Published May 1, 1967
Binding of Ca Ions by Paramecium caudatum
Y U T A K A N A I T O H and I K U O Y A S U M A S U
From the Zoological Institute, Faculty of Science, University of Tokyo, Tokyo, Japan, and
the Biological Laboratory, College of Education, Waseda University, Tokyo, Japan. Dr.
Naitoh's present address is the Marine Biological Laboratory, Woods Hole, Massachmetts,
and the Department of Zoology, Syracuse University, Syracuse, New York
INTRODUCTION
A t e m p o r a r y c h a n g e in b e a t d i r e c t i o n of cilia (ciliary reversal o r reversal
response of cilia) occurs in Paramecium caudatum a n d m a n y o t h e r ciliated
p r o t o z o a n s in response to various stimuli (1). T h e i m p o r t a n c e of a c c u m u l a t e d
C a ions in the cell to the ciliary response was first e m p h a s i z e d b y K a m a d a
(2, 3). J a h n (4) a n a l y z e d the d a t a of K a m a d a a n d K i n o s i t a (5) a n d suggested the applicability of G i b b s - D o n n a n ' s rule to the distribution of C a
ions o n the cell surface of Paramecium, a n d e m p h a s i z e d the i m p o r t a n c e of the
surface c a l c i u m to the response. I n fact, d u r a t i o n of the ciliary response to a
K stimulation is f o u n d to be identical b e t w e e n different organisms equilib r a t e d in different m e d i a respectively w h e n the r a t i o of the p o t a s s i u m conc e n t r a t i o n to the s q u a r e r o o t o f c a l c i u m c o n c e n t r a t i o n , [ K + ] / % / [ C a + + ] , of
these m e d i a was held constant, regardless of their ionic c o n c e n t r a t i o n s (4). 1
T h i s strongly suggests t h a t the response is closely c o r r e l a t e d w i t h the a m o u n t
of C a ions b o u n d at the cell surface in a c c o r d a n c e w i t h the G i b b s - D o n n a n
rule or the law of mass action.
I o n e x c h a n g e type b i n d i n g systems h a v e b e e n d e m o n s t r a t e d in a n u m b e r of
1y. Naitoh. 1967. Ionic control of reversal response of cilia in Paramecium:A Ca-hypothesis. Manuscript in preparation.
I3o3
The Journal of General Physiology
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ABSTRACT Binding of 45Ca by live Paramecium caudatum was determined
under various external ionie conditions. It was found that calcium uptake was
separable into at least two components, a rapid and a slow one. The rapid
component was influenced by the presence of certain other ions in a manner
which agrees with the law of mass action. It appears that an ion exchange
system may be involved in a binding equilibrium established between Paramecium, Ca ++, and certain other ions. K +, Rb +, and Ba ++ in the equilibrium
medium are among those ions which inhibit calcium uptake. It is proposed that
liberation of Ca ++ from binding sites on Paramecium by an exchange reaction
with competing ions is the first step in the mechanism of ciliary reversal in the
response to external application of these ions.
Published May 1, 1967
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THE 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 • VOLUME 50 • I 9 6 7
cellular constituents, such as liver microsomes (6), muscle microsomes (7, 8),
red cell ghost (9), a n d also artificial p h o s p h o l i p i d m e m b r a n e (8).
B i n d i n g of C a ++ b y m e m b r a n e fractions of various cells has b e e n f o u n d
to o b e y the law of mass action. M o r e o v e r , it is clear t h a t C a ions p l a y a n
i m p o r t a n t role in m e m b r a n e excitation (10).
I n the present e x p e r i m e n t s , we used 46Ca to e x a m i n e the kinetics of C a ++
b i n d i n g b y live Paramecium. T h e results show t h a t one kinetic c o m p o n e n t of
C a ++ uptake, w h i c h reaches e q u i l i b r i u m relatively rapidly, does so in c o m p e tition w i t h c e r t a i n o t h e r ions, a n d obeys the law of mass action.
MATERIAL
AND
METHODS
RESULTS
AND
DISCUSSION
A. Time Course of 45Ca Binding and Effect of Coexisting K Ions
T o e x a m i n e the kinetics of 4~Ca a c c u m u l a t i o n b y Paramecium a n d the influence of coexisting K ions, eight aliquots of the Paramecium suspension w e r e
2 T h e p H of all t h e e x p e r i m e n t a l m e d i a in these e x p e r i m e n t s was a d j u s t e d to 7.2 by 1 n ~ (in final
concentration) Tris buffer.
s I t is desirable to r e m o v e all C a ions f r o m t h e cell surface, however, w a s h i n g the o r g a n i s m s w i t h a
Ca-free p o t a s s i u m solution rapidly killed the organisms.
