1Sussex Centre for Neuroscience,
Yeoman, M. S. and
P. R. Benjamin.
Two Types of Voltage-Gated K+ Currents in Dissociated
Heart Ventricular Muscle Cells of the Snail Lymnaea
stagnalis.
J. Neurophysiol. 82: 2415-2427, 1999.
We have used a combination of current-clamp and voltage-clamp
techniques to characterize the electrophysiological properties of
enzymatically dissociated Lymnaea heart ventricle cells.
Dissociated ventricular muscle cells had average resting membrane
potentials of Invertebrate preparations with their identified
neurons and accessible muscle systems have proved suitable preparations
for understanding transmitter complexity and relating cellular changes to behavioral plasticity (Kupfermann 1991 Action potentials have also been recorded from single muscle fibers of
the auricle of another type of mollusk, the snail
Lymnaea (Buckett et al. 1990c The innervation of the heart of Lymnaea is extremely
complex, and previous work by Buckett (1990a To determine the detailed effects of this complex set of neuropeptides,
it was important to first characterize the ionic channels present in
the Lymnaea heart muscle fibers. To date, nothing is known about the voltage-gated currents present in these cells and their
relationship to the generation of the spontaneous action potentials.
The data presented in this and the following paper (Yeoman et
al. 1999 Preparation
Membrane currents were characterized in dissociated heart
ventricle cells. These were chosen in preference to auricle cells because they are larger and easier to record using the single-electrode voltage-clamp (SEVC) technique employed here.
Dissociation of ventricle muscle fibers
Ventricle muscle fibers were dissociated from hearts of 2- to
3-g Lymnaea stagnalis (supplied by Blades Biological, Kent) kept on a 12 h/12 h light/dark cycle at 19°C and fed ad libitum on
lettuce. The technique for producing dissociated cells was modified
from that described by Brezden and Gardner (1986) Agar embedding of muscle fibers and drug application
The fibers were immobilized for recording as described
originally by Brezina et al. (1994a) Drug application
During recordings the chamber was continually perfused with
normal Lymnaea saline (composition shown below). The level
of fluid in the bath was kept constant by removing the excess using a
suction pipette attached to a vacuum pump. Drugs and modified salines
were applied by means of a local superfusion pipette, tip diameter 75 µm, placed ~100 µm away from the cell. Quantitatively similar
effects were obtained when the effects of drugs applied via the
superfusion pipette were compared with those applied grossly to the
bath, indicating that the perfusate from the superfusion pipette was
reaching the whole cell. Using dyes in the superfusate, solutions were
seen to be fully exchanged within 15 s. In these experiments drugs
were applied for at least 30 s before commencing voltage-clamp recording.
Electrophysiological techniques
CURRENT-CLAMP RECORDINGS.
Isolated cells were impaled with glass microelectrodes pulled from 1-mm
borosilicate glass (100FT Clark Electromedical) and filled with 3 M KCl
to give final resistances of 20-30 M VOLTAGE-CLAMP EXPERIMENTS.
The surfaces of dissociated Lymnaea ventricle cells have
numerous invaginations, making whole cell recordings using a patch pipette extremely difficult. It was therefore decided to use sharp microelectrodes to perform a voltage-clamp analysis of membrane ion
currents (cf. Brezina et al. 1994a
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
55 ± 5 mV. When hyperpolarized to potentials
between
70 and
63 mV, ventricle cells were capable of firing
repetitive action potentials (8.5 ± 1.2 spikes/min) that failed
to overshoot 0 mV. The action potentials were either simple spikes or
more complex spike/plateau events. The latter were always accompanied
by strong contractions of the muscle cell. The waveform of the action
potentials were shown to be dependent on the presence of extracellular
Ca2+ and K+ ions. With the use of the
single-electrode voltage-clamp technique, two types of voltage-gated
K+ currents were identified that could be separated by
differences in their voltage sensitivity and time-dependent kinetics.
The first current activated between
50 and
40 mV. It was relatively fast to activate (time-to-peak; 13.7 ± 0.7 ms at +40 mV) and
inactivated by 53.3 ± 4.9% during a maintained 200-ms
depolarization. It was fully available for activation below
80 mV and
was completely inactivated by holding potentials more positive than
40 mV. It was completely blocked by 5 mM 4-aminopyridine (4-AP) and
by concentrations of tetraethylammonium chloride (TEA) >10 mM. These
properties characterize this current as a member of the A-type family
of voltage-dependent K+ currents. The second voltage-gated
K+ current activated at more depolarized potentials (
30
to
20 mV). It activated slower than the A-type current (time-to-peak; 74.1 ± 3.9 ms at +40 mV) and showed little inactivation (6.2 ± 2.1%) during a maintained 200-ms depolarization. The current was fully available for activation below
80 mV with a proportion of the
current still available for activation at potentials as positive as 0 mV. The current was completely blocked by 1-3 mM TEA. These properties
characterize this current as a member of the delayed rectifier family
of voltage-dependent K+ currents. The slow activation rates
and relatively depolarized activation thresholds of the two
K+ currents are suggestive that their main role is to
contribute to the repolarization phase of the action potential.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). A large
number of studies have examined how transmitters can alter the gross
contractile properties of a wide variety of different muscle types, but
how these substances affect the detailed ionic conductances of muscle cells has been relatively little studied, particularly in rhythmically active systems like the heart. The recent development of methods for
dissociating muscles in to their constitutive fibers in invertebrate preparations (Brezden and Gardner 1986
;
Brezina et al. 1994a
; Dorsett and Evans
1991
; Laurienti and Blankenship 1996a
) has
facilitated the study of muscle electrophysiology, but there are still
very few detailed reports of the ion current complement of invertebrate muscle cells. Those that have been published have concentrated on the
unstriated muscles of invertebrates, which are most similar to
vertebrate smooth muscle. These include studies on the accessory radula
closer (ARC) muscle of Aplysia (Brezina et al.
