Published December 1, 1977
Two Levels of Resting Potential
in Cardiac Purkinje Fibers
D A V I D C. G A D S B Y and P A U L F. C R A N E F I E L D
From The Rockefeller University, New York 10021
INTRODUCTION
It is well k n o w n that as the e x t e r n a l potassium c o n c e n t r a t i o n , [K]0, is raised
above a b o u t 10 m M , the resting potentials o f both skeletal a n d cardiac muscle
fibers fall a p p r o x i m a t e l y as p r e d i c t e d by the N e r n s t relation for a potassium
electrode. As [K]0 is gradually r e d u c e d below this level, however, the resting
potential, Vr, b e c o m e s progressively less negative than the potassium equilibr i u m potential because o f a small inward c u r r e n t , usually carried by s o d i u m
ions, a n d is b e t t e r a p p r o x i m a t e d by the G o l d m a n (1943), H o d g k i n a n d Katz
(1949) equation (Adrian, 1956; W e i d m a n n , 1956). A c c o r d i n g to that equation,
Vr will monotonically increase to a m a x i m u m (negative) value as [K]0 a p p r o a c h e s
zero. H o w e v e r , the resting potentials o f cardiac Purkinje fibers ( W e i d m a n n ,
1956) a n d frog skeletal muscle fibers ( A k i y a m a a n d G r u n d f e s t , 1971) may fall to
a b o u t - 5 0 mV w h e n potassium ions are o m i t t e d f r o m the external solution.
This b e h a v i o r is not p r e d i c t e d by the G o l d m a n equation, a l t h o u g h it m a y be
a c c o u n t e d for if, for e x a m p l e , the potassium permeability is a s s u m e d not to
r e m a i n constant, b u t to decline at low values o f [K]0.
THE JOURNAL OF GENERAL PHYSIOLOGY
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VOLUME 70, 1977 " p a g e s 7 2 5 - 7 4 6
725
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A B S T R A C T In an appropriate ionic environment, the resting potential Of canine
cardiac Purkinje fibers may have either of two values. By changing the external K
concentration, [K]0, in small steps, it was shown that, in the low (1 mM) CI, Nacontaining solutions used in this study, the two levels of resting potential could be
obtained only within a narrow range of [K]0 values; that range was usually found
between 1 and 4 mM. Within the critical [K]0 range the resting potential could be
shifted from either level to the other by the application of small current pulses. It
was shown that under these conditions the steady-state current-voltage relationship was "N-shaped," and that a region of both negative slope, and negative chord,
conductance lay between the two stable zero-current potentials. The negative chord
conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord
conductance could be abolished by several experimental interventions and the
membrane potential thereby shifted from the lower to the higher resting level:
those interventions which were effective by presumably diminishing the steadystate inward current included reducing the external sodium concentration, adding
T T X , or adding lidocaine; those which presumably increased the steady-state
outward current included small increases in [K]0, brief depolarizations to around
- 2 0 mV, or the addition of acetylcholine chloride.
Published December 1, 1977
726
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 7 0 " 1 9 7 7
MATERIALS
AND
METHODS
Small bundles of Purkinje fibers, usually 100-300 tzm in diameter and 2-5 mm long,
dissected from the right ventricles of dog hearts, were used in these experiments. The
bundles were suspended between two fine entomological pins in the narrow channel of
a flow chamber similar to that designed by Hodgkin and Horowicz (1959). The rapid
perfusion system described here was initially developed for use with skeletal muscle
fibers (Gadsby et al., 1977). The pins were passed through connective tissue at each end
of the bundle and pushed into the Sylgard (no. 184, Dow Corning Corp., Midland,
Mich.) floor of the channel. The preparation was positioned in midstream so that it was
surrounded by flowing solution except where it rested lightly on two 100-/zm diam,
stainless steel wires which afforded support during the insertion of microelectrodes.
The chamber was connected by a short section of silicone rubber tubing to one output
of a two-position valve (no. 86410, Hamilton Company, Reno, Nev.). This valve had
two inputs and two outputs; the second output served as a drain for the removal of
stagnant fluid from an input line immediately before switching that line to the chamber.
The Teflon rotor of the valve was modified to permit last switching between the two
inputs to occur without any concomitant change in the rate of perfusion through the
chamber. This minimized the mechanical disturbance associated with rapid solution
changes and thus greatly facilitated continuous intracellular recording. Each input to
this "final" valve was supplied by the output from a four-position distribution valve (no.
86414, Hamilton). Each of these valves had four inputs and one output, and was fitted
with spring-loaded stops, allowing any one of several test solutions to be quickly selected
for switching to the experimental chamber. Flow-limiting bypasses in the fluid supply
lines to the distribution valves allowed selection of two different flow rates: perfusion
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A n o t h e r experimental finding which is not predicted by the G o l d m a n
equation, and which is probably related to the depolarization seen when [K]o is
lowered, is the possible existence o f two levels o f resting potential at a single
fixed value o f [K]o (see Wiggins a n d Cranefield, 1976). T h u s it is k n o w n that,
partly as a result o f inward-going rectification, the steady-state m e m b r a n e
current-voltage relationship in these fibers is " N - s h a p e d " (Adrian and Freygang,
1962; Dudel et al., 1967b) and may t h e r e f o r e , u n d e r certain conditions, intersect
the voltage axis at three points. T w o o f these z e r o - c u r r e n t points may then be
possible resting potentials, between which the m e m b r a n e potential may be
shifted by the application o f c u r r e n t pulses o f a p p r o p r i a t e m a g n i t u d e and
polarity.
T h e aims o f the present study were to d e t e r m i n e changes in resting potential
following small changes in [K]o over a wide concentration r a n g e in o r d e r
to define the experimental conditions necessary for the d e m o n s t r a t i o n of two
levels o f resting potential, and to investigate the n a t u r e o f the steady-state,
" b a c k g r o u n d " inward c u r r e n t responsible for the depolarization at low [K]o.
For this p u r p o s e , very thin u n b r a n c h e d bundles of Purkinje fibers were
perfused in a modified Hodgkin-Horowicz (1959) fast-flow system with solutions
in which c h l o r i d e ions were usually replaced by the l a r g e r isethionate a n d
methylsulfate ions. T h e major advantages o f this p r o c e d u r e are that: [K]o may
be c h a n g e d rapidly and, therefore, i n d e p e n d e n t l y o f the intracellular potassium
concentration; complications arising f r o m cellular chloride m o v e m e n t s are
absent; and p a c e m a k e r activity is, apparently, largely abolished.
Published December 1, 1977
GADSBY AND CRANErlELD Two Levels of Resting Potential
727
RESULTS
The Dependence of the Resting Potential on [K]o
Fig. 1 s h o w s t y p i c a l r e s p o n s e s o f t h e m e m b r a n e p o t e n t i a l o f a f i b e r in a t h i n
b u n d l e to s m a l l s t e p c h a n g e s in [K]o o v e r t h e r a n g e 0 - 4 m M in l o w - C l s o l u t i o n s .
A t t h e b e g i n n i n g o f t h e s w e e p [K]o was 4 m M a n d t h e r e s t i n g p o t e n t i a l was
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was maintained at the lower rate o f 5 ml/min but the higher rate of 20 ml/min could be
used when fast solution changes were required. Fluid changes near the middle o f the
channel occurred with a half-time of less than 50 ms at the higher flow rate, and were
about 10 times slower at the lower rate. This was measured by switching between C1containing and Cl-free solutions while recording the time course of establishment of the
liquid junction potential between two broken microelectrodes, one filled with agarTyrode's, and the other with 3 M KCI, positioned in midstream with their tips in close
proximity.
Conventional glass microelectrodes filled with 3 M KC1 (resistances, 15-30 Mfl; tip
potentials less negative than - 5 mV) were used for potential recording; similar microelectrodes were filled with 2-3 M K-citrate for current injection. T h e reference half-cell
incorporated a sintered Ag/AgCl/Pt-black pellet (Annex Research) and was connected to
the chamber, downstream from the preparation, via a flowing 3 M KCI junction. This
electrode was placed alongside the suction tube which continuously removed perfusate
from the chamber. Also adjacent to this tube was a 3 M KCl-agar bridge containing a
Ag/AgCI electrode; the latter was attached to an operational amplifier which held the
chamber at virtual g r o u n d and served to monitor applied currents. A constant-current
generating circuit delivered current pulses o f variable waveform: e.g., rectangular, and
either rising or falling linear ramps. In some experiments (see, e.g., Fig. 4) currents
which increased linearly with time to a m a x i m u m and then declined linearly at the same
rate were used; these will be r e f e r r e d to as "double ramps." In addition to p h o t o g r a p h i n g
oscilloscope traces, records were made with a rectilinear pen r e c o r d e r (Gilson Medical
Electronics, Middleton, Wis.; frequency response, 75 Hz).
