The Action of Certain Polyvalent Cations
on the Voltage-Clamped Lobster Axon
M. P. B L A U S T E I N and D. E. G O L D M A N
From the Naval Medical Research Institute, Bethesda, Maryland 20014. Dr. Blanstein's
present address is Department of Physiology, Cambridge University, England. Dr.
Goldman's present address is Department of Physiology, Woman's Medical College,
Philadelphia, Pennsylvania 19129
ABSTP.ACT Calcium appears to be an essential participant in axon excitation
processes. Many other polyvalent metal ions have calcium-like actions on
axons. We have used the voltage-clamped lobster giant axon to test the effect
of several of these cations on the position of the peak inidal (sodium) and steadystate (potassium) conductance vs. voltage curves on the voltage axis as well as
on the rate parameters for excitation processes. Among the alkaline earth
metals, Mg +9 is a very poor substitute for Ca +s, while Ba +a behaves like "high
calcium" when substituted for Ca +~ on a mole-for-mole basis. The transition
metal ions, Ni +~, Co +s, and Cd +~ also act like high calcium when substituted
mole-for-mole. Among the trivalent ions, La +8 is a very effective Ca +~ replacement. AI+a and Fe +8 are extremely active and seem to have some similar effects.
A1+8 is effective at concentrations as low as 10-5 u. The data suggest that many
of these ions may interact with the same cation-binding sites on the axon m e m brane, and that the relative effects on the membrane conductance and rate
parameters depend on the relative binding constants of the ions. The total
amount of Na + transferred during a large depolarizing transient is nearly independent of the kind or amount of polyvalent ion applied.
INTRODUCTION
E v i d e n c e has long been available t h a t c a l c i u m ions are i n t i m a t e l y associated
with the m e c h a n i s m of a x o n c o n d u c t i o n (see Brink, 1954). Studies such as
those of H 6 b e r (1920), a n d of G u t t m a n (1940) showed t h a t the resting p o t e n tial a n d excitability of axons c a n be effectively m a i n t a i n e d w h e n c e r t a i n o t h e r
d i v a l e n t cations, p a r t i c u l a r l y those of the alkaline e a r t h series, are substituted
for calcium. T h e effects of alkali m e t a l ions on n e r v e excitability h a v e b e e n
reviewed b y N a r a h a s h i (1966). F r a n k e n h a e u s e r a n d H o d g k i n (I 957) studied
the effects of c a l c i u m on the m e m b r a n e ionic c o n d u c t a n c e s of voltagec l a m p e d squid axons. R e c e n t studies b y D o d g e (1961) a n d b y T a k a t a et al.
(1966 b) d e m o n s t r a t e t h a t nickel a n d l a n t h a n u m h a v e c e r t a i n effects on the
electrical properties of voltage-clamped axons w h i c h are qualitatively similar
279
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to those of calcium. These similarities suggest the presence in the axon
m e m b r a n e of negatively charged sites with which calcium or its substituents
interact and that the interaction plays a controlling role in the conduction
process. It thus seems of interest to study in some detail the effects of a n u m b e r
of polyvalent cations on the electrical properties of axons using the voltageclamped giant axon of the lobster. A preliminary report of some of these
results has appeared (Blaustein and Goldman, 1966 a).
MATERIALS
AND
METHODS
T h e sucrose g a p t e c h n i q u e , v o l t a g e c l a m p a n d dissection m e t h o d for t h e lobster
g i a n t a x o n h a v e b e e n p r e v i o u s l y d e s c r i b e d ( J u l i a n , M o o r e , a n d G o l d m a n , 1962). O n l y
TABLE I
E F F E C T OF P O L Y V A L E N T C A T I O N S ON
S O D I U M AND P O T A S S I U M C O N D U C T A N C E VS.
MEMBRANE POTENTIAL CURVES
Sodium conductancc~
AVM[[
Ion
EcI-Eq exchange
with Ca+S
Av~
Avy¶
ECl-Eq exchange
with Ca +~
Fivcfold concerttration change
AV~
Fivcfoldconcentration change
my
Ca +2
Mg +~
Co +2
Cd +2
Ba +~
Zn +2
Ni +2
La +~
Potassium conductancc~
tnv
rag
12 (17)
10 (1)
--10 (5)
+ 9 (2)
+ 7 (4)
+ 7 (5)
+ 9 (1)
-t-12 (10)
+22 (12)
8
(2)
12
10
(4)
(4)
--9
+17
-t-23
-t-13
m~
(5)
(2)
(4)
(5)
-t-20 (10)
+37 (12)
10 (17)
4 (1)
13 (2)
10
21
(4)
(4)
:~ Sodium conductance at large depolarizations calculated as:
g'N,,
Ipl
VM -
V~
'
where Ipx is the corrected peak initial current; VNa is the apparent sodium equilibrium potential; i.e., the positive potential at which IPr = 0, and V~ is the membrane potential.
§ potassium slope conductance calculated as:
d/ss
g*K -- d V ~ '
where I , , is the corrected steady-state current.
[[ The magnitude of the shift of the normalized conductance vs. voltage curve along the voltage
axis when an equimolar concentration of another cation is substituted for 50 or 60 m E q / l i t e r
of calcium. Positive values mean that the conductance curve is shifted in the direction of depolarization.
¶ The magnitude of the shift of the normalized conductance curve along the voltage axis when
the polyvalent cation concentration is changed fivcfold. Numbers in parentheses indicate the
number of measurements.
M. P. BLAUSaXmAND D. E. GOLDMAN Aaion of Polyvahnt Cations on Nerve
281
axons with resting potentials greater than 95 mv, as measured in the sucrose gap,
were voltage-clamped. Clamping (holding) potentials were greater than I00 mv and
no correction was made for resting sodium inactivation (cf. Blaustein and Goldman,
1966 c).
T h e seawater used was Dalton's (1958) lobster solution, modified by the addition
of 4 rnM Tris(hydroxymethyl)aminomethane, adjusted to p H 7.4. This solution normally includes 50 m E q of calcium and 16 m E q of magnesium per liter. I n most of
our studies only a single polyvalent cation was used, as the chloride, at a concentration
of 60 m E q per liter. In some instances, the polyvalent cation concentration was
reduced to 12 mEq/liter and osmolarity maintained by the addition of sucrose.
Control experiments showed that the sucrose had a negligible effect. I n several
experiments, the magnesium was not omitted and the various polyvalent cations were
then used to replace the 50 mEq/liter of calcium in the controls. T h e solutions containing 60 m E q / l i t e r of trivalent ion were slightly hypotonic since 20 m_~ of trivalent
cation replaced 30 m ~ of CaCI2.
ASW
(5OmEq/liter Co+z ) 50mEq/liter Lo+ ~
A SW
I00 m v
5OmEq/liler Ni +2
ASW
A SW
Fxou~ 1. Axon L-50-65. T -5°C. Effects of La +3 and of Ni+2
at 50 mEq/liter on the action
potential.
t KC
T h e voltage and current-time data were recorded on film and analyzed with the
aid of a film-reading device and a digital computer. The current-time curves were
corrected for leakage using as a basis the current-time curves from hyperpolarizing
pulses and assuming that these were essentially linearly dependent on the voltage.
These curves normally decay to a steady-state value with a time constant of the order
of 0.3 msec (cf. Moore et al., 1966).
T h e reduced data were plotted as peak initial and steady-state currents, peak
initial and steady-state conductances, and certain time parameters, all as functions
of m e m b r a n e potential. Peak initial conductances (g~a) were referred to that potential at which the early transient current appeared to reverse direction (cf. Table I,
first footnote). T h e steady-state conductances (g~*) were calculated as slope rather
than chord conductances since the potassium equilibrium potential was not obtained
(cf. Table I, second footnote). I n certain solutions such as lanthanum seawater, the
time to reach the steady state was greatly prolonged so that it was difficult to be
certain that the steady-state values obtained were completely reliable. Thus, the
actual values of g* m a y sometimes have been greater than the measured values.
T h e time parameters used were the time from the beginning of the step to the
peak transient current and the time required to reach two-thirds of the corrected
steady-state value.
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RESULTS
Effects on Action Potentials
I n c o n f i r m a t i o n of studies b y M o o r e et al. (1966) on the effects of l a n t h a n u m
on the squid a x o n a n d those of D o d g e (1961) o n the action of nickel o n the
4 -
E
O
E
0
/ rs sE
qJ
r-
-2
E
-4
-6
I
-60
I
-40
I
-20
0
I
+20
I
+40
+60
Membrane Potentiol (my)
FIOURE 2. Axon L-58-65. T = 7°C. Current-voltage curves for peak transient and
steady state for normal calcium in artificial seawater and for two concentrations of Ni +2
substituted for the calcium.
frog node, b o t h of these ions, w h e n substituted for c a l c i u m in the lobster axon,
slow the rates of rise a n d fall of the action p o t e n t i a l (Fig. 1). N o t e t h a t the
voltage at the p e a k of the action p o t e n t i a l is also s o m e w h a t r e d u c e d d u r i n g
t r e a t m e n t with either of these ions.
