J. Exp. Biol. (197*). 56, SSi-563
With 8 text-figures
Printed in Great Britain
IONIC DISTRIBUTION IN AMOEBA PROTEUS
BY ROBERT D. PRUSCH* AND PHILIP B. DUNHAM
Department of Zoology, Syracuse University, Syracuse, New York
(Received 18 October 1971)
INTRODUCTION
In amoebae the concentration of K greatly exceeds that of Na, but both are generally
greater than the external concentrations. The factors determining the distribution of
inorganic ions and maintenance of ionic gradients in protozoa are by no means clear.
Klein (1959, 1961, 1964) showed in a series of studies with Acanthamoeba sp. that both
Na and K were actively accumulated from the environment as well as being bound
intracellularly. Chapman-Andresen & Dick (1962) postulated the active expulsion of
Na and passive distribution of Cl ions in Chaos chaos. They felt that ion binding,
especially of Na, played little in the role of ionic regulation of this animal. On the
other hand, Bruce & Marshall (1965) discount the active transport of any ionic species
across the plasmalemma of Chaos chaos. The present study was undertaken to investigate the distribution of inorganic ions across the plasmalemma of Amoeba proteus
and how this distribution is maintained.
The results of this study demonstrate that both Na and K are actively transported
in Amoeba proteus. K is actively accumulated from the environment and Na is actively
eliminated from the cell, although other factors are probably partially responsible for
the distribution of both Na and K. The effect of Ca on Amoeba proteus in a high-Na
medium is to decrease the internal concentration of Na by decreasing the Na permeability of the plasmalemma. Ca acts by associating with binding sites, perhaps in the
plasmalemma.
MATERIALS AND METHODS
Culture techniques
Samples of Amoeba proteus were obtained from mass cultures grown in PrescottJames medium (1955) and fed on Tetrahymenapyriformis. The composition of freshly
made Prescott-James medium (control medium) and that from which the amoebae
have been harvested is given in Table 1. The cultures were kept at 17 °C in the dark,
and were fed and washed every 48 h. The generation time of the amoebae under these
conditions was approximately 48 h; 1 g of amoebae could be harvested from a culture
dish (14 x 9 in.) after 4 days. Before being used in an experiment the amoebae were
starved for 24 h and washed twice in fresh culture medium.
Cell weight and volume
After experimental treatment a 25 ml suspension of cells was concentrated by gentle
centrifugation for 1 min. The cells were then packed into pre-weighed 10 ml Kolmer
• Present address: Division of Biological and Medical Sciences, Brown University, Providence,
Rhode Island 03912.
35
B I B 56
55 2
ROBERT D. PRUSCH AND PHILIP B. DUNHAM
Table i. Intracellular K, Na, Ca and Cl concentrations of Amoeba proteus and PrescottJames medium, both fresh (control medium) and that from which the cells have been
harvested
(Standard errors and number of determinations are given.)
Cells
mM/kg cells
K
Na
Ca
Cl
24-8310-43(63)
i-o8±o-O7 (60)
2-93 ±0-03 (26)
973 ±0-13 (45)
mM/1
Medium
Control
K
Na
Ca
Cl
008
o-o
0-03
008
Culture
oi4±o-os
o-o2±o-o2
o-io±o-O4
016±008
(35)
(45)
(20)
(20)
tubes by centrifugation at i6oog for 5 min. Wet weight of each sample was at least
100 mg. Since sample volume and thus the number of cells per sample was kept constant throughout each experiment, changes in the wet weight of the packed pellets
were proportional to changes in cell volume.
Extracellular space
Extracellular space of the packed cell pellets was determined by adding 0-02 fiCi of
[14C]inulin (New England Nuclear Corp.) to the Kolmer tube containing the cells just
prior to centrifugation. Exposure of the cells to inulin for up to 30 min did not change
the apparent extracellular space. Therefore the cells did not accumulate inulin to a
degree that would give erroneous estimates of the extracellular space. Radioactivities
of samples of the cell extracts (see below) and dried samples of the supernatant were
determined in a gas-flow counter. The mean extracellular space used in all experiments
to calculate intracellular concentration was 8-3 ± 1-4 (s.D., N = 65).
