Jf. exp. Biol. (1980), 88, 305-325
ith :2 figures
'inted in Great Britain
305
C
A Ca-INDUCED Na-CURRENT IN PARAMECIUM
BY YOSHIRO SAEVII AND CHING KUNG
Laboratory of Molecular Biology and Department of Genetics, University of WisconsinMadison, Madison, Wisconsin 53706 U.S.A.
(Received 12 March 1980)
SUMMARY
Under a voltage clamp, step depolarization and repolarization can induce
a sustained inward current and a tail inward current in Paramecium tetraurelia
bathed in a solution containing 8 mM-Na+. These currents are best seen in
the ' paranoiac' mutant. The I-V plot of the sustained inward current can
have a region of negative resistance around — 20 mV. This current is absent
when Na+ is excluded from the bath solution, and it increases as the Na+ concentration increases from 2 to 8 mM. Injection of Na+ into the cell suppresses
this inward current. This current develops very slowly, reaching its maximum
seconds after the step depolarization and decays with a time constant of hundreds of milliseconds after the repolarization. This slow current is dependent
on Ca2+. It can be suppressed by reduction or deletion of external Ca2+ or
by iontophoretic injection of EGTA. 'Pawn' mutants with defective Caconductance also lack this current. We conclude that Paramecium has
a Ca-induced conductance through which the Na-current flows. Although
more prominent in the 'paranoiac' mutant, this Ca-induced Na-current is
also seen in the wild type. This conductance may function in generating
plateau depolarizations lasting seconds or even minutes and the corresponding prolonged backward swimming away from sources of irritation
and stress.
INTRODUCTION
The locomotion of a paramecium is regulated by its membrane potential. When
the membrane is at rest, the cilia beat in their normal direction and propel the paramecium forward. Upon proper stimulation, a Ca-action potential can be triggered
which increases the internal Ca2+ concentration which, in turn, causes the cilia to
beat in the reverse direction and the cell to swim backward for a short distance (the
'avoiding reaction'). Like the 'tumbling' in motile bacteria (Springer, Goy & Adler,
1979), the avoiding reaction is necessary for certain chemotaxes (Van Houten, 1978),
thermotaxes (Hennessey & Nelson, 1979) and the response to mechanical stimulation
at the anterior end (Naitoh & Eckert, 1969). Mutational blocks on the action potential
also block the avoiding reaction and cause a failure in these taxes but not in normal
ciliary motion (Nelson & Kung, 1978; Kung, 1979).
The electrophysiology of Paramecium has advanced greatly since the first electrode
»ietration by Kamada in 1934 (see Naitoh & Eckert, 1974; Eckert & Brehm, 1979;
306
Y. SAIMI AND C. K U N G
Kung, 1979 for reviews). Much previous work concentrates on the Ca-action potentjB
which involves a voltage-sensitive Ca-channel and a delayed rectifying K-channeT
However, several electrophysiological problems related to the behaviour of this
organism have not been resolved. For example, the basis for the natural membrane
discharges, which correspond to the 'spontaneous' avoiding reactions in the culture
medium, is not fully understood. A paramecium can also spin backward rapidly and
continuously for minutes upon certain stimuli. The electrophysiological basis of this
continuous backing is also poorly understood.
Na + is a natural cation in fresh water and in various culture media for Paramecium.
Spontaneous avoiding reactions are easily observed in solutions containing Na+, such
as the commonly used Cerophyl culture medium buffered with sodium phosphates
(Sonneborn, 1970). The membrane discharges corresponding to such avoiding
reactions have been recorded from paramecia bathed in Na+-containing solutions
(Satow & Kung, 1974). Several mutants have been isolated which are best distinguished by their peculiar behaviour in these solutions (Kung, 1971a; Satow &
Kung, 1974, 19766; Satow, Hansma & Kung, 1976). 22Na influx has been measured
in Paramecium, particularly in the ' paranoiac' mutants which exhibit prolonged backward swimming and plateau depolarizations in solutions containing Na + (Hansma &
Kung, 1976; Satow et al. 1976; Hansma, 1979). Nevertheless, the questions of
whether there is a Na-conductance in this membrane and what the functional role of
Na + is have not been settled electrophysiologically. We show in this paper that the
Paramecium membrane has indeed a Na-conductance. The magnitude, kinetics and
triggering mechanism of the Na-current under a voltage clamp are described and the
functional significance and behavioural implications of this current are discussed.
MATERIALS AND METHODS
Stocks and Cultures
P. tetraurelia, formerly P. aurelia species 4, was used. The stocks used included 51s
(wild type), d4-9O (genotype PaA/PaA, a 'paranoiac mutant'), d4~94 and d4-5oo
and pwA^^/pzvA500,
respectively, 'pawn A mutants'), d4-95 and
and pu>B501/pwB501, respectively, 'pawn B mutants') (see for
the genetical descriptions: Kung, 19716; Van Houten, Chang & Kung, 1977; Kung,
1979), and three constructed paranoiac-pawn double mutants (PaA/PaA
pwA9i/
pwA9i, PaA/PaA pwB9h/pwB™, PaA/PaA pwB^/pwB™1 (Van Houten et al. 1977;
Chang, Thiede & Kung, unpublished)). d4~94 is a phenotypically slightly leaky,
d4-5oo a nearly non-leaky mutant of the pwA complementation group (Chang et'al.
1974; Satow & Kung, 1980a) and d4>95 and d4-5oi are mutants of thepwB group with
little leakiness (Chang et al. 1974; Schein, Bennett & Katz, 1976).
Paramecia were ordinarily cultured in Cerophyl medium buffered with Naphosphates and bacterized with Enterobacter aerogenes (Sonneborn, 1970). To delete
the cellular Na+, the enterobacters were first grown overnight in a defined glucoseTris medium without Na+ (Linn & Forte, personal communication). Paramecia were
then transferred into a ten-fold dilution of this medium to a final concentration of
8-9 mM-K+, 12-13 mM-Tris, 0-09 mM-Ca2+, 0-02-O-03 mM-Mg2+ and ca. 5
stigmasterol.
Ca induced Na-current in Paramecium
307
well-fed cells that had been grown in some medium for more than 20 hours
ere used for experimentation.
Recording techniques
The methods of capturing, rinsing, immobilizing, penetrating and recording from
paramecia were basically those described by Naitoh & Eckert (1972). Cells were withdrawn from their culture media, washed in the ' K-solution' (see below) and put into
a drop hanging under a glass slip. The slip was then put on the experimental chamber
on the stage of a compound microscope with electrodes poised for penetration.
A syringe-controlled suction device was used to regulate the size of the hanging drop
to immobilize the cell without undue desiccation. One electrode was then inserted by
micromanipulation into the paramecium before the chamber was flooded with the
K-solution to immerse the cell. A second electrode was then inserted. These electrodes
were filled with 3 M-KC1 with resistances around 50 MQ.
