Download LVA and HVA Ca Currents in Ventricular Muscle Cells of the

LVA and HVA Ca21 Currents in Ventricular Muscle Cells of the
Lymnaea Heart
M. S. YEOMAN,1,2 B. L. BREZDEN,1 AND P. R. BENJAMIN1
Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG; and
2
School of Pharmacy and Biomolecular Sciences, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, United Kingdom
1
INTRODUCTION
In the previous paper Yeoman and Benjamin (1999) described two voltage-gated K1 currents [IK(A) and IK(V)] as part
of a study aimed at characterizing ion currents in the myogenic
molluscan heart. In this paper, further electrophysiological
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analysis shows the presence of two voltage-gated calcium
currents. We propose that these currents may have a role in
pacemaking as well as providing part of the calcium influx
necessary for excitation-contraction coupling.
Voltage-gated calcium channels are a common feature of all
invertebrate and vertebrate muscles (McDonald et al. 1994). A
variety of different Ca21 currents have been characterized
(Kits and Mansvelder 1996). In mollusks, voltage-gated Ca21
currents have been classified into two distinct groups depending on their voltage sensitivity. Those currents that are activated at relatively hyperpolarized voltage ranges (less than or
equal to 240 mV) have been termed low-voltage activated
(LVA), whereas those that are activated at potentials more
positive to 240 mV have been termed high-voltage activated
(HVA). The majority of invertebrate muscles studied to date
contain only the HVA type of voltage-activated Ca21 current.
These HVA currents vary slightly in their voltage sensitivity,
but all could be blocked with nifedipine. Substitution of Ba21
for Ca21 as the permeant ion led to an increase in the amplitude
of these currents and a slowing in the inactivation kinetics
(Brezina et al. 1994; Laurienti and Blankenship 1996a; Ram
and Liu 1991). Consequently, these currents have been classified as L-type. Examples of LVA currents in invertebrate
muscle are scarce. The only example to date is from the vas
deferens muscle of Lymnaea (Van Kesteren et al. 1995). This
current activates at 240 mV, is fast to activate, and has rapid
inactivation kinetics. These properties are characteristic of
vertebrate T-type Ca21 currents, although a conclusive pharmacological characterization of this current was not performed.
Thus in the invertebrate muscles studied so far, the most
prevalent voltage-gated Ca21 current is the L-type current.
In vertebrates, the situation is more complex. Cardiac, skeletal, and smooth muscle all possess both T- and L-type Ca21
currents (McDonald et al. 1994). Of the remaining classes of
Ca21 current, neuronal P/Q and R types have no known muscle
counterpart, whereas the existence of the N-type channels are
extremely rare or absent (Bean 1989b).
In vertebrate cardiac muscle, both T- and L-type calcium
currents have been shown to be responsible for action potential
generation (Hagiwara et al. 1988). In invertebrate hearts, very
little is known about the role of Ca21 currents in the generation
of the myogenic beat. Previous work has shown that the action
potentials generated by molluscan heart tissue were strongly
dependent on the influx of Ca21 (Hagiwara and Byerly 1981),
but there are no studies where the currents responsible for
action potential generation have been characterized. Two types
of Ca21 channel have previously been characterized in disso-
0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society
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Yeoman, M. S., B. L. Brezden, and P. R. Benjamin. LVA and HVA
Ca21 currents in ventricular muscle cells of the Lymnaea heart. J.
Neurophysiol. 82: 2428 –2440, 1999. The single-electrode voltageclamp technique was used to characterize voltage-gated Ca21 currents
in dissociated Lymnaea heart ventricular cells. In the presence of 30
mM tetraethylammonium (TEA), two distinct Ca21 currents could be
identified. The first current activated between 270 and 260 mV. It
was fully available for activation at potentials more negative than 280
mV. The current was fast to activate and inactivate. The inactivation
of the current was voltage dependent. The current was larger when it
was carried by Ca21 compared with Ba21, although changing the
permeant ion had no observable effect on the kinetics of the evoked
currents. The current was blocked by Co21 and La31 (1 mM) but was
particularly sensitive to Ni21 ions ('50% block with 100 mM Ni21)
and insensitive to low doses of the dihydropyridine Ca21 channel
antagonist, nifedipine. All these properties classify this current as a
member of the low-voltage–activated (LVA) T-type family of Ca21
currents. The activation threshold of the current (270 mV) suggests
that it has a role in pacemaking and action potential generation.
Muscle contractions were first seen at 250 mV, indicating that this
current might supply some of the Ca21 necessary for excitationcontraction coupling. The second, a high-voltage–activated (HVA)
current, activated at potentials between 240 and 230 mV and was
fully available for activation at potentials more negative than 260
mV. