchanged from the
control provided that the fiber starts from a constant level o f conditioning or a
long rest and provided that inactivation is calculated from the relative INa on the
first pulse after a change to a new prepulse level. On the other hand, he also
found that if the inactivation curve with GEA 968 is calculated from the steady
level o f peak current reached after a train of impulses at each prepulse level, the
curve appears shifted to the left by m o r e than 20 mV and has a much smaller
slope than the control. In these same terms the present results (Fig. 2 vs. Fig. 6)
may be described by saying that benzocaine gives its full shift on the first test
pulse and shows no cumulative effect of stimulation, lidocaine shows some shift
on the first pulse and a further shift on fast repetitive testing, and the quater-
nary molecules tested show almost no shift on the first pulse and a considerable
but slowly developing shift on repetitive testing.

TABLE I
BLOCKING PROPERTIES OF THE THREE CHARGE C L A S S E S OF LOCAL
ANESTHETICS AND ANALOGS
Molecular type

Property Quaternary Amine Neutral

Modulation by h o l d i n g potential No Yes No
Modulation by [Ca ++] No Yes -
h® shift on first pulse No Yes or N o Yes
F u r t h e r h® shift with m a n y pulses Yes Yes No
Block e n h a n c e d by large depolarizations Yes Yes No
Persistence time o f m e m o r y in seconds >20 0.4-12 <0.2

T h e various blocking properties described here for the three charge classes o f
local anesthetics are summarized in Table I.
DISCUSSION

T h e degree of block of Na channels measured in voltage clamp experiments

with local anesthetics varies over a wide range. T h e block depends on whether
the membrane is held at rest or is hyperpolarized electrically or pharmacologi-
cally for longer or shorter periods, and it depends on the frequency and type of
recent stimulation (Strichartz, 1973; Courtney, 1974, 1975; Hille et al., 1975;
Khodorov et al., 1974, 1976). These complexities create a problem of even
defining what should be meant by the potency of a local anesthetic drug in such
measurements and suggest that reported parameters like "half-blocking concen-
trations" will vary from laboratory to laboratory. As already pointed out by
Courtney (1974, 1975), frequency- and use-dependent inhibitions limit the maxi-
mum rate of firing or the minimum interspike interval of a nerve or muscle cell
and may play a role in specific antiarrhythmic and analgesic actions. T h e
remainder of the Discussion is devoted to developing a model to explain fre-
quency- and use-dependent inhibition.
506 THE JOURNAL OF GENERAL PHYSIOLOGY" VOLUME 6 9 ' 1977

