Long-lasting potassium channel inactivation in myoepithelial fibres is related to characteristics of swimming in diphyid siphonophores
1 Ine Marine Laboratory of National Institute for Physiological Sciences,
Ine, Kyoto 626-0424, Japan
2 Institute for Enzyme Research, Tokushima University, Tokushima 770-8503,
Japan
3 Laboratory of Biology, Graduate School of Commerce and Management,
Hitotsubashi University, Kunitachi, Tokyo 186-8601, Japan
4 Marine Biological Association of UK, Plymouth, PL1 2PB, UK
* Author for correspondence (e-mail: iinoue{at}ier.tokushima-u.ac.jp)
Accepted 13 October 2005
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Summary |
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Key words: diphyid, behaviour, striated muscle, K+ channel, inactivation, Diphyes chamissonis
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Introduction |
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A curious feature of their swimming behaviour is that the force generating
the propulsive jets is weak at first and then successively increases during
the initial jets. Previous studies of the electrical properties of the
myoepithelium showed that action potentials that propagate through the muscle
sheet were carried by both Na+ and Ca2+ and possessed
unusual features: during a short burst of action potentials, the initial
action potentials were of conventional rapidly rising and rapidly falling
form, but subsequent action potentials showed both a greatly increased
overshoot and the formation of a plateau or hump on the falling phase, leading
to greatly prolonged action potentials
(Chain et al., 1981;
Bone, 1981
;
Bone et al., 1999
). It has
therefore been suggested that this augmentation of the action potential evokes
successive increases in myoepithelial tension by increased Ca2+
influx during the action potentials (Bone,
1981
; Bone et al.,
1999
). However, in order to investigate the ionic mechanisms of
the action potential augmentation, potential recordings are not sufficient and
it is necessary to develop an experimental system enabling ionic currents
passing through voltage-gated ionic channels to be recorded under controlled
membrane voltage. In the present study, we developed a protocol whereby single
myoepithelial fibres were isolated, and we independently recorded
Na+, Ca2+ and K+ currents by subjecting the
fibre to whole-cell voltage clamp. We describe here the experimental results
showing that the action potential augmentation is produced by
voltage-dependent long-lasting inactivation of K+ channels, not by
a successive increase in activities of Na+ or Ca2+
channels. The whole-cell clamp experiments on single fibres also revealed the
pharmacological properties of the ion channels that could not be detected in
in vivo preparations.
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Materials and methods |
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Solutions and chemicals
Artificial seawater (ASW) consisted of (in mmol l1) 450
NaCl, 9 KCl, 10 CaCl2, 50 MgCl2 and 15 Na-Hepes buffer
(pH 7.8). An ASW containing a lower (4 mmol l1)
concentration of Ca2+ (4Ca-ASW) was also prepared to avoid damage
to the cell in whole-cell voltage clamp experiments. Nominally
Ca2+-free ASW (N-Ca-free-ASW) was made by replacing
CaCl2 in ASW with MgCl2. Ca2+-free ASW
(Ca-free-ASW) was made by replacing CaCl2 in ASW with Na-EGTA. The
pipette (intracellular) solution for whole-cell voltage clamp experiments
consisted of (in mmol l1) 450 Cs-D-aspartate, 15
MgCl2, 15 EGTA, 15 Cs-MOPS buffer (pH 7.2). This solution is
referred to as Cs-asp. For K+ current measurements,
Cs-D-aspartate and Cs-MOPS were replaced with
K-D-aspartate and K-MOPS, respectively (K-asp). Stock solutions of
100 mmol l1 CoCl2, 1 mmol l1
tetrodotoxin (TTX; Sankyo, Tokyo, Japan) dissolved in distilled water and 10
mmol l1 nifedipine (Sigma, St Louis, MO, USA) dissolved in
ethanol were kept in the dark at 4°C. The experiments were carried out at
a room temperature of 2022°C, which was close to the sea
temperature.
Isolation of single locomotor muscle fibres
The anterior nectophore of D. chamissonis was cut open and
incubated in a dissociation medium (Ca-free-ASW containing 5 mg
ml1 of trypsin; Boehringer, Mannheim, Germany) at 25°C.
When the myoepithelium was detached from the nectophore, it was taken and
incubated in a new dissociation medium until the connection between muscle
fibres became loose. Then the myoepithelium was gently agitated in an
experimental chamber filled with an external solution for each experiment.