The bath solution could be changed by a gravity-fed perfusion system. The first
solution in our experiments was always the K-solution in which a fixed reference
level was established for the entire experiment. After the penetration of the electrodes
in the K-solution, the specimens gradually attained membrane potentials of about
— 35 mV in 1-2 min. Experimentation began after this period of adjustment in this
solution. The bath was changed several times as necessary.
The voltage clamp was that of Oertel, Schein & Kung (1977), with slight modifications. The holding potential was — 30 mV unless otherwise stated. The voltage
stabilized within 1 msec after a command step change. However, a notch of less than
4 mV was usually observed when the transient Ca inward current was triggered by
the depolatization. The open-loop gain was most often 100 x (80 x to 200 x ). The
series resistance of the clamp circuit was less than 200 K£2.
Unless noted otherwise, each set of experiments was repeated on at least three cells.
Inotophoresis
Ions were injected into the paramecium through a third electrode rilled with the
desired ion by a current of appropriate polarity. The iontophoresis was performed
when the membrane was under the voltage clamp. Thus, current flowed largely
between this third electrode and the current electrode of the clamp, both inside the
cell. Since relatively little current actually passed through the membrane, we were able
to inject large iontophoretic currents without damaging the specimens. The deflection
of the membrane potential was as small as 6 mV (up to 20 mV in some cases) with
a 10 nA injection by this method, even though the total membrane resistance of
a paramecium was about 50 MQ. A 10 nA, 1 min injection changes the internal concentration of the injected ion by 20 DIM as caluclated by the method of previous
workers (Satow & Kung, 1976 a).
Solutions
Standard salt solutions used in this study were the ' K-solution' with 4 mM-K+,
1 mM-Ca2+, 1 mM-HEPES, 5-6 mM-Cl", the ' Na-solution' with 8 mM-Na+, 1 mM2
+, 1 mM-HEPES, 9-iomM-Cl- and the ' Ca-solution' with 1 mM-Ca2+, 1 mM£PES, 2-4 mM-Cl~. Solutions with different ionic compositions were also used as
R
308
Y. SAIMI AND C. K U N G
specified in the text. All solutions included I O / J M - E D T A to chelate any possihB
toxic heavy-metal ions and were adjusted to pH 7-1-7-3.
Tris [Tris(hydroxymethyl)aminomethane], HEPES (N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid), EDTA (ethylenediaminetetraacetic acid) were from Sigma.
All chemicals were of reagent grade.
Experiments were performed at room temperatures (20-23 °QRESULTS
+
Effect of Na
on the behaviour of Paramecium
When paramecia are transferred to a solution of different ionic strength and species,
they respond behaviourally. The nature, intensity and duration of the response depend
on the ionic compositions of both of the solutions (Jennings, 1906; Naitoh, 1968).
The paramecia that have been washed into and incubated in the ' K-solution' (4 mMK+, 1 mM-Ca2+; see Materials and Methods) swim forward in this solution, rarely
showing an avoiding reaction. The mechanical disturbance of transferring them from
one pool of K-solution to another also induces little change in this behaviour. Transferring these ' adapted' cells into solutions containing Na+, however, induces reversal
of the ciliary beat and therefore backward swimming. When the wild-type paramecia
in the K-solution are transferred into a solution of high Na + or low Ca2+ concentrations, e.g. the ' Na-solution' (8 mM-Na+, 1 mM-Ca2+), the first few responses are
variable but often consist of longer backward swimming for several seconds (up to
30 s in some cases). These prolonged backings are followed by jerks and do not recur
later.
Sustained backward swimming is easily observed in the 'paranoiac' mutants of
P. tetraurelia. Without any apparent trigger, these ' paranoiac' paramecia sporadically
swim backward for up to 60 s or more in the culture medium (bacterized Cerophyl
infusion buffered with Na phosphates). However, they show no such backward swimming in the K-solution. When transferred from the K-solution into various solutions
containing Na + , they again swim backward up to 2 min or more. Such bouts of backward swimming are repeated as long as the cells are in the Na-containing solution.
The following is a study of the electrophysiological correlates of the prolonged backward swimming in the Na-solution.
Electrophysiological effects of Na+ on the mild-type membrane
Corresponding to the simple forward swimming in the K-solution the membrane
of the wild-type paramecium is found to be quiescent, resting at —36-1+5-5 mV
(mean + s.D., n = 16) with no spontaneous discharge (Fig. iA). Injection of an outward square pulse triggers an action potential followed by a steady-state depolarization (Fig. iB), as has been previously reported (Naitoh & Eckert, 1968; Satow &
Kung, 1976 a). There is an after-potential of about + 5 mV (up to + 10 mV from the
resting level) that follows the response to the large square pulses. The ionic nature of
this small after-potential is not known.
In paramecia bathed in the K-solution, under voltage clamp, a step depolarization
from the holding potential of — 30 mV to — 26 mV or higher, induces a transi
inward current lasting about 5 ms (Oertel et al. 1977; Brehm & Eckert, 1978; S
Ca induced Na-current in Paramecium
3°9
30 s
400 ms
Fig. 1. Electrical activities from a wild-type cell in the K- and the Na-solutions. A: Chart
record of the membrane potential in the K-solution. The regularly appearing spikes are
responses to injected current of various intensities. B: An oscillograph of a response in A
(asterisk). C: Membrane potential in the Na-solution. Note that the stimulations elicit low
plateau depolarizations (arrows) and the membrane potential fluctuates spontaneously more
than that in A. Stimulations are not delivered periodically. D: An oscillograph of the response
marked with asterisks in C to current injection. Note the large positive after-potential which
leads to the plateau depolarization (arrows in C). The interrupted lines are the reference potential level determined in the K-solution. The upper traces are voltages and the lower traces
currents in B and D.
& Kung, 1979), and followed by an outward current sustained throughout the depolarization step (Fig. 2 A). The plot of current measured at 1 sec vs. the voltage step
is nearly linear from —45 to around — 17 mV. The current is clearly rectified beyond
this range (Fig. 2E). A step depolarization to above — 20 mV induces a slowly growing
outward current after the inward transient (Fig. 2B). Correspondingly, a tail current
appears after the step depolarization. This tail current is outward after a depolarization above — 20 mV and it decays nearly completely within 200 ms. This slowly
growing outward current and the corresponding outward tail current are similar to
those identified as the Ca-induced K-current and its tail by Satow & Kung (19806).
It has also been reported that there is a depolarization sensitive K-current with a
fast activation kinetics (Oertel et al. 197; Satow & Kung, 1980 a), though its voltage
sensitivity is not studied here.