Chemical analyses
Cell pellets were extracted for chemical analysis in 10 ml of distilled water plus
2 drops of glacial acetic acid. These suspensions were heated near boiling in a water
bath for 10 min and allowed to stand overnight. Cellular debris was then removed by
centrifugation. Na, K and Ca concentrations were analysed with a Coleman flame
photometer. Cl concentrations were determined with an Amino-Cotlove Cl titrator.
Amounts were expressed as mM/original kg cells, after correction for extracellular
space. In most experiments cell volume was constant within 10%.
Flux measurements
Unidirectional influx and extent of exchange of Na, K and Cl were determined by
adding trace amounts of ^Na, 42K (Cambridge Nuclear Corp.) or MC1 (Nuclear
Chicago) to the medium. The wet pellet and samples of the medium comparable to
the pellet weight were counted in a Nal-TI crystal scintillation well counter. All
counts were at least 10 times background.
Ionic distribution in Amoeba proteus
553
Electrical measurements
Membrane potentials were measured using glass micropipettes filled with 3 M-KC1.
Resistance of these micropipettes ranged from 15 to 30 MO. The recording chamber
consisted of a glass slide with a circular depression 3 mm deep and 16 mm across.
An Ag-AgCl wire circled the base of the chamber and served as the indifferent electrode. No means were employed to restrict the movement of the amoebae.
Resistance of the plasmalemma was determined using two of the micropipettes
inserted into a cell. Current in the form of a depolarizing or hyperpolarizing ramp
(5 x io" 9 A/sec) of about 3 sec duration was applied to one micropipette through
a 100 MXi resistor from a modified Tektronix 162 waveform generator. The ramp
voltage also served to trigger and drive the horizontal sweep of a Tektronix 564
storage oscilloscope. The other micropipette, used to record changes in membrane
potential, was connected through a d.c.-pre-amplifier to the vertical amplifier of the
oscilloscope. The resulting current-voltage tracings on the oscilloscope were photographed with a Polaroid camera. The slopes of the tracings were used to calculate the
resistance of the plasmalemma.
Surface-area measurements
The surface area of the amoeba was determined after mechanical stimulation of
the slide containing the amoebae until they assumed a roughly spherical shape. The
diameter of the amoeba was then measured with a calibrated ocular micrometer. The
resistance of the plasmalemma in Mii per cell was converted into specific resistance by
multiplying by the surface area of the cell to give kii. cm2. The mean surface area
(25 measurements) of Amoeba proteus was 5 x io~* cma and the mean volume of
a single cell was 1 x io" 6 cm8.
Calculations
(a) Unidirectional fluxes. In a closed two-compartment system the rate coefficients,
&! and k0, of unidirectional efflux, Mo and influx, M,, respectively in moles/time are:
where [C]o and [C]l are the outside and inside concentrations of the substance and Vo
and Fj are the volumes of the two compartments. At steady state where M = M, = Mo,
The rate of change in isotope concentration in the inside compartment is (Sheppard
& Martin, 1950):
d[C]t _
dt ~ V0[C]0
where [C]f and [C]* are the isotope concentrations in the inside and outside compartments.
With constant volume, equation (2) can be rearranged and integrated between
t = 0 and t = 00 to
[CIV, = [Ctf(»,(i-«-*)•
(3)
Equation (3) is used to determine the value of (^1 + ^,).
35-3
ROBERT D. PRUSCH AND PHILIP B. DUNHAM
554
120 r
100
80
IT 60
s
40
20
20
40
60
80
100
[Na]u(mM/l)
Fig. i. [Na]j as a function of [Nal,. Cells were equilibrated for 4 h in control medium plus the
Na,, indicated. Each point represents the mean of 12 determinations in mM/kg cells, and the
brackets are the S.E. of the mean. The broken line connects points of equal cellular and external
Na concentration.
From £j + k0, and the known volumes and concentrations, the unidirectional influx
in moles/time. volume, Ml[V1, can be calculated by rearranging equation (1):
(4)
RESULTS
Intracellular concentrations
The intracellular concentrations of K, Na, Ca and Cl in Amoeba proteus are given
in Table 1. The cells were grown in Prescott-James medium, the composition of
which is also given in Table 1. Concentrations of K, Na, Ca and Cl are all higher in the
medium in which the cells were grown than in the fresh medium, probable due to the
addition of Tetrahymena to the culture medium. All of the measured internal concentrations are higher than those of the medium, but as in most other cells K is the
predominant inorganic cation.