Single muscle fibres that attached to the bottom of the chamber were used for
experiments. The isolated fibres gradually became spherical and lost the
structure of cross-striation. We therefore finished an experiment shortly (45
min) after fresh preparations were made.
Whole-cell current recordings
An experimental chamber was placed on the mechanical stage of an inverted
microscope (TMD, Nikon, Tokyo, Japan), and whole-cell voltage clamp was
carried out with a patch clamp amplifier (EPC-7, List, Darmstadt, Germany).
Electrode resistance was 12 M, and the series resistance of 1
M
was compensated. Pulse generation and data acquisition were performed
with a 12-bit D/A and A/D converter (Labmaster/DMA, Scientific Solution,
Solon, OH, USA) and an IBM/AT compatible computer using pCLAMP software (Axon
Instruments, Chicago, IL, USA). The cell membrane capacitance was calculated
from the area under a transient capacitive current produced by a 10 mV step
depolarisation from a holding potential of 70 mV. Linear currents were
subtracted digitally using a P/4 pulse protocol.
Recordings of action potentials and contractions from the myoepithelium sheet of C. appendiculata
The animals were pinned to the SylgardTM base of a small dish, using
Opuntia spines. The anterior nectophore was partially cut open to
permit access of microelectrodes to the myoepithelial fibres. Conventional 3
mol l1 KCl-filled microelectrodes (1530 M)
with long flexible shanks were led via a laboratory-made follower
amplifier. The preparations were stimulated at the basal nerve rings using
fine polyethylene suction electrodes fed by a stimulator (S48, Grass, Quincy,
MA, USA) via an isolating unit. A single stimulus usually evoked a
short burst of action potentials and associated contractions in the
myoepithelium. Contractions were detected with a fine probe attached to a
strain gauge (N801; SensoNor, Horteu, Norway). Both electrical and mechanical
signals were simultaneously recorded with a PCM recorder (PC204; Sony, Tokyo,
Japan).
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Results |
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Membrane ion currents in Diphyes
Na+, Ca2+ and K+ currents under whole-cell voltage clamp
Isolated single myoepithelial fibres were whole-cell voltage clamped, and
membrane currents were recorded under different internal and external salt
compositions. The mean value of the cell membrane capacitance was
147.9±101.2 pF (mean ± S.D.,
N=77). To detect Na+ currents, fibres bathed in
N-Ca-free-ASW were dialysed with Cs-asp. Note that internal Cs+
blocks currents through K+ channels. Short (10 ms) depolarising
voltage pulses from 50 mV to +50 mV in 10 mV increments were applied to
the membrane from a holding potential of 70 mV (pulse protocol is shown
in Fig. 3A). Transient inward
currents appeared at voltages more positive than 20 mV
(Fig. 3).
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To detect Ca2+ currents, fibres were bathed in 4Ca-ASW containing 410 µmol l1 TTX and dialysed with Cs-asp. Depolarising pulses of 50 to +50 mV with 25 ms duration evoked long-lasting inward currents (Fig. 4A). No distinct spontaneous run-down of the current was observed. The IV relationship of the peak currents obtained from eight experiments (Fig. 4B) shows that the inward current appeared at voltages higher than 40 mV and peaked at +10 mV. Whole-cell voltage clamp experiments revealed that the currents were sensitive to nifedipine, a dihydropyridine derivative (N=5). Fig. 4C superimposes five current records at +10 mV pulses obtained from the same fibre before (1, 2) and after (35) external application of 10 µmol l1 nifedipine; records 1 and 2 were obtained 3 and 6 min after the whole-cell configuration was made, and records 35 were obtained 2, 4 and 6.5 min after the nifedipine application. The inward currents were also blocked by 5 mmol l1 Co2+ applied to the bath (N=6; data not shown). The results show that these currents were carried by Ca2+ through L-type Ca2+ channels.
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When fibres were dialysed with K-asp instead of Cs-asp in N-Ca-free-ASW
containing 410 µmol l1 TTX, outward currents
appeared associated with depolarisations to more than 10 mV
(Fig. 5). Because this current
component did not appear when the internal cation was Cs+, these
currents are thought to be carried by K+ through K+
channels. The outward current appeared 1 ms after each depolarisation,
reached a peak in 310 ms, depending on the voltage, and greatly
inactivated during each depolarisation
(Fig. 5A). Note that recovery
of this current from inactivation was very slow (time constant of recovery was
13 s at 70 mV; see Fig.