T h e Tyrode's solution with which the preparations were equilibrated d u r i n g dissection
and mounting in the chamber contained (mM): NaCI, 137; KCI, 4; NaHCOs, 12;
NaH2PO4, 1.8; MgCI2, 0.5; CaC12, 2.7; dextrose, 5.5. This solution was bubbled with 95%
02-5% CO2. T h e virtually Cl-free solution contained (mM): Na-isethionate (Koch-Light,
Colnbrook, Bucks., England), 146; K-methylsulfate (Hopkin & Williams, Chadwell
Heath, Essex, England), 4; MgCI,, 0.5; Ca-methanesulfonate (made with methanesulfonic acid; Eastman Kodak Corp., Rochester, N. Y.), 2.7; dextrose, 5.5; HEPES (N-2hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid; Sigma Chemical Co., St. Louis, Mo.;
adjusted to p H 7.3), 5. This solution was bubbled with pure oxygen. To alter the K
concentration, Na-isethionate was replaced with K-methylsulfate, or vice versa. T h e Na
concentration was sometimes lowered, in CI-containing solution buffered with 5 mM
Tris or HEPES, by substitution with Tris + (Tris [hydroxymethyl] aminomethane; Sigma).
T h e following drugs, when used, were a d d e d from refrigerated, concentrated stock
solutions: tetrodotoxin ( T T X , Sigma); acetylcholine chloride (Sigma); atropine sulfate
(Amend, Irvington, N. J.); lidocaine hydrochloride m o n o h y d r a t e (xylocaine; Astra
Pharmaceuticals, Worcester, Mass.).
T h e t e m p e r a t u r e o f the solutions was monitored close to the preparation by means o f
a small thermistor bead (no. 32A 130, VECO, Springfield, N.J.) and was kept between
35 ° and 37°C.
Published December 1, 1977
728
THE JOURNAL OF GENERAL P H Y S I O L O G Y ' V O L U M E 70
"1977
4
k"jrvlo [raM) ~
2
L
1
iO~
1
i
4
2 F
(mv)
_100 L.
,-~lOs
FIGURE 1. Membrane potential changes in response to small step changes in [K]0
as indicated by the upper line. The first 10 s of the trace includes the start of a
second sweep. Low CI solutions; preparation AI2-1.
T h e relationship between the resting potential a n d log [K]o o b t a i n e d o v e r a
m o r e extensive concentration r a n g e in a n o t h e r fiber is shown in Fig. 2. Each
point r e p r e s e n t s a single m e a s u r e m e n t . A given i m p a l e m e n t was m a i n t a i n e d
t h r o u g h o u t several solution changes but the electrode was periodically withd r a w n to m o n i t o r possible changes in its tip potential. A f t e r each c h a n g e in
[K]o at least 30 s were allowed for equilibration in the extracellular spaces; the
steady potential was t h e n n o t e d b e f o r e switching to a d i f f e r e n t [K]o. It can be
seen that the m e m b r a n e potential increased f r o m - 0 to - 1 0 0 m V as [K]o was
r e d u c e d o v e r the r a n g e 150-2 m M . At zero [K]o, the m e m b r a n e potential was
n e a r - 3 0 m V . At both 1 a n d 2 m M [K]o, however, two levels o f resting
potential, differing by a b o u t 60 m V , could be r e c o r d e d .
T h e b r o k e n line shows EK as a . f u n c t i o n o f log [K]o a n d was d e t e r m i n e d by
means o f the N e r n s t equation, taking the intracellular potassium c o n c e n t r a t i o n ,
[K]l, to be 155 mM and the t e m p e r a t u r e to be 36.5°C; its slope is 61.5 m V per
10-fold c h a n g e in [K]o. T h e m e a s u r e d potentials a p p r o a c h this line w h e n [K]o
exceeds 8 raM. T h e continuous line shows the resting potential, Vr, calculated
by using the G o l d m a n , H o d g k i n , Katz equation,
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steady at - 9 0 m V . T h e m e m b r a n e h y p e r p o l a r i z e d when [K]o was r e d u c e d to 2
m M but f u r t h e r r e d u c t i o n , to 1 m M , was followed by an a b r u p t depolarization
which gave rise, at a b o u t - 6 0 m V , to an action potential. After the u p s t r o k e o f
that action potential the m e m b r a n e repolarized only to a b o u t - 3 5 inV. O n
switching to K-free solution a f u r t h e r depolarization was seen which was -readily
reversed on r e t u r n i n g to 1 m M [K]o. W h e n [K]o was raised to 2 m M , h o w e v e r ,
the m e m b r a n e potential did not r e t u r n to the level previously seen in that
solution (close to - 1 0 0 mV) but increased by only a few millivolts. I n contrast,
the subsequent c h a n g e in [K]o f r o m 2 mM to 4 mM was followed by a large,
a b r u p t increase in m e m b r a n e potential a n d , within a few seconds, the resting
potential was again steady at - 9 0 mV; this can be seen f r o m the thickening o f
the initial 10 s o f the potential trace which includes the start o f a second
sweep. T h e resting potential o f this fiber t h e r e f o r e could have one o f two
values, 55 mV a p a r t , at a [K]o o f 2 raM, d e p e n d i n g on w h e t h e r that concentration was r e a c h e d via 4 mM or 1 m M .
Published December 1, 1977
GADSBYAND CRANEFIELD Two Levels of Resting Potential
Vr = ? l n [ K ] o
729
+ ~[Na]o
[K], + o,,[Na],'
in which R, T, and F have their usual meanings, the sum o f the external
concentrations o f potassium a n d sodium, [K]o + [Na]o, was taken to be 150
mM, and the intracellular concentrations o f potassium and sodium, [K]l and
[Na]l, were assumed to be 155 and 20 mM, respectively. T h e constant, a,
represents the ratio o f the constant-field permeability coefficients for sodium
and potassium, PNa/PK, and was taken to be 0.01 (cf. H o d g k i n and Horowicz,
1959). This curve provides a better fit to the e x p e r i m e n t a l points over a wider
Vm (mY)
0
-40
/
J
-60
-80
$,J~ /
-100
-120
J
-----4
,
0
//
ii/i
/
i
1
i
2
L
4
[K] o
,
8
i
l
I
16 32 64
I
150
(mM)
FIGURE 2. Relation between membrane potential and log [K]0 in low CI solutions.
Results from five impalements (each represented by a different symbol) in a single
bundle of Purkinje fibers. The microelectrode remained in the cell during several
solution changes. Each point represents a single steady potential measurement.
Open circles have been corrected for a - 5 mV change in tip potential which
occurred during that impalement. Preparation A12-9.
[K]o range than the Nernst relation but clearly cannot, if 0t is constant,
simultaneously fit both the h i g h e r a n d the lower levels o f m e m b r a n e potential
r e c o r d e d at 1 mM and 2 mM [K]o.
Two Levels of Resting Potential and the Steady-State Current-Voltage Relationship
As shown in Fig. 1, a small change in [K]o may be sufficient to shift the resting
potential f r o m the h i g h e r to the lower level, or vice versa, but this change must
be to a concentration outside the limited range o f [K]o within which both levels
may be f o u n d . In the e x p e r i m e n t o f Fig. 1, for example, it was necessary to
depolarize the fiber in 1 mM [K]o b e f o r e the lower o f the two levels o f resting
potential could be obtained at 2 mM [K]o. T h e critical r a n g e o f [K]o over which
both potential levels could be r e c o r d e d varied between preparations. For
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./
-20
/
dD
Published December 1, 1977
730
THE
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example, two levels were seen at both 1 mM and 2 mM in the fiber of Fig. 2,
but only at 2 mM in that o f Fig. 1; more usually the r a n g e e x t e n d e d to 4 mM.