To investigate the effect of Na + on the wild-type paramecium membrane, the bath
of K-solution is replaced by solutions containing Na+. The undamped membrane
responds to this change of solution with a series of rapid discharges, i.e. it repeatedly
depolarizes and repolarizes (Fig. 1C). These discharges correspond to the jerks behaviourally observed (Satow & Kung, 1974). When 8 mM-Na+ (the Na-solution) is
used to replace the K-solution, the membrane depolarizes to about — 10 mV ( — 3 to
— 23 mV) for tens of seconds. This first depolarization is often followed by a few
isodes of shorter and smaller depolarizations from the new ' resting level' (— 26-0
3-6 mV, n = 14). These long depolarizations are highly variable in magnitude and
f
3io
Y. SAIMI AND C. KUNG
A
zlL
c
-l
400 ms
B
-9
-9
•r
Vm (mV)
JI
20 ms
-60
-8
Fig. 2. Membrane currents, under voltage clamp, from a typical wild-type cell in the Ksolution (A, B) and in the Na-solution (C, D). There is little difference between membrane
currents in the two solutions with a small (A, C) or a large (B, D) voltage step, except for the
tail current. Note that the tail current after a large voltage step turns inward in the Na-solution
(D: arrow), on which a faster outward component (also seen in B) is superimposed. The Catransients shown beneath B and D are simultaneous records of a faster sweep speed of B and D,
respectively (arrows point at the peak Ca-transients). Interrupted lines refer to zero current
levels: numbers on the left shoulder of each trace are the membrane potential of the test pulse
(in mV): the duration of a voltage step is indicated by asterisks on each trace throughout this
paper. E shows the I-V relationship at i s during the voltage step (closed symbols) and the
peak Ca-transients within 5 ms (open symbols). Circles are the currents in the K-solution and
squares those in the Na-solution. These plots are obtained from the same cell of A to D. The
curves in the two solutions are similar. The inward current upon hyperpolarization in the
Na-solution appears more prominent in other cells.
Ca-induced Na-current in Paramecium
311
•luration from cell to cell and are often separated by clusters of rapid discharges, each
lasting less than 1 s (Satow & Kung, 1974). The long depolarization and the rapid
discharges most likely correspond respectively to the backward swimming and the
jerks when adapted paramecia are transferred to the Na-solution. Both the spontanous membrane activities and the behaviour upon the introduction of Na + vary from
specimen to specimen.
In the Na-solution, an outward current injected across the membrane induces an
action potential and the steady-state depolarization (Fig. 1C, D) as in the K-solution
(Fig. 1 A, B). However, unlike those in the K-solution, the after-potentials in the
Na-solution are always depolarizations that can be very long (up to 2 min) and very
large (up to — 5 mV) (arrows, Fig. 1C, D). The level and duration of such depolarizations vary greatly from cell to cell. The trigger needed to generate such depolarizations
also varies. In some cases, plateau depolarizations spontaneously occur; in others,
they cannot be triggered even with outward currents up to 1 nA for 1 s. It is, therefore,
difficult to study the properties of the membrane during the prolonged depolarization
induced by Na + , using the wild-type membrane.
The membrane currents under voltage clamp in the Na-solution are similar to those
in the K-solution. Upon step depolarizations, the inward transient, the steady-state
outward current, and the slowly developing outward currents are all observed in cells
bathed in the Na-solution (Fig. 2C, D). The anomalous rectification is much stronger
in the Na-solution, a phenomenon to be dealt with elsewhere.
Although there is no large difference in the depolarization half of the I-V curve
(Fig. 2E) in the two solutions, the tail current after the step depolarization is more
complex in the Na-solution. When the step is above — 17 mV, this tail has a prominent
inward component (arrow, Fig. 2D). The inward current decays very slowly over
hundreds of milliseconds. Before this inward tail current, an outward tail current
which decays faster can be observed. The kinetics of this outward tail current are
similar to those found in cells bathed in the K-solution. These complex tail currents
are found in all wild-type paramecia bathed in the Na-solution and most likely
represent the conductances responsible for the positive after-potentials which sometimes appear as plateau depolarizations of the undamped wild-type membranes.
Although the membrane behaviour is better controlled under the voltage clamp,
the variability and the small size of the inward tail current from wild-type specimens
make the analyses more difficult. We therefore chose to study the 'paranoiac' mutant
where prominent plateau depolarizations can always be triggered in the Na-solution
and the inward tail currents are larger.
The 'Paranoiac' mutant
The electrical properties of the membrane of the paranoiac mutant bathed in the
K-solution are similar to those of the wild type in the same solution. There is no
spontaneous membrane activity and the membrane remains at — 39-3 + 4-4 mV
(n = 24). Proper current injections elicit action potentials, steady-state depolarizations
and the small positive after-potentials in the mutant (Fig. 3, left) as in the wild type
(Fig. 1, left). In the K-solution and under voltage clamp, the mutant membrane also
behaves similar to that of the wild type. The inward transient, the steady-state outward current, the delayed rectification current and the tail current are seen in the
312
Y. SAIMI AND C. KUNG
-A-~r—jvjw
_
lo
30 s
h
:
_J
400 ms
Fig. 3. Electrical activities from one 'paranoiac' mutant cell in the K-solution and the Nasolution. A: Chart record of the membrane potential in the K-solution. Various amount of
current was injected into the cell every 20 s. B: An oscillograph of a response (asterisk in A).
In the K-solution, the membrane activities of the mutant are similar to those of the wild type
(Fig. 1 A, B). C: Membrane potential in the Na-solution. The membrane of the mutant shows
well-defined plateau depolarizations either spontaneously or triggered by current injections.
The initial part of the trace shown in C is the end of a previous plateau depolarization. The
plateau depolarization marked with asterisks is elicited by a current injection. The initial part
of this depolarization is shown in D. 4-5 min of the plateau depolarization are omitted in C
where marked. The order of traces is as Fig. 1.
mutant (Fig. 4A, B) as in the wild type (Fig. 2A, B). The I-V plot of the mutant at
1 s of the voltage step shows a rectification above — 17 mV as in the wild type. Correspondingly, slow tail outward currents are seen after such steps (Fig. 4B).
When the bath is changed from the K-solution to the Na-solution, the membrane
of the paranoiac mutant first depolarizes to — 2-6 ± 4-9 mV (n — 24). Although in some
cases, this first plateau depolarization can last for more than 5 min, typically the membrane repolarizes within 3 min to a lower 'resting' level ( — 27-1 ±6-o mV, n = 10).
After the first episode, plateau depolarizations of up to several minutes take place
spontaneously and can be triggered by injected current (Fig. 3, right). These repeated
prolonged plateau depolarizations correspond to the bouts of backward swimming in
the Na-solution, which constitute the diagnostic phenotype of paranoiac mutants
(Kung, 1971 a, b).
Under voltage clamp, a step depolarization from the — 30 mV holding level induces
the inward transient in the Na-solution. The peak inward transient and the voltage at
which this peak occurs are similar to those in the K-solution. However, when the
voltage step is less than — 14 mV, the late current in inward (Fig. 4C, arrow) instead
of outward. This inward current develops very slowly during the depolarization (see
below on kinetics). At the end of the voltage step, the sustained inward current is
followed by a prominent inward tail current (Fig. 4C, double arrow), which decays
slowly (see below and Fig. 7 for kinetics). A voltage step above —14 mV induces
a large net outward current followed by a complex tail current. Nevertheless, a very
slow inward component is clearly seen in the tail current after the step (arrow.