M . P. B~UST~XN AND D. E. GOLD~AN
Action of Polyvalent Cations on Nerve
283
Effects on Ion Conductances
As has been shown in squid axons (Frankenhaeuser and Hodgkin, 1957), in
myelinated fibers (Frankenhaeuser, 1957), and in lobster axons (Julian et al.,
1962; Blaustein and Goldman, 1966 b), an increase in external calcium shifts
the sodium conductance curve to the right along the voltage axis so that a
larger depolarization step is needed to obtain any given level of conductance.
Conversely, a reduction in external calcium shifts the conductance curve in
the opposite direction. The" m a x i m u m conductance is little affected b y these
changes. There is a b o u t a 10-12 my shift along the voltage axis for a fivefold
A
:iEqilr
~'~-
J
J~ ~s
-J6o
l
/
0
,
0 60
0
0.5-
r
-4o
-210
+210
Membrane Potentiol(my)
I
+40
I
+60
0.5-
FTOuRJz 3. A x o n L-58-65. T = 7°C. S o d i u m c o n d u c t a n c e vs. m e m b r a n e potential
curves for the same artificial seawaters as in Fig. 2.
change in calcium concentration (6-8 m y per e-fold change). Similar effects
also occur with respect to the potassium conductance.
Current-voltage relations for an axon in which nickel was substituted for
calcium (Fig. 2) show that 12 m E q / l i t e r of nickel appear to be a good replacement for 60" m E q / l i t e r of calcium. O n the other hand, the peak initial and
steady-state current curves for the 60 m E q / l i t e r of nickel resemble the curves
one would expect to obtain with a large increase in external calcium. Data
from this experiment plotted as sodium conductance vs. voltage (Fig. 3)
indicate that while the curves for the 60 m E q / l i t e r of calcium in the seawater
and 12 m E q / l i t e r of nickel in the seawater are nearly superposable, the
sodium conductance curve for the 6 0 m E q / l i t e r nickel seawater is shifted
a b o u t 10 mv to the right along the voltage axis. This is roughly where one
would expect the curve to lie for a four- to fivefold increase in calcium concentration as compared with the normal concentration. When 60 m E q / l i t e r of
284
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m a g n e s i u m are substituted for c a l c i u m ( T a b l e I), the s o d i u m c o n d u c t a n c e
c u r v e shifts a b o u t 10 m v to the left along the voltage axis, r o u g h l y paralleling a
fivefold decrease in e x t e r n a l calcium. If the m a g n e s i u m c o n c e n t r a t i o n is t h e n
r e d u c e d fivefold, t h e r e is a f u r t h e r 10-12 m v shift to the left. Corresponding:
d a t a were o b t a i n e d for several o t h e r ions ( T a b l e I).
•
°
.A.*.
!
.~"
o
•
-2.0
a.
•
~o
a
_A a
a•
Z~
t
~
- - /
~ {
i ''0 g "~
¢J E
CO*:' Le÷3 Sucrose
mEq / liter
mM
60
0
0
o
E'¢-
A
I
-80 -
-60
-4~-
-20
Membrane
Fmuzu~ 4.
Axon L-56-65. T
w i t h ca]Glum o r l a n t h a n u m
ION
Ca+z
Ca+~
La+3
LQ+3
VM
Conc,
(mEq/liJer)
60
12
60
12
-40mv
l
*20
0
Potential
0
I
*=tO
60
0
I
*60
+80
(my)
= 5°C. Sodium conductance vs. membrane potent:&
i n a r t i f i c i a l s e a w a t e r at i n d i c a t e d c o n c e n t r a t i o n s .
o
•
8.0
°
a
•
0
+40my
FmURE 5. Axon L-56-65. T = 5°C. Steady-state current vs. membrane potential with,
calcium or lanthanum in artificial seawater at indicated concentrations.