4 R a d i o e a l c i u m solution was p r e p a r e d by m i x i n g 1 m e 4~la w i t h 1 m l of 0.1 M CaClz solution.
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Specimens of Paramecium caudatum reared in hay infusion were washed well with a
solution containing 20 mM KC1 and 0.1 nna CaC12 (pH 7.2) 2 to minimize the amount
of bound Ca ions in the organism a and reduce bacterial contamination. Then, the
organisms were suspended in the solution (1-mg organisms in dry weight/ml) for 30
min or more prior to experimentation.
A 1-ml aliquot of the suspension was pipetted into a filtrating glass vessel at the
bottom of which a sheet of Millipore filter was mounted (Millipore Filter Corp.,
Bedford, Mass.). Then, 10 ml of radioactive solution containing adequate amounts of
46Ca4 and other ions were pipetted into the vessel and mixed well with the organism
suspension. After an adequate equilibration time, the mixed suspension medium in
the vessel was sucked out through the Millipore filter rapidly (1 ml/sec) by a vacuum
pump. Then, in order to wash out the 45Ca remaining in the filter, 10 ml of an unlabeled mixture having the same ionic composition as the labeled suspension medium
was poured into the vessel at a rate of about 1 ml/sec while the suction was maintained.
T h e Millipore filter was dried by infrared illumination, and the radioactivity of
the filter due to accumulated 45Ca by Paramecium was counted by a G-M counter.
The same procedure without Paramecium proved that residual 45Ca in the filter was
almost negligible.
At least three measurements were made to determine each point presented in the
figures of the present paper. Fluctuations of measured values were so small that the
standard errors did not exceed the diameter of circles presenting the mean values.
All the experiments were performed at room temperatures of 19-21 °C.
Published May 1, 1967
Y. NAITOHANDI. YASUMASU Binding of Ca Ions b~ Paramecium¢audatum
~3o5
equilibrated in two kinds of radioactive media with identical Ca concentrations (1 rn~ in final concentration; the amount of 45Ca was I0 # c / m l ) b u t
varied K concentrations (20.4 and 2.4 rnM in final concentration) for periods
of 1, 5, 10, and 20 rain. Then, the radioactivities of each aliquot were detern-fined.
T h e results shown in Fig. 1 indicate that a m u c h greater a m o u n t of 4"Ca
was accumulated within 1 min equilibration in the presence of low-potassium
than in high-potassium concentration. This rapid uptake was followed b y a
rather slow phase, radioactivity reaching a plateau a b o u t 20 min after the
I- 2 0
3:
ld
2.4raM K I I m M Co
,/,,,,,,,~ ""-~
15
w
/
f
0
20.4 mMK/ImM C,
E
r~
o
M
5
o
O't
0 1
t
5
TIME
I
I0
(rain)
I
20
FIoLmE 1. Time course of ~Ca++ binding by live Parameduraeaudatumand the effect of
coexisting K ions in the equilibrium medium on the binding (open circles, 2.4 nm
K+; solid circles, 20.4 nm K+). Ca++ concentration in the media was 1 mM (~Ca++,
10 pc/ml). Notice the rapid binding of 4r~a'V+within 1 rain from the start of equilibration (dotted lines). See text for discussion.
start of the equilibration. T h e slow-uptake component exhibits a time course
which is independent of the potassium concentration.
It is proposed that the rapid initial component of 45Ca uptake is due to
binding of ~ C a by anionic sites on Paramecium, and the K ions compete for
the same binding sites. The slow increase in radioactivity, which seems to be
unaffected by the presence of K ions, m a y be due to a diffusion of 46Ca into
the cell, passive exchange of 45Ca with preexisting Ca ions in the cell, or active
Ca accumulation by the cell.
B. The Initial Rapid Binding of '~Ca and the Effect of Coexisting K Ions
If an ion exchange type system is involved in the initial rapid binding of Ca
ions b y Paramecium, the equilibrium between the binding sites of Paramecium,
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n..