Fig. 4D). This inward component is preceded by an outward component that decay|
faster (as estimated from the end of the voltage pulse to the peak of the tail current
Ca-induced Na-current in Paramecium
C
A -17.
-17.
313
4--
400 ms
n -J
20 ms
Vm (mV)
Fig. 4. Membrane currents under voltage clamp from a typical 'paranoiac' mutant cell in the
K-solution (A, B) and in the Na-solution (C, D). The membrane currents in the K-solution
are similar to those in the wild-type cell (see Fig. 2 A, B). The currents in the Na-solution,
however, are different. With a lower voltage step (C), the total current during the step gradually
develops inwardly (arrow) and accompanies a large inward tail current (double arrow). A higher
voltage step which induces a large outward current and an outward tail in the K-solution
(B) also gives rise to a huge inward tail together with an outward component (D: arrow) in
the Na-solution. Ca-transients inactivate too rapidly to be photographed clearly in A to D.
The traces under B and D are the Ca-transients simultaneously taken with B and D. Arrows
show the peak Ca-transients. I-V relationships at 1 s during steps (closed symbols) from the
same cell of A to D are shown in E. Circles are currents in the K-solution: the curve is similar
to that of the wild type (Fig. 2 E). Squares denote currents in the Na-solution. Note a prominent
N-shaped curve of I-V relationship in the late current (arrow). The peak Ca-transients,
however, are similar in the two solutions (open symbols).
314
Y. SAIMI AND C. KUNG
complex). The outward tail current seen in the Na-solution is similar in kinetics tq
that in the K-solution.
A plot of the late current at 1 s vs. step voltage level shows a prominent region of
negative resistance around — 20 mV (Fig. 4E, arrow). This region of negative resistance is not seen in the K-solution or in the ' Ca-solution' devoid of both K+ and Na+.
The N-shaped I-V plot suggests that depolarization of the undamped membrane can
become regenerative because of this slow inward current.
The kinetics of the slow inward current in the Na-solution
To study the kinetics of the slow inward current, we compare the membrane
currents in solutions with and without Na+. In the K-solution and in the Na-free
Ca-solution the I-V curve is nearly linear from —45 to about —17 mV (Fig. 4E),
showing that the late outward current develops very little, while the slow inward
current is induced (Fig. 4). Fig. 5 B is a typical result showing the magnitude of the
inward current at different times during a 1 min voltage step from the holding level
of — 30 mV to — 21 or — 17 mV. The inward current increases in the first 9 s (4-9 s in
three cases) to about 1 nA (0-9-1-2 nA) and then slowly declines to less than 0-7 nA
(0-3-0-7 nA) in 60 s during the clamp depolarization.
The development of the slow current was also studied by measuring the size of the
tail inward current upon repolarization from the voltage step of various durations.
Since the tail inward current decays exponentially after the return from a low voltage
step (see below), we are presumably dealing with a single conductance in isolation
(unlike the situation during the measurement of total current (Fig. 5 B) which may
contain more than one component). The size of the tail current shows a rise and a fall
(Fig. 5 A) when plotted against the duration of the voltage pulses which parallel those
of the total slow inward current itself; the maximal tail current is obtained after
a voltage pulse of 4-9 s in duration (three cells). Therefore we conclude that the
development of the slow inward current in the Na-solution is due to a change of one
conductance which activates and inactivates very slowly.
The slow inward current begins its development within 10 ms of the onset of
depolarization to a low ( < — 17 mV) or a high (^ — 14 mV) potentials. On the other
hand, the slow outward current, most probably the Ca-induced K-current (Satow
& Kung, 19806), which is induced only at the high potential, appears > 200 ms
after the onset of the voltage step (Fig. 6A-D). The outward component of the tail
current following the pulse also occurs only when the step is long. When the step
is < 200 ms (Fig. 6C), neither the outward current or its tail is observed. Thus, the
slow outward current has a different threshold and different kinetics from those of
the slow inward current.
The time course of the tail of the inward current during the step is also slow. The
inward tail current (Fig. 4C) after a small depolarization ( — 21 to—17 mV in different
cases) decays almost exponentially, with a time constant of approximately 0-4 s
(0-38 ±0-15 s, n = 6) after the intial 20 ms during which an additional small inward
component is occasionally seen. The slower inward component of the compound tail
(Fig. 4D) seen after a larger depolarization (— 10 to —9 mV) also decays with similar
kinetics (time constant 0-43 ±o-io s, n = 6). An example of a semi-logarithmic plot
of the tail currents is shown in Fig. 7. At higher voltage steps, the slow inwara
Ca-induced Na-current in Paramecium
315
Time (s)
0
-1-
10
20
30
40
SO
1
1
1
1
1
1
60
1
o
•
_
•
"
O
T3—O-"
-2-
Fig. 5. The rise and fall of the slow inward current during a 60-s voltage step from a ' paranoiac'
mutant bathed in the Na-solution. B: The total membrane current from a chart record during
the step at a low voltage ( — 17 mV), where there is little sign of outward current in the Ksolution and the Ca-solution. A: Tail current 20 ms after return from the voltage step (at
—17 mV) of various duration in the same cell of B as diagrammed in the inset. The tail
currents are plotted against the duration of the voltage step. Note that the curve in A also
describes the activation and inactivation of the slow inward current in B.
600 ms
Fig. 6. Development of the slow current of the ' paranoiac' mutant in the Na-solution examined
with various duration of the voltage step ( — 14 mV). In each frame the upper trace is the membrane potential and the lower the membrane current. In the Na-solution, this voltage step
triggers first a slow inward then an outward net current followed by a tail current complex (A).
When examined with various duration of the voltage step (1 s in A to 10 ms in D), the inward
component of the tail current complex increases slowly with the duration of the voltage step.
Also note that the faster outward tail component, probably that of Ca-induced K-current, in
the complex (A) is lost by shortening the duration (C), while the inward tail component
remains. The Ca-transients of similar size are present in every case, but are not shown.
316
Y. SAIMI AND C. KUNG
s -
0-5
I
01
100
I
300
200
Time (ms)
400
500
Fig. 7. Semi-log plots of typical slow tail current obtained in the Na-solution. An inward tail
after a 1 s-voltage step at —17 mV is shown by closed circles. Note that this inward tail
decays almost exponentially for several hundred milliseconds with a time constant of 320 ms.
The open circles represent the tail current after a voltage step to —10 mV, which induces
a large outward current and also an outward component in the tail. The curve has a kink reflecting a faster decay of the outward component than the inward component in the tail
complex. The slower inward component of the tail well fits an exponential curve which nearly
parallels that from a lower voltage step (closed circles) in the same cell. Time o denotes the
end of the voltage step. Any component in the tail faster than 10 ms is not analysed in this
experiment because of the capacitative surge.
component of the tail is preceded by an outward component (Figs. 4D, 6 A and the
upper curve of Fig. 7) which decays faster, with a time constant of less than o-1 s. In
contrast to these slow currents, the tail currents associated with the Ca-transient and
the voltage-sensitive K-current decay so fast that they cannot be clearly discriminated
from the capacitative surge.