8). Therefore, under the present pulse protocol (5 s interval
between depolarisations), the peak value was smaller than that it would have
attained after full recovery from inactivation (only 30% at +30 mV). Two other
sets of records were taken from the same fibre at different holding potentials
of 90 and 40 mV. Fig.
5B compares three traces associated with depolarisations to +30 mV
from holding potentials of 40 mV, 70 mV and 90 mV. There
was no significant difference in the current time courses and their voltage
dependencies between the holding potentials of 70 and 90 mV (see
also Fig. 5C). This means that
there is no difference in the rate of recovery of the K+ channel
from inactivation between the two voltages. On the other hand, the
K+ currents were more inactivated at the holding potential of
40 mV (Fig. 5B,C).
Externally applied EGTA (10 mmol l1, N=2),
Co2+ (up to 6 mmol l1, N=3) or
nifedipine (5 µmol l1, N=1) to the external
N-Ca-free ASW had no effect on these properties of the K+
current.
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Changes in the Na+, Ca2+ and K+ currents during repetitive depolarisations
There are several possible causes for the augmentation of action potentials
during repetitive firings; i.e. (1) an increase in the activities of
Na+ and Ca2+ channels during repetitive depolarisations,
(2) a decrease in the activity of K+ channels or (3) both the cases
of (1) and (2) together. We measured changes in the Na+,
Ca2+ and K+ currents during repetitive depolarisations
under whole-cell voltage clamp. Ten depolarising pulses of 100 mV from the
holding potential of 70 mV were given at a 200 ms interval (5 Hz; see
Fig. 6A). As can be seen from
Fig. 6A,B, there was no
augmentation of Na+ or Ca2+ current during the
repetitive depolarisations. Instead, both currents slightly decreased, and the
final amplitude of Na+ and Ca2+ currents at their peak
(relative to the initial values) became approximately 86% and 93%,
respectively. On the other hand, the K+ current completely
inactivated during the first depolarisation with a time constant of 9 ms
(Fig. 6C) and did not recover
from inactivation during the repetitive depolarisations.
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Recovery of the K+ current from inactivation
The time constant of recovery of the K+ current from
inactivation was determined by changing the interval (t) of
two depolarising pulses from 70 to + 30 mV (pulses protocol is shown in
the upper panel of Fig. 8A).
The lower panel of Fig. 8A
shows the currents at
t=10 s. The peak amplitude of the second
current was
50% of the initial one
(Fig. 8A).
Fig. 8B plots the peak value of
K+ current amplitude (IK) during the second
pulse relative to that during the first pulse against
t, and
the plots were fitted with a single exponential function,
1exp(
t/
). The time constant (
) of the
recovery from inactivation was calculated to be 13.2 s.
K+ current inactivation during repetitive short depolarisations
We investigated whether the properties of the K+ channel can
explain the augmentation of the initial few action potentials during a burst
using the potential records obtained from Chelophyes appendiculata.
Fig. 9A superimposes the
initial six action potentials of the burst shown in
Fig. 2. It is seen from the
traces that the augmentation was mainly brought about by the growth of the
slower Ca2+ spike. As the duration of the action potential,
especially the overshooting time, increased and as the action potential peak
became higher, the subsequent action potential was further augmented. The
overshooting times of the first to sixth action potentials were 1.5, 2.0, 2.5,
3.4, 14.9 and 20.4 ms, respectively, and the peak potentials were 9.3, 12.6,
16.3, 21.6, 29.3 and 47.1 mV, respectively, in this specific case. To test if
K+ channel inactivation during the action potentials could produce
the action potential augmentation, 10 short (5 ms) depolarising pulses to +10
mV were applied at 200 ms intervals, and the decay of K+ currents
during the short depolarisations was measured.
Fig. 9B superimposes
K+ current records produced by the ten repetitive depolarisations.