Previous Work on Sodium Inactivation and Anesthetics

SLOW SODIUM INACTXVATION. Block of Na channels by local anesthetic
analogs and inactivation of channels by potential changes seem to be interacting
processes. Block changes inactivation and inactivation changes block. The early
literature reports a relief of block by hyperpolarization in myelinated fibers
treated with procaine, urethane, or pentothal (Takeuchi and Tasaki, 1942;
Posternak and Arnold, 1954; Schoepfle, 1957; Khodorov and Beljaev, 1964a;
Khodorov, 1974); however, without voltage clamp measurements these results
were difficult to interpret. The relief could have been due to the simple removal
of resting inactivation that happens even in normal axons rather than to the
reversal of some special drug-induced effect.
Subsequent voltage clamp work in Khodorov's laboratory in particular has
demonstrated that the changes of inactivation are actually profound. Khodorov
et al. (1974, 1976) described what they called slow sodium inactivation during
treatment with procaine or trimecaine. The extra inactivation was produced by
1.0 s depolarizations and could be removed only very slowly upon hyperpolariza-
tion. The recovery time constants rs were 200-900 ms at room temperature.
Similarly, with GEA 968 Courtney (1974, 1975) found an extra block of Na
channels produced by repetitive depolarization with short pulses which reversed
with time constants as long as 10 s (10°C) when the fiber was held at rest.
Khodorov et al. found that the steady-state voltage dependence of the extra
inactivation (described by their s~ parameter) is qualitatively similar to that of
ordinary inactivation (described by the h~ parameter) and that it is shifted
towards more positive potentials by adding extra Ca ++, Ni ++, or H + ions to the
external solution. Adding H + ions but not Ca ++ ions also appreciably lengthened
the time constant ~'s of recovery. Khodorov et al. envisioned the new inactivated
state as being reached from the normal inactivated state by a slow transition
involving the binding of an anesthetic molecule to an "inactivation gating parti-
cle" of the Na channel. This receptor causing slow sodium inactivation was
placed on the extracellular side of the membrane to account for the greater
potency of amine anesthetic agents applied externally than of those applied at
the cut end. However, the previous paper (Hille, 1977) shows that the potency
difference is only a consequence of the relative thickness of internal and external
unstirred layers, so the receptor for amine molecules could well be identical to
that for quaternary molecules which is accessible only from the inside.
ORDINARY INACTIVATION. Weidmann (1955) found a negative shift of the
inactivation curve in cardiac Purkinje fibers treated with cocaine. However,
similar shifts were not considered to be likely in axons after Taylor (1959) found
none in squid giant axons treated with procaine. Recently, however, Courtney
(1974, 1975) reported an apparent negative voltage shift in axons with local
anesthetics. As was already described, he found that inactivation is not shifted by
GEA 968 when measured from the first pulse at each conditioning level but then,
during trains of conditioning pulses combined with test pulses, a shift develops.
I have found a shift of the inactivation curve on the first 50-ms prepulse with
lidocaine, tetracaine, and benzocaine (Fig. 2, Table I) and further apparent
shifts after trains of pulses with both amine and quaternary drugs. In agreement
HILLE ModulatedReceptorfor Local Anesthetics 507

with my observations, Strichartz (1973) found no shift on the first test pulse with
quaternary analogs, but because his experiments did not include protocols like
that of Fig. 6, the need to postulate shifts with multiple pulses was not so evident.
He did, however, study the influence o f prepulses on the rate o f block or
unblock caused by depolarizing test pulses and found a prepulse dependence
resembling the normal h= curve for block and resembling a 15 mV shifted 1~
curve for unblock. Khodorov et al. (1974, 1976) report inactivation measure-
ments on procaine- and trimecaine-treated fibers, but as there was no compari-
son to normal fibers, it is not clear whether they find a shift. T h e i r "h~" and "s~"
curves might in fact be incomplete since the most negative pulses they show ( - 8 0
to - 9 0 mV) would not have removed all the inactivation seen in my work (Figs. 3
and 4).
CALCIUM AND ANESTHETIC BLOCK. T h e literature is confusing on the
question of whether Ca ++ ions alter the blocking effects of local anesthetics.
Several studies show recovery of excitability in single blocked vertebrate cells
upon elevation of the external divalent cation concentration (Aceves and
Machne, 1963; Khodorov and Beljaev, 1964b; Seeman et al., 1974). T h e drugs
tested include procaine, lidocaine, chlorpromazine, heptanol, and barbital, but
again without voltage clamp methods those results were not easily interpreted.
Four previous voltage clamp studies looked for relief o f procaine block by added
Ca ++ ions. In two studies with myelinated fibers (Khodorov et al., 1974, 1976;
~ r h e m and Frankenhaeuser, 1974) no variation of Psa was found on changing
the Ca ++ concentration in the range 0.74-20 raM. T h e nodes were held hyperpo-
larized in the range - 9 0 to - l l 0 mV between test pulses in an effort to avoid
complications from resting sodium inactivation. However, Khodorov et al.
(1974, 1976) did observe that Ca ++ ions shift the voltage dependence of their
d r u g - d e p e n d e n t slow blocking process, giving a relief of block at the normal
resting potential comparable to that shown in Fig. 4 A and D of this paper. In
summary, Ca ++ ions unblock Na channels o f drug-treated myelinated fibers held
at the resting potential but not of fibers that are already strongly hyperpolarized.
With squid giant axons held at - 7 0 mV, Narahashi et al. (1976) found no
recovery o f Na channel block upon raising the Ca ++ concentration from 10 to 100
mM but there was a small enhancement o f block on lowering the Ca ++ concen-
tration to 5 or 2 raM. By contrast, with lobster giant axons held at - 9 0 to -130
mV Blaustein and Goldman (1966) obtained a large decrease of block on raising
the Ca ++ from 50 to 183 mM. Blaustein and Goldman (1966) and Khodorov et al.
(1974) conclude that Ca ++ and procaine compete directly for an external site
important in gating, while/~rhem and Frankenhaeuser (1974) and Narahashi et
al. (1976) conclude that the actions o f Ca ++ and procaine are independent. In a
model described in the next section, I conclude that the binding sites for the two
agents are on opposite sides o f the membrane but that the anesthetic receptor is
strongly coupled to the sodium inactivation mechanism which is in turn sensitive
to the membrane potential and to external Ca ++ ions.
Modulated Receptor Model
A major question that led to the experiments described in this paper is whether
local anesthetics and their neutral and quaternary analogs act by common or by
508 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 69 • 1977