Ions which, w h e n substituted for c a l c i u m on a mole-for-mole basis act like
high calcium, often seem to p r o d u c e a decrease in the m a x i m u m s o d i u m
c o n d u c t a n c e (plateau level of the c o n d u c t a n c e curve). T h i s is also n o t i c e a b l e
to a slight e x t e n t with high c o n c e n t r a t i o n s of c a l c i u m itself ( T a k a t a et al.,
1966 a; Blaustein a n d G o l d m a n , 1966 b). In o r d e r to separate the h o r i z o n t a l
M. P. BI~,Us'rEL~AND D. E. GOLDMAN Action of Polyvalent Cations on Nerve
285
shifts in the conductance curves from the vertical shifts (depression of conductance), we have measured the horizontal shifts after displacing the sodium
c o n d u c t a n c e curves vertically to make the plateau regions coincide. W h e n this
is done, it turns out that 60 m E q / l i t e r of c a d m i u m shift the sodium conductance curve about 5 m y to the right along the voltage axis. O n the other hand,
a fivefold reduction in c a d m i u m concentration shifts the curve 8-10 m y to
-..the left along the voltage axis. W h e n l a n t h a n u m is substituted for calcium
[ON
Ca
Ni
\t,
\
\
\
Ni
£
60
:
1.6o
"TIME TO PEAK OF I~--=U SCALE
&,TME TO 2/3 ISS . . . . RISCALE
&
•
CONC (mEcl/liter)
60
o
12
A\
\
2,O
\o
hJ
~oo
•
I--
•
I,£
xo
hi
"-%.
°~ o
20
FIGURE 6. Axon L-58-65. T --7°C. T i m e parameters vs. m e m b r a n e potential for axon in calcium
and in nickel solutions.
e•
-40
o
+4b
MEMBRANE POTENTIAl (my)
+8o
(equivalent-for-equivalent) even larger displacements are found (Fig. 4),
.although the shift due to changes in the concentration of l a n t h a n u m ion
itself is approximately the same as for the other polyvalent ions.
T h e effects of two concentrations of l a n t h a n u m and two concentrations of
.calcium on the steady-state current vs. voltage curve are shown in Fig. 5. T h e
curve shifts progressively to the right as the calcium concentration is increased
from 12 m E q / l i t e r calcium to 60 mEq/liter, then exchanged for 12 m E q / l i t e r
of lanthanum, and then the latter is increased to 60 mEq/liter. T h e m a x i m u m
slope conductance (i.e., the slope of the current-voltage curve at large depolarization steps) is only slightly reduced in the l a n t h a n u m solutions.
Table I summarizes some of these effects. Note that all the ions tested give
roughly a 10-12 m y shift in the sodium conductance curve along the voltage
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axis for a fivefold concentration change. For the most part, this also holds
true for the potassium conductance curve (cf. Figs. 2 and 5). T h e apparently
large deviations in the steady-state conductances during l a n t h a n u m treatment
(Table I) are in part attributable to the smaller ratio of Na and K currents
to the teakage current, and to the possibility that a "steady state" m a y not
have been reached by the end of a 10 or 20 msec pulse.
The data in Table I also show that replacement of one ion by another
(equivalent-for-equivalent) shifts the conductance curves to a varying degree.
°°%°'°",~
?
'.
Go /
"
3o
~ .
~ 4
o./~/
] Co"2 LO"3
J .~q/,,e,
I 60
0
o
12
.
o
I m o
I o
~\
-/~°
\ ,
\
to
2,
x
"o
\
"
"~
~
I 0 60
o
o~ ~
1 T~METO PEAK OF Ipi--=Lt scole
" I= ~' T I M E T 0 2 / 3 Iss. . . . Rtscde
2
o
~\
\o
~ °O oDO
LO
-
-40
O
+40
MEMBRANE POTENTIAL (my)
+80
+120
FmUR~ 7. Axon L-56-65. T = 5°C. Time parameters vs. membrane potential for
axon in calcium and in lanthanum solutions.
Magnesium, for example, shifts the sodium conductance curve about 10 mv
to the left; whereas the other ions shift the sodium conductance curve to the
right. The effects of cobalt, cadmium, barium, and zinc are equivalent to
about a t h r e e - t o fourfold increase in calcium concentration; while nickel is
about 5 times and l a n t h a n u m 20-25 times as effective as calcium. In a few
instances, two polyvalent cations (e.g., calcium and nickel or calcium and
l a n t h a n u m ) were present in the seawater and the effects of these ions on the
conductance curves appeared to be additive (cf. Dodge, 1961).
