J. Physiol. (1981), 314, pp.

377-393 377
With 11 text-figures
Printed in Great Britain

A STUDY OF THE IONIC NATURE OF THE PACE-MAKER CURRENT IN

CALF PURKINJE FIBRES
BY D. DiFRANCESCO
From the Istituto di Fisiologia Generale e Chimica Biologica,
Sez. Elettrofisiologia, via Mangiagalli 32, 20133 Milano, Italy
(Received 29 July 1980)
SUMMARY
1. Properties of the pace-maker current (if) in Purkinje fibres were studied in the
presence of Ba, which by partially blocking the iK channel reduces K depletion
during hyperpolarizing voltage-clamp pulses, and eliminates the main cause of
distortion in the current time course.
2. On raising the external potassium concentration (Kb), the if fully activated
current-voltage relation (tf(E)) increases in the inward direction. In the range
3-36 mM-Kb and negative to -50 mV the current is inward, and no cross-over is
observed.
3. In normal conditions, the reversal potential (Ef) for if lies in the voltage region
positive to - 50 mV, and can be observed on lowering the external sodium
concentration (Nab). Ef shifts to the negative direction when Nab is decreased. Slopes
ranging between 29 and 35 mV/decade are found for Nernst plots of Ef against Nab.
Changing Nab in the range 140-4'4 mM causes the if(E) relation to undergo a simple
shift along the voltage axis, without significant change in its slope.
4. Ef also depends on Kb, as can be observed in low Nab (35 mM), and shifts to
the positive direction by about 26 mV for every 10-fold change in Kb. The fully
activated slope conductance increases when Kb is increased.
5. It is concluded that Na and K both participate in carrying if. The slope of the
fully activated if (E) relation increases with Kb, but is unchanged in different Nab,
indicating that the channel conductance depends on Kb, but not appreciably on Nab.

INTRODUCTION
Evidence has been presented that the 'pace-maker' current in Purkinje fibres is
not, as previously thought, a pure K current deactivated by hyperpolarizations
(Noble & Tsien, 1968; Peper & Trautwein, 1969) but is an inward current activated
by hyperpolarizations negative to the threshold of about - 50/-60 mV (DiFrancesco,
1980a, 1981). The apparent current reversal obtained near EK during hyperpolari-
zations is produced by the superimposition of an inward-decreasing 'depletion'
component, due to the K depletion effect on iK , with the inward-increasing pace-
maker component (itself modified by the K depletion process), which cancel each
other and give rise to a nearly flat current (DiFrancesco & Noble, 1980; DiFrancesco,
0022-3751/81/4830-1115 $07.50 (D 1981 The Physiological Society
378 D. DiFRANCESCO
1981). According to this new interpretation, the properties of the pace-maker current
in Purkinje fibres are identical to those of the current if in the SA node (Brown &
DiFrancesco, 1980; DiFrancesco & Ojeda, 1980). This suggests a possible direct
correlation between normal pace-maker activity in heart cells and the presence of the
if channel. An obvious problem arising from the evidence that the pace-maker current
in Purkinje fibres is not a pure K current, concerns its ionic nature. In view of the
known dependence of 'iK2' on Nab and Kb (McAllister & Noble, 1966; Noble & Tsien,
1968) the following questions arise: Is the pace-maker current carried by Na? Does
the K- dependence of the pace-maker current reflect an activation mechanism, or does
K actually cross the channel, or both? These questions prompted the experiments
described in this paper. The results indicate that both Na and K participate in
carrying the pace-maker current in Purkinje fibres, and while changes in Nab only
affect the driving force, increasing Kb is also associated with an increasing channel
conductance. A preliminary report of this work has appeared (DiFrancesco, 1980b).
Nomenclature
The pacemaker current is here referred to as if rather than 'iK,' to distinguish
between the more recent interpretation of this current as an inward component
activated by hyperpolarization in the pace-maker range, like if in the SA node
(DiFrancesco, 1981), and the 'classical' pure K current interpretation (Noble & Tsien,
1968). For a discussion of the relation between the two descriptions see DiFrancesco
(1981)). The symbols Kb, Nab and Kc have been used to indicate the external (bulk)
potassium and sodium concentrations, and the cleft potassium concentration
respectively.
METHODS
Methods for isolating and voltage-clamping short Purkinje fibres from calves' hearts have been
described elsewhere (see for example DiFrancesco, 1981). After dissection the fibres were soaked in
Tyrode solution with the following composition (concentrations in mM): NaCl, 140; KCI, 3; CaCl2,
1-8; MgCl2, 1; NaHCO3, 12; NaH2PO4, 0-4; Glucose 1 g/l. The solution was maintained at room
temperature and continuously bubbled with 02 +5% C02. Tyrode solution used for experiments
was buffered at pH 7-4 (35 + 02°C) with Tris-HCI (2-5 mM) and contained 5 mM-MnCI2, used
routinely to limit interference from inward current during voltage-clamp depolarizations. TTX
(5 x 10-6 g/ml.) was also used when fast inward current was particularly large. When needed, BaCl2
(5-10 mM) and KCl were added to normal Tyrode without correcting for the change in osmolarity.
In the Na-replacement experiments NaCl was substituted with equimolar quantities of Tris
(hydroxymethyl) aminomethane-HCI (pH 7 4) or of choline-Cl. Activation curves for the current
if (see Figs. 2 and 7) have been obtained by the conventional method of applying voltage-clamp
pulses of different amplitudes and durations, and measuring the tail amplitudes on return to a fixed
potential. Fully activated current-voltage relations can be obtained by dividing the if time-depen-
dent change during a pulse from a holding potential (EH) to a test potential (ET) by the change
in the steady degree of activation between EH and ET. Fig. 1 shows an alternative method also
used in this paper: test pulses are preceded by either a hyperpolarizing or a depolarizing prepulse.
If durations and amplitudes of the two prepulses are sufficient to cover the whole if activation range,
the difference between tail amplitudes on clamping at the following test potential (measured
between arrows as in Fig. 1) represents the fully activated current amplitude at the test potential
itself. This method does not avoid errors from interference of components other than if and/or from
distortion induced by accumulation/depletion, but has the advantage of being a direct measure
of tf and therefore of eliminating any error coming from calculations. Also, changes in the tf(E)
relation can be followed during the experiment itself, and a relatively precise measurement of the
 IONIC NATURE OF if 379
if reversal potential can be obtained by taking the voltage where the (algebraic) difference between
the tail amplitude following the pre-hyperpolarization and that following the pre-depolarization
changes its sign.
a

50 mV
==b
c

_ - _

-0 25 <

2 sec
Fig. 1. Example of the method used for the determination of the f(E) relation. From a
holding potential of -39 mV, 2 sec prepulses to -119 mV or to -19 mV are followed
by pulses to various levels. Size and duration of prepulses are sufficient to bring the
activation variable y to 1 (hyperpolarization) or to 0 (depolarization), as can be checked
by previous measurement of the steady-state activation curve y"x,(E). The time-dependent
current change at a potential E will therefore be ,(E) (y.(E) -yo) where yo is 1 or 0. The
algebraic sum of the amplitudes of the two tails corresponding to the same potential
E (a, b, c) yields f(E). In the case a the pulse from -19 mV is not needed. 5 mM-MnCl2,
10 mM-BaCl2 present in the 9 mM-K, 35 mM-Na Tyrode solution.

RESULTS
The effect of low concentrations of Cs and the behaviour of the current in the
presence of Ba indicate that during hyperpolarizations the pace-maker current in
Purkinje fibres behaves like an inward-activating current (if) rather than an outward-
deactivating current ('iK2') (DiFrancesco, 1981). In particular, the presence of Ba
allows the properties of if to be studied with little or no interference from
accumulation/depletion processes due to the iKj changes during voltage-clamp pulses.
When Kb is varied in the presence of Ba, the behaviour of if is radically different from
that seen in normal Tyrode. When Ba is present, no current reversal is observed
negative to about -50 mV even at high Kb (48 mM) and conductance measurements
in normal and high Kb show that on hyperpolarization the current is inward-activating
(DiFrancesco, 1981), and that it cannot therefore be carried by K only. A more direct
approach to the problem ofthe K dependence of if can be made by studying the effects
of Kb on the activation curve and the fully activated relation of if in Ba-containing
solutions.
380 D. DiFRANCESCO
Activation curve and fully activated if(E) relation in different K concentrations in
Ba-containing solutions
Fig. 2A shows the activation curves for if obtained in 5 mM-Ba by plotting tail
amplitudes after pulsing to different voltage levels, for Kb values of 3, 9, 18 and
36 mm. The curves are plotted according to the new interpretation of if as an inward