d i f f e r e n t mechanisms. T h e blocking properties s u m m a r i z e d in Table I for each

o f the charge classes are not identical, and for no criterion listed do the three
molecular types show exactly the same behavior. Nevertheless, a single hypothe-
sis building on those o f Strichartz (1973), C o u r t n e y (1974, 1975), and K h o d o r o v
et al. (1974, 1976) seems to reconcile most differences. As a first a p p r o x i m a t i o n ,
all inhibitory actions o f the drugs are attributed to binding to a single receptor.
Slow sodium inactivation, cumulative f r e q u e n c y - d e p e n d e n t block, and voltage-
d e p e n d e n t block are all seen as manifestations o f a single inhibitory mechanism.
T h e special features o f the model include that the r e c e p t o r has a voltage- and

Modulated Receptor Hypothesis

Normal R = Resting
states 0 = Open
I = Tnactivated
Modified -,,x-= Drug
states

FIGURE 7. Modulated receptor hypothesis represented as a kinetic scheme with

interconversions among normal states of the channel (R, O, and I) and drug-
modified states (R*, O*, and I*). All interconversions may in principle be voltage
dependent and the equilibria and rates for the R-O-I-R pathway may be different
from those for the R*-O*-I*-R* pathway. When this scheme is applied to local
anesthetics, the O* state is not electrically conducting, the R-R* and I-I* pathways
are accessible primarily to hydrophobic drug forms and the O-O* pathway is
accessible to hydrophilic as well as hydrophobic forms.

t i m e - d e p e n d e n t c o n f o r m a t i o n and that there may be several physical pathways

for the d r u g to reach the receptor.
THE CENERAL MODEL. A schematic diagram o f a quite general type o f
d r u g - r e c e p t o r hypotheses is given in Fig. 7. T h e normal Na channel is repre-
sented as making transitions between several states: resting, o p e n , and inacti-
vated. T h e exact n u m b e r o f normal states is certainly larger than three but the
three shown h e r e e m b o d y the i m p o r t a n t features n e e d e d for this discussion. In
principle each channel f o r m may react with a d r u g molecule (*) to give modified
forms which themselves can mark transitions between resting, o p e n , and inacti-
vated states. T h e s e transitions o f the modified channels may have rates and
equilibria d i f f e r e n t f r o m those for normal channels. Consequently, the reaction
o f d r u g with one f o r m may also have d i f f e r e n t rates and equilibria from the
HILLE ModulatedReceptorfor Local Anesthetics 509