Within that critical range the existence o f two resting potentials at a single
value o f [K]o could be d e m o n s t r a t e d m o r e directly by applying small c u r r e n t
pulses o f a p p r o p r i a t e polarity via a second microelectrode inserted, within 100
p.m o f the r e c o r d i n g electrode, in the midregion o f the fiber.
Fig. 3 A shows the result o f injecting 15 nA c o n s t a n t - c u r r e n t pulses o f 1 s
duration into a fiber exposed to 4 mM [K]o whose resting potential was initially
- 4 9 mV. D u r i n g the first pulse the c u r r e n t was inward and the m e m b r a n e
potential increased to - 9 6 mV; the potential subsequently declined to - 9 1 mV
and r e m a i n e d at that level until the start o f the second pulse. This c u r r e n t was
A +20
0
i
)
-100
I-1
1 (~A)*I5E ---u
-15
B
-40f
Vm
I (nA)_30[ ~[
Z'-
ls
[
FIGURE 3. Switching between two stable resting potentials by means of small
rectangular current pulses. In both A and B the membrane potential is shown on
the upper trace and the applied current on the lower trace. In A the first 15-nA
current pulse was inward and the second, outward (preparation A7-28), The
inward current in B was 30 nA (preparation A7-15). Both preparations were
exposed to 4 mM K, low Cl solution.
outward, and it caused a regenerative depolarization which gave rise to an
action potential. T h e end o f the pulse o c c u r r e d d u r i n g the "plateau" o f this
action potential and was a c c o m p a n i e d by an a b r u p t repolarization of only 15
mV after which the m e m b r a n e potential very slowly r e t u r n e d to its initial level
o f - 4 9 mV. (Note that the upstroke o f the action potential was followed by a
m a r k e d '~notch" even t h o u g h the external solution contained only 1 mM C1.
This confirms recent findings in solutions in which large anions o t h e r than
isethionate were substituted for Cl ions [Kenyon and Gibbons, 1977].)
Fig. 3 B shows a similar response to a hyperpolarizing c u r r e n t pulse and was
r e c o r d e d , in a different fiber, at a faster sweep speed to illustrate the time
course o f the potential changes. T h e resting potential of this fiber was initially
- 4 8 mV. D u r i n g the c u r r e n t pulse the m e m b r a n e potential increased slowly
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\
Vm
(my)
Published December 1, 1977
GADSBYAND CRANEFIELD TWO Levels of Resting Potential
731
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and then m o r e rapidly, before reaching a steady level o f - 1 0 2 mV. Shortly
after the pulse, the potential was steady at the new resting level o f - 9 2 inV.
T h e ability o f the m e m b r a n e potential to show two resting levels, i.e, two
levels at which the net m e m b r a n e c u r r e n t is zero (Figs. 1-3), t o g e t h e r with the
characteristic, "regenerative," n a t u r e o f the time course o f the potential change
f r o m the lower to the h i g h e r o f these levels (Fig. 3 B) suggests that, u n d e r these
conditions, the steady-state current-voltage relationship crosses the voltage axis
with a positive slope at two points, between which it crosses a third time in a
region o f negative slope conductance. In o r d e r to verify this suggestion the
steady-state current-voltage characteristic was d e t e r m i n e d u n d e r conditions in
which two levels o f resting potential could be obtained. For this p u r p o s e , small,
slowly rising and falling, double ramps o f c u r r e n t (see Materials and Methods)
were applied via an intracellular microelectrode, while the resulting potential
changes were r e c o r d e d nearby with a second microelectrode. Typical results
f r o m an e x p e r i m e n t o f this kind are shown in Fig. 4. T h e m a x i m u m rate o f
change o f c u r r e n t in this e x p e r i m e n t was about 1.5 nA/s. Each o f the insets, a
and b, shows s u p e r i m p o s e d records o f the c u r r e n t and potential changes
d u r i n g two runs (1 and 2, inset a; 3 and 4, inset b) using currents o f opposite
polarity. T h e c u r r e n t was outward t h r o u g h o u t runs 1 and 4, and inward
t h r o u g h o u t runs 2 and 3. T h e current-voltage relationship was simultaneously
r e c o r d e d with an X-Y plotter and the g r a p h in Fig. 4 is a tracing o f the
resulting curves. T h e fiber was continuously s u p e r f u s e d with 4 mM K, low C1
solution; the resting potential was initially - 4 7 mV. D u r i n g the rising phase o f
the first double r a m p (Fig. 4a, 1; amplitude +30 hA) the m e m b r a n e slowly
depolarized to - 3 0 mV. D u r i n g the declining phase this change was reversed,
although the potential was some 2-3 mV m o r e negative at all currents than it
was d u r i n g the rising phase (Fig. 4, graph) and r e t u r n e d to - 4 7 mV only some
30 s after the e n d o f the r a m p . T h e second c u r r e n t pulse (Fig. 4a, 2; amplitude
- 7 5 nA) caused the potential to increase steadily to about - 5 5 mV, then
change abruptly to - 9 0 mV, and t h e r e a f t e r rise slowly to - 1 0 2 inV. As the
c u r r e n t r e t u r n e d to z e r o , - t h e potential slowly fell to - 8 8 mV a n d r e m a i n e d
steady at that level until the start o f the third pulse. D u r i n g this double r a m p
(Fig. 4b, 3; a m p l i t u d e - 3 0 nA) the potential increased to - 9 5 m V and t h e n
r e t u r n e d to - 8 8 inV. At the resting potential o f - 8 8 mV a pulse o f outward
c u r r e n t was applied (Fig. 4b, 4; amplitude +30 nA), initially causing the
potential to decline slowly until, at about - 8 0 mV, a m o r e rapid depolarization
gave rise to an action potential; the m e m b r a n e repolarized only to - 3 5 mV,
however, and t h e r e a f t e r the potential changes were similar to those observed
d u r i n g the previous pulse o f outward c u r r e n t (run 1). For simplicity these
changes have been omitted f r o m the graph. Shortly after the e n d o f the pulse
the resting potential was close to its initial value o f - 4 7 mV.
T h e shifts in the z e r o - c u r r e n t potential associated with pulses 2 and 4 are
directly comparable to those caused by the rectangular pulses in Fig. 3 A, but
additional i n f o r m a t i o n is p r o v i d e d by the steady-state current-voltage relationship shown in Fig. 4. A major point o f interest is the hysteresis between c u r r e n t
ramps 2 and 4. T h e hysteresis results f r o m the presence o f a region o f negative
Published December 1, 1977
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slope c o n d u c t a n c e a n d allows the extent o f that region to be defined, at least
a p p r o x i m a t e l y . I n the p r e s e n t e x a m p l e , the a b r u p t potential changes seen at
a b o u t - 1 0 a n d + 10 nA indicate that the negative slope extends f r o m a b o u t - 8 0
to - 6 0 inV. Since a p p r o p r i a t e steady-state m e a s u r e m e n t s cannot be m a d e in
this region by the p r e s e n t m e t h o d , the short b r o k e n line c o n n e c t i n g these
coordinates was a d d e d by h a n d in o r d e r to c o m p l e t e the e x p e r i m e n t a l l y
d e t e r m i n e d steady-state c u r r e n t - v o l t a g e relationship.
T h e net ionic c u r r e n t at any potential is the s u m o f inward a n d o u t w a r d
c o m p o n e n t s . Since the equilibrium potential for potassium ions is p r o b a b l y
I nA)
*30
a
1
-~
°I
~
-~o[
I (nA)
~
~
~40
Vm
/
,20
~ . ,,,4
i
(mY) ~ ~ ~ v - 4 ' O
'-2b
/i
.""
...'
'
0
~-20
I(.A)
*30E
-30 - ' - - ' ~
0
b
Y
-40
~
4
I
FIGURE 4. The steady-state current-voltage relationship obtained in 4 mM K,
low C1 solution (same preparation as in Fig. 3 A). Insets (a) and (b) show photographically superimposed chart recordings of double ramps of current (upper
traces) and resulting potential changes (lower traces), numbered sequentially. The
main graph is a tracing of the corresponding current-voltage relationship recorded
with an X-Y plotter. Ordinate, applied current; abscissa, membrane voltage. The
arrows indicate the direction of potential change. The numbers relate each limb
of the current-voltage curve to the appropriate double ramps, viz.: 1, outward
current, solid line; 2, inward current, dotted line; 3, inward current, dashed line;
4, outward current, solid line. The short broken line which crosses the voltage axis
near - 7 0 mV indicates the negative slope conductance region and was added by
hand to complete the experimental curve (see text).
close to - 1 0 0 m V u n d e r the p r e s e n t conditions (see Fig. 2, b r o k e n line), at
potentials positive to this, any K c u r r e n t m u s t be o u t w a r d . T h e net ionic
c u r r e n t at - 6 0 m V in Fig. 4, however, is inward; its m a g n i t u d e , a b o u t - 1 0 nA
in this instance, t.herefore provides a m i n i m u m estimate o f the inward c u r r e n t
at that potential. Since that c u r r e n t m u s t be carried by an ion with an
equilibrium potential positive to - 4 7 m V , the most likely candidates are Na +
a n d Ca ++ . E x p e r i m e n t s c o n c e r n i n g the n a t u r e o f that c u r r e n t are described
below.
The Nature of the Steady-State Inward Current
I f appreciable inward c u r r e n t is carried by sodium ions t h e n their r e p l a c e m e n t
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3o~
Published December 1, 1977
733
GADSBYAND CRANEFIELD Two Levels of Resting Potential
by the larger, less p e r m e a n t Tris ions should result in a r e d u c t i o n o f that
c u r r e n t a n d , consequently, in a h y p e r p o l a r i z a t i o n o f the m e m b r a n e . Fig. 5 A
illustrates the effect, on a fiber with an initially low resting potential, o f b r i e f
e x p o s u r e s to a solution in which Na ions were r e p l a c e d by Tris +. D u r i n g the
first e x p o s u r e , which lasted 2 s, the m e m b r a n e rapidly h y p e r p o l a r i z e d by ~40
mV a n d , a few seconds later, the potential was steady at - 9 0 m V . A m u c h
smaller, transient h y p e r p o l a r i z a t i o n o f - 2 m V a c c o m p a n i e d a s u b s e q u e n t 5-s
e x p o s u r e to N a - f r e e solution, a f t e r which a small transient depolarization
occurred.
A
[Na]o 146r
(mM) 0 L "~j
-40
II
-201
Vm
-
-100L
-
(mV)
10~-~
_100L
Q
lOs
i
C
[Na]o(rnM)
146
1 109,5
Vm -40[
(mV) _80L
12xlO-6g/rnl TTXI
lO~'-s
FIGURE 5. Effects of reducing [Na]0 and/or adding 2 x 10-e g/ml T T X on the
lower level of resting potential. In A, the downward deflections in the upper line
show when Na ions were briefly replaced by Tris+; the lower trace is a chart
recording of the membrane potential (4 mM K, Cl-containing solution, Tris
buffer; preparation A7-29). In B, the bar indicates the period of exposure to T T X
(4 mM K, low CI solution; preparation A7-16). In C, the upper line shows when
[Na]0 was reduced from 146 mM to 109.5 mM by partial replacement with Tris +.
The potential trace is a chart recording and the bar below it indicates the period
of exposure to T T X (4 mM K, Cl-containing solution, HEPES buffer, [Ca]o 0.5
mM; preparation A9-14).
T h e a b r u p t shift in resting potential in Na-free solution suggests that the net
inward c u r r e n t n e a r - 6 0 m V was abolished w h e n s o d i u m ions were o m i t t e d
f r o m the e x t e r n a l solution, t h e r e b y shifting u p w a r d s a c u r r e n t - v o l t a g e relationship like that illustrated in Fig. 4 until only a single z e r o - c u r r e n t intercept, n e a r
- 9 0 m V , r e m a i n e d . This result shows that at least s o m e o f the inward c u r r e n t
in the r a n g e - 6 0 to - 9 0 m V is c a r r i e d by s o d i u m ions. T h e m a i n t e n a n c e o f the
h i g h e r resting potential after r e a d m i s s i o n o f Na indicates that the steady-state
c u r r e n t - v o l t a g e relationship in that solution did i n d e e d have two stable zeroc u r r e n t potentials; the smaller potential changes resulting f r o m the s u b s e q u e n t
b r i e f e x p o s u r e to N a - f r e e solution are consistent with the m e m b r a n e conductance being g r e a t e r at the m o r e negative o f those z e r o - c u r r e n t potentials. T h e
e x p l a n a t i o n for the transient depolarization o n r e a d m i s s i o n o f Na ions is not
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(mV)
B
2xlO-6g/ml TTX
I ~
Published December 1, 1977
734
THE
JOURNAL
Or
GENERAL
PHYSIOLOGY
' VOLUME
70
• 1977
Downloaded from on June 18, 2017
k n o w n but it may reflect changes in intracellular ionic composition since its
m a g n i t u d e was f o u n d to increase with the duration o f the e x p o s u r e to Na-free
solution.
Since the t h r e s h o l d for activation o f the excitable "fast" sodium c h a n n e l is
n e a r - 6 0 m V , a steady inward c u r r e n t c o m p o n e n t could flow at potentials just
positive to this if some or all o f those channels u n d e r g o only i n c o m p l e t e
inactivation. It was t h e r e f o r e o f interest to investigate the effect o f a high
concentration o f t e t r o d o t o x i n ( T T X ) on the "lower" level o f resting potential.
As shown in Fig. 5 B, the application o f 2 × 10 -6 g/ml T T X for 15 s to a fiber
e x p o s e d to a 4 m M K, low CI solution, in which its resting potential was initially
- 3 6 m V , caused an i m m e d i a t e hyperpolarization; the potential increased by
almost 20 mV b e f o r e a b r u p t l y shifting to the "higher" resting level w h e r e it
r e m a i n e d after r e m o v a l o f the T T X . Reapplication o f T T X to this fiber 4 rain
later at the new resting potential o f - 8 7 mV caused no ft~rther change in that
potential (not illustrated). In t e r m s o f the a r g u m e n t s used above, this result
suggests that in this instance T T X t e m p o r a r i l y r e d u c e d the steady inward
c u r r e n t by an a m o u n t sufficient to abolish the m o r e positive zero c u r r e n t
intercepts.
Additional e x p e r i m e n t s were carried out to d e t e r m i n e w h e t h e r the effects o f
a reduction in [Na]0 a n d the addition o f T T X are equivalent or s u p p l e m e n t a r y .
An e x a m p l e is shown in Fig. 5 C. This fiber was equilibrated in 4 m M K, CIcontaining T y r o d e ' s solution; its resting potential was - 5 0 m V . T h e addition o f
T T X (2 × 10 -6 g/ml) caused the m e m b r a n e to h y p e r p o l a r i z e by 8 mV. T h e
fiber was then e x p o s e d to a solution in which 25% o f the s o d i u m ions had been
replaced by Tris ions; that solution contained no T T X , in spite o f which a
f u r t h e r 15-mV h y p e r p o l a r i z a t i o n was r e c o r d e d . In a subsequent r u n on the
same fiber, the c o r r e s p o n d i n g potential increases were 8.5 m V a n d 18 m V ,
respectively. T h e i m m e d i a t e f u r t h e r h y p e r p o l a r i z a t i o n seen in Fig. 5C on
r e d u c i n g [Na]o suggests that this intervention caused the decline o f an inward
c u r r e n t which h a d not b e e n blocked by 2 × 10 -6 g/ml T T X . H o w e v e r , a
f u r t h e r question concerns the relative m a g n i t u d e s o f these two effects. Since
the h y p e r p o l a r i z a t i o n in r e s p o n s e to r e d u c i n g [Na]o was initiated at a m o r e
negative m e m b r a n e potential t h a n that in response to a d d i n g T T X , it m i g h t be
a r g u e d that the l a r g e r m a g n i t u d e o f the f o r m e r effect results f r o m the
nonlinearity in this region o f the current-voltage relationship. It is unlikely that
the difference can be entirely a c c o u n t e d for in this way, however, since in two
control runs the 25% reduction in [Na]o by itself h y p e r p o l a r i z e d this fiber f r o m
the - 5 0 mV resting potential by 13 mV a n d 13.5 m V , respectively (not
illustrated).