It is clear, therefore, that during long depolarization a conductance is slowly
activated for several seconds to pass the inward current, but only when the paramecium is bathed in a solution containing Na+. The slow current and its associated
tail current, found in the K-solution and in the Ca-solution, are both outward and
clearly differ from the inward currents in their kinetics and apparent voltage dependency. The results in this section further strengthen the notion that the slow inward
current with a voltage range of negative resistance is due to the presence of external
Na+.
Effects of external and internal Na+ concentration
Since the long plateau depolarization in the undamped membrane and the slow
inward current in depolarized membrane under the voltage clamp are all Nadependent, we investigated the effects of external concentrations of Na+ on these
parameters.
The level of the first plateau depolarization following Na + perfusion, usually trg
Ca-induced Na-current in Paramecium
317
Table 1. Electrical parameters of the 'paranoiac '-mutant cells in various solutions
()
Solution
K-solution
Ca-solution
Na-solution
Na-solution
Na-solution
(d)
(e)
(/)
Membrane
Maximal
()
Membrane
W
Concentration
Potential Slow Inward
Potential
Maximal
Current
of (c)
of
of (e)
Ca-transient
+
(nA)
K or Na+
(mV)
(mV)
(nA)
4 mM K +
no K+ or Na +
2 mM Na +
4 DIM Na +
8 mM Na+
-7-I±I-2
-7-5±i'7
— 9-2±o-7*
-7-5 + 07
— 8-4±i-8
(g)
Holding
Current
At - 3 0 m V
(nA)
(h)
n
-7'3±i-o
f
+ 0-33+012
+ 046 ±0-29
-7'4±i-'
t
-6-7±3-2* —oo6±o-O9 — 20-2 ± I-I + 004 +015
— 6 - 2 + 1-0 — 0 1 8 + 0 1 3 -2O'8±2-6
-5-5 ±2-3 —0-65 ±0-49 - i 6 - 8 ± 3 - 5 — 0i6±0'3O
« = 3There is no slow inward current in these solutions.
These data show that the slow inward current is dependent on the Na + concentration in the solution.
The maximal peak Ca-transient inward currents and their corresponding membrane potentials are
given in columns (c) and (d), respectively. Similarly, the maxima of the slow currents in the negativeresistant region (e.g. Fig. 4E: arrow) and their corresponding membrane potentials are listed in columns
(e) and (/), respectively. The holding currents at — 30 mV are used as reference, because the left
shoulder of the N-shaped I-V curve (e.g. Fig. 4E) lies at around —30 mV in various concentrations of
Na + . Parameters for the Ca-transients stay similar in different solutions. See Materials and Methods for
the composition of the solutions. Data are given as mean + s.D.
longest and largest, is dependent on the external Na concentration. In 2, 4 and 8 mMNaCl the depolarizations were — 9-1 + 6-1 (n = 18), —8-3 + 3-0 (n = 24) and —2-6
± 4-9 mV (w = 24), respectively. (In 2 out of 20 cases, there was no typical plateau
depolarization in the solution containing 2 mM-Na+. All cases gave plateau depolarizations with higher Na + concentrations.) At 2 mM-Na+, there is little inward current and
the region of negative resistance of this current is less prominent. This current and the
region of negative resistance appear and become bigger in solutions of higher Na +
concentrations, although the variation is large. Changes in external Na + concentration, however, have only minor effects on the fast inward transient (the Ca-current)
and the region of negative resistance of this transient Ca-current (Table 1).
If the Na-dependent, slow, inward current is carried by Na + , one would expect
internally added Na + to inhibit this current. We, therefore, iontophoretically injected
Na + into paranoiac mutants bathed in Na-solution (8 mM-Na+) in which the slow
inward current had been observed. A 10 nA injection of Na+ for 1 min suppresses
the slow inward current during a small depolarization step, and the tail inward
current after it. With a larger depolarization (> —14 mV) Na+ injection increases
the apparent outward current and decreases the tail inward current (Fig. 8 A, B),
suggesting a suppression of the inward component in the complex. To isolate the
suppressible inward component the membrane current after the Na-injection was
subtracted from that before the injection. The isolated inward component, which
corresponds to a lower depolarization step (—17 mV), is more than 0-2 nA and
increases throughout the 1 s period. At a higher voltage ( - i 4 m V ) this current is
larger (> o-6 nA) (Fig. 9 A). This isolated current is always associated with an inward
tail current after the depolarization step. The magnitudes and the kinetics of the
inward current and the tail current isolated by this method are similar to those induced
by lower voltage steps (Fig. 4C, 5B). In control experiments, iontophoresis of K +
Pith a current identical to the one used to inject Na+ (10 nA, 1 min) has little effect
EXB 88
Y. SAIMI AND C. KUNG
Na injected
K injected
Fig. 8. Suppression of the slow inward current and its associated tail by the injection of Na +
into the cell. A: Before injection. B: After injection of Na + (10 nA, i min). C, D: Control
experiment with a different cell. C: Before injection. D: After injection of K + (10 nA, i min).
0
•^
200
400
Time (ms)
600 800
r
1
1000 1200
1500
p » f t
TT
tt—°
2
Fig. 9. Changes in the slow membrane currents after iontophoresis of Na + . Each curve shows
the subtracted change in the membrane current with an identical voltage step (its duration is
indicated by arrows) due to a 10 nA, 1 min iontophoresis. A: From Na + injection into a cell
bathed in the Na-solution (the current before injection (Fig. 8 A) minus that after injection
(Fig. 8B)). B: From K + injection into a cell bathed in the Na-solution (the current before
injection (Fig. 8C) minus that after injection (Fig. 8D)). C: From Na + injection into a cell
bathed in the K-solution (Na-free) (the current after injection minus that before injection).
D: From K + injection into a cell bathed in the K-solution (the current after injection minus
that before injection). Voltage steps: —14 mV in A, B and D; — 9 mV in C.
A is therefore the slow inward current suppressible by the Na+-injection in the presence of
external Na + . C is the slow outward current induced by the Na+-injection in the absence of
external Na + . Although of opposite signs, these two currents and their associated tails are
similar. The current in A is also similar to the current directly observed upon a lower step
depolarization (—17 mV) where the outward current is not induced (Fig. 4C). Control
experiments with K+-injection, B and D, show much less effect on the slow membrane
current. The broken lines and the circles before time o denote the change in the holding current
(at — 30 mV) by the iontophoreses.
Ca-induced Na-current in Paramecium
319
M t h e slow membrane currents (Fig. 8C, D). Subtraction of the membrane current
alter the K+ injection from that before yields only a small change in currents
(< 0-5 nA at — 14 mV) that changes little in time (Fig. 9B).