reaction with another form. This last statement is equivalent to saying that the
receptor for drug changes state when the channel changes state and is suffi-
ciently general that it will be given the name "modulated receptor" model. The
concepts of modulated receptors and of allosteric sites seem closely related.
The first modulated receptor model for ionic channels was developed by
Armstrong (1966, 1974) to explain block of K channels by internal quaternary
ammonium ions. Such models were subsequently used to explain the Na chan-
nel-opening effects of DDT (Hille, 1968) and a scorpion venom (Cahalan, 1975)
and the Na channel-blocking effects of quaternary (Strichartz, 1973) and amine
(Courtney, 1974, 1975) local anesthetic analogs.
The action of local anesthetic analogs is now discussed in terms of Fig. 7 and
the modulated receptor model. Throughout the Discussion the conversion of a
normal form of the channel (R, O, or I) into a modified form (R*, O*, or I*) is
called the drug-receptor reaction. As a general principle, voltage- or frequency-
dependent inhibition will arise in the scheme of Fig. 7 only if at least one of the
drug-receptor reactions has rate constants that can be varied by stimulation or if
the voltage dependence of some of the modified R*-O*-I* transitions differs
from that of the normal R-O-I transitions. Examples of both cases are given
below. The R*, O*, and I* states are all considered to be nonconducting, drug-
blocked states.
MODELS OF STRICHARTZAND COURTNEY. In a normal (untreated) fiber at
rest, roughly 60% of the Na channels are in state R and the remainder in state I.
If the membrane is then depolarized, most of the channels in state R proceed to
the transient open state O and then inactivate to state I. Strichartz (1973) found
that Na channels are most quickly blocked by internally applied quaternary QX-
314 when the channels are cycled through transient state O by repetitive depo-
larizing pulses. Presumably, axoplasmic drug has access to the receptor only
when the channel is open. In addition, channels recover most quickly from block
when repetitive pulses are applied that are thought to cycle them through
transient state O*. Without such pulses recovery is much slower, taking minutes.
Hence with quaternary anesthetic analogs, the fast rate constants for the drug-
receptor reaction are associated with open channels reacting via the O-O*
pathways, while the reaction with closed channels (I-I* and R-R* pathways) has
comparatively slow or possibly negligible rate constants. According to Strichartz
the entire modulation of block by stimulation (as in Fig. 6 F with quaternary
RAD 250 I) is attributed to voltage dependence of the O-O* and O*-O reactions
thought to arise from the electrostatic potential energy changes of the cationic
drug within the electric potential profile of the membrane. This voltage depend-
ence is seen in Fig. 6 F as the sizable extra steady-state block obtained with pulse
waveform 3 over that with waveform 4. Strichartz did not realize that in addition
the inactivation mechanism in blocked channels is modified, meaning that the
R*-O*-I* transitions of blocked channels are changed from the corresponding
transitions of unblocked channels.
Courtney (1974, 1975) studied the exceptionally hydrophilic amine lidocaine
analog GEA 968. As with Strichartz's work on QX-314, the fastest drug-receptor
reactions of GEA 968 required depolarizing pulses that open channels and favor
510 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69 • 1977

the O - O * pathway. H o w e v e r , C o u r t n e y also f o u n d several differences. With

GEA 968 the blocked f o r m s recover at the resting potential with a time constant
n e a r 10 s, so pulses need to be applied at rates higher than 0.1 s -1 for cumulative
block to be studied. In t e r m s o f Fig. 7, the recovery implies that the channel
need not be o p e n for reaction to occur, i.e. the R*-R or I*-I rate constants are
appreciable. Also, with GEA 968 the O-O* reaction is not strongly voltage
d e p e n d e n t . Finally, C o u r t n e y concluded that the h= curve o f m o d i f i e d (blocked)
channels is shifted to m o r e negative potentials, m a k i n g the I* state relatively
m o r e stable than the I state. T h e n , unless s t r o n g h y p e r p o l a r i z i n g prepulses are
given, Na channels eventually b e c o m e t r a p p e d in state I* d u r i n g repetitive
stimulation.
T h e changes o f inactivation might be physically i n t e r p r e t e d as in Fig. 8. T h e

membrane phase membrane phase

/ W) ;,.
m-gatin ~
subunit \\ )
la

h-gate open h-gate closed

FIGURE 8. Diagram of a local anesthetic molecule binding in the pore of an Na
channel in a manner that promotes Na inactivation. The molecule can reach its
binding site from the intracellular solution if activation and inactivation gates are
both open. It can also reach the site from the membrane phase even if one or both
of the gates are closed. The binding site has an important hydrophobic component
(shading) and closure of the inactivation gate enhances the hydrophobic interac-
tion.