A f u r t h e r possible c o m p o n e n t o f the steady-state inward c u r r e n t must be
c o n s i d e r e d since e x p e r i m e n t a l evidence suggesting i n c o m p l e t e inactivation of
the slow inward c u r r e n t has recently been obtained (Gibbons a n d Fozzard,
1975; Kass et al., 1976). T h e threshold for activation o f that c u r r e n t , however,
is t h o u g h t to be positive to - 4 0 mV (see, e.g., Reuter, 1973) a n d , for this
reason, its contribution to the steady-state inward c u r r e n t n e a r - 6 0 mV (Fig. 4)
m i g h t be e x p e c t e d to be negligible (but cf. McAllister et al., 1975). I n d e e d , in
fibers at the "lower" level o f resting potential in low chloride solutions, the
Published December 1, 1977
GADSBY AND CRANEFIELD
735
Two Levels of Resting Potential
e q u i m o l a r r e p l a c e m e n t o f Ca ++ by either Mn ++ or Mg ++, b o t h o f which are
p r o b a b l y relatively p o o r carriers o f slow inward c u r r e n t ( K o h l h a r d t et al., 1973;
Delahayes, 1975), caused only small a n d slow changes in m e m b r a n e potential,
Mn ++ t e n d i n g to h y p e r p o l a r i z e a n d Mg ++ to depolarize. Explanations based on
the ability o f these ions to carry slow inward c u r r e n t would s e e m to be
unnecessary since these small effects are consistent with the efficacy with which
these ions mimic the "stabilizing" influence o f calcium ions on cell m e m b r a n e s ,
viz., Mn ++ > Ca ++ > Mg ++ (see, e. g., J e n d e n a n d Reger, 1963).
A p p a r e n t l y , t h e n , in the p r e s e n c e o f n o r m a l [Na]o, the inward c u r r e n t which
helps to maintain the lower level o f resting potential is carried p r e d o m i n a n t l y
by sodium ions via at least two pathways, only one o f which is sensitive to T T X .
H o w e v e r , Fig. 6 serves to illustrate that even in the absence o f extracellular
sodium a n d calcium ions, the m a g n i t u d e o f the r e m a i n i n g inward c u r r e n t may
0
]
4
0
Vm
(my)
-100
V---4
lOs
FIGURE 6. Depolarization in K-free solution in the absence of Na and Ca ions.
The upper line indicates the level of [K]0. The potential trace is a chart recording.
In this experiment 146 mM Na-isethionate was replaced by 248 mM sucrose and
Ca-methanesulfonate was replaced by MnCI2. The cation content of the K-free
solution was thus Mn ++, 2.7 meq/liter and Mg ++, 0.5 meq/liter (preparation A8-31).
be sufficient to depolarize the m e m b r a n e w h e n potassium ions are also o m i t t e d
f r o m the external solution. In this e x p e r i m e n t s o d i u m isethionate was replaced
with an osmotic equivalent o f sucrose, and calcium ions were r e p l a c e d with
m a n g a n o u s ions so that, a p a r t f r o m K +, the cation content o f the e x t e r n a l
solution was 2.7 meq/liter Mn ++ a n d 0.5 meq/liter Mg ++. At 4 mM [K]o the
resting potential, after equilibration in this solution, was a b o u t - 8 0 m V .
I m m e d i a t e l y a f t e r the switch to zero [K]o the m e m b r a n e rapidly h y p e r p o l a r i z e d
by a b o u t 12 mV a n d t h e n d e p o l a r i z e d with a c o m p l e x time course d u r i n g the
next 30 s to a new steady level o f a b o u t - 2 5 m V . T h e h y p e r p o l a r i z a t i o n
p r e s u m a b l y reflects the increase in EK d u r i n g the initial m o d e r a t e decline o f
the K + c o n c e n t r a t i o n in the extracellular space (cf. the c h a n g e f r o m 4 mM to 2
m M [K]o in Fig. 1), j u s t as the s u b s e q u e n t depolarization p r e s u m a b l y reflects
the r e d u c t i o n in PK as the K + concentration declined f u r t h e r . O n switching
back to 4 m M [K]o this sequence is r e v e r s e d so that the a b r u p t repolarization
results f r o m the increase in PK, a n d the subsequent slower decline in potential
f r o m the r e t u r n o f the K + c o n c e n t r a t i o n in the extracellular space to its original
level. In o r d e r to account for the depolarization at zero [K]o u n d e r these
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4
[K]o (mM) "-7
Published December 1, 1977
736
THE JOURNAL
OF GENERAL PHYSIOLOGY
" VOLUME
70 " 1977
conditions, an inward c u r r e n t must be assumed to flow, and it is presumably
carried by either Mn ++ or Mg ++, or by both.
Outward Current and the Lower Level of Resting Potential
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We have seen that the resting potential can be shifted from the lower to the
higher level by interventions which r e d u c e the steady-state inward c u r r e n t ,
e.g., by lowering [Na]o, or by applying T T X . Since, as a result of inward-going
rectification, the steady-state outward potassium c u r r e n t is e x p e c t e d to be small
at the lower level o f resting potential, interventions which e n h a n c e that outward
c u r r e n t will t e n d to increase the resting potential, and a sufficient e n h a n c e m e n t
may cause the potential to shift f r o m the lower to the h i g h e r resting level.
It is known, for example, that the characteristics o f inward-going rectification
in cardiac and skeletal muscle fibers are such that when the electrochemical
potential gradient for K ions is large and outward, an elevation o f [K]o may
result in an increase in outward K c u r r e n t at a given potential, in spite o f the
smaller driving force (Hall and Noble, 1963; Noble, 1965). In this context, the
small increase in resting potential seen when [K]o was raised from 0 to 1 mM,
or f r o m 1 mM to 2 mM (Figs. 1 and 2) might be recalled. This change was seen
consistently and may have been caused, at least in part, by a small increase in
PK. A m o r e striking example o f this behavior is p r o v i d e d by the greater
hyperpolarization seen after raising [K]0 from 2 mM to 4 mM in Fig. 1; this
potential change is presumably associated with a larger increase in PK.
An increase in outward c u r r e n t o f some seconds' duration may also be
elicited by applying pulses o f depolarizing c u r r e n t in the "plateau" range o f
m e m b r a n e potential, as was d e m o n s t r a t e d by McAllister and Noble (1966, see
their Fig. 7). Using the same technique we were often able to induce a
maintained hyperpolarization in fibers whose resting potential was initially
steady at the lower level, as illustrated in Fig. 7 A. D u r i n g the constant-current
pulses the depolarization rose to a peak and then declined, presumably as the
potassium conductance increased. T h e first pulse was followed by an afterhyperpolarization o f almost 20 mV which decayed via a d a m p e d oscillation
about the steady potential level; the second, larger pulse was followed by a
greater hyperpolarization which caused an a b r u p t and maintained shift o f the
m e m b r a n e potential to the m o r e negative resting level.
A large and specific increase in potassium permeability o f a d i f f e r e n t n a t u r e
may be p r o d u c e d in cells o f the sino-atrial node and atrium by the muscarinic
action o f acetylcholine (e.g., B u r g e n and T e r r o u x , 1953; Harris and H u t t e r ,
1956). Acetylcholine also has been r e p o r t e d to decrease the automaticity o f
Purkinje fibers (Bailey et al., 1972; Tse et al., 1976). It seemed worthwhile,
t h e r e f o r e , to see whether acetylcholine would hyperpolarize Purkinje fibers
whose resting potentials were at the lower level. As shown in Fig. 7 B ( u p p e r
record) the application o f 1.1 x 10-4 M acetylcholine (ACh) for 20 s to a fiber in
4 mM K, low CI solution caused a rapid and maintained hyperpolarization o f
about 45 mV f r o m the initial resting potential o f - 4 2 mV. Reapplication o f the
same concentration o f acetylcholine at the new, higher level o f resting potential
p r o d u c e d a f u r t h e r hyperpolarization o f less than 2 mV (not illustrated). T h e
Published December 1, 1977
737
GADSBY AND CRANEFIELD Two Levels of Resting Potential
lower r e c o r d (Fig. 7 B) shows the r e s p o n s e o f the s a m e fiber to a m u c h lower
concentration o f acetylcholine (2.2 x 10 -e M), this time in 2 m M K, low CI
solution. T h e resting potential was initially - 4 0 m V , a n d the acetylcholine
caused a r a p i d h y p e r p o l a r i z a t i o n o f 10 m V which was readily r e v e r s e d u p o n
washing out the d r u g . N e i t h e r o f these concentrations o f acetylcholine caused
any c h a n g e in m e m b r a n e potential w h e n applied in the p r e s e n c e o f 7.2 x 10 -e
M a t r o p i n e (5 p~g/ml); that c o n c e n t r a t i o n o f a t r o p i n e did not itself have any
effect on the resting potential. T h e results o f Fig. 7 B suggest that acetylcholine
m a y have an action on Purkinje fibers qualitatively similar to its well-known
action on atrial cells, namely, to increase potassium permeability.