The effect of injected Na+ can also be demonstrated in cells bathed in the Na^-free
' K-solution'. Iontophoresis of Na + for 1 min with 10 nA greatly increases the outward
currents induced by voltage steps above —14 mV. Subtraction of currents reveals
a slow outward current, which develops slowly during the 1 s step, and the corresponding slow outward tail current, which decays over several 100 ms (Fig. 9C).
Iontophoretic injection of K+ into cells bathed with the K-solution has virtually no
effect (Fig. 9D).
Paramecia grown in the Na+-free medium can be expected to have very low internal
Na + . The wild types grown in the Na+-free medium, show 'paranoiac' behaviour
(i.e. they swim backward repeatedly for tens to hundreds of seconds when exposed to
external Na + ). However, their normal behaviour is re-established within a few
minutes, possibly due to the accumulation of internal Na+. This rapid readjustment
and the complex nature of the tail current makes very difficult a quantitative analysis
of the slow inward current and its tail current from 'Na+-depleted cells'. Addition of
5 mM-Na+, in place of K + , in the 'Na+-free medium' (see Materials and Methods)
suppressess part of the ' paranoia' induced by Na + deficiency in the wild types. The
size of the tail inward current of these cells is smaller than that of the Na+-depleted
cells.
The results in this section are consistent with the view that prolonged depolarization
induces a slowly developing current carried by Na + . This current is inward when the
cell is bathed in the Na-solution but is outwardly directed in the Na-free K-solution.
It inactivates slowly after the repolarization. Since depolarization first induces a fast
Ca-response, which increases the internal concentration of Ca2+, it is possible that the
Na-current is induced by internal Ca2+ and not by the depolarization directly. Therefore, we tested the effect of Ca2+ on this current.
The effect of external and internal Ca2* concentrations on the Na inward current
To measure the external Ca2+ requirements for the inward Na + current, we replaced
most of the Ca2+ in the Na-solution with Mg2+. When this Mg-Na solution (8 mMNa + , 0-99 mM-Mg2+, o-oi mM-Ca2+) replaces the Na-solution (8 mM-Na+, 1 mMCa2+), the Ca-transient is reduced. However, the presence of an inward tail current
after the step depolarizations indicates that a residual, though greatly reduced, slow
inward current persists in cells bathed in the Mg-Na solution (data not shown).
Correspondingly, most undamped cells still show repeated spontaneous or triggered
plateau depolarizations, albeit smaller and shorter.
For a more stringent test, paramceia were bathed in a 8 mM-Na+ solution with
o-1 mM-EGTA and no added Ca2+. The cells were thus in a critical condition. However, experiments could be performed, rapidly, before severe deterioration occurred.
Step depolarizations with the voltage clamp, revealed little sign of the inward Catransient or of the slowly developing current, as judged by the lack of a tail current in
this Na-EGTA solution (Fig. 10 A). Upon return to the Ca-containing Na-solution,
Ca-transient and the slow currents are partially restored (Fig. 10 B). The re-
320
Y. SAIMI AND C. KUNG
B
_z5
400 ms
400 ms
20 ms
Fig. 10. The effect of removing Ca a+ from the Na-solution on the slow inward current. The
cell is first incubated in Ca-free (EGTA added) Na-solution and given a voltage step (A). The
Ca-transient is lost (see the trace below A). The slow tail current is also lost. The membrane
is leaky in this solution, since the holding current at — 30 mV is large. Broken line shows zero
current level. B: Partial recovery of the same cell in the normal Ca-containing Na-solution.
Note the slow tail inward current as well as the fast Ca-transient (the trace below B) is restored.
EGTA injected
-17 ,
A-I
400 ms
-14
.-14
Fig. 11. Suppression of the slow inward current by EGTA-injection. A: Before injection.
B: After injection of EGTA (5 nA, 30 s). The slow inward current and its tail are reduced.
C and D: An experiment in the same cell similar to A and B, but with a large voltage step.
After injection of EGTA (5 nA, 30 s), the inward tail becomes smaller (D). Also note the large
activating outward current during the step is removed together with the outward tail component by the EGTA-injection. This result suggest that the activating outward current and
the associated outward tail, like the slow inward current and the inward tail, are Ca2+triggered. E and F: Effect of injection of larger amount of EGTA (10 nA, 1 min) through
a low resistant electrode. After injection of EGTA the current following the Ca-transient
(too faint in these photographs) is held constant inwardly during the voltage step (see text for
interpretation), while the inward tail is greatly reduced.
appearance of the inward tail current indicates the slow inward current in the slow
current complex.
The results above suggest that a small quantity of external Ca2+ is needed to
initiate the Na-current and the plateau depolarizations. EGTA was, therefore, injected
into the cells to discover whether internal Ca2+ induces the Na-current.
EGTA (injected with a current of 5 nA for 30 s) increases the inward Ca-transient
slightly. In contrast, the slow Na inward current and its corresponding inward tail
current are suppressed after EGTA injection (Fig. 11 A, B). The injected EGTA also
suppresses the slow outward K-current, and its corresponding outward tail current,
after the voltage step change (Fig. 11C, D).
Larger EGTA injection (10 nA, 60 s) has an additional effect. It induces a sustain
inward current when the voltage step is at about —14 mV. Unlike the slow
Ca-induced Na-current in Paramecium
321
-17
400 ms
J;
20 ms
H8
E
I
- 4
/
-40
c
- 2 0 ^
m
- -4
Fig. 12. Membrane currents of a'pawn'-'paranoiac' double mutant
(PaA/PaApwa^/pwA").
A and B show currents induced in the K-solution and C and D in the Na-solution. Note that
there is no sign of the slow inward current in the Na-solution, despite the presence of the
'paranoiac' mutation. Also note the nearly completely lost Ca-transients due to the 'pawn'
mutation (traces below B and D). The tail after a larger voltage step in the Na-solution (D)
is slightly inward in some cases, probably because of leakiness of this ' pawn' mutation. An
I-V relationship of this cell is shown in E. Only the late currents at i s during the voltage step
are plotted. Circles denote currents in the K-solution and squares represent those in the Nasolution. The negative resistant region of the I-V plot for the slow current seen in the ' paranoiac' mutant (Fig. 4E) is abolished by adding the 'pawn' mutation which blocks the Ca2+entry.
inward current, that develops during seconds after the Ca inward transient, this
sustained inward current appears immediately after the Ca-transient. This observation in P. tetraurelia is similar to that in P. caudatum by Eckert & Brehm (1979), who
attribute this current to the residual Ca2+ flow through an incompletely inactivated
Ca-conductance due to the low internal concentration of Ca2+. Unlike the Na-current
this Ca-current is not followed by a slow tail after the step depolarization (Fig. 11E, F).
The above experiments show that the slow Na-current requires a high internal
Ca 2+ concentration. It is, therefore, reasonable to suppose that this Na-current is not
Brectly induced by depolarizations but by the high internal concentration of Ca2+
322
Y. SAIMI AND C. KUNG
which may come from the Ca-transient triggered by the depolarization. To test tltf
idea we examined mutants which do not have the Ca-transient.