anesthetic r e c e p t o r is in the channel n e a r the inactivation gating subunit. W h e n

the h gate is o p e n , b i n d i n g to the r e c e p t o r is not very firm, but when the gate is
closed the r e c e p t o r is m o d i f i e d a n d the b i n d i n g is stronger. This m u t u a l interde-
p e n d e n c e o f d r u g b i n d i n g a n d sodium inactivation is equivalent to saying that
anesthetic binding increases the probability o f the inactivated state I*. In the
d r a w i n g the surface area o f d r u g - r e c e p t o r interactions is shown to increase as
the channel inactivates. T h e interactions m u s t include an i m p o r t a n t h y d r o p h o -
bic c o m p o n e n t r e q u i r e d to account for the O v e r t o n - M e y e r rule plus, very
probably, some polar c o m p o n e n t . H o w e v e r , since p e r m a n e n t l y cationic and
p e r m a n e n t l y neutral d r u g molecules both appreciably shift the inactivation
curve, neither the d r u g - r e c e p t o r reaction n o r the extra interaction u p o n inacti-
vation has an i m p o r t a n t c h a r g e - c h a r g e c o m p o n e n t .
HILLE Modulated Receptorfor Local Anesthetics 511

RECONCILING THE ACTIONS OF THREE CHARGE TYPES. My observations

seem best explained by s u p p l e m e n t i n g the ideas o f Strichartz and C o u r t n e y with
the concept that while hydrophilic molecules in their charged f o r m are obliged
to bind and u n b i n d f r o m o p e n channels (O-O* pathway), neutral molecules o r
h y d r o p h o b i c cations may also use the R-R* or I-I* pathways. T h e m o r e h y d r o -
phobic the molecule, the m o r e rapid are the transitions in these alternative
recovery pathways and the s h o r t e r is the persistence o f the influence o f previous
stimulation. A kinetic diagram o f this concept is shown in Fig. 9. Lipid-soluble
molecules are shown e x c h a n g i n g between the i n n e r and o u t e r solutions and the
h y d r o p h o b i c m e m b r a n e . Since they are amphipathic, d r u g molecules accumu-
late preferentially at the two boundaries o f the n o n p o l a r m e m b r a n e phase and
the polar m e d i u m (e.g. McLaughlin, 1975). T h e d r u g has access to the r e c e p t o r
both f r o m the m e m b r a n e pool and f r o m the internal solution. T h e dashed

Basic Amine Neutral or Quaternary

' Bmo-- BmiJ Nm° ?Nmi ~1~

B/ ~N ........ Ni
H*4 o
+ + + + +
BH~ BHr. . . . "-BHi Qo Qr ....... Qi
Out Membrone I In Out Membrane In
FIGURE 9. Diagram of the movements of local anesthetic molecules through the
membrane phase and to and from the receptor. Dashed lines are transitions
requiring open gates in the Na channel. Solid lines are transitions possible any time.
Charged forms are drawn as unable to cross the membrane. Amine molecules can
protonate or deprotonate while in the channel or in the aqueous medium. Meaning
of subscripts: 0, outside medium; mo, outer surface of membrane; r, receptor; mi,
inner surface of membrane; i, inside medium.