0
10m
E I0
'"
Published May 1, 1967
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THE 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 • VOLUME 5 ° • I 9 6 7
Ca ions, and K ions may be formulated according to the law of mass action as:
PCa½ + K + ~ P K +
½Ca++
[PKI
[K+]
- k
[PCa½]
~/[Ca++]
(1)
(2) ~
in which P represents the binding site of Paramecium; [PCa½] represents the
concentration of Ca bound by Paramecium; [PK] represents the concentration
of K bound by Paramecium; [K +] and [Ca ++] represent the concentrations of
K and Ca ions in the equilibrium medium; and k represents the equilibrium
constant.
If it can be assumed that all the binding sites are filled with Ca and K,
the total binding capacity of Paramecium, Pt, can be represented as:
Pt = [PK] + [PCa{]
(3)
Pt
[PCa½] - kJa + 1
(4)
in which Ja ° represents the ratio [K +]/x/[Ca ++] in the equilibrium medium.
By rearranging, Equation 4 can be put in the form of a straight line as:
1
k
[P~a½] - ~ J a
1
+~
(5)
Equation 4 (or Equation 5) shows that if Ja in the equilibrium m e d i u m is
kept constant, the amount of Ca bound by Paramecium should remain constant,
regardless of the ionic concentration in the equilibrium medium. In order to
test this prediction, three aliquots of the Paramecium suspension were equilibrated for 1 rain in three different radioactive media having the same Ja
value (1.41) and different ionic concentrations [1, 1 mM KC1 + 0.5 rnM
CaCI~ (5 uc/ml); 2, 2 rnM KC1 + 2 mM CaCI~ (20 #c/ml); 3, 4 mM KCI +
8 mM CaCI2 (80/~c/ml) all in final concentration] respectively; then, radioactivities were determined on each aliquot.
Results shown in Fig. 2 clearly indicate that 45Ca uptake by Paramecium
during 1 rain equilibration was almost identical in each of the aliquots in
5 Activity coefficients a r e neglected in all t h e e q u a t i o n s in the p r e s e n t p a p e r because of low ionic
c o n c e n t r a t i o n s e m p l o y e d in these experiments.
e I n h o n o r of Dr. T . L. J a h n w h o suggested first the i m p o r t a n c e of the ratio to t h e b i n d i n g of C a in
Paramecium a n d its ciliary reversal. T h e concentrations of e a c h ion species in the solutions e m p l o y e d
in these e x p e r i m e n t s a r e all expressed in millimoles per liter.
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and the following expression for the amount of bound calcium can be derived
from Equations 2 and 3:
Published May 1, 1967
Y. NAITOHANDI. YASUMASU Bindingof Ca Ions by Parameciumcaudatum
~3o7
spite of differences in the total concentration (1.5-12 mM) and the amount
of 45Ca (5-80 #c/ml) between three equilibrium media.
Equation 5 implies that the reciprocal value of the a m o u n t of b o u n d Ca
b y Paramecium must have a linear relationship with Ja in the equilibrium
medium. T o ascertain this, radioactivities were determined on aliquots of
the Paramecium suspension equilibrated in various media having various
K concentrations and the same Ca concentration (1 mM, 10 /~c/ml). T h e
reciprocal values of each radioactivity plotted against the Ja value were
found to form a straight line as shown in Fig. 3 (open circles).
o
15
LU
10
"
--0
0
E
o
I
I
I
2
I
4
5
I0
o
K
Ca 0 5
(Spc/ml)
2
(20Fc/ml)
(raM)
8
(80Fc/ml)
FIGURE2. Amount of 4sCa++ bound by live P. caudatumduring 1 rain of equilibration
inthree kinds of solutions having the same ratio, [K+]/-~v/[C-C--a-a~ -- 1.41. Concentration
of K+ and Ca"H- and the amount of ~6Ca++ are represented in abscissa. The amounts of
45Ca++ bound by P. caudatumare equal between the three ionic conditions of the equilibrium media, in spite of large differences in the ionic concentrations and amounts of
4~Ca++.
These observations provide strong evidence for the applicability of the law
of mass action to the uptake equilibrium between Paramecium, Ca ions, and
K ions, and saturation of the assumed binding sites of Paramecium with Ca and
K ions, at least under the conditions employed.