Effects on Time Parameters
An obvious effect of nickel and lanthanum, for example, on the action potential is a slowing of the rates of both rise and fall. Time parameter data for
Ni+2 and La +~ are shown as functions of the m e m b r a n e potential in Figs. 6 and
M . P. BLAUSTEIN AND D. E. GOLDMAN Action of Polyvalent Cations on Nerve
~87
7. Most of the ions tested when substituted for calcium mole-for-mole
appear to displace these curves to the right; i.e., they increase the time to the
peak and to two-thirds of the steady state at a given membrane potential. A
similar effect is produced by raising polyvalent ion concentration (Frankenhaeuser and Hodgkin, 1957; Goldman and Blaustein, 1966). Magnesium
displaces the curves to the left as does the lowering of the polyvalent cation
concentration (Figs. 6 and 7).
•
•
O
-
1.5
&
•
A
0
E
Ca+2 Lo+3 Sucrose
mEqlliter
mM
~,
o
"~
o
60
0
0
0.5 "~ ._c
•
t2
0
25
,~,
~
"o
o~
i
I
-40
I
I
-20
I
t
A
0
60
0
•
o
;2
25
I
+20
I
J
+40
Membrane Potential (my)
FIGURE 8. T i m e integrals o f s o d i u m c o n d u c t a n c e vs. m e m b r a n e potential. S a m e a x o n
as in Fig. 4. See text for m e t h o d o f calculation.
The multiple effects of these different ions may be further analyzed by
calculating the time integrals of the transient current. These represent approximately the amount of sodium transferred during the lifetime of the
transient. 1 Using lanthanum as an example, the data have been plotted (Fig.
t T h e time integrals were calculated from the early transient part of the current-time curves after
subtraction of the leakage currents. Because a single axon rarely lasts more t h a n an hour, and bec a m e the time required to change solutions, reach a steady state, and perform a voltage-clamp r u n
takes about 10 rain, the separation of sodium and potassium contributions to the early current was
not carried out. If, as the data in Table I suggest, the time- and voltage-dependent parameters for
sodium and potassium are affected by about the same amount, we would expect that at
large depolarizing voltages the relative contributions of the late (potassium) conductance to the
early transient should remain about the same w h e n the polyvalent ion, or its concentration, is
changed.
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8) in terms of conductances rather than currents, i.e.
f IN, dt---- (V,,-- VN,) I gN, dt
where V~ is the step potential and V•, is the potential at which the early
current reverses direction. T h e integration has been carried out over the
duration of the transient. Despite the apparent concentration-dependent
depression of sodium conductance by lanthanum (cf. Fig. 4), the sodium
transferred at large depolarizations appears to be unaffected by the substitution of lanthanum for calcium, and is independent of the polyvalent cation
concentration.
In addition to La +3, several other trivalent cations have been tested, inIon
(60mEq/liter)
CO+2
Time (min: sec)
0.00
1:05
1:05
2:48
AI+~
[~[[[
5:09
1:16
5:04
_=r-'-...__ __j~..____
5:15
5:16
Ca +2
i0:42
lOOmv
FIOURE9. Axon L-62-65. T
= 7°C. Effect on action potential of substitutions of Cr+3 or
A1+3 for calcium. Numbers
under tracings are times (in
minutes and seconds) after the
start of the experiment.
IK C
cludign Al+3, Cr+3, and Fe +3. When substituted for Ca +~ on an equivalent-forequivalent basis, these ions block the action potential. T h e effects are reversible
if the trivalent ion is washed out within a minute or less (Fig. 9). A significant
effect of A1+3 m a y be obtained when this ion is added to normal seawater at
concentrations at least as low as 0.03 mEq/liter. There is also a marked
reduction of early transient current resulting from the addition of 0.03 m E q /
liter of A1+3 to seawater. This ion also reduces the steady-state current.
Cr+8 and Fe +3 have similar effects on the voltage-clamped axon. These trivalent ions also appear to delay the time-to-peak b u t the effects are partially
obscured b y the large reduction in the magnitude of the currents.