36
A

-150 mV

501-

nA

0
L mV B
I I I I I I I
-150 -100 -50
Fig. 2. A: if activation curves (y.(E)) in 3, 9, 18 and 36 mM-Kb solutions all containing
5 UM-Ba, plotted from tail amplitudes at -72 mV after pulses to various potentials.
Positions of half-activation points (arrows) are: -91, -91, -86 and -81 mV, respec-
tively, for 3, 9, 18 and 36 mM-Kb (curves drawn by eye). B: y00 (E) activation curve at
3 mM-Kb in Ba (O. same points as in A) compared with the curve 1 - 8 (E) before
perfusion with Ba at 3 mM-Kb (0). The ratio between amplitudes of the two curves is
1 12. Half-activation point for the 1 -8. (E) curve is approximately -8 mV. Curves drawn
by eye.
 IONIC NATURE OF if 381
current activated on hyperpolarization. In comparison with the "iK2' interpretation
the following relation holds:

(here the symbol y represents the if activation variable).

~~~~~~~/K
~~~~~~~*s
$/0~~~~~~~~~~0
* -100 -50 mV 0
/t t
0 0 0

/0 3~~~~~~~~

0
o A

0
18/

3/6 ~ ~ ~ -0-5
Fig. 3. 'zK2(E)' relation obtained in 3 mM-Kb without Ba (@) (Noble & Tsien's (1968)
method) and tf(E) relations in 3 (0), 9 (U), 18 (El) and (A) 36 mM-Kb, all obtained in
5 mM-BaCI2 using the protocol of Fig. 1. Half-filled circles (E) refer to return to 3 mM-Kb.
Curves in 3 mM-Kb drawn by eye. Least-squares linear fitting slopes of 2-7, 50 and
6-6 nA/mV at 9, 18 and 36 mM-Kb respectively.

A single curve fitting the experimental values at all Kb concentrations is not found.
The half-activation point shifts in the positive direction on changing Kb from 9 to
36 mm. Comparing (at 3 mM-Kb) the if activation curve in Ba with the curve 1-s. (E),
deduced according to the Noble & Tsien (1968) method in normal Tyrode before Ba
was applied (Fig. 2B), shows that the current is slightly reduced after perfusion with
Ba. A similar small reduction after Ba has been reported for the current if in the SA
node (Yanagihara & Irisawa, 1980). A current decrease is expected in Ba, at potentials
where distortion by K depletion is negligible, i.e. for small hyperpolarizing voltage-
clamp pulses, as a secondary effect of the iK, block. By blocking iK1, Ba will cause
the steady-state cleft K concentration (Kc) to decrease as a consequence of the
smaller, outwardly directed K flow. Therefore, given the K dependence of if (see Figs.
3 and 10), the absolute value of if(E) will also decrease. The size of the if reduction
induced by Ba indicates only a small change in the steady-state Kc in this experiment,
as can be seen by comparing the change in the amplitude of the activation curve on
adding Ba (Fig. 2 B) with the change in amplitude brought about by altering Kb (Fig.
2 A). However, the degree by which if is reduced after Ba, and the voltage range where
it is observed, vary from fibre to fibre (in the experiment of Fig. 3, for example, the
reduction is about 2-fold in the voltage range -50 to -90 mV, as seen by comparing
382 D. DiFRANCESCO
the 3 mM-Kb curve before Ba (filled circles) with that in Ba (open circles). This
variability is expected, as it corresponds to the variable balance between amplitude
of ir and the amount of K depletion present during a hyperpolarization in normal
conditions.
HP -38
50 mV -
~= -- -68

S/
flCA
A

. D

16 sec
Fig. 4. Pulses of different amplitudes and durations, followed by 8 sec pulses to - _8 mV,
are applied from -38 mV in 140 (A), 105 (B), 70 (C) and 35 (D) mm-Nab (all 9 mM-Kb)
solutions. Na replaced by equimolar quantities of Tris. Note reversal of current at - 38 mV
just visible in 70 mm-Nab, and more evident in 35 mM-Nab. 5 mm-MnCl2 and 8 mm-BaCl2
present throughout.