Lidocaine is an a n t i a r r h y t h m i c a g e n t which is t h o u g h t to have several effects
B
A
+40
._n
1t xlO-4M ACh
t
I
+50
,,N
(my
-100 L
(~v)
-20
10s
2.2x10-6M ACh
I
t
-100 L
2s
-60
Vrn(r.V)
10s
FIGURE 7. Switching from the lower to the higher level of resting potential with
outward current pulses or acetylcholine (ACh). In A, current pulses are shown on
the upper trace, membrane potential on the lower trace (4 mM K, low Cl solution;
same preparation as in Fig. 5B). B shows the membrane potential changes
resulting from the application of two different ACh concentrations. Bars indicate
the periods of exposure to ACh. The potential traces are chart recordings. The
upper record was obtained in 4 mM [K]0, the lower record in 2 mM [K]0; both
were low Cl solutions (preparation A7-23).
o n Purkinje fibers: in addition to its well-known "local anesthetic" effect o f
r e d u c i n g the steady-state, v o l t a g e - d e p e n d e n t availability o f excitable s o d i u m
channels (e.g., Davis a n d T e m t e , 1969; Strichartz, 1973; Hille, 1977), it may
increase t i m e - i n d e p e n d e n t p o t a s s i u m c o n d u c t a n c e (Wittig et al., 1973; W e l d
a n d Bigger, 1976), a n d diminish b o t h the b a c k g r o u n d inward c u r r e n t a n d the
p a c e m a k e r potassium c u r r e n t (Weld a n d Bigger, 1976). Any or all o f the first
t h r e e o f these effects would cause a net decline in inward c u r r e n t a n d ,
consequently, a h y p e r p o l a r i z a t i o n o f the m e m b r a n e if lidocaine were applied to
a fiber with a resting potential at the lower level. Lidocaine was i n d e e d f o u n d
to cause a r a p i d a n d reversible increase in m e m b r a n e potential in fibers that
were partially d e p o l a r i z e d , in Cl-free solutions containing 4 m M K. As shown
in Fig. 8, the h y p e r p o l a r i z a t i o n resulting f r o m the addition o f a relatively high
concentration o f lidocaine (3.5 x 10 -5 M) was often o f sufficient m a g n i t u d e to
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I (hA)
-20
Published December 1, 1977
738
THE JOURNAL OF GENERAL P H Y S I O L O G Y ' V O L U M E 7 0 .
1977
carry the m e m b r a n e potential to the higher resting level. W h e n r e a p p l i e d to
the same fiber at the high level o f resting potential, however, this concentration
o f lidocaine caused no f u r t h e r c h a n g e in m e m b r a n e potential (not illustrated).
DISCUSSION
Low Chloride Solutions
I
Vm
(mV)
I
lidocaine
I
i
-I00 L
5s
FXGURE 8. The effect of 3.5 x 10-5 M lidocaine (10 /zg/ml) on the lower level of
resting potential. The bar indicates the duration of exposure to lidocaine. 4 mM
K, low C| solution; preparation A8-10.
inward anion c u r r e n t contributes to the p a c e m a k e r depolarization, and that
this c u r r e n t is smaller for less p e r m e a n t anions. Since the anion equilibrium
potential may shift in a negative direction as repetitive activity slows, the
resulting f u r t h e r decline in that inward c u r r e n t m i g h t eventually lead to
quiescence. More recent results suggest that P~ might decline when CI ions are
replaced by larger anions (Carmeliet and V e r d o n c k , 1977). Such a m e c h a n i s m
could provide an alternative e x p l a n a t i o n for the depolarization and e n h a n c e d
spontaneity initially observed after such solution changes, but cannot account
for the s u b s e q u e n t lowering o f the rate o f s p o n t a n e o u s discharge to below
control values (cf. H u t t e r and Noble, 1961). T h e f u r t h e r possibility that anion
substitution in s o m e way modifies p a c e m a k e r currents cannot at p r e s e n t be
excluded. A p a r t f r o m the absence o f s p o n t a n e o u s activity, o t h e r a d v a n t a g e s in
the present context accrue f r o m the r e p l a c e m e n t o f C1- by larger anions,
namely a reduction in the overall m e m b r a n e c o n d u c t a n c e , and r e m o v a l o f the
slowing or "stabilizing" effect e x e r t e d by cellular chloride m o v e m e n t s on
potential changes initiated by alterations in o t h e r c o m p o n e n t s of m e m b r a n e
c u r r e n t (Adrian, 1960; H o d g k i n a n d Horowicz, 1959; Carmeliet, 1961; Wiggins
and Cranefield, 1976). A l t h o u g h the d e m o n s t r a t i o n o f two levels o f resting
potential is facilitated by the absence o f C1 ions, this is not a necessary condition
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Since this study was primarily c o n c e r n e d with resting potentials, p r e p a r a t i o n s
in which p a c e m a k e r activity was either absent or easily s u p p r e s s e d at both levels
o f resting potential were selected for study. T h e majority of p r e p a r a t i o n s
e x p o s e d to virtually Cl-free solutions qualified in this way; in still others
quiescence could be induced, particularly at the lower level of resting potential,
by a brief, t e m p o r a r y increase in [K]o. An eventual decline in the f r e q u e n c y o f
s p o n t a n e o u s activity o f Purkinje fibers when C1 ions were replaced by the less
p e r m e a n t methylsulfate ions was o b s e r v e d by H u t t e r and Noble (1961); their
explanation was that, in a spontaneously active fiber, the equilibrium potential
for a p e r m e a n t anion lies positive to the m a x i m u m diastolic potential, so that
Published December 1, 1977
GADSBY AND CRANEFIELD Two Levels of Resting Potential
and the membrane potential o f some
solution may be switched between the
current pulses (see Fig. 6 of Cranefield,
that two levels of resting potential may be
739
fibers exposed to normal Tyrode's
two resting levels with small, 1-2-s
1977). Fig. 5 A above also illustrates
obtained in Cl-containing solution.
The Resting Potential as a Function of [K]o
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T h e results presented in this paper confirm the linear dependence of the
membrane potential, Vm, on log [K]o at relatively high potassium concentrations,
and the large depolarization at very low concentrations, as previously described
by Weidmann (1956). T h e existence o f two separate levels of resting potential
at moderately low potassium concentrations will be discussed later. Previous
studies showing two levels o f resting potential in Purkinje fibers, e.g., those of
Chang and Schmidt (1960) and of Carmeliet (1961) were recently reviewed by
Wiggins and Cranefield (1976).
As already mentioned, the presence o f a small inward sodium current causes
the resting potential to become progressively less negative than EK as [K]o is
lowered, so that the outward driving force on K ions, Vm-EK, progressively
increases. Since the potassium permeability of cardiac and skeletal muscle
fibers is known to decline to a low value when Vm-EK becomes large and
positive (e.g., Hodgkin and Horowicz, 1959; Noble, 1965; see Hagiwara et al.,
1976, for further references), the sodium to potassium permeability ratio, c~,
may be expected to increase when [K]o is very low; that increase in a may
provide an explanation for the depolarization which is observed u n d e r these
conditions. An attempt to reconstruct the dependence of Vm on [K]o, using
empirical equations to take some account of the dependence of PK on Vm-EK,
was made by Noble (1965). At low values of [K]o, the resulting curves showed a
progressive depolarization as [K]o declined, the extent of which d e p e n d e d on
the assumed magnitude o f the constant sodium conductance. In contrast to
that calculated behavior, however, the observed depolarization as [K]o is lowered
is quite abrupt, as is the greater part o f the repolarization when [K]o is
subsequently raised (Fig. 1). These abrupt potential changes result from the
presence o f a region o f marked negative slope conductance in the net membrane
current-voltage relationship (see Fig. 4, and below). It is probably mainly in this
respect that the calculated and observed behaviors differ since the net (sodium
plus potassium) current-voltage curve used by Noble has a negative slope
region only when the sodium conductance is negligibly small (see Fig. 1 of
Noble, 1965).