Net Na influx
Net Na influx was measured after adding NaCl to the control medium. When
external Na is increased to 20 mM, the new steady-state (Na), (25 mM/kg cells) is
reached after approximately 3 h. There was a concomitant net efflux of K which
levelled off after 8 h when [K]j was 10 mM/kg. A semi-logarithmic plot of 1 — [Na]t/
[Najoo demonstrates two separate components of the net flux, an initial fast component
which accounts for 20% of the total flux and a second slower component. Fig. 1 shows
Ionic distribution in Amoeba proteus
555
Co.
1
2
3
4
5
6
Time (h)
Fig. 2. Kinetics of change in (Na)i when the NaCl concentration of the control medium is
increased by 20 mM at zero time. Closed points: [Ca]0 increased to 5 nm at 3 h. Open points:
(Ca)0 increased to 5 mM at time zero. Points represent single determinations from one experiment.
the steady-state [Na]t in cells equilibrated in various [Na]0 in the control medium for
4 h. Internal [Na] varied linearly with [Na]0 and was always slightly higher than
[Na]0.
Effect of [Ca]0 on [Na]{
Fig. 2 demonstrates the effect of Ca on [Na]j during a net Na influx. External Na
was increased to 20 mM initially, with Na, increasing to 25 mM/kg. CaCla was added
to 5 mM after 3 h while keeping [Na]0 constant. Internal [Na] fell to approximately
13 mM 2 h after the addition of Ca^ If 5 mM-CaCl2 is added at the same time as the
NBQ (lower curve in Fig. 2), [Na], increases to only 11 mM. When either MgClj (3 mM)
or KC1 (10 mM) was added instead of CaCla there was no reduction in [Na]t.
The effect of various concentrations of Ca,, on the steady-state [Na], is shown in
Fig. 3. The cells were equilibrated in 30 mM-NaCl in the control medium with various
concentrations of CaCl^ from 003 to 5-0 mM for 3 h. [Na]0 decreases with increasing
[Ca]0. The [Ca]0 at half maximal effect is o-i mM. In the following experiments 3 mM
CaCls was used to assure a maximum Ca effect.
Effect of inhibitors on [Na\ and \K\
The effects of ouabain, CN, and 2,4-dinitrophenol (DNP) on [Na], and [K], in the
amoeba were examined. In Fig. 4 the effect of 05 mM CN on both [Na], and [K], is
shown. The cells were exposed to 20 mM-NaCl in the high-Ca medium (3 mM) for
2h, at which time 0-5 mM-CN was added to the medium. In 90 min [K], decreased
from 29 to 18 mM and [Na]t increased from 11-5 to 16-5 mM/kg. The effect of CN was
reversible. After the cells were exposed to CN for 90 min the CN was removed from
556
ROBERT D. PRUSCH AND PHILIP B. DUNHAM
35
30
25
20
15
10
j_
0-1
0-3
JL
0-7
0-5
20
1-5
10
[Gi]o(mM/l)
Fig. 3. Steady-state [Na]i as a function of [Ca]o. Cells were equilibrated for 4 h in control
medium with 30 mM Na» and the Ca<, indicated. Each point represents the mean of 8 determinations ±S.E.M.
-CN
30
r
1
2
3
Time (h)
4
S
6
Fig. 4. Effect of CN on [K], and [Na]i. The NaCl concentration of the control medium was
increased to 30 nun and the CaCl, concentration was increased to 3 mM at zero time. Closed
points represent [K]| and the open points [Na]i. NaCN, was added to the medium to a concentration of 5 x io~* M at 2 h and was removed at 3i h. Each point is the mean of 8 determinations ±S.E.
Ionic distribution in Amoeba proteus
557
50
40
a, 30
c
20
10
10
12
14
Time (h)
16
18
20
22
24
Fig. 5. Unidirectional Na influx in Amoeba prottus in steady state. la Na was added to the control
medium at zero time. Closed points, exchange in control medium with 3 miu-Ca; open points,
exchange in the control medium. Curves were calculated from the expression C, «= C«o(i —«"**)•
Each point represents the meon of two determinations.
the medium and [Na], and [K], returned close to their previous levels. It should be
noted that, as shown above, during a net Na influx when Ca,, is low, [K], decreases.
When Ca,, is increased, a net Na influx is accompanied by an increase of [K],. Ouabain
and DNP, both in concentrations up to io" 1 M, had no observable effect on either
[Na], or [K], under the conditions described above.