In Fig. 3 the voltage protocol of Fig. 1 has been used to construct the fully activated
(if(E)) relations at different Kb, varying from 3 to 36 mm. Theb'8 relations, obtained
in the absence of Ba according to the Noble & Tsien (1968) method, is also shown
at 3 mm-Kb. In the Ba-containing solutions no current reversal is obtained negative
to - 50 mV, while in the absence of Ba the current-voltage relation is grossly
distorted negative to -100 mV, and an 'apparent' reversal potential is observed at
about -114 mV. In Ba the if(E) relations at 9, 18 and 36 mm-Kb do not show evident
deviation from linearity in the voltage range analysed, while at 3 mm-Kb outward-
going rectification is apparent. It also appears that raising Kb does not simply
produce a shift of the ia(E) curve along the voltage axis, as expected from a change
in the driving force only, but also increases the slope conductance. The increase in
slope conductance is also present in low Nab (see Fig. 10). Results similar to those
observed in Figs. 2 and 3 have been obtained in three experiments in normal Nab
(140 mm), for Kb varying from 3 to 54 mm o.
 IONIC NATURE OF if 383
Effect of Nab
Experiments of the type shown in Fig. 3 lead to the conclusion that the 'apparent'
reversal potential observed under normal conditions does not correspond to the true
reversal potential of the current and suggest that at least one ion with an equilibrium
potential more positive than EK must be involved in carrying if. Given the known
dependence of the pace-maker current on Nab (McAllister & Noble, 1966), an obvious
candidate for carrying if is Na. The experiment of Fig. 4 shows that Na does indeed
contribute to carrying if.

50 mVC -A .i

0F
pA~ \
2

-0.5 L A
A _B

3 sec

-32 - ' -42

<C~~~~~
D

Fig. 5. Determination of if reversal potential (Er) in 140 (A), 105 (B), 70 (C) and 35 (D)
mM-Nab. Current reversal obtained by pulsing to different levels after a 3 sec, 70 mV
hyperpolarization from -30 mV. In A and B the reversal potential has been determined
by applying progressively more depolarized 2-3 mV steps (not all pulses are drawn) and
choosing the voltage just negative to that at which the it tail becomes outward-decaying.
Reversal potentials marked near corresponding traces. 9 mM-Kb Tyrode solutions all
containing 5 mM-MnCl2, 5 x 10-6 g/ml. TTX and 5 mM-BaCl2

In the presence of Ba, Nab was changed from 140 to 105, 70 and 35 mm. Kb was
kept higher than normal (9 mM) in order to have a large time-depenident current
even at low Nab. As expected, the amplitude of the current activated during a
hyperpolarization decreased on decreasing Nab. The behaviour of if at the potentials
of -68 mV and -38 mV indicates that Na crosses the channel. On return to the
384 D. DiFRANCESCO
holding potential of -38 mV, small but detectable inward-decreasing tails are
observed in 140 and 105 mM-Nab, but outward-decreasing tails appear in 70 mM-Nab
and more clearly in 35 mM-Nab, indicating that at these Nab values the current
reverses at a potential more negative than -38 mV. Correspondingly, the current
tails at -68 mV decrease on decreasing Nab, until in 35 mM-Nab (D) they have nearly
vanished.
Reversal potential in different Nab
The protocol of Fig. 5 has been used to give a better indication of the if reversal
potential (Ef) in different Nab. In 9 mM-Kb, after a 3 sec, 70 mV hyperpolarization
0
-30
50mV[ -
0-

uA F ~~~~~d
b

C
tails
eA
-0.5L\/
f
I |
3 sec tl
a

b
O.1 ;AL b *W=C
d

1 sec

Fig. 6. Separation of two current components during depolarizations to 0 mV. A: 3 sec

hyperpolarizations of different amplitudes are applied from -30 mV before stepping to
0 mV at time t. Trace a corresponds to the reference current level at the holding potential
of -30 mV, while traces c-f correspond to hyperpolarizations of 20, 40, 50, 60 and 70 mV
respectively. B: current records obtained on depolarizing to 0 mV after the various
hyperpolarizing pulses (a-f as indicated). The traces have been displaced vertically by
arbitrary amounts for clarity. Note expanded time scale. Traces e andf before time t are
off the scale. The outward-increasing component apparent in trace c corresponds to a
hyperpolarization where no if was activated (see A). Activation of if during the preceding
hyperpolarization is related to the appearance at 0 mV of a relatively fast outward-
decreasing tail, corresponding to if deactivation (traces e and f). Same experiment as in
Fig. 5.
 IONIC NATURE OF if 385
from a holding potential of -30 mV to activate if, the potential is stepped into a
region where reversal occurs. The current trace is nearly flat at -25 mV in
105 mM-Nab, at -32 mV in 70 mM-Nab and at -42 mV in 35 mM-Nab. In the normal
Nab (140 mM) the determination of Er is more complicated and requires more detailed
analysis. In general, currents other than if can contribute to the total time-dependent
current (for example the slow inward current, isj and the plateau outward current,
ix see Noble, 1979). The degree to which the records in Fig. 5 reflect the deactivation
of if alone needs therefore to be verified. This is particularly important in high Nab,