C. Concentration Effect of Various Ions on the Initial Rapid Binding of 4"Ca
It is interesting to know whether ions other than K+, for example N a +, R b +,
M g ++, and Ba ++, act to compete for the same calcium-potassium binding sites
on Paramecium, since these ions (exception of M g ++) are known to induce
ciliary reversal in Paramecium (1 1). 1 Aliquots of the Paramecium suspension
were equilibrated for 1 rain in several different media, each of which consisted of 1 mM CaCI~ (10 #c/ml) plus one of the test ions in one of several
concentrations. T h e radioactivities due to the b o u n d 4~Ca were determined
in each case, and the reciprocal values of the radioactivities were plotted
against the Ja value of each medium. T h e Ja value, in the present cases,
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0
E
Published May 1, 1967
~3o8
were
calculated
THE
as
JOURNAL
OF
GENERAL
[monovalent
PHYSIOLOGY
• VOLUME
i o n ] / ~ / [ C a ++] or
50
x/[bivalent
•
~967
ion]/
~v/[Ca++].'
As is clearly shown in Fig. 3, all the data showed linear relationships with
the Ja values, and each ion species gave a line with a different slope. All the
lines crossed the ordinate at a point which corresponds to 1/Pt, as is expected
from Equation 5. These findings indicate that the law of mass action is also
applicable to the equilibrium established between the proposed binding sites,
Ca ++, and the other ions used in the present experiments.
3O
K.- °
o
--
Na"e
IO
/~.~~1_
I-
5
0
0
I
I
[
2
I
3
l
4
(Mono)/J('Co)
I
5
I
6
I
7
I
8
I
9
I
I0
OR (,/(-8ii/./~'~
FIout~ 3. EffEt~ of various coexisting ions in the equilibrium medium (O, K+; A,
Rb+; 0, Na+; O, Ba++; l , Mg++) on the binding of °Ca++ by live P. caudatum. Abscissa represents the ratio, [monovalent i o n ] / ~ / ~ - ~ or %/[bivalent iou]/%/[Ca++J.
Ordinate represents the reciprocal value of the counts per minute from the 4~a++
bound by I nag dry weight of Paramecium. See text for discussion.
F r o m the value of 1~It and the slope of each line, the value of the equilibrium constant, k, for each ion was calculated, and listed in T a b l e I. T h e
equilibrium constant represents the ability of the given ion to prevent the
binding of Ca ++ to the sites. In other words, those ions having the large
equilibrium constants as K +, R b +, and Ba ++ bind comparatively easily to
the sites in exchange with Ca ions, whereas those with small constants as
the M g ion have a low affinity for the site.
T h e equilibrium between the binding sites, Ca ions, and bivalent ions can be formulatext as:
PCa½ -F ½B++ .~-PB½ + ½Ca++
(1')
CP~½1
[PCa½]
~ X/[C--~J
(2')
in which B ++ represents bivalent ion and [PB½] represents the amount of hound bivalent ions by
Paramecium. Thus, in the case of bivalent ions, Ja = % / ~ ] / V ~ - ' ~ .
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15
Published May 1, 1967
Y. NAITOH AND I. YASuuAsu
Binding of Ca Ions by Paramecium caudatum
1309
Reversal response of cilia in Paramecium can be elicited by the external
application of certain monovalent (K +, R b +, Na +) and certain bivalent (Ba ++)
ions. Magnesium ions, on the other hand, do not induce reversal (11). 1 T h e
effectiveness of ions in eliciting the reversal response of cilia seems to be correlated with their equilibrium constant of binding on Paramecium. T h a t is,
those ions having relatively large constants such as K +, R b +, and Ba ++ induce prolonged responses, while the response to Na+ is significantly shorter
in duration than that to K +. Magnesium, which has a very small constant,
does not induce reversal? These correlations suggest that the liberation of
bound Ca ++ from the binding sites of Paramecium by exchange with other ions
is directly concerned with the reversal response of cilia.
TABLE
I
Ion
Bpeci~
Sodium
Potassium
Rubidium
Magnesium
Barium
Equilibrium
comtant
0.19
0.35
0.28
0.033
0.32
Liberation of Ca ++ from the sarcoplasmic reticulum is thought to be concerned with the control of contraction in striated muscle fibers (12). Displacem e n t of bound calcium has also been proposed as a step underlying
permeability changes during m e m b r a n e excitation (10). In the glycerol-extracted Paramecium model, ciliary reversal occurs only in the presence of
Ca ++ and A T P . ' All present evidence indicates that ciliary reversal is dependent on calcium?