Unidirectional Na, K and Cl influxes
The unidirectional influx of Na was measured in steady state by the addition of
15 /iCi of ^Na to approximately 1 1 of control medium, or to 1 1 of control medium
with 3 iriM-Ca, and taking cell samples at various times, as shown in Fig. 5. The
maximum percentage exchange [CJ was approximated by finding the C^ which gave
the best fit to the data. The curves in Fig. 5 were calculated using these values of C^
from the expression Ct = C^i — e~u). A good fit for Na exchange was obtained
using a Cm of 48-4 in the control medium and 24-3 in the high-Ca medium.
Unidirectional influxes for 4SK are i-55mM/kg.h in the control medium and
i-iomM/kg.h in the high-Ca medium. The maximum percentage exchange of Kt
with *2K was 76-5 in the control medium and 49-1 in the high-Ca medium. Fig. 6
shows ^Cl steady-state exchange with cells in the control medium. The unidirectional
influx was 0-43 mM/kg.h and the maximum percent exchange was 45*5. Bruce &
Marshall (1965) maintain that the amoeba Chaos chaos is impermeable to Cl. The Cl
exchange data presented here indicates that the plasmalemma of Amoeba proteus is
permeable to Cl. Percentage exchanges and unidirectional influxes for Na, K and Cl
are summarized in Table 2. All steady-state isotope exchange data fit first-order
kinetics and one-compartment exchange.
558
ROBERT D. PRUSCH AND PHILIP B. DUNHAM
40 h
30
I
•5
20
u
10
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
Fig. 6. Kinetics of "Cl influx in Amoeba protein in steady state. "Cl was added to the control
medium at zero time. The curve was calculated from the expression, C< => C<o(i — e~*'). Each
point represents the mean of two determinations.
Table 2. Unidirectional influxes and percentage exchange of K, Na and Cl in the control
medium and in control medium with 3 mM Ca0
K
Af, (mM/kg.h)
% exchangeable
Control
Control + Ca
1-55
I-IO
76-5
49-1
Na
Control
Control+ Ca
0-07
C025
48-4
24-3
0-43
45-4
a
Control
Membrane potentials
The membrane potential (Em) of Amoebaproteus in the control medium is — 89-5 mV
(mean, 45 measurements). The membrane potential decreases when either Na or K is
added to the control medium. Increasing [Na]0 to 20 mM causes an immediate decrease
in the potential from 89-5 to 25 mV, followed by a further depolarization to 17 mV
over the following 3 h period.
Addition of Ca to the control medium, to 3 mM, causes the membrane potential to
depolarize from 89-5 to 20 mV. However, after cells have been equilibrated in a highNa medium, the addition of Ca causes a hyperpolarization of Em. Fig. 7 shows the
membrane potential of cells equilibrated in 30 mM-[Na]0 with various Ca concentrations. Under these conditions the membrane potential increases with increasing Ca
concentration in the medium. The Ca concentration at half maximal effect is 0-2 mM.
Membrane resistance
The effective resistanceoftheplasmalemmaof^4moe6a/)rotettf in the control medium
is 2*2 x io 7 Cl, and the specific resistance is 11 k£2.cm2 (mean value of 10 measurements). Addition of 3 mM-Ca to the control medium increases the resistance of the
plasmalemma to 3-6 x io 7 Cl, while addition of 5 mM-Na to the control medium
Ionic distribution in Amoeba proteus
559
22 r
20
18
14
12
10
0-2
0-4
0-6
0-8
10
[Ca]o(mM)
20
Fig. 7. Effect of [Ca]0 on the membrane potential of Amoeba proteus in 30 mM [Na]0. Cells were
equilibrated for 3 h in the control medium with 30 mM-Nao and [Ca]0 from o to 3 mM. Each
point represents the mean of 11 measurements ± s.E.
decreases the resistance to 1 x io7 Q. (Fig. 8). In the presence of both 3 mM-Ca and
5 mM-Na in the control medium the resistance of the plasmalemma was 3'3 x io7 Q.
DISCUSSION
The evidence indicates that both Na and K are actively transported in Amoeba
proteus. Evidence for active K influx consists of the reversible decrease of [K]t caused
by CN and the fact that K, is not distributed according to its equilibrium potential.
The K equilibrium potential in the control medium, as calculated from the Nernst
equation, is —145 mV while the measured potential is about —90 mV. Glynn (1959)
has pointed out that the discrepancy between the calculated and experimentally
determined membrane potential may be due to either ion binding or active transport.