HP -38 23
_ - ~~~~~~A
100 mVKA

ef e Id \c

0 a

-0L25L>L

16 sec

0 25 4 [<
K-

0*5

-100 mV -50 0
Fig. 7. A: if tail amplitude at -23 mV as a function of the preceding pulse level. In the
9 mM-Kb, 35 mM-Nab (Tris substitute) solution if is outward at -23 mV. Expanding the
time scale 10-fold (A, lower) makes the relation between tail amplitude and preceding pulse
amplitude more evident (the relative positions of the tails in this case are arbitrary). Traces
a to f correspond to hyperpolarizations of increasing amplitude from -68 to -118 mV,
in 10 mV steps. B: activation curve (y (E)) obtained from tails at -38 (@), -28 (0),
-23 (A, data as in A) and -18 (A) mV. Same experiment as in Fig. 4. Curve drawn
by eye.
13 PH Y 314
386 D. DiFRANCESCO
where on depolarization inward transients are larger and the degree of activation of
kx at or near Ef, more positive than in low Nab solutions, is also larger. Fig. 6 shows
the correlation between degree of activation of if and the outward-decreasing tail
already observed in the largest depolarization in Fig. 5A, at 140 mM-Nab. When
hyperpolarizations are given down to -70 mV (traces a to c) no if current is activated.
The time course ofthe current during the following depolarization to 0 mV (particularly
trace c in Fig. 6B) is therefore attributable to components other than if. Only
following larger hyperpolarizations (traces d to f) does the outward-decreasing tail
appear and grow with the degree of if activation, indicating that the two are
correlated. In the presence of contamination of if by other components, the deter-
-38 -28
,t 1
I
-138

IA

-0-5

103 0 8 sec

nA

12
10 0~~~~~~

10~~~~~~

01

I , ,t ,

0 02 0*4
Time (sec)
Fig. 8. A: envelope test at- 138 mV (same experiment as in Fig. 4). Tail amplitudes
measured at -28 mV, where if is outward in the 35 mM-Nab, 9 mM-Kb Tyrode. B:
semilogarithmic plots of current onset at - 138 mV (@) and of tail amplitudes at -38
(A), -28 (0) and -18 (El) mV. Time constants from least squares fitting are 166 (@),
179 (A), 167 (0) and 171 (El) msec.
 IONIC NATURE OF if 387
mination of Ef can only be done approximately by selecting, as done in Fig. 5,
the voltage level just negative to the range where the outward-decreasing deflexion
starts to appear.
The time course of if-tails in the voltage range positive to reversal potential can
be further analyzed by plotting activation curves and by the use of envelope tests.
Fig. 7 shows how the amplitude of the outward-deactivating tail depends on the
preceding voltage level. Plotting the tail amplitude against voltage yields the if
activation curve. The curve in 35 mM-Nab has been obtained by plotting (after
scaling) tail amplitudes at -38, -28, -23 and -18 mV. All points can be fitted by a
single curve, confirming that the total current change observed in this voltage range
is indeed if. Results similar to those shown in Fig. 7 have been obtained in 140, 105
and 70 mM-Nab (three experiments). As observed with Kb changes (see Fig. 2), a
decrease in Nab has been observed to cause a small shift to the positive direction of
the y. curve half-activation point (+ 6 mV from 140 to 35 mM-Nab in the experiment
of Fig. 7). From the experiment of Fig. 5, the time course of the current appears to
become slower in low Nab. For example, half-times of 0-20, 0-29, 0-38 and 0 50 sec
are found for the if tails on depolarizing to -50 mV in 140, 105, 70 and 35 mM-Nab
respectively. This effect has not been investigated in detail, but a positive shift of
the time constant (rf(E)) curve along the voltage axis in low Nab, similar to that
observed for the y. (E) curve, could be partly responsible for it.
In Fig. 8 an example of envelope test for if activation at - 138 mV is shown. Tails
have been measured in this case at -28 mV, where the current is outward-deactivating,
after a -100 mv pulse of variable duration. The time courses of the tail amplitude
at -28, -38 and at -18 mV parallel that of the current activation (Fig. 8B),
indicating that the current tail observed on pulsing in the range -38 to -18 mV reflects
deactivation of it. Envelope tests performed in the range 35-140 mM-Nab, and
9-12 mM-Kb (four experiments) have yielded results similar to those shown in Fig. 8.
It is important to point out that the reliability of the Er measurements is dependent on the
assumption that interference from currents other than if is negligible during depolarizations as those
shown in Fig. 5. Plots of activation curves at potentials where if is positive and envelope tests as
those shown in Figs. 7 and 8 are consistent with the view that within the range investigated the
time course of the current on depolarizations reflects if decay. Interference from other components
cannot, however, be excluded (see for example Fig. 6), especially during high depolarizations when
activation of the transient outward current (ito) is reported to normally occur. This can, in principle,
affect the measurement of Ef. Until the relation between the time course attributable to ito and
that attributable to if during large depolarizations is clarified, Ef measurements as those shown
in Fig. 5 have to be interpreted with some caution.
Fully activated if(E) relations in different Nab
The fully activated if(E) relations in Fig. 9 have been obtained using the protocol
shown in Fig. 1, in different Nab in the range 4-4-140 mm. The current-voltage
relations do not show any significant deviation from linearity and are remarkably
parallel. Similar behaviour has been obtained in one other experiment in the range
35-140 mM-Nab. The major effect on if of varying Nab seems to be simply related
to a change in the driving force, with no detectable effect on the i(E) slope
conductance.