DISCUSSION
It is clear that a large n u m b e r of polyvalent cations can replace calcium in
the environmental m e d i u m in the sense that the axon m a y remain excitable
and give rise to voltage clamp curves which qualitatively resemble those of
M. P.
BLAUSTEIN AND
D. E.
GOLDMAN
Action of Polyvalent Cations on Nerve
289
axons in a normal medium. There are, however, important quantitative
differences. The current-time curves appear to be, roughly at least, stretched
out or shrunken along the time scale, as well as along the amplitude scale. For
myelinated fibers, there is considerable evidence (Del Castillo-Nicolau and
Hufschmidt, 1957; Dodge, 1961; Takahashi et al., 1960; Meves, 1963) that
several polyvalent cations including nickel and cobalt increase the duration
of the action potential just as they do in the present studies. However, the
list of ions tried is by no means complete. We have used Mg, Ca, Ba, Zn, Co,
Ni, and Cd among divalent ions and A1, La, Cr, and Fe among trivalent ions.
There are" many more, but except when they have a strong heavy metal
poisoning or other special effect, such as may be produced by Pb or Hg, most
polyvalent ions could be expected to behave similarly. It should be noted that
A1, Cr, and Fe are so effective as to raise the question whether they behave
only as calcium replacements even though trivalent ions may be expected to
be more potent, possibly because of the greater charge. In this respect, more
work needs to be done.
The marked similarity of qualitative effects of these ions strongly suggests
that if there are anionic sites in the membrane which are essential participants
in the excitability control mechanisms, any of these ions may interact with
these sites in a similar fashion. The quantitative differences can be attributed
primarily to differences in the rate constants of association and dissociation for
the various cations. Further, dissociation of the site-cation complex may well
be a first step in the changes subsequent to membrane depolarization. Thus,
for example, an increase in the time parameters for the transient and steadystate currents could arise from a relative decrease in the dissociation rate
.constant and this would then lead to a lengthened action potential. If the
"sodium-on" process is slowed more than the "sodium-off'' process, there
would also be a decrease in the plateau value of the sodium conductance,
even though, as has been shown, the total sodium transferred during a transient remains nearly unchanged at large potential steps. This lack of effect of
polyvalent cations on sodium transfer" may be contrasted with the action of
.certain anesthetics such as tropines or barbiturates, which reduce sodium
transfer at all membrane potentials (Blaustein, unpublished data). A possible
interpretation of this phenomenon would be that polyvalent ions affect the
speed of operation and the voltage response of the mechanisms that control
the flow of Na and K without, however, reducing the total number of sites
involved. The anesthetics, on the other hand, may also reduce the number of
available sites. However, more data are needed before any definite conclusions
can be drawn.
We have also found that although each ion has its own degree of relative
activity, the displacement of the conductance curves per fractional change of
ion concentration is nearly the same for all the ions tested. This may be inter-
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preted in the following way. When the axon is sufficiently polarized, both the
sodium and potassium conductances appear to be exponential functions of the
potential. T h e y are then also approximately inversely proportional to the
concentration of the polyvalent ion and independent of its binding strength.
However, when the ion is changed, so is the binding constant and with it the
position of the conductance curve on the voltage axis. The amount of displacement should also be related to the value of any coefficient which occurs
in the exponent (see Frankenhaeuser and Hodgkin, 1957; Goldman, 1964).
In the presence of more than one ion species, competition for the sites
should occur. It has been shown (Blaustein and Goldman, 1966 b) that calcium
and procaine appear to compete for these anionic sites. We would expect that
other polyvalent cations would likewise compete. T h e data obtained by
Khodorov and Beliaev (1964) and Takahashi et al. (1960) indicating that Ni,
Cd, and Co interact with procaine in the frog myelinated fiber are thus
consistent with the view expressed here. Carvalho and Sandow (1966) and
Sandow and Isaacson (1966) have discussed similar effects on the excitation
and contraction of muscle. In addition, a n u m b e r of polyvalent ions have
been shown to compete both with each other and with procaine for binding
to phospholipids in vitro (Blaustein, 1967). It would be very useful to know
the binding parameters for eacff ion. T h e material presented here gives only
a rough indication; more data are required for characterizing the anionic
sites and for helping in their exact identification.
The authors are grateful to Professor A. L. Hodgkin for helpful suggestions concerning the manuscript.
From the Bureau of Medicine and Surgery, Navy Department, Research Task MR-005.08-0020.02.
The opinions or assertions contained herein are the private ones of the authors and are not to be
construed as official or reflecting the views of the Navy Department or the naval service at large.
Received.for publication 12 October 1966.
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