pathway is the direct r o u t e for axoplasmic anesthetic to pass by the open gates
and t h r o u g h the channel to the receptor. It will be called the hydrophilic
pathway to distinguish it f r o m the h y d r o p h o b i c pathways which bypass the gates
by diffusion t h r o u g h the m e m b r a n e . T h e dashed hydrophilic pathway is closed
when gating closes the channel and is the only route for q u a t e r n a r y c o m p o u n d s .
T h e h y d r o p h o b i c pathways are available at all times to lipid-soluble molecules.
T h e time constants for recovery f r o m d r u g - i n d u c e d slow sodium inactivation
show strong parallels with the half-block times for s u d d e n application o f d r u g
described in the previous p a p e r (Hille, 1977), probably because both processes
require diffusion o f the d r u g t h r o u g h part o f the m e m b r a n e . According to this
hypothesis different time constants for removal o f slow sodium inactivation
reflect differing rates o f d e p a r t u r e o f d r u g molecules via the h y d r o p h o b i c
pathway d u r i n g the maintained hyperpolarization. A p e r m a n e n t l y neutral mol-
ecule such as benzocaine can leave in times well u n d e r 50 ms so that one finds
512 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69 • 1977

only a shifted inactivation curve but no long time constants o f inactivation.

However, amine molecules are in the lipid-diffusible neutral f o r m for only a
small fraction o f the time at neutral p H , so they leave less quickly than benzo-
caine and show slower recovery d u r i n g hyperpolarization. I n d e e d , K h o d o r o v et
al. (1976) showed that ~'s, the time constant o f recovery f r o m slow inactivation, is
l e n g t h e n e d f u r t h e r by acidifying the external m e d i u m , as expected if the d r u g
cation f o r m e d in acid media does not escape t h r o u g h the h y d r o p h o b i c pathway.
Again, the p H effects on rate o f recovery f r o m inactivation correlate well with
the p H effects on half-times o f block in the previous p a p e r (Hille, 1977).
Probably, the pK a o f the d r u g , which determines the lifetime o f the p r o t o n a t e d
f o r m , and the difference between the pKa and the local p H , which determines
the average fraction o f molecules in the p r o t o n a t e d form, will both be i m p o r t a n t
d e t e r m i n a n t s o f the time constants 7s. Finally, q u a t e r n a r y molecules show no
recovery with a 1-min hyperpolarization presumably because the p e r m a n e n t
cation is t r a p p e d in the channel by closed activation gates. T h e same arguments
explain why a relief o f block by high calcium occurs with amine but not with
q u a t e r n a r y molecules. Namely, when external Ca ++ ions relieve some of the
resting inactivation t h r o u g h their effect on the electric field within the mem-
brane ( F r a n k e n h a e u s e r and Hodgkin, 1957; Hille et al., 1975), amine molecules
on the r e c e p t o r can escape gradually via the R*-R pathway while q u a t e r n a r y
molecules cannot and remain t r a p p e d b e h i n d closed activation gates.
All o f the c o m p o u n d s tested give a p p a r e n t shifts o f the inactivation curve
(Table I). With the more h y d r o p h o b i c molecules the shift is evident on the first
test pulse, while with the hydrophilic molecules the shift requires several pulses.
T h e n u m b e r o f pulses r e q u i r e d to obtain the shift increases in the sequence:
benzocaine < lidocaine < GEA 968 < QX-572 < RAD 250 I. T h e s e observations
seem well explained by Courtney's idea that blocked channels have their h~
curve shifted by the b o u n d d r u g , while the channels without d r u g are normal.
T h e more h y d r o p h o b i c molecules reveal the shift on the first prepulse since with
them the blocked channels in the I* state can be restored to the conducting pool
rapidly via the h y d r o p h o b i c pathway. With benzocaine, for example, d u r i n g a
single 50-ms hyperpolarizing prepulse, all the channels in state I* might revert
first to R* and then lose d r u g fast e n o u g h to make R. With more hydrophilic
drugs, however, less R is f o r m e d from R* in 50 ms o f hyperpolarization because
they move less well in the h y d r o p h o b i c pathway and must wait for the conversion
o f R* to O* and thence partly to O d u r i n g each depolarizing test pulse.
T h e r e f o r e , the channels o p e n i n g after a single short prepulse are mostly those
that were unaffected by d r u g in the first place.
T o summarize, then, in my view there is a single specific r e c e p t o r in the Na
channel for all local anesthetics. Occupancy per se may be equated with block o f
the channel, and occupancy also has a p r o f o u n d influence on sodium inactiva-
tion. T h e d r u g - r e c e p t o r interactions change as the channel changes a m o n g its
various voltage-dependent states, and the physical pathways used for the drug-
r e c e p t o r reaction change as well. While an axon is being stimulated, the d r u g
never reaches equilibrium with the r e c e p t o r but is constantly being driven on
and o f f as Na channels are cycled t h r o u g h their changes o f state. In this
HXLLE Modulated Receptor for Local Anesthetics 513