13-2
388 D. DIFRANCESCO

I'l .
mI'll
0-2

mV -1007

0~£

17-5,

05&
o 01
70 105~
140 mM-Nab
Fig. 9. Fully activated i,(E) relations in different Nab (concentrations marked in mm near
each curve). All 9 mM-Kb solutions containing 5 mM-MnCl2, 5 mm-BaCl2 and 5 x 10-6 g/ml.
TTX. Nal substitutedwith cholineCl. 2 sec, - 70/ -80 mVand2 sec. + 30/ + 40 mVpulses
were applied before clamping to the test potential (see Fig. 1). Least-squares linear fitting
gives slopes of 4 6, 4 9, 5'0, 5 1, 4-9, 4 9, 4 6 nA/mV (from 140 to 4'4 mM-Nab).

Effect of Kb in low Nab solutions

The hypothesis that K flows through the if channel is supported by the evidence
that Na cannot be the only ion carrying if, the values of Ef found in different Nab
being substantially more negative than the expected ENa (Figs. 5 and 9). An ion with
a more negative equilibrium potential must therefore also participate in carrying if.
The influence of Kb on Ef can be better analysed in a low Nab solution, as in this
case Ef lies in a relatively negative range. The experiment of Fig. 10 shows that
changing Kb does indeed alter Er in a way which implies that K ions also contribute
to carrying if. Ef was measured using a protocol similar to that in Fig. 5, in 3, 9, 18
and 36 mM-Kb in a solution containing 35 mM-Nab. Values obtained for Ef are -49,
-39, -34 and -26 mV respectively. In a low Nab solution the reconstruction of the
fully activated if(E) relationship in different Kb can be extended over a potential
range including Ef. This is shown in Fig. lOB for 3, 9, 18 and 36 mM-Kb. As in the
experiment of Fig. 3, linearity is observed over a wide voltage range at all values of
Kb, while outward-going rectification is apparent especially at negative potentials
and low Kb. Again as in the experiment in normal Nab of Fig. 3, increasing Kb
increases the f4(E) slope, in contrast to the absence of change in slope conductance
on changing Nab (Fig. 9). The increase in the channel slope conductance when Kb
is raised in the Nab range 35-140 mm is a constant finding (four experiments). An
interesting feature of the data in Fig. 10 is the tendency of the curves in different
Kb to converge to a common point.
Fig. 11 summarizes the results obtained in experiments where Ef is measured at
 IONIC NATURE OF if 389

A
50 mV [ _ __

I~~~~~~~~~~~~~

0 r -399 -3-4
-A0
-0-25 L a SMb
. s
.2 sec

-26 Ad -49 ---

N
c d

B 0/

-100 mV

pA

36r

Fig. 10. A: hyperpolarizations to activate if are applied from -39 mV in 9 (a), 18 (b), 36 (c)
and 3(d) mh-Kb, before the membrane is clamped at different levels in the range of current
reversal. Nearest values to Er marked near corresponding current traces. B: if(E) in
different Kb. Straight lines are drawn only in the range used for best fitting. Slopes of 2t2,
2-8, 3-7 and 5-4 nA/mV respectively for 3, 9, 18 and 36 mM-Kb. 35 mm-Nab (Tris
substitute) solution containing 5 mm-MnCl2 and 10 mm-BaCl2.
390 D. DiFRANCESCO
different values of Nab and Kb. In both cases, slopes of the Nernst plots are
substantially smaller than the -60 mV/decade required for a channel carrying a
single ion.
A serious problem for a quantitative analysis of the effects of changes in any
external cation concentration, is posed by the related changes in internal concentra-
tions. Even if pump activity counteracts changes in bulk concentrations, internal