1994a
-c
; Brezina and Weiss 1995
;
Ram et al. 1990
, 1991
) and its antagonist the accessory radula opener (ARO) (Scott et al. 1997
),
the buccal mass retractor muscles of Philine
(Dorsett and Evans 1991
) and most recently the
parapodial swim muscles of Aplysia (Laurienti and
Blankenship 1996a
,b
). These muscles contained a mixture of Ca2+ and K+ currents that was similar to those
recorded in invertebrate neurons and are related to those present in
vertebrate muscle. These muscles were conspicuous by the absence of a
voltage-gated Na+ current, a property they have in common
with the majority of vertebrate smooth muscles that have been
characterized to date. On the other hand, invertebrate heart muscle has
been classified by Hoyle (1964)
as
cross-striated because it appears identical under the light microscope
to the skeletal muscle of vertebrates and arthropods (Brezden
and Gardner 1992
). It is therefore different from those
invertebrate muscles that have been studied previously. Early work
using extracellular recordings from the whole heart of a variety of
bivalve mollusks showed that this organ like its vertebrate counterpart
fired regular action potentials (for review see Deaton and
Greenberg 1980
). The shape of these action potentials was
extremely variable, but two components were usually identifiable. These
were a fast spike followed by a slower plateau. Ionic substitution experiments have shown that in the majority of species tested the spike
phase was Ca2+-dependent, whereas the plateau phase was
dependent on the presence of extracellular Na+ ions. Thus
it appears that the majority of bivalve hearts contain both
Na+-permeable and Ca2+-permeable ion channels.
). Using
intracellular recording techniques on the whole heart, these authors
demonstrated the presence of simple spikelike events, although the
ionic dependence of these events was not examined. Other work by
Brezden and colleagues, using the cell-attached patch-clamp technique,
has demonstrated the presence of several ion channel types in
dissociated Lymnaea ventricle cells (for review see
Brezden and Gardner 1992
). These included at least one
type of K+ current that probably contributed significantly
to the resting membrane potential of the cell. This channel also showed
some sensitivity to stretch, indicating that it may have other
functions (Brezden and Gardner 1986
).
-c
) has
shown that this organ is regulated by a variety of classical and
peptide neurotransmitters. Our detailed knowledge of the motor neurons
innervating the heart and their transmitter content makes this system
extremely tractable as a model for examining the mechanisms of action
of transmitters that modulate muscle contractility. In particular, we
are interested in understanding the role of multiple co-localized
peptides that are encoded by a single neuropeptide gene. The gene and
its products have been shown to be present in a single motor neuron
that innervates the heart (Benjamin and Burke 1994
).
) on Lymnaea provides the first
detailed description of the voltage-gated ionic currents present in the
heart of an invertebrate and provides the basis for a future detailed
examination of the mechanism of action of multiple peptides in cell signaling.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
.
Briefly, the heart was dissected from the animal, and the auricle and
the remains of the aorta removed. The ventricle was carefully cut in to
small pieces (
1 mm square). The cells were
then dissociated by digestion for 20 min in 0.25% trypsin wt/vol
(Sigma Type XII S) and subsequently for 75 min in 0.1% wt/vol
collagenase (Sigma Type II S), both made up in 0.5 mM
Ca2+ Leibowitz medium containing gentamycin
(Sigma, 500 µg/ml) and 30 mM glucose. After the digestion the cells
were centrifuged at 300 rpm for 5 min, the supernatant removed, and the
pellet resuspended in 3.5 mM Ca2+ Leibowitz
medium containing gentamycin (Sigma, 500 µg/ml), 30 mM glucose, and
2% vol/vol fetal calf serum. This process was repeated three times.
The cells were then stored overnight at 19°C or until required. Cells
remained viable in suspension for up to 5 days.
. Briefly, an
aliquot of the cell suspension was mixed with an equal quantity of low
melting point agarose gel (GIBCO-BRL; 0.8% wt/vol) and the mixture
pipetted into the experimental chamber (35-mm Falcon 3001 Petri
dishes). The chamber was then inverted to allow the fibers to move to
the surface of the agar, thus facilitating recording. Agar embedding did not appear to alter the properties of ventricle cells.
Morphologically, dissociated ventricle cells embedded in agar did not
differ from those cells plated directly on glass cover slips
(Brezden et al. 1986
). Fibers were 40-120 µm long and
10-20 µm wide. The surface of healthy relaxed fibers, unlike those
described recently by Brezina et al. (1994a)
and
Laurienti and Blankenship (1996a)
, were covered with
numerous invaginations as previously described (Brezden and
Gardner 1986
) and contracted repeatedly in response to
injection of short (200 ms) depolarizing current pulses. Injection of
large amounts of current (
0.5 nA) caused the cells to become irreversibly contracted with their membrane taking on a "bleblike" appearance. However, this irreversible contraction did not appear to
disrupt the integrity of the cells because both their input resistance
and the amplitude of evoked voltage-gated currents were unaltered (data
not shown). Further confirmation that the agar embedding was not having
any deleterious effects on the cells came from recordings of the
resting membrane potential. Resting membrane potentials of the cells
varied between
40 and
60 mV with a mean of
55 ± 5 mV
(mean ± SE, n = 25). This mean value was similar
to that recorded in the intact ventricle (Yeoman, unpublished
observations) (
53 mV) and Brezden and Gardner (1986)
(
59 mV), who recorded from dissociated ventricle cells in
Lymnaea. Cells that appeared to be physically damaged
usually had membrane potentials much lower than
55 mV and were not
used for further experimental analysis. The input resistance of healthy
cells calculated over a hyperpolarizing range of potentials
80 to
120 mV varied between 450 and 1,890 M
(939 ± 73.9 M
,
n = 46). The input resistance was dramatically reduced
over the depolarized range of potentials (
20 to +50 mV) where
currents as large as 20 nA were routinely recorded.
. Cells were impaled, and
recordings of the voltage changes in the cell were made using the
discontinuous current-clamp configuration on the Axoclamp 2B (Axon
Instrument). Data were recorded simultaneously on a Gould 2 channel pen
recorder and on to digital audio tape (DAT) using a Biologic 8 channel
recorder. Spontaneous action potentials were only generated from
membrane potentials between
70 and
63 mV. In the majority of cells
this meant injecting a small amount of constant hyperpolarizing
current, via the recording electrode.
). Due to the small
size of the muscle fibers (
40-120 µm long by 10-20 µm wide),
it was extremely difficult to impale these cells with two
microelectrodes and maintain the integrity of the cell for more than a
few seconds. We therefore chose to use the discontinuous SEVC technique
to analyze the ion currents in these fibers. Electrodes were pulled from 1-mm, thin-walled filamented borosilicate glass (100FT, Clark Electromedical) and filled with 3 M KCl. Electrodes had resistances of
between 20 and 30 M
. Unlike the ARC muscle of Aplysia
where isolated muscle cells showed a slowly developing
hyperpolarization-activated Cl
current
following prolonged recording with KCl electrodes (Brezina et
al. 1994a
), we did not observe any change in current amplitude during our recordings, and it was therefore decided to use 3 M KCl as
our pipette solution. The bath was grounded via an agar bridge
containing 3% wt/vol agar made up in 3 M KCl, connected to a Ag-AgCl
half-cell that was filled with 3 M KCl. Voltage-clamp recordings were
made using an Axoclamp 2B amplifier with data sampled at 20 kHz and
recorded directly on to the computer using a Digidata 1200 data
acquisition system and pClamp software (Axon Instruments, Foster City,
CA). The switching frequency used in these experiments varied between
13 and 18 kHz depending on the cell, producing voltage steps that
settled within 2-3 ms of the depolarizing pulse. However, to eliminate
any disparities between the command voltage and the actual voltage of
the cell, all records of currents shown in this and the following paper
use the command voltages. The final statistical analysis of the
currents based on current-voltage (I-V) curves were
constructed using actual voltages. Voltage-activated currents were
initially identified using ramp protocols and then further
characterized using voltage step protocols. Voltage protocols were
given at regular intervals (1 s). This time allowed identical
I-V relationships with sequential voltage protocols.
Currents of different types were separated both by means of voltage
protocols where appropriate and/or by the use of pharmacological
blocking agents.
120 to +50 mV). Therefore
where possible, leakage currents were subtracted using the
pharmacological method. For clarity, capacitance artifacts have been
removed from all records.
Adequacy of space clamp
Lymnaea ventricle muscle cells are long and thin, and
it was therefore important to check whether they could be adequately space clamped. Fibers were injected with hyperpolarizing square-wave current pulses and the shape of the voltage response during the onset
of the current pulse was examined (Brezina et al.
1994a). In general the voltage trace could be fitted by a
single exponential at both depolarized (
30 mV) and hyperpolarized
(
80 mV) potentials, indicating that the fibers were being adequately
clamped. It would have been preferable to have confirmed this finding
by directly recording the voltage response of the cell at two different
locations along the length of the fiber under voltage-clamp conditions. However, due to the small size of the fibers, it proved impossible to
impale them with more than one electrode.
To substantiate these results, we used the following equation to
estimate the length constant () in an average Lymnaea
ventricle muscle cell
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Solutions and drugs
The normal Lymnaea saline used in all the experiments described in this paper contained (in mM) 50 NaCl, 1.7 KCl, 3.5 CaCl2, 4 MgCl2, and 10 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES). The pH was adjusted to 7.8 using NaOH. In current-clamp experiments where we examined the effects of removing either Na+ or Ca2+ ions on action potential generation, Na+ ions were replaced by an equimolar concentration of choline (zero Na+ saline), or Ca2+ ions were replaced by an equimolar concentration of Cd2+ (zero Ca2+ saline). For the voltage-clamp experiments, pharmacological agents (TEA or 4-AP) were added to the saline at the expense of an equimolar concentration of Na+ ions. To calculate the selectivity of channels for K+ ions, extracellular K+ concentrations were raised from 1.7 mM to either 4.76 or 17 mM at the expense of Na+ ions, to give 4.76K or 17K saline. In some experiments extracellular K+ concentrations were raised to 100 mM (100K saline). The osmolarity change associated with this increase was partly offset by removing all of the Na+ (50 mM) and Ca2+ (3.5 mM) ions from the normal saline. However, it should be noted that the final osmolarity of this saline is approximately one-third higher than that of the normal saline. All chemicals and drugs used in this paper were obtained from Sigma Chemical Co., Poole, U.K.
Statistical analysis of data
All values quoted in the text represent means ± SE (standard error of the mean). Unless stated otherwise in the text, groups of data were compared using an unpaired t-test assuming unequal variances. Probability values <0.05 were taken as significant.
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RESULTS |
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Dissociated Lymnaea heart ventricle cells generate two types of action potential
Isolated heart ventricle cells were capable of generating
repetitive spontaneous action potential-like events at membrane potentials between 70 and
63 mV (Fig.
1A1; n = 20).