The K+ current did not fully inactivate during the first
depolarisation; it decreased successively during the repetitive
depolarisations. Fig. 9C plots
relative peak values to the first K+ current peak during the
repetitive depolarisations. Each point was obtained by averaging seven data
points, and the error bar indicates the magnitude of
S.D. The curve was drawn by fitting the averaged values
with an exponential decay function. The time constant was 0.46 s. The
depolarising pulse seemed to mimic the overshooting voltage of the fourth
action potential. Therefore, this depolarisation would inactivate the
K+ channels more strongly than the first to third action potentials
and less strongly than the fifth and sixth action potentials. For example, the
initial action potential had much shorter overshooting time than the pulse
duration. Therefore, the rate of inactivation produced by the initial action
potential would be much smaller than that produced by the initial voltage
pulse; hence, the second action potential was not much augmented. This may
explain the characteristic property of the action potential augmentation.
Because no other factor that produces the augmentation was found, we concluded
that the action potential augmentation of diphyid siphonophores was due to
K+ channel inactivation during a burst of action potentials. It is
also noted in Fig. 9A that the
resting potential did not change during the action potential augmentation,
indicating that this K+ channel did not contribute to the resting
potential generation.
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Discussion |
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Muscle electrical responses
As Spencer and Satterlie
(1981) pointed out, action
potentials in the subumbrella swimming muscle sheet of the medusa
Polyorchis not only propagate through the tissue to evoke contraction
but also carry information about the required duration of contraction. The
action potentials of the muscle sheet in large individuals last longer than
those in small individuals, hence contraction assumed (reasonably) to result
from Ca2+ entry during the action potential lasts longer in the
larger individuals. A similar situation evidently occurs in diphyids, where
whatever the size of the specimen, the first few action potentials of a burst
differ from those succeeding (Chain et al.,
1981
). These latter authors used drugs and substitution
experiments to show that the action potentials were carried both by
Ca2+ and Na+, recognising that further studies with
whole-cell voltage clamp techniques were required. Until now, this latter
approach has only been used in cnidarian muscle to examine isolated
Polyorchis muscle cells (Lin and
Spencer, 2001
). Here, although previous experiments
(Spencer and Satterlie, 1981
)
had suggested that Na+ was required to support action potentials,
the only inward current observed was a T-type Ca2+ current.
Excitationcontraction coupling in diphyids is apparently solely
dependent upon Ca2+ entry across the sarcolemma
(Bone et al., 1999) and
intracellular stores are absent. Presumably, a Na+/Ca2+
exchange mechanism to extrude Ca2+ is found across the sarcolemma,
similar to that in the small tunicate Doliolum
(Bone et al., 1997
), which also
has no internal Ca2+ stores within the muscle cell
(Inoue et al., 2002
).
The striking feature of the diphyid action potential is its change in
amplitude and form during repetitive stimulation. Our records show that this
is brought about in an unique way, by long-lasting inactivation of the
K+ channel. During a train of action potentials, the first few do
not inactivate the K+ channel completely so that the first few
action potentials gradually change to the form of the subsequent potentials
during the train. (Note that this K+ channel plays a leading role
in the action potential termination of only the initial few action potentials.
It is not clear how the action potentials are regularly terminated after this
K+ channel has been fully inactivated. This ionic mechanism remains
unsolved.) Simultaneous records of action potential form during stimulated and
`spontaneous' series of contractions show that, just as in
Polyorchis, contractions increase as the action potentials increase
in duration (Bone, 1981). In
these experiments, muscle contractions were measured with a strain gauge
linked to the edge of the cut nectophore, hence no propulsive jets were
produced and changes in the hydrodynamics of the propulsive jet or of flow
around the intact free-swimming animal are not relevant. Our interest has been
solely in the way in which membrane currents underlie the striking change in
action potential form during successive potentials. It is, however, not
unnatural to ask how such changes affect the swimming ability of diphyids.
In diphyids, rapid swimming almost always involves several powerful jet
pulses in succession, as it does in the hydromedusan Aglantha, which
is capable of very rapid swimming
(Donaldson et al., 1980). There
is, however, a striking difference between the two. The first jet pulse of
Aglantha is the most effective in driving it forward, since
subsequent pulses begin before the bell has completely refilled and hence are
less effective, driving it forward only 4060% of the distance given by
the first pulse. By contrast, the first few pulses in diphyids are the least
effective (Bone and Trueman,
1982
), since their contractions are the least strong, because
during the first few shorter action potentials less Ca2+ crosses
the sarcolemma than it does during later potentials of the swimming burst.
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Acknowledgments |
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References |
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