£ mV

-50 A

A>

5 10 50 100 5 10 50
mmb
mM-Nab mM-Kb
Fig. 11. Nernst plots of Ef as a function of Nab (left) and of Kb (right) from four
experiments. Same symbols refer to the same experiment. The two curves in different Kb
are in 35 mM-Nab. In the same experiments (A and 0), Nab was also changed in 9 mM-Kb
Tyrode (Tris substitute). Points from these experiments have been fitted by single lines.
Filled circles (0) are from an experiment in 12 mM-Kb, and filled triangles (A) from an
experiment where choline Cl was used as a substitute for NaCl in 9 mM-Kb (see Fig. 9).
Least squares fitting slopes are (in mV/decade): 33-7 (A), 34-7 (A, 0), 29-1 (@) for the
Nab plots, and 25-7 for the Kb plot.

concentrations are bound to vary accordingly. Particularly for Nab changes, it has
been shown (Ellis, 1977) that in a certain range changes in Nab are followed by
proportional changes in the internal sodium concentration (Na1). From Ellis' data
(Fig. 10: see also Fig. 3 in Ellis & Deitmer, 1978) the general relation between Nai
and Nab seems to be Nai = aNab + b with b * 0. Thus, even if Nai cannot be regarded
as constant in a Na-replacement experiment, ENa will still vary, while this would be
possible only transiently if b were zero. Changes in Nai following Nab changes will
therefore decrease the slope of the Nernst plot calculated at steady-state conditions,
when only external ion concentration changes are taken into account. In the absence
of continuous monitoring of internal ionic activities, results such as those in Fig. 11.
can only be regarded as qualitative.