Note that whereas the negative chord conductance in Fig. 4 is readily
attributable to some other ion, the negative slope conductance may be a property
of the inwardly rectifying potassium ion channel (see, e.g., McAllister and
Noble, 1966; Dudel et al., 1967b). Such a negative slope region was previously
demonstrated in the current-voltage characteristic of the inward-going rectifier
in frog skeletal muscle fibers by use o f electrical conductance measurements
(Adrian and Freygang, 1962). This negative slope has also been demonstrated
by measurements of tracer-potassium flux in both Purkinje fibers (Haas and
Kern, 1966) and skeletal muscle fibers (Horowicz et al., 1968).
An additional point concerns the reversibility of the membrane potential
Published December 1, 1977
740
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 7 0 " 1 9 7 7
changes at low [K]o illustrated in Fig. 1. O n close inspection it can be seen that
after the b r i e f e x p o s u r e to K-free solution, the m e m b r a n e potential was about
2'mV more negative shortly after the r e t u r n to 1 mM [K]o than it was some 15 s
later in that solution. In o t h e r experiments it has been shown that the
m a g n i t u d e o f this " u n d e r s h o o t " increases with [K]o in a low range o f concentrations, increases with the duration o f the e x p o s u r e to K-free solution, and is
abolished by cardiac steroids (Gadsby and Cranefield, 1977). T h e u n d e r s h o o t
probably reflects a tI'ansient increase in outward m e m b r a n e c u r r e n t caused by
the e n h a n c e d activity o f an electrogenic Na-K p u m p following its t e m p o r a r y
arrest in pota_';,:,n-free solution.
Two Levels of Resting Potential
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Over a limited r a n g e o f [K]o (1-4 mM in these experiments) two sets of values
were obtained for the resting potential. As shown in Fig. 2, the more negative
values are reasonably well a p p r o x i m a t e d by the G o l d m a n , Hodgkin, Katz
equation with a constant permeability ratio, ~x, o f 0.01, whereas to fit the lower
values, at 1 and 2 mM [K]o, it would be necessary to increase ot about 25-fold.
T h e increase in a probably results partly f r o m a decline in PK and partly f r o m
an increase in PNa- Some increase in PNa may be expected in partially depolarized
fibers since the present results suggest that, u n d e r these conditions, steadystate inward sodium c u r r e n t flows t h r o u g h TTX-sensitive, "excitable" sodium
channels (see discussion below o f the n a t u r e o f the steady-state inward current).
A reduction in PK is also to be expected: the tracer-potassium efflux data
obtained u n d e r voltage-clamp conditions by Hass and Kern (1966) indicate that
PK declines about sevenfold in response to a m e m b r a n e depolarization of 55
mV f r o m near EK. In the present study, the slope conductance was f o u n d to be
smaller about the lower than about the higher resting potential (see Fig. 4).
This finding is consistent with a decline in PK on depolarization if it can be
assumed that t h e r e are no large changes in the slope o f the steady-state inward
current-voltage curve between the two resting potential levels.
As previously stated, the existence o f two possible stable resting potentials at
a given value o f [K]o requires that the steady-state net current-voltage relationship be "N-shaped," with two z e r o - c u r r e n t intercepts in regions of positive
slope conductance. T h e third, unstable intercept occurs in a region o f negative
slope conductance. T h e s e features are seen in the g r a p h of applied c u r r e n t
against r e c o r d e d voltage illustrated in Fig. 4. Conductance measurements were
made on fibers that were short and thin so that, in the steady state, a p p r o x i m a t e
spatial uniformity of the m e m b r a n e potential is likely to have been achieved
over the voltage range o f interest. Although it is possible that the m e m b r a n e
potential was not always u n i f o r m t h r o u g h o u t the fiber (for example, n e a r - 1 0 0
mV, where the conductance is greatest) the i m p o r t a n t features, i.e. the two
resting potentials, the greater slope conductance at the more negative o f these
potentials, and the negative slope between them, are i n d e p e n d e n t o f the small
correction for cable properties which might apply. Such a cable correction
might, however, r e d u c e the peak-to-peak amplitude o f the negative slope
conductance region, i.e. the m a g n i t u d e o f the hysteresis. It is in any event clear
Published December 1, 1977
GADSBY AND CRANEFIELD Two Levels
of Resting Potential
741
The Nature of the Steady-State Inward Current
T h e net current-voltage relationship may also be shifted in an u p w a r d (i.e.
outward) direction by a decrease in inward current; the hyperpolarization
caused by the r e p l a c e m e n t o f sodium ions by Tris ions, or by the addition o f
T T X (Fig. 5) and possibly also that caused by the addition o f lidocaine (Fig. 8),
presumably resulted f r o m such a shift. In cooled Purkinje fibers r e d u c t i o n o f
Nao also t e n d e d to stabilize the m e m b r a n e potential at the more negative o f the
two possible levels, presumably because o f a reduction in inward Na c u r r e n t
(Chang and Schmidt, 1960).
T h e hyperpolarization r e c o r d e d in T T X strongly suggests that at least some
o f the fast sodium channels u n d e r g o only incomplete inactivation in the plateau
range o f potentials, if it can be assumed that T T X is without effect on o t h e r
c o m p o n e n t s o f m e m b r a n e c u r r e n t . T h e existence o f a TTX-sensitive steadystate sodium c u r r e n t in this voltage range is implicit in the model o f McAllister
et al. (1975) and might in part explain the s h o r t e n i n g o f the action potential
and loss o f plateau caused by the addition o f T T X (Dudel et al., 1967a). T h e
fiber o f Fig. 5 C was f o u n d to hyperpolarize more, f r o m the - 5 0 mV resting
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from Fig. 4 that relatively small c u r r e n t changes would suffice to displace the
current-voltage relationship vertically in either direction so that only a single,
high o r low, z e r o - c u r r e n t potential remains. For e x a m p l e , some o f the large
shifts in resting potential caused by small changes in [K]o (Figs. 1 and 2) may be
explained in this way. T h u s it is known that raising [K]o increases the K c u r r e n t
for a given potential displacement, whereas lowering [K]o reduces the K
currents (Hall et al., 1963; Noble, 1965; Dudel et al., 1967b). I f the steady-state
inward c u r r e n t is little influenced by small changes in [K]o, then the changes in
K c u r r e n t just described may be sufficient to account for the loss o f the two
more positive z e r o - c u r r e n t potentials at higher values o f [K]o (e.g., Fig. 2, [K]o
> 2 mM), and the loss o f the two more negative intercepts at very low [K]o
(e.g., Fig. 2, [K]o < 1 raM).
T h e large shifts in resting potential shown in Fig. 7 which resulted f r o m the
application o f ACh, or outward c u r r e n t pulses, may be explained in similar
terms: i.e. a t e m p o r a r y u p w a r d shift o f the net current-voltage curve secondary
to the increase in steady-state outward c u r r e n t . T h e failure o f many previous
studies on Purkinje fibers to d e m o n s t r a t e such large hyperpolarizations in
response to the application o f ACh (cf. H o f f m a n and Cranefield, 1960; Cranefield, 1975) n e e d not be surprising. As discussed above, a very small change in
net m e m b r a n e c u r r e n t in the outward direction may suffice to shift the resting
potential f r o m the lower to the higher level, whereas the same c u r r e n t change
may cause only a small hyperpolarization f r o m the h i g h e r resting potential
where the m e m b r a n e conductance is greater (cf. Fig. 4). It would also be
expected that in Cl-containing solutions, such small potential changes as do
occur would be obscured to some extent by the accompanying cellular chloride
ion movements. O n the o t h e r h a n d , the diminished rate o f p a c e m a k e r depolarization in Purkinje fibers, r e p o r t e d by Bailey et al. (1972) and by Tse et al.
(1976), is consistent with the present results.
Published December 1, 1977
742
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potential, when [Na]o was r e d u c e d by 25% than it did w h e n T T X was a d d e d . I f
three simplifying assumptions are m a d e , namely, that inward Na c u r r e n t is
p r o p o r t i o n a l to [Na]o, that only s o d i u m c u r r e n t is sensitive to T T X , a n d that 2
x 10 -6 g/ml is a s u p r a m a x i m a l concentration o f T T X , t h e n this result suggests
that in this p r e p a r a t i o n less t h a n 25% o f the steady-state sodium c u r r e n t n e a r
- 5 0 mV was sensitive to T T X . It is clear, however, that changes in even such
small c o m p o n e n t s o f m e m b r a n e c u r r e n t may have significant effects on plateau
potentials (see, e.g., Fig. 5 B).