Pawn mutations eliminate the Ca-induced slow Na-current
'Pawns' are mutants that have little or no transient Ca2+ inward current (Kung &
Eckert, 1972; Oertel et al., 1977; Satow & Kung, 1980a). Voltage-clamp experiments
in four different pawn mutants show that, in all cases (^ 2 cells of each group), the
mutations suppress not only the Ca inward transient, but also the slow Ca-induced
Na inward current and its tail. The slow outward current (the Ca-induced K-current)
is also suppressed.
As noted above, the inward Na-current is best observed in the paranoiac mutants.
Therefore, we used pawn-paranoiac double mutants to test whether the Ca-transient
is really a prerequisite for this Na-current in a genetic background more favourable
for its appearance. As shown in Fig. 12, both the pawn A-paranoiac double mutant
and the pawn B-paranoiac double mutants are also devoid of any late currents and
their associated tail currents. In all cases of such double mutants, there is also no
spontaneous or triggered prolonged depolarizations in the Na-solution characteristic
of the paranoiac single mutant. These observations further support the view that the
outward K-current and the inward Na-current that develop slowly upon depolarization in the wild type are induced by internal Ca2+ entered through the voltagesensitive Ca-channel.
DISCUSSION
We have discovered a slow inward current in Paramecium. It is most likely carried
by Na* because: (1) this current and the tail current associated with it are observed
only when there is external Na+; (2) the current and its tail are larger when external
Na + is more concentrated; (3) they are smaller or absent when internal Na + is
increased by iontophoresis.
A large influx of Na + associated with prolonged depolarizations has been demonstrated by 22Na accumulation experiments in Paramecium (Hansma & Kung, 1976;
Hansma, 1979). During step depolarizations of the paramecium membrane the Cacurrent does not inactivate completely (Brehm & Eckert, 1978) and there is a Cainduced K-current (Satow & Kung, 19806), although both currents should be small
at voltage steps of less than —17 mV. This complexity, and the small size of the
Na-current, make it very difficult to determine accurately the reversal potential of
this Na-current. This reversal potential, though variable from cell to cell, is definitely
above — 15 mV in the 8 mM-Na^-solution.
The inward current has very slow kinetics. It develops, on depolarization for several
seconds, to a peak and decays upon a repolarization with a time constant of hundreds
of ms. The slow kinetics of this current exclude the possibility that the current flows
through the voltage-sensitive Ca channel which activates and nearly completely
inactivates in a few ms. Furthermore, EGTA-injection, which would be expected to
enhance and prolong Ca-current or any current through the Ca-channel (Eckert &
Brehm, 1979), in fact, reduces the slow inward current (Fig. 11). It is also unlikely that
this inward current is a residual current after a hypothetical suppression of the
ward current by Na + . Such a hypothesis entails that Na + suppresses a conductan
Ca-induced Na-current in Paramecium
323
fti fact, during the Na-induced plateau depolarizations, the membrane is far more
conductive (i.e. having very low resistance, Satow et al. 1976) and large fluxes of ions
occur (Hansma & Kung, 1976; Hansma, 1979). Thus, the conductance for this slow
inward current in Na-specific in physiological conditions. K + , Ca2+, Mg2+ and Ba2+
cannot substitute for Na+ to carry this current. Only Li^ can replace Na+ to trigger
the characteristic behavioural responses and spontaneous or triggered membrane discharges of wild type and 'paranoiac' (unpublished data).
The Na-current in Paramecium is very different from the well-known Na-current
in axons. Besides its slow kinetics, it is not affected by tetrodotoxin or saxitoxin (unpublished data). Furthermore, its triggering mechanism is very different from the
conventional Na-current. It is not directly triggered by depolarization but by internal
Ca2+, since depletion of external Ca2+ and injection of EGTA suppress or eliminate
this Na-current even under depolarization. It appears to be indirectly depolarizationsensitive, because the increase in internal Ca2+ is normally induced by the activation
of the voltage-sensitive Ca-conductance. This is demonstrated by the lack of this Nacurrent in the single or double mutants carrying a defect which makes the Ca-channel
nonfunctional (Fig. 12).
The Ca-induced Na-current can have a region of negative resistance in the I-V
plot (Fig. 4E). It is, therefore, potentially a mechanism for regenerative depolarization
in the undamped membrane. A form of slowly rising depolarization lasting tens to
hundreds of seconds has been recorded in the paranoiac mutants (Fig. 3C; see also
Satow et al. 1976). The slow rise and the long duration of such depolarizations of the
undamped mutant membrane parallel the slow activation and the persistence of the
Na inward current under the voltage clamp (Fig. 5). Continuous ciliary reversal during
these long depolarizations indicates a lasting increase in internal Ca2+ concentration
which should be correlated with activations of the Ca-induced Na inward current as
well as the Ca-induced K outward current. Such prolonged depolarizations have
indeed been shown to be correlated with a large 22Na influx and a large K + loss
(Hansma & Kung, 1976; Hansma, 1979). During the long depolarizations the membrane is electrically very conductive but gains resistance gradually over tens of
seconds (Satow et al. 1976). This change parallels the gradual inactivation of the Na
inward current, ~ 10 s after the step depolarization under the voltage clamp (Fig. 6).
A decrease in the Na-conductance could be part of the mechanism which terminates
the long depolarization and, therefore, long backward swimming, probably augmented
by the loss of the electromotive force for Na + resulting from internal accumulation
(Hansma, 1979).
The Ca-induced Na-current and the spontaneous long depolarizations are easily
triggered in the paranoiac mutants. We have, therefore, exploited this opportunity to
study these phenomena using these mutants. However, such phenomena also exist
in the wild type. Although the observable slow inward current appears to be smaller
and is often obscured by the outward current, the slow inward tail current after the
repolarization is clearly seen in the wild type and such tails have similar decay kinetics
as those of the paranoiac mutant (Fig. 2). Such tail inward currents become larger
when the wild-type cells are cultured in a Na-free medium. Na-related long depolarition and prolonged backward swimming can also be observed in the wild type
pecially when first exposed to a high concentration of external Na+ and/or after
R
324
Y. SAIMI AND C. K U N G
growing in Na+-free medium. Prolonged depolarizations in Na-solutions can bj
spontaneous or can be triggered by either a train of closely-spaced short pulses ol
prolonged current injection in the wild type. Such prolonged depolarizations are
retained although the long backward swimming is seldom observed in free-swimming
wild types, in near physiological conditions. This difference suggests that certain constraints or damage by the low-resistant electrodes favour the generation of the prolonged depolarizations. It is commonly observed that extreme ionic imbalances (e.g.
relatively low external Ca2+ concentration), noxious chemicals (detergents, proteases,
fixatives, etc.), excessive mechanical stress (microsurgery, microinjection, forcing
through micropipette) or attacks of predators (suctorians, Didinium) all cause the
wild-type paramecium to spin rapidly backward continuously for seconds to minutes
in various Na+-containing culture media (Cerophyl, hay-infusions, fresh water) or
wash solutions (Dryl's, Pringsheim's). A common trigger is likely to be increased
internal Ca2* which is prolonged by repeated Ca-action potentials or by membrane
damage. The adaptive value of this rapid backward swimming could be in mediating
direct escape from a source of threat. This behaviour is distinct from the transient
avoiding reactions, which, like the tumblings in bacteria (Springer et al. 1979)
accounts for various taxes through the biased random walk (Jennings, 1906; Dryl,
1973; Van Houten, 1978; Hennessey & Nelson, 1979). The continuous backward
swimming and the transient avoiding reaction are mechanistically different. The
former is based on the long depolarization in second or minute time scale, is due to
Ca-induced conductances and involves external Na + , while the latter is in millisecond
scale, due to voltage-sensitive channels and does not involve Na + .