Two distinct types of spikelike events could be recorded at these
membrane potentials. The first had a simple waveform with a "fast"
rising and slower repolarizing phases and was 2-4 s in duration (Fig. 1A2). These events were never associated with visually
observed muscle contractions. The second type (Fig. 1A3) had
a more complicated waveform. It consisted of an initial brief spike
followed by a sustained plateau phase of varying duration (2-4 s). The
presence of the plateau phase was always associated with a strong
contraction of the muscle fiber, which lasted for the duration of the
plateau. Every muscle fiber that we recorded under current-clamp
conditions (n = 20 fibers) showed both types of action
potential-like event, indicating that these two different events were
not cell specific. In 13 of the 20 cells recorded, there appeared to be
no regularity in the pattern of firing. However, in the remaining seven
cells, action potentials were generated with extreme regularity with rates ranging between 1 spike/5 s and 1 spike/15 s in different preparations (mean of 8.5 ± 1.2 spikes/min, n = 7 fibers). In all cases the rate in isolated cells was slower than the
rate of spontaneous heart beat recorded in the intact animal which is
~1 beat/3 s but was comparable to rates seen in the isolated heart
that was constantly perfused with saline in an organ bath (Buckett et al. 1990a
-c
). The ionic dependence of these
action potentials was examined under current-clamp conditions.
Initially, the effects of applying the K+ channel
blocker TEA on the waveform of spontaneously generated action
potentials was examined (Fig. 1B). Ten seconds after
superfusion of the cell with 5 mM TEA, a noticeable broadening of the
action potential was observed (Fig. 1B2), when compared with
controls (Fig. 1B1). This effect reached a maximum by
30 s (Fig. 1B3) with spike widths increasing by
260 ± 32% (mean ± SE; n = 6;
P < 0.01). The effects of the TEA were almost
completely reversible after a 60-s wash with normal saline (Fig.
1B4). To test whether the influx of
Ca2+ was important in generating the spike, the
heart was perfused with a saline in which the
Ca2+ ions had been replaced by 3.5 mM
Cd2+, a selective Ca2+
channel blocker. An example of one such experiment (n = 5) is shown in Fig. 1C. Before the application of the
Cd2+ saline, cells were capable of generating
fast action potentials (Fig. 1C1). Application of the
Cd2+ saline prevented the cells generating action
potentials. Instead we observed a number of low-amplitude (10 mV)
membrane depolarizations (Fig. 1C2). These lacked the fast
rise and fall characteristic of the action potentials. On washing the
preparation with normal saline, the fast action potentials returned. It
appeared therefore that the fast spikelike events were dependent on
extracellular Ca2+ ions. Despite these results,
it was still possible that Na+ ions were
important for spike generation. However, perfusion with a saline
containing zero Na+ ions had no observable effect
on the amplitude of an evoked muscle cell spike (Fig. 1D2)
when compared with controls (Fig. 1D1). In these experiments
action potentials were reproducibly generated by applying a
hyperpolarizing square-wave current pulse to the cell and generating an
action potential on the rebound. As in the previous experiment, action
potentials generated by this method were reversibly blocked by
Cd2+ ions (Fig. 1, D3 and
D4). The ionic basis of the plateau potential was not
investigated in the present experiments.
|
Characterization of K+ currents present in dissociated Lymnaea ventricle cells by voltage clamp
Having determined that the efflux of K+ ions was important in determining the duration of the spontaneously generated action potentials, we characterized the types of voltage-gated K+ currents that might underlie this phase of the action potential.
In an initial series of experiments aimed at isolating the currents
present in dissociated ventricle muscle fibers, a series of voltage
ramp protocols were used. Although these types of protocol preclude a
characterization of time-dependent currents, they can provide
information about whether or not the cells show inward or outward
rectification at different membrane potentials. Cells were held at 60
mV and stepped briefly to
90 mV (25 ms) to remove any channel
inactivation that might have been present at the holding potential of
the cell. Cells were then ramped to +40 mV over a 2.5-s period. The
I-V plot shown in Fig. 2 shows
a record of the ramp current from a single ventricle muscle cell. Over
the hyperpolarized range (
90 to
60 mV), the I-V plot is
flat, indicating that there was no voltage-dependent current flow
across the membrane. This therefore excludes the presence of an inward
rectifier, which would appear as a steepening downward deflection of
the I-V curve at these potentials. Hyperpolarizing the cell
from its resting membrane potential using a series of voltage steps to
potentials produced small linear leakage currents, again confirming the
absence of an inward rectifier current. At more depolarized potentials (
50 mV) the cells started to show a pronounced outward current that
increased as the membrane potential became more depolarized. The
majority of cells (
85%, n = 30) also had two
regions of inward rectification. The first occurred around
60 mV
(Fig. 2; I1), whereas the second
occurred at more depolarized potentials,
30 mV(Fig. 2;
I2). Although
I2 is difficult to visualize in ramps performed in normal saline, we will show in the following paper (Yeoman et al. 1999
) that these regions of inward
rectification are due to activation of two different classes of
voltage-gated Ca2+ current and that
I2 recorded in normal saline is
essentially masked by the large voltage-gated K+
currents and is thus barely evident.