DISCUSSION
The new interpretation for the pace-maker current in the Purkinje fibre as an
inward current activated by hyperpolarizations in the pace-maker range (DiFran-
cesco, 1981) is based on the observation that the analysis of the properties of this
 IONIC NATURE OF if 391
current is distorted by K depletion occurring during hyperpolarizations. In normal
conditions, when a large iK, current flows, hyperpolarization-induced depletion gives
rise to an apparently time-dependent inward-decreasing current ('depletion'
component) (Baumgarten & Isenberg, 1977; DiFrancesco, Ohba & Ojeda, 1979). This
component overlaps with and distorts the time course of 'iK2' in such a way that
an apparent current reversal is observed on hyperpolarization. The reversal potential
(Erev) is close to, even if somewhat more negative than, the expected EK (Cohen, Daut
& Noble, 1976). Moreover, on changing Kb, Erev displays a -60 mf/decade slope in
the Nernst plot, as expected for a pure K current (Noble & Tsien, 1968; Peper &
Trautwein, 1969). The behaviour of 'iK2' in different Kb concentrations seems thus
to support the view that the current is a pure K current.
The 'if' hypothesis is, however, also consistent with the above experimental
observations. Computations using a non-uniform three-compartment model show in
fact that the appearance of a Erev slightly more negative than EK is an indirect
consequence of the presence of marked K depletion during a hyperpolarization
(DiFrancesco & Noble, 1980). Curiously enough, the Nernst plot of Erev against Kb
displays approximately a -60 mV/decade slope. Again, this is not simply a
coincidence, and it is due to the high dependence of iK, on voltage and on Kb in the
voltage range near Erev. The inward nature of if, normally masked by K depletion,
becomes apparent only when iK,, and therefore the K depletion associated with
it, is blocked by Ba. Thus the use of Ba allows the properties of if to be investi-
gated with the interference from accumulation/depletion processes much reduced
(DiFrancesco, 1981).
Effect of Kb in normal Nab
The behaviour of if when Kb is changed in the range 3-36 mm, as seen in Fig. 3,
suggests the possibility that the if reversal potential (Er) lies in the voltage region
positive to -50 mV, at all Kb concentrations. Reversal of the current is not found
in the voltage range negative to -50 mV at any Kb. The fully activated i(E)
relations obtained for all Kb values have a slope which increases with Kb, suggesting
that raising Kb involves not only a change in the driving force, but also an increase
in the channel conductance. This result is confirmed in lower Nab (Fig. 10).
Outward-going rectification is displayed by the i(E) curve in 3 mM-Kb in Fig. 3. Thus,
it looks as if the inward-going rectification of the ' K2' curve could be not only caused
by depletion, but also be a true channel property. However, it is difficult to assess
how much the K depletion still occurring during hyperpolarization when Ba is
present, both because of incomplete iK1 block and because if also, by carrying K, itself
contributes to depletion, can still distort the fully activated current-voltage relation.
The hypothesis that K depletion, although much reduced, is still present in
Ba-containing solutions, is favoured by the finding that in another complete
experiment like the one in Fig. 3 (not shown), pronounced outward-going rectification
was found in a similar voltage range over all the Kb range analysed (3-36 mm).
Effect of Nab
As seen in the experiment of Fig. 5, decreasing Nab causes Ef to shift to the negative
direction, indicating that Na crosses the if channel. A change in Nab simply causes
the i(E) relation to shift along the voltage axis (Fig. 9). In another experiment where
392 D. DiFRANCESCO
Nab was changed between 140 and 35 mm, the if(E) curve was also observed to
undergo simple shifts along the voltage axis (slope conductance of least squares fit
was 4-80, 4-32, 4-32 and 4 40 nA/mV, respectively, for the 140, 105, 70 and 35 mM-Nab
curves). These results agree with the hypothesis that on changing Nab, at least in
the concentration range analysed (see Fig. 9), the fully activated current depends only
on the Na driving force. The finding that if is partially carried by Na provides a
straightforward explanation for the known Na dependence of 'iK2' (McAllister &
Noble, 1966).
Effects of Kb in low Nab solutions
In their analysis, Noble & Tsien (1968) observed that the 'iK2(E)' relationships for
two different Kb concentrations display the cross-over phenomenon, i.e. positive to
a certain voltage the current corresponding to the higher Kb is larger than that
corresponding to the lower Kb. The present data indicate that the cross-over
phenomenon is an artifact due to K depletion and its influence on the analysis of
the properties of the pace-maker current. The dependence of the if(E) relation on Kb
shown in Fig. 10, where distortion by the iK,-induced depletion is limited, indicates
that as in the case for Nab changes, changing Kb causes Ef to shift, in agreement with
the view that K does contribute to carrying if. The tf(E) relations show a good degree
of linearity, and no cross-over is visible in the voltage range negative to -50 mV.
It is interesting to note that all the if(E) curves converge towards a common point
at about -10 mV. As observed in normal Nab (Fig. 3), raising Kb not only shifts
Ef in the positive direction, but also increases the channel conductance.
It is now possible to predict how the cross-over phenomenon described by Noble
& Tsien comes about. The dependence of iK, on voltage and on Kb is extremely steep
in the voltage range near EK. Thus, when Kb is increased, hyperpolarizing to a fixed
potential will produce a much larger iK1 change, and hence a much larger inward-
decreasing 'depletion' component. Even if the inward-activating if also augments
during the same hyperpolarization, due to its dependence on Kb, its increase is offset
by the 'depletion' component. This results, as observed experimentally, in a more
positive Erev In higher Kb, therefore, the positive shift of Erev combined with the
increased pace-maker current at more positive potentials, will cause the appearance
of a cross-over.
In conclusion, the pace-maker current if in Purkinje fibres is a mixed current,
carried partly by Na and partly by K. Under normal conditions, the current activates
on hyperpolarization from a threshold ranging between -40 and -60 mV, and is
completely saturated at about -100 to -110 mV. The evidence that Na participates
directly in carrying if clarifies the dependence of the current on Nab, which was not
satisfactorily explained in the original 'iK2' model. The fully activated if(E) relation
is linear over a relatively wide voltage range around the reversal potential, and tends
to display outward-going rectification in the far negative region. It is possible that
accumulation/depletion processes associated with the K component of if contribute
to distorting the if(E) relation. Changes in Nab produce only a shift in the position
of the if(E) curve along the voltage axis, while changing Kb also affects the if(E) slope
conductance.
 IONIC NATURE OF if 393
I should like to thank A. Ferroni and D. Janigro for assistance at various stages during this work,
and D. Noble for comments on the manuscript. Equipment was provided by the University of
Milano and the C.N.R.
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