This study takes advantage of 'paranoiac' mutant because it is able to generate
prolonged depolarizations easily in the Na-solution and therefore allows us to investigate the electrophysiological parameters related to prolonged depolarizations. The
findings apply to the wild type since it also exhibits such a current under certain
circumstances. They do not, however, identify the specific defect in the paranoiac
mutant since a variety of factors (from the retardation of the Ca-pump to alterations
of the number or topography of the Na channel) can lead to ' paranoia' according to
the above hypothesis of the generation of prolonged depolarization.
We thank Drs M. Forte, A. D. Murphy and Y. Satow for their comments and
suggestions. This work was supported by NSF grants BNS 77-20440 and BNS
REFERENCES
BREHM, P. & ECKERT, R. (1978). Calcium entry leads to inactivation of calcium channel in Paramecium.
Science, N.Y. Z02, 1203-1206.
CHANG, S. Y., VAN HOUTEN, J., ROBLES, L., LUI, S. & KUNG, C. (1974). An extensive behavioral and
genetic analysis of the Pawn mutants of Paramecium aurelia. Genet. Res. 23, 165-173.
DRYL, S. (1973). Chemotaxis in ciliated protozoa. In Behavior of Micro-organisms (ed. A. Perez-Miravete),
pp. 16-30. New York: Plenum Press.
ECKERT, R. & BREHM, P. (1979). Ionic mechanisms of excitation in Paramecium. Ann Rev. of Biophy.
and Bioeng. 8, 353-383.
HANSMA, H. (1979). Sodium uptake and membrane excitation in Paramecium. J. Cell Biol. 81, 374-381.
HANSMA, H. & KUNG, C. (1976). Defective ion regulation in a class of membrane-excitation mutations
in Paramecium. Biochim. biophys. Ada 463, 128-139.
Ca-induced Na-current in Paramecium
325
IIENNESSEY, T. & NELSON, D. L. (1979). Thermosensory behavior in Paramecium tetraurelia: a quantitative assay and some factors that influence thermal avoidance. J. gen. Microbiol. 112, 337-347.
JENNINGS, H. S. (1906). Behavior of Lower Organisms. Bloomington: Indiana University Press.
KUNG, C. (1971a). Genie mutations with altered system of excitation in Paramecium aurelia. I. Phenotypes of the behavioral mutants. Zeitschrift fur vergleichende Physiologie 71, 142-164.
KUNG, C. (1971 6). Genie mutations with altered system of excitation in Paramecium aurelia. II. Mutagenesis, screening and genetic analysis of the mutants. Genetics 69, 29—45.
KUNG, C. (1979). The biology and genetics of paramecium behavior. In Topics in Neurogenetics.
(ed. X. O. Breakfield), pp. 1-26. New York: Elsevier North-Holland, Inc.
NAITOH, Y. (1968). Ionic control of the reversal response of cilia in Paramecium caudatum - a calcium
hypothesis.,?. gen. Physiol. 51, 85-103.
NAITOH, Y. & ECKERT, R. (1968). Electrical properties of Paramecium caudatum: Modification by bound
and free cations. Z. vergl. Physiol. 61, 427—452.
NAITOH, Y. & ECKERT, R. (1969). Ionic mechanism controlling behavioral responses of Paramecium
to mechanical stimulation. Science, N.Y. 164, 963-965.
NAITOH, Y. & ECKERT, R. (1972). Electrophysiology of ciliate protozoa. Exp. in Physiol. and Biochem.
5, 17-31NAITOH, Y. & ECKERT, R. (1974). The control of ciliary activity in protozoa. In Cilia and Flagella.
(ed. M. A. Sleigh), pp. 305-352. New York: Academic Press.
NELSON, D. L. & KUNG, C. (1978). Behavior of Paramecium: chemical, physiological and genetic
studies. In Taxis and Behavior (Receptors and Recognition, Series B. volume 5) (ed. G. L. Hazelbauer), pp. 75-100. London: Chapman and Hall.
OERTEL, D., SCHEIN, S. J. & KUNG, C. (1977). Separation of membrane currents using a Paramecium
mutant. Nature, Lond. 268, 120—124.
SATOW, Y., HANSMA, H. G. & KUNO, C. (1976). The effect of sodium on 'paranoiac'— a membrane
mutant of Paramecium. Comp. Biochem. Physiol. 54A, 323-329.
SATOW, Y. & KUNG, C. (1974). Genetic dissection of active electrogenesis in Paramecium aurelia.
Nature, Lond. 247, 69-71.
SATOW, Y. & KUNG, C. (1976a). A TEA+-insensitive mutant with increased potassium conductance in
Paramecium aurelia. J. exp. Biol. 65, 51-63.
SATOW, Y. & KUNG, C. (19766). A mutant of Paramecium with increased relative resting potassium
permeability. J. Neurobiol. 7, 325-338.
SATOW, Y. & KUNG, C. (1979). Voltage sensitive Ca-channels and the transient inward current in
Paramecium tetraurelia. J. exp. Biol. 78, 149-161.
SATOW, Y. & KUNG, C. (1980a). Membrane currents of pawn mutants of the pivA group in Paramecium
tetraurelia. J. exp. Biol. 84, 57-71.
SATOW, Y. & KUNG, C. (19806). Ca-induced K+-outward current in Paramecium tetraurelia. J. exp. Biol.
88, 293-303.
SCHEIN, S. J., BENNETT, M. V. L. & KATZ, G. M. (1976). Altered calcium conductance in pawns,
behavioral mutants of Paramecium aurelia. J. exp. Biol. 65, 699-724.
SONNEBORN, T. M. (1970). Methods in Paramecium research. In Methods of Cell Physiology, vol. 4
(ed. D. M. Prescott), pp. 243-339. New York: Academic Press.
SPRINGER, M. S., GOY, M. F. & ADLER, J. (1979). Protein methylation in behavioral control mechanism
and signal transduction. Nature, N. Y. 280, 279-284.
VAN HOUTEN, J. (1978). Membrane potential control chemotaxis in Paramecium. J. comp. Physiol. 127,
167-174.
VAN HOUTEN, J., CHANG, S. Y. & KUNG, C. (1977). Genetic analysis of 'Paranoiac' mutants of Paramecium aurelia. Genetics 86, 113-120.