|
Muscle fiber types
Although there was no obvious morphological differences between
the muscle fibers, an analysis of the membrane ion currents in
Lymnaea heart ventricle cells indicated the presence of two different fiber types (N.B. both these types of fiber could generate both the spike and spike/plateau type of action potentials). These differences did not manifest themselves clearly during voltage-ramp protocols but were clearly evident in cells that had undergone a series
of voltage-step protocols designed to characterize time-dependent currents. An example of the current profile of each of the two types of
fiber is shown in Fig. 3, A1
and A2. These cells were initially held at 90 mV and
stepped to
120 mV for 25 ms and then in 10-mV steps from
70 to +40
mV (200 ms). Although there was no statistical difference in the peak
amplitude of the outward currents recorded in the two cell types,
several differences were observed in the current complement of the two
fibers. Outward currents present in type I muscle cells were relatively
slow to reach a peak (23 ms with voltage steps to +40 mV) and showed
marked inactivation during the duration of the 200-ms step that became increasingly more obvious as the membrane potential was stepped to more
positive potentials (25.4 ± 2.9% at +40 mV). Inactivation was
measured as the difference between the size of the peak current and the
current flowing at the end of the voltage step expressed as a
percentage of the peak current. Outward currents in the type II muscle
fibers were faster to reach a peak (11 ms for voltage step to +40 mV)
and showed less current inactivation than type I cells (13.2 ± 2.5% at +40 mV). Although the magnitude of the differences in the
time-to-peak and current inactivation for the two cell types changed
with alterations in the membrane potential of the cells, they were
qualitatively similar at all recorded membrane potentials (
50 to +40
mV). I-V plots of the peak current versus membrane potential
for examples of the two cell types are shown in Fig. 3B.
Outward currents in both cell types activated between
50 and
40 mV
and increased to the same peak amplitudes as the cells were stepped to
increasingly more positive potentials. We will show later that the
outward current in type I cells is composed of an A-type current
[IK(A)] and a delayed rectifier type
current [IK(V)], whereas that in the
type II cells is composed of IK(A),
IK(V) and at least one type of
Ca2+-dependent current. Both cell types have
small inward currents that can be seen as the negative-going waveforms
at the beginning of the voltage step (arrows, Fig. 3, A1 and
A2). The size of these inward currents varied considerably
between cells. However, there was no correlation between their size and
the fiber type (type I or type II), when cells were recorded in normal
saline.
|
A further difference between the two fiber types was the degree to
which they each contracted in response to the depolarizing voltage
steps described above. By clamping the muscle cells at 90 mV,
prestepping the membrane potential briefly to
120 mV (25 ms) and then
stepping the membrane sequentially in 10-mV steps from
70 to +40 mV,
we were able to elicit a series of contractions in the muscle fiber.
The strength of these contractions was measured by noting the amount by
which each muscle fiber shortened after each voltage step, using a
micrometer graticule placed in the eye piece of the microscope. An
indication of the strength of contraction was gained by calculating how
much the fiber had shortened as a percentage of the relaxed fiber
length. Figure 3C shows a plot of the combined data from
five experiments where %contraction was plotted against membrane
potential of the cell. Two lines are plotted, based on the
electrophysiological characterization of the fibers detailed above.
Type I fibers (
) showed a maximum contraction of 11 ± 1% of
their original length (n = 5), whereas type II fibers
(
) showed average contractions of 48 ± 4% (n = 5). In both cases the muscle cells began to contract at around
50
mV and reached their peak contraction with voltage steps to
20 mV. At
potentials more positive than this, the degree of contraction became
weaker presumably due to a combination of the increasing size of the
outward current (see Fig. 3) and the decreasing size of the inward
Ca2+ current (see Yeoman et al.
1999
). These experiments were all performed on the largest
cells with lengths between 100 and 120 µm. The technique thus
allowed us to resolve changes
1 µm.
The majority of cells we recorded could be classified as either type I or type II (82%). However, 18% of cells had intermediate properties, suggesting that this classification was not complete.
Isolation of IK(A)
IK(A) was isolated from the total
whole cell current in type I fibers by superfusing the cells with low
concentrations of TEA (1-5 mM). An example of one such experiment is
shown in Fig. 4A. Figure
4A1 shows the series of currents evoked in a type I muscle
cell initially held at 90 mV, stepped briefly to
120 mV (25 ms),
and then depolarized in 10-mV steps from
70 to +40 mV (200 ms).
Application of either 1 mM (Fig. 4A2) or 5 mM (Fig. 4A3) TEA, left a series of smaller transient currents that
inactivated faster than the whole cell current recorded in Fig.
4A1. The lack of sensitivity to TEA and the significant
inactivation seen during the 200-ms voltage steps are both
characteristics of an A-type current. The residual current activated at
potentials between
50 and
40 mV (
45 ± 1 mV,
n = 14) and increased in amplitude as the cell was
stepped to more positive potentials (Fig. 4B). This
activation threshold was within the range of potentials seen for
A-currents in a variety of tissues in different organisms (Rudy
1988
). Increasing the TEA concentration to 10 mM blocked the
residual transient current almost completely. (Fig. 4A5). Unlike a number of A-currents described in the literature, the time-to-peak current for IK(A) was
relatively slow, increasing from 50.4 ± 5.2 ms at
10 mV to
12 ± 1.4 ms at +40 mV (n = 8; Fig. 4,
A2 and A3). A decrease in the time-to-peak as the
voltage step becomes more depolarized is characteristic of all
"A"-type currents (Rudy 1988
). The inactivation of
IK(A) in type I cells was calculated
by fitting an exponential curve to the declining phase of the current
from its peak to the end of the voltage step. This inactivation phase
fitted best to a single exponential with a mean time constant (
) of
145 ± 31.5 ms (n = 12). The current blocked by
the lower concentrations of TEA (1-5 mM) in type I cells showed all
the characteristics of a delayed rectifier-type current
[IK(V); Fig. 4A4]. It was
slow to activate, showed very little inactivation during the 200-ms
voltage pulse, and was sensitive to low concentrations of TEA. This
current will be described in detail in the next section. Washing the
preparation with normal saline removed the TEA block (cf. Fig. 4,
A1 and A6).