Recalculation of the K equilibrium potential, assuming that 25% of K, is bound
(Table 2), gives a value of —135 mV which is still considerably higher than the
measured potential.
Active Na efflux is indicated by the effects of Ca on Na concentrations, fluxes and
membrane resistance. When Ca is added to cells suspended in a high-Na medium,
there is a net efflux of Na against an electrochemical potential gradient. This net
efflux can be explained by the presence of a Na-pump and a large decrease in P Na
(Na permeability) caused by Ca. In low Ca the P Na is sufficiently high that active Na
efflux does not maintain an appreciable gradient. The reduction in i^j a is demonstrated
by a 60 % decrease in unidirectional Na influx caused by Ca. Under the same conditions
560
ROBERT D. PRUSCH AND PHILIP B. DUNHAM
140
I"
Depolarizing
ft
mM . CaClj
100
/ Control
60
5niM-NaCl
6
4
2
4
xlO-'A
1
1
6
8
20
60
Hyperpolarizing
JQO L
Fig. 8. Current-voltage relationships of the plaamalemma of Amoeba proteus. Abscissa, polarizing current in io~* A. Ordinate, membrane potential. Closed points, cells in control medium;
open points, cells in high-Ca medium; crossed points, cells in control medium with 5 mM-NaCl.
Points were taken from photographed oscilloscope tracings of continuous current—voltage plots as
described in the Materials and Methods. Each point represents the mean of six determinations.
K influx is reduced only 30%, probably due for the most part to the depolarization of
the membrane potential which was observed after the addition of Ca to the control
medium. The greater reduction in Na influx caused by Ca when both [K]o and [Na]0
are constant indicates a decrease in P Na . The addition of Na to the high-Ca medium
fails to decrease the membrane resistance while in low-Ca medium Na does decrease
the membrane resistance. This is consistent with a reduction of PN& by Ca.
The membrane potential in high-Na medium is hyperpolarized by the addition of
Ca. This can be interpreted to indicate a reduction in PNa by Ca, if the hyperpolarization
reflects an approach of the membrane potential toward the equilibrium potential for K.
A hyperpolarization would be expected if P Na were decreased and Em approached the
K equilibrium potential. In addition, the effect of Ca on the membrane resistance would
appear to be a specific effect on PNa. The reduction of P$a by Ca may be brought about
by the competition of Ca for Na binding sites in the plasmalemma as has been suggested in HeLa cells by Morrill & Robbins (1967). Calcium binding by the surface of
Ionic distribution in Amoeba proteus
561
Paramecium caudatum has been shown by Naitoh & Yasumasu (1967). The effect of Ca
by association with binding sites is consistent with the saturation effect of Ca on
(Na), and Em, both of which incidentally showed a half-maximal effect at a (Ca)0 of
O-I-O-2 mM.
While it is clear that there is an active Na efflux in the amoeba, which is particularly
manifest when .PNa is reduced by Ca, there are obviously additional mechanisms
involved in the Na regulation in these animals. In the control medium, as well as in
medium with elevated [Na]0, [Na][ can exceed [Na]0 when [Ca]0 is low. It is possible
that this relatively high [Na], under these conditions due to a high P Na and to the
electrical potential difference across the membrane (inside negative in respect to the
outside). However, active Na influx in addition to active Na efflux cannot be ruled out,
as has been suggested in a ciliated protozoan (Kropp & Dunham, 1971). The active
efflux of Na may be associated with the expulsion of Na by the contractile vacuole of
Amoeba proteus. Riddick (1968) has shown that the contractile vacuole of Pelomyxa
carolinensis (= Chaos chaos) eliminates Na and conserves K. The active transport of
Na out of the amoeba may therefore be necessary for the osmotic regulation of this
animal.
Bruce & Marshall (1965) state that there is no active transport of any ionic species
directly across the plasmalemma of Chaos chaos. It was stated that Na and K are
distributed according to a Donnan equilibrium and that the plasmalemma of C. chaos
was impermeable to Cl. Chaos presumably obtains its Cl from its food. In Amoeba
proteus Na and K are both actively transported and intracellular Cl exchanges with
36
C1 added to the medium. The differences in the ionic distribution between A. proteus
and C. chaos may be due to basic differences between species, or the differences may
only be apparent due to differences in experimental techniques used to evaluate the
ionic distribution in the two animals. For example, Bruce and Marshall failed to
follow net fluxes or determine permeability by the use of isotopes.