A n o t h e r e x a m p l e o f the effects o f p r e s u m a b l y small inward currents is the
depolarization in K-free solution shown in Fig. 6, which o c c u r r e d in a Na +- and
Ca++-free solution containing sucrose a n d 2.7 meq/liter Mn ++ and 0.5 meq/liter
Mg ++. T w o features of the time course of those potential changes deserve
c o m m e n t . T h u s the m e m b r a n e potential took m o r e than 30 s to reach a new
steady level after [K]o was c h a n g e d f r o m 4 mM to 0, but less than 15 s to r e t u r n
to its original level after reversing this change. A similar a s y m m e t r y in the time
course of the m e m b r a n e potential changes in r e s p o n s e to raising or lowering
[K]o was seen in isolated skeletal muscle fibers by H o d g k i n and Horowicz
(1960). T h e i r e x p l a n a t i o n , which is based on a retention o f K ions n e a r the
m e m b r a n e in a space f r o m which diffusion occurs only slowly, may also apply
in the present case: immediately a f t e r the c h a n g e in [K]o in Fig. 6 the K ion
concentration at the p e r i p h e r y o f the fiber falls rapidly towards zero while that
d e e p e r in the clefts between adjacent cells is, as yet, essentially u n c h a n g e d . E~
at the surface o f the fiber t h e r e f o r e increases m o r e rapidly than the intracellular
potential a n d , as a result o f i n w a r d - g o i n g rectification, PK at the surface
m e m b r a n e becomes very small. U n d e r these conditions changes in resting
potential are primarily d e t e r m i n e d by changes in the concentration of K + in
the clefts as these ions slowly diffuse out o f the bundle. I m m e d i a t e l y after
switching back to 4 mM [K]o, h o w e v e r , the situation is r e v e r s e d so that both the
K ion concentration a n d PK b e c o m e g r e a t e r at the surface o f the fiber t h a n in
the clefts. T h e r e c o r d e d changes in potential thus largely reflect the m o r e
r a p i d increase in K concentration which occurs towards the p e r i p h e r y o f the
fiber; the slower rise in K concentration in the clefts affects the m e m b r a n e
potential m u c h less since PK r e m a i n s smaller there until the potassium concentration becomes u n i f o r m t h r o u g h o u t the extracellular spaces.
T h e second p o i n t concerns the n a t u r e o f that depolarization in K-free
solution. T h e decline with time o f the K ion concentration in the clefts should
be m o n o t o n i c if the rate is d e t e r m i n e d primarily by diffusion. T h e m o r e
complicated time course of the resulting potential changes then p r e s u m a b l y
results f r o m the nonlinearity o f the steady-state c u r r e n t - v o l t a g e relationships in
this r a n g e o f K concentrations. As already m e n t i o n e d , a reduction o f the
external K concentration has two principal effects on the c u r r e n t - v o l t a g e curve
for that ion: in the first place, the K c u r r e n t for any potential d i s p l a c e m e n t is
r e d u c e d a n d , second, t h e r e is a shift of+ the curve to m o r e negative potentials.
T h e latter effect is p r o b a b l y responsible for the initial h y p e r p o l a r i z a t i o n seen in
Fig. 6. T h e progressive decline in K c u r r e n t , however, eventually causes a
positive shift o f the zero net c u r r e n t (i.e. resting) potential because o f the
Published December 1, 1977
GADSBYAND CRANEFIELD Two Levels of Resting Potential
743
Implications for Excitation and Conduction
It has been well established that two distinct types of p r o p a g a t e d action
potential may be r e c o r d e d , u n d e r d i f f e r e n t conditions, in cardiac Purkinje
fibers (see Cranefield, 1975). T h e m o r e "normal" action potential has a rapid
upstroke caused by inward c u r r e n t flowing in "fast" sodium channels and it is
most readily elicited in cells showing a high resting potential (e.g., - 9 0 mV).
T h e upstroke o f the "slow response" action potential results f r o m a "stow"
inward c u r r e n t presumably carried by sodium and calcium ions and is most
readily elicited f r o m a lower resting potential (e.g., - 5 0 mV), at which level the
"fast" channels are largely inactivated. In fibers depolarized to - 5 0 mV by
elevation o f [K] o, however, PK is high and the m e m b r a n e resistance is correspondingly low so that slow response activity may usually be seen only if the
slow inward c u r r e n t is e n h a n c e d by the addition of catecholamines. On the
o t h e r hand, at moderately low [K]o values it is relatively easy to initiate slow
responses f r o m the lower o f the two possible resting potential levels since, u n d e r
these conditions, PK is low and the m e m b r a n e resistance is high. Since fibers
showing slow response activity can generate abnormal rhythmic activity or be a
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presence o f a small inward c u r r e n t c o m p o n e n t . T h e resting potential would be
expected to fall most rapidly over the voltage range in which the currentvoltage relationships are shallowest. T h e more rapid depolarization f r o m near
- 9 0 mV to - 5 0 mV in Fig. 6 is consistent with this interpretation since inwardgoing rectification is m a r k e d in this region (e.g., Dudel et al., 1967b; cf.
current-voltage relationship in Fig. 4, above). It seems possible, then, that the
later phase o f depolarization, f r o m about - 4 0 mV to - 2 5 mV, indicates the
presence o f a second shallow (low conductance) region of the current-voltage
relationship. One might f u r t h e r speculate that the c u r r e n t responsible for this
decline in m e m b r a n e slope c o n d u c t a n c e flows via incompletely inactivated slow
inward c u r r e n t channels (Gibbons and Fozzard, 1975; Kass et al., 1976). In this
context it should be m e n t i o n e d that Mn ions may carry some slow inward
c u r r e n t in cardiac cells (Ochi, 1970, 1976; Delahayes, 1975). Since the depolarization began near - 9 0 mV, however, there must have been some pathway for
steady-state inward c u r r e n t o t h e r than t h r o u g h "excitable" channels. T h u s
Mn ++ and Mg ++ may carry c u r r e n t via the channel t h r o u g h which the n o r m a l
inward leak o f sodium ions occurs; the ionic specificity o f this channel is not yet
known. O t h e r , presumably nonspecific, c u r r e n t paths may exist a r o u n d the
impaling microelectrode and at the cut ends o f the fiber bundle.
Carmeliet (1961) f o u n d that omission o f external K ions did not result in
depolarization o f Purkinje fibers e x p o s e d to a Na-free, Cl-containing solution
even in the presence o f Ca ++. In Cl-containing solutions, if the m e m b r a n e
conductance to o t h e r ions becomes very small, the m e m b r a n e potential may be
d e t e r m i n e d for a time by the existing t r a n s m e m b r a n e distribution o f CI ions
( H o d g k i n and Horowicz, 1959; Adrian, 1960). T h e absence o f this "stabilizing"
effect o f CI ions u n d e r the conditions o f the present e x p e r i m e n t s could p e r m i t
a very small inward c u r r e n t to cause a relatively rapid depolarization o f the sort
seen in Fig. 6.
Published December 1, 1977
744
THE JOURNAL OF GENERAL PHYSIOLOGY ' VOLUME 70 ' 1977
locus o f slow c o n d u c t i o n w h i c h m a y p e r m i t c i r c u s m o v e m e n t o f e x c i t a t i o n , a
fall in m e m b r a n e p o t e n t i a l f r o m t h e h i g h e r to t h e l o w e r level o c c u r r i n g o v e r a
p o r t i o n o f t h e c a r d i a c s y n c y t i u m m i g h t b e a n i m p o r t a n t s t e p in t h e g e n e r a t i o n
o f c a r d i a c a r r h y t h m i a s . I n t h a t case t h e r e t u r n o f t h e m e m b r a n e p o t e n t i a l to
t h e h i g h e r level w o u l d t e n d to a b o l i s h s u c h a r r h y t h m i a s .
We thank Joan Leary for her excellent technical assistance and Drs. Frank Brink and C. M.
Connelly for helpful discussions.
This work was supported by United States Public Health Service grant HL 14899.
Receivedfor publication 29 April 1977.
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