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Isolation of IK(A) from type II fibers was achieved in a similar way to type I fibers. Application of low concentrations of TEA (5 mM) altered the waveform of the outward currents from one of a type II cell (Fig. 5A1) to that representative of a type I cell (see Fig. 5A2). Addition of 10 mM TEA allowed us to resolve IK(A) in this cell type (Fig. 5A3). The complex waveform of the current blocked by 5 mM TEA (Fig. 5A4) in the type II cell is suggestive that this is in fact two separate currents. Superfusion of type II cells with 3.5 mM Cd2+ (Fig. 5B2) a specific Ca2+ channel blocker was also shown to block current(s) with the same waveform as those blocked by 5 mM TEA (compare Figs. 5A4 and 5B3). We therefore conclude that as well as having both IK(A) and IK(V) type II cells also possess one or more Ca2+-dependent currents whose properties were not investigated further in the present experiments.
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A comparison of I(KA) currents
isolated from type I cells and type II cells demonstrated that there
was no significant difference in the activation thresholds of the two
currents (41 and
45 mV, respectively; P > 0.05).
Similarly, there were no differences in the time-to-peak with voltage
steps to +40 mV (12 ± 1.4 ms and 15 ± 0.8 ms, respectively;
P > 0.05; mean for both fiber types 13.7 ± 0.7 ms) or the inactivation rate recorded at the same potential (
= 145 ± 31.5 and 94 ± 10.3 ms, respectively, for the 2 different cell types; P > 0.05; mean for both fiber
types 102.6 ± 10.6 ms) of the two currents. This indicated that
IK(A) isolated from both type I and
type II fibers were probably the same current.
Holding potential dependence of IK(A)
A-type currents, like all voltage-dependent currents, show
increasing steady-state inactivation when the holding potential from
which they are evoked becomes increasingly more positive (Hille
1992). To determine the holding-potential dependence of IK(A) the current was first isolated
by perfusing type I cells with 5 mM TEA. Muscle fibers were then
stepped from
70 to +50 mV from a series of different holding
potentials (
90 to
30 mV) and I-V plots constructed from
the peak current evoked at each membrane potential. Currents evoked
from a variety of holding potentials are illustrated in Fig.
6. At a holding potential of
90 mV,
IK(A) was fully available for
activation (Fig. 6A1). However, as the holding potential
became more positive the amplitude of IK(A) was reduced (Fig. 6,
A2-A5) until at potentials more positive than
40 mV
IK(A) was almost completely eliminated
(Fig. 6A7). Figure 6B shows I-V plots
for the currents evoked at four different holding potentials (
90,
70,
50, and
30 mV). The overall holding potential dependence of
IK(A) is illustrated in Fig.
6C, indicating maximal activation of the current from
90-mV holding potentials and complete loss of the current in cells
stepped from a holding potential of
40 mV. The data were fitted with
a Boltzmann curve giving a V1/2 of
66 mV and a slope factor of 11.4 ± 2.1 mV. The residual current
seen in Fig. 6, A6 and A7, probably represents an
unblocked component of IK(V).
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Sensitivity of IK(A) to 4-AP
Another characteristic of A-type currents is their
sensitivity to the potassium channel blocker, 4-AP. To test the
sensitivity of IK(A) to 4-AP, the
current was initially isolated by perfusing a type I muscle fiber with
5 mM TEA. Figure 7 shows an example of an
experiment in which a muscle fiber was superfused with increasing concentrations of 4-AP. In normal saline, cells were initially held at
90 mV and stepped briefly to
120 mV (25 ms) and then in 10-mV
depolarizing voltage steps from
70 to +30 mV to evoke a series of
currents that showed inactivation during the 200-ms voltage step
representative of a type I cell (Fig. 7A1). Application of 5 mM TEA left a transient outward current (Fig. 7A2) that was partially blocked by the application of 1 mM 4-AP (Fig.
7A3). Increasing the concentration of 4-AP to 5 mM caused a
complete block of the outward current (Fig. 7A4).
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The majority of transient currents isolated from Lymnaea
heart ventricle cells were sensitive to low concentrations of 4-AP (100% block <5 mM, n = 30 fibers) and showed complete
steady-state inactivation from holding potentials more positive than
40 mV. However, in a small subset of cells (10%, n = 3), the currents appeared relatively insensitive to 4-AP (50% block at
10 mM) but were completely inactivated by holding potentials of
60
mV. The combined data from these 33 cells is illustrated in Fig.
7B, where the mean %block of
IK(A) by 1 mM and 5 mM 4-AP is
plotted. In the presence of 1 mM 4-AP, the peak amplitude of
IK(A) was reduced by 52.5 ± 9.6%, with the %block increasing to 84.5 ± 15.2% in the
presence of 5 mM 4-AP (P < 0.05).
K+ selectivity of IK(A)
A-type currents show a high selectivity for
K+ ions. Usually, this can be determined by
examining the reversal potentials of their tail currents in bath
solutions containing different concentrations of
K+ ions [see section on characterization of
IK(V)]. However, the tail currents of
IK(A) were extremely fast and
impossible to resolve using the single-electrode voltage-clamp
technique. We have therefore used two methods to indirectly illustrate
the K+ selectivity of these channels. The current
flowing through IK(A) channels could
either be due to an efflux of positive ions, presumably K+ or an influx of negative ions, namely
Cl. To test whether a significant proportion of
the current flowing through IK(A)
channels was carried by Cl
ions, we substituted
85% of the extracellular Cl
ions with
methanesulphonate and examined the effects on the amplitude of
IK(A). Figure
8B shows an example of one
such experiment. IK(A) has been
isolated from a type I fiber by superfusing the cell with 5 mM TEA. The
current was evoked by holding the cell at
90 mV, prestepping the
voltage to
120 mV for 25 ms and then stepping the membrane potential
to +40 mV. Substitution of 85% of the Cl
ions
with methanesulphonate had no effect on the amplitude of IK(A) (n = 4).
Therefore Cl
is unlikely to be the main charge
carrier. Further confirmation of the K+
selectivity of the channel would be obtained if we could show that
IK(A) could be reversed by elevating
the levels of K+ ions bathing the cell. In Fig.