In Amoeba proteus, equilibrated in the control medium, considerable fractions of
Naj, K, and Cl, are unexchangeable with their isotopes in the external medium. These
unexchangeable compartments may represent ion binding or restriction to various
impermeable intracellular structures. However, bound substances could conceivably
exchange. An additional compartment of Na, is indicated by the two kinetic components observed during a net N influx. In Tetrahymena pyriformis ionic regulation
may be accomplished in part by the compartmentalization of Na, and K, (Dunham &
Child, 1961). According to Klein (1961) one of the mechanisms for regulating internal
cation levels in Acanthamoeba is a shift between bound and free ions in response to
external osmotic conditions. This has also been shown for amino acids in Miamiensis
avidus, a marine ciliate (Kaneshiro, Holz & Dunham, 1969), and in Tetrahymena
(Stoner & Dunham, 1970). It should be pointed out that the addition of Ca to the
control medium reduces both the exchangeable compartments for both Na and K in
the amoeba. It is not known how this is brought about.
The membrane potential of Amoeba proteus has been reported to be sensitive to
changes in both Na and K in the external medium. Table 3 presents the values from
the literature for changes in membrane potential with a tenfold change in either Na,,
or Ko in several protozoa. For a tenfold change in Kg there is approximately a 40 mV
change in the membrane potential of both Chaos chaos and Amoeba proteus.
562
ROBERT D. PRUSCH AND PHILIP B. DUNHAM
Table 3. Change in membrane potential in various protozoa for either a tenfold change in
\K\, or [Na]0 and the [Ca]0 at which the potentials were recorded
Paramecium
Opalina
Chaos chaos
Amoeba proteus
K
(mV)
Na
(mV)
26
297
40
44
26
20
40
57
0
[Ca].
(mil)
o-o
o-6
0
085
0
—
o-s
0-07
35
57
o-o
o-o
Reference
Yamaguchi (i960)
Ueda (1961)
Bruce & Marshall (1965)
Riddle (1962)
Bingley (1962)
Josefeson (1966)
Batueva & Lev (1967)
Changes in the membrane potential are more variable for a tenfold change in Na0.
Included in Table 3 are the concentrations of Ca present when the membrane potential,
as a function of Na,,, was recorded. In the presence of relatively high Ca (> 0-05 mM)
the membrane potential is insensitive to changes in Na,,. At lower concentrations of Ca
(< 0-05 mM) the membrane potential is sensitive to changes in Na0. This provides
additional evidence that Ca reduces the Na permeability of the plasmalemma.
Brandt & Freeman (1967) were able to correlate changes in the structure of the
plasmalemma of Chaos chaos with resistance decreases and pinocytosis by the addition
of Na to the external medium. Ca blocked the permeability change and the pinocytosis
generally caused by increased (Na)0. Josefsson (1968) also demonstrated that the
amount of Na required to ehcit pinocytosis in Amoeba proteus was increased as the Ca
concentration of the medium was increased. These findings are consistent with
reduction of the permeability of the plasmalemma to Na caused by Ca. This correlation
suggests that a specific increase in Na permeability may be associated with pinocytosis.
SUMMARY
1. The intracellular concentrations of K, Na, Ca and Cl in Amoeba proteus are
24-83, 1-08, 2-93 and 9-73 mM/kg cells respectively.
2. Intracellular Na is always slightly higher than that of the external medium when
external Ca is low.
3. Addition of Ca to the medium reduces the intracellular concentration of Na by
decreasing the permeability of the plasmalemma to Na.
4. The membrane potential of Amoeba proteus in Prescott-James medium is
-89-5 mV and is sensitive to changes in both external K and external Na when
external Ca is low.
5. Intracellular Na and K are both sensitive to the addition of CN to the external
medium.
6. Both Na and K are actively transported in the amoeba; K is actively accumulated
from the external medium and Na is actively expelled from the cell.
7. It has been suggested that the effects of Ca on Amoeba proteus, i.e. reduction of
i^j a , reduced internal concentration of Na, reduced unidirectional Na and K influxes,
and increased membrane resistance are due to Ca binding to the plasmalemma.
This investigation was supported by United States Public Health Grant NS 08089.
Ionic distribution in Amoeba proteus
563
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