It is proposed, on the basis of these observations, that Ca ++ liberated from
cellular binding sites activates, directly or indirectly, a contractile system
which is energized by ATP. Contraction of this proposed contractile component in turn results in the reversal of effective beat direction of the cilia.
In this model ciliary reversal is closely analogous in fundamental mechanism
and its control to contraction of muscle.
T h e locality of the binding sites is still unknown. However, it is highly
probable that the sites m a y be on the surface or at least, near the surface of
s y . Naitoh. 1967. Reversal response of cilia in glycerol-extracted model of Paramecium. I n preparation.
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CALCULATED EQUILIBRIUM CONSTANTS
IN T H E E Q U I L I B R I U M B E T W E E N B I N D I N G S I T E S O F
PARAMECIUM, CA ++ , AND O T H E R I O N S
T h e e q u i l i b r i u m between binding sites, Ca ++ , a n d other ions is formulated
in accordance w i t h the law of mass action as in Equations 1, 1', 2, a n d 2'.
Calculation was made from the d a t a in Fig. 3.
Published May 1, 1967
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•
i967
the organism because Ca binding reaches an equilibrium relatively rapidly
and because the bound Ca is intimately concerned with the behavior of the
cilia, which, of course, are located at the cell surface.
Ciliary reversal of Paramecium is closely associated with the depolarization
of the membrane, regardless of whether depolarization is the result of applied
current, application of KC1 (13, 14), or spontaneous (13, 14, 15). It will be
of great interest to our understanding of ciliary reversal to determine in subsequent studies the relationship between depolarization and the release of
Ca ++ from binding sites on Paramecium.
We are grateful to Dr. R. Eckert of Syracuse University and Dr. H. Sanlai of the University of
California, Berkeley,for their critical reading of the manuscript.
Receivedfor publication 8 August z966.
REFERENCES
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1. JENNINGS, H. S. 1906. Behavior of the Lower Organism. Columbia University
Press, New York.
2. KA~tADA, T. 1938. Intracellular calcium and ciliary reversal in Paramecium.
Proc. 1rap. Acad. Tokyo. 14:260.
3. KAMADA,T. 1940. Ciliary reversal of Paramecium. Proc. Imp. Acad. Tokyo. 16:241.
4. JAHN, T. L. 1962. The mechanism of ciliary movement II. Ciliary reversal and
ion antagonism. J. Cellular Physiol. 60:217.
5. KAMADA,T., and H. KINOSITA. 1940. Calcium-potassium factor in ciliary reversal
of Paramecium. Proc. 1rap. Acad. Tokyo. 16:125.
6. CARVALHO,A. P., H. SANta, and N. PACE. 1963. Calcium and magnesium binding
properties of cell membrane materials. J. Cellular PhysioL 62:311.
7. KOKETSU, K., R. KITA~OJRA,and R. TANAKA. 1964. Binding of calcium ions to
cell membrane isolated from bullfrog skeletal muscle. Am. J. Physiol. 207:509.
8. MIKULEGKV,D. C., and J. M. TOBIAS. 1964. Phospholipid-cholesterol membrane
model. J. Cellular Physiol. 64:151.
9. SANta, H., A. P. CARVALHO,and N. PACE. 1962. Relationship of hydrogen ion
binding to sodium and potassium binding by rat liver cell microsomes and
human erythrocyte ghosts. J. Cellular PhysioL 59:241.
10. TOBIAS, J. M. 1964. A chemically specified molecular mechanism underlying
excitation in nerve: A hypothesis. Nature. 203:13.
11. OLIPHANT,J. F. 1942. Reversal of ciliary action in Paramecium induced by chemicals. Physiol. Zool. 15:443.
12. HUXLEy, A. F. 1964. Muscle. Ann. Rev. Physiol. 24:131.
13. KINOSITA, H., S. DRYL, and Y. NAITOH. 1964. Change in membrane potential
and the response to stimuli in Paramecium. J. Fac. Sci., Univ. Tokyo, Sect. 1V.
10:291.
14. NAITOH, Y. 1966. The reversal response elicited in nonbeating cilia of Paramecium
by membrane depolarization. Science. 154:660.
15. KINOSITA, H., A. MtrRAKAMr, and MOMOKO, YAStmA. 1965. Interval between
membrane potential change and ciliary reversal in Paramecium immersed in
Ba-Ca mixture. J. Fac. Sci., Univ. Tokyo, Sect. 1V. 10:421.
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