8A2 IK(A) is reduced
dramatically in amplitude when the cell is bathed with a saline
containing 100 mM K+ when compared with the
control currents evoked in Fig. 8A1. Elevating extracellular
levels of K+ ions to 100 mM caused a siginificant
increase in the osmolarity of the saline bathing the cells, and it was
possible that this may have had an effect on the amplitude of
I(KA). However, the data presented are
unlikley to be an artifact of the experimental procedure because all
the other evidence presented earlier suggests that this current is a
member of the A-type family of potassium currents.
|
Characterization of IK(V)
We have used a variety of methods to isolate and characterize
IK(V). As we have shown previously
(see Fig. 4), application of 1-5 mM TEA to type I cells leaves a
transient current with properties that indicate that it is an A-type
current. The current that is blocked by TEA is markedly different, and
we believe that this current falls into the category of delayed
rectifier K+ currents that have previously been
identified in a variety of cell types (Rudy 1988).
TEA sensitivity of IK(V)
First, the current was markedly more sensitive to block by TEA
than IK(A), being completely blocked
by 1 mM TEA, while a substantial proportion of the
IK(A) still remained active (see Fig.
4). The currents that remained after application of TEA
[IK(A)] are illustrated in Fig. 4,
A2 and A3, whereas the TEA-sensitive current is
illustrated in Fig. 4A4. This sensitivity to TEA is one of
the characterizing properties of delayed rectifier type currents in a
variety of cell types (Rudy 1988). Second, the
TEA-sensitive current activated at potentials that were significantly
more positive than IK(A) (P < 0.001). Currents were first resolved at
potentials between
30 and
20 mV (
27 ± 1 mV) compared with
IK(A) that activated at potentials
between
50 and
40 mV (
46 ± 1 mV; Figs. 4B and 9B). This more depolarized
activation threshold is again characteristic of the delayed rectifier
type currents (Brezina et al. 1994b
; Laurienti
and Blankenship 1996a
; Miyoshi et al.
1991
; Yamamoto et al. 1989
). Third, the
activation and inactivation of the TEA-sensitive current were much
slower than those seen for IK(A). The
time-to-peak for TEA-sensitive currents ranged between 175 ± 11.4 ms at
10 mV to 74.1 ± 3.9 ms at +40 mV. Because
IK(V) showed little time-dependent inactivation during the 200-ms voltage step, it was impossible to fit
the declining phase of the current to an exponential. We have therefore
calculated the %reduction in the amplitude of the current. With
voltage steps to +40 mV the TEA-sensitive current showed very little
inactivation during (6.2 ± 2.1%) compared with the 53.3 ± 4.9% inactivation seen with IK(A)
(P < 0.001). We cannot exclude the possibility
that the small amount of inactivation observed in the TEA-sensitive
current was due to a residual IK(A). Comparison with I(KV) isolated from type I
cells and type II cells showed there to be no significant difference in
the activation threshold (
46 ± 1 mV compared with
45 ± 1 mV; P > 0.05), the time-to-peak (74.1 ± 3.9 ms compared with 70.1 ± 4.5 ms, +40 mV; P > 0.05) and the reduction in the amplitude of the current during the
voltage step (6.2 ± 2.1% compared with 7.1 ± 2.5%;
P > 0.05) for type I and type II cells,
respectively.
|
Holding potential dependence of IK(V)
A further property that separates the delayed rectifier type
currents from the A-type currents is their holding potential dependence. Although A-type currents are completely inactivated by
holding potentials in the range of 60 to
40 mV, the delayed rectifier currents are less sensitive to these low holding potentials. An example of the holding potential dependence of
IK(V) is shown in Fig. 9. This current
was isolated from a type I cell by perfusing the cell with 5 mM 4-AP to
block IK(A). Figure 9,
A1-A6, illustrates the holding potential dependence of the
current. Unlike IK(A) that is
completely inactivated by holding the cell at
40 mV (see Fig. 6),
IK(V) can still be activated by
voltage steps to +50 mV from holding potentials as positive as 0 mV.
I-V plots of the peak current at various holding potentials
are shown in Fig. 9B. The activation thresholds for currents
isolated in this way were not significantly different from those of the
TEA-sensitive current (see Fig. 4B). Figure 9C
shows a plot of the holding potential dependence of
IK(V) for three separate cells. The
plot clearly demonstrates that, as the holding potential is made more
positive, there is a reduction in the amplitude of the peak-evoked
current in all three cells. A Boltzmann fit of the three sets of data gave a V1/2 of
43 mV and a slope
factor of 20.8 ± 5.6 mV.
K+ selectivity of the delayed rectifier current
Delayed rectifier currents are highly selective for
K+ ions. To test whether the Lymnaea
current was similarly sensitive to K+ ions, we
examined the change in the reversal potential of the tail currents
following superfusion of the ventricle cells with salines containing
different concentrations of K+ ions. Three
concentrations were studied: 1.7 mM K+ (normal
saline), 4.76 mM K+, and 17 mM
K+. IK(V) tail
currents were evoked by holding the cell at 40 mV to remove any
residual IK(A) current, and stepping
the cell to 0 mV and then stepping down to a series of potentials
between
30 and
140 mV (see Fig.
10A1, top). Figure 10 shows
an example of the tail currents evoked in a 1.7K (Fig. 10A1)
or a 17K (Fig. 10A2) saline. A plot of the reversal
potential of the tail currents versus the log of the
K+ concentration of the bathing solution is shown
in Fig. 10B. The reversal potential increased linearly with
increasing concentrations of K+ and showed a
41-mV change for a 10-fold increase in the concentration of
K+ ions bathing the cell (
). This compares
favorably with Goldman-Hodgkin-Katz prediction for a purely
K+-selective current of 58 mV (
), indicating
that a significant proportion of the current carried through the
delayed rectifier channels is carried by K+.
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DISCUSSION |
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Dissociated Lymnaea heart ventricle cells are capable of generating regenerative action potentials
Current-clamp recordings were made from acutely dissociated
Lymnaea heart ventricle cells. All cells were capable of
generating two types of spontaneous action potentials. The first had a
prepotential with fast rising and slow repolarizing phases and was the
most common type seen. The second consisted of a prepotential and a fast spike that was followed by a plateau phase. The fast spikelike action potentials appear to be dependent on the influx of
Ca2+ ions, whereas the repolarization phase is
sensitive to TEA and is therefore presumably due to the efflux of
K+ ions. The ionic basis of the plateau
phase is unclear because we were unable to find a means of repeatedly
activating this type of action potential. In the
Mercenaria ventricle (Devlin 1993) the
fast spikelike action potentials and the repolarization phase were also
due to the influx of Ca2+ and efflux of K+
ions, respectively. The plateau phase, however, was shown to be the
result of an influx of Na+ ions. This was a similar result
to that found in other bivalve mollusks that had spike/plateau-like
action potentials (Deaton and Greenberg 1980
;
Huddart and Hill 1996
). Classical Na+ entry
via voltage-gated channels can be ruled out as a possible mechanism of
plateau generation in Lymnaea ventricle cells as previous work by Brezden and Gardner (1992)
using a
cell-attached patch technique and our work (see also Yeoman et
al. 1999
) failed to show the presence of any voltage-gated
Na+ channels or currents, respectively. If Na+
is responsible for the plateau phase, the most likely routes of entry
is via calcium-activated nonspecific cationic channels. Our observation
that spike/plateau action potentials evoke relatively stronger muscle
contractions compared with those evoked by the spike type potentials is
suggestive that spike/plateau potentials are associated with a larger
increase in intracellular Ca2+, which in turn may activate
the nonspecific cation channels. The lack of voltage-gated
Na+ currents in Lymnaea ventricle cells
identifies these cells with those recorded in the sinoatrial and
atrioventricular nodes of mammalian hearts in which the upstroke of the
action potential is solely dependent on the influx of Ca2+
(Hagiwara et al. 1988
; reviewed in Irisawa et al.
1993
).
We believe that this is the first time that spontaneous action
potentials have been recorded from dissociated ventricle cells of a
mollusk. This general ability of all the Lymnaea
ventricle cells to generate action potentials is significant,
particularly as the rate of action potential generation (8.5 ± 1.2 spikes/min) is not dissimilar to beat rates recorded from intact
isolated hearts (Buckett et al. 1990a-c
). This
observation is suggestive that the heart of Lymnaea may
possess a distributed pacemaker system. However, further work
characterizing the pacemaking properties of the auricle are required
before this can be confirmed. Previous work on intact molluscan hearts
have described three types of action potential, fast and slow spikelike
action potentials, and spike and plateau action potentials
(Jones 1983
). Lymnaea ventricle cells
appear to possess two of these action potential types and therefore
appears to be most similar to the Mercenaria ventricle, which is also capable of generating the same two types of action potential (Devlin 1993
). Both these types of action
potential are seen in mammalian heart tissue, although not in the same
region of the heart (Katz 1977
). Both the sinoatrial
node and atrioventricular node (pacemaker regions of the mammalian
heart) show classical spikelike action potentials, whereas the auricle,
ventricle, bundle of His and Purkinje network show the spike and
plateau type of action potential.
Characterization of the potassium currents in Lymnaea ventricle cells
ABSENCE OF AN INWARD RECTIFIER CURRENT.
Ramp protocols produced current profiles that were flat in the voltage
region between 90 and
50 mV, indicating the absence of an inward
rectifier potassium current. This type of current has been
characterized in a noncardiac molluscan muscle, the ARC muscle of
Aplysia (Brezina et al. 1994a
), and similar
currents have been recorded in mammalian ventricle cells
(Irisawa et al. 1993
). However, inward rectifying
currents are absent from the sinoatrial node pacemaker in the mammalian
heart (Giles and Van Ginneken 1985
;
Irisawa 1987
). The absence of the inward rectifier in
this region results in a high-input resistance compared with other
cardiac cells, and this is thought to be important in increasing the
sensitivity of the cell to small current fluxes. Lymnaea
ventricle cells also have high-input resistances (939 ± 73.9 M
) presumably for the same reason, and this is consistent with their
proposed role as pacemaker cells in the heart.
CHARACTERIZATION OF OUTWARD RECTIFYING POTASSIUM CURRENTS.
The large outward current seen in both the ramp and voltage-step
protocols activated between 50 and
40 mV and increased in magnitude
as the membrane potential became more positive. Based on our
voltage-clamp analysis, we have classified Lymnaea ventricle cells into two groups, termed type I and type II cells. We have shown
that in type I cells the outward current consists primarily of two
types of current; an A-type current
[IK(A)] and a delayed rectifier
current [IK(V)]. Type II cells also
contained IK(A) and
IK(V) as well as an outward
Ca2+-dependent current that was not studied in
detail in this paper. We found no quantitative differences between the
IK(A) and
IK(V) currents recorded in the two
different cell types.
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ACKNOWLEDGMENTS |
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This work was supported by Grant IR3521-1 from the Biotechnology and Biological Sciences Research Council, U.K.
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FOOTNOTES |
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Address for reprint requests: M. S. Yeoman, School of Pharmacy and Biomolecular Sciences, Cockroft Building, University of Brighton, Moulsecoomb, Brighton, East Sussex BN2 4GJ, UK.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 April 1999; accepted in final form 22 July 1999.
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REFERENCES |
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