hypothesis the a p p a r e n t differences in the actions o f neutral, a m i n e , a n d quater-

n a r y molecules are largely explained by the differences in the rates o f various
allowed on and o f f reactions. T h e recovery time constant % d e f i n e d by K h o d o -
rov et al. (1974, 1976) would then describe the time r e q u i r e d for a sequence o f
events including reversal o f the inactivation o f blocked channels at the holding
potential and d e p a r t u r e o f the d r u g via the h y d r o p h o b i c pathway into the
m e m b r a n e phase. T o the old question o f inside vs. outside action, the answer is
within the m e m b r a n e , a n d to the old question o f cation vs. free base the answer
is both, but with d i f f e r e n t pathways a n d hence with d i f f e r e n t p h e n o m e n o l o g y .
T h e idea p r o p o s e d h e r e o f a m o d u l a t e d r e c e p t o r with alternate h y d r o p h o b i c
and hydrophilic pathways is p r o b a b l y applicable to o t h e r cases. T h e rate-de-
p e n d e n t action a n d the inactivation shift with various a n t i a r r h y t h m i c d r u g s
applied to the m y o c a r d i u m ( W e i d m a n n , 1955; a n d r e f e r e n c e s in Gettes a n d
Reuter, 1974) are so similar to the actions o f local anesthetics on Na channels o f
n e r v e that the u n d e r l y i n g m e c h a n i s m s may be very similar. It would t h e r e f o r e
be interesting to study the d r u g - t r e a t e d m y o c a r d i u m again by using d i f f e r e n t
repetitive w a v e f o r m s as in Fig. 6 a n d c o m p a r i n g d r u g s o f d i f f e r e n t lipid solubil-
ity a n d pKa in o r d e r to test if the m e m o r y o f previous stimulation is again related
to the difficulty o f escape t h r o u g h the m e m b r a n e phase. A n o t h e r e x a m p l e o f a
d r u g - r e c e p t o r interaction varying with the state o f a channel a n d p r o b a b l y
having a hydrophilic a n d h y d r o p h o b i c reaction pathway is the block o f K
channels by the a m i n o p y r i d i n e isomers (Meves a n d Pichon, 1975; Yeh et al.,
1976a, b). T h e analogies are extensive. Repetitive or l o n g depolarization m o d u -
lates the blocking effect with time constants which are longer for c o m p o u n d s o f
higher pKa a n d for d r u g solutions o f low p H . T h e lipid-soluble molecules may
be applied f r o m the outside, but are quite possibly acting f r o m the insde or f r o m
the m e m b r a n e itself. T h e m a j o r difference is that a m i n o p y r i d i n e s bind m o r e
firmly to resting K channels than to activated ones, but the analogy is n o n e t h e -
less striking.

Much of the writing of these papers was done while I was visiting the I Institute of Physiology,
University of Saarland, Homburg, Germany, as an awardee of the Alexander von Humboldt
Foundation. I thank Dr. Robert St/impfli and his staff for their help and hospitality during that visit.
I am grateful to Drs. W. Schwarz and P. T. Palade for commenting on the manuscripts, and to Mrs.
R. Stolz and Miss R. Jackson for typing.
This research was supported by grants NS 08174 and FR 00374 from the National Institutes of
Health.
Received for publication 1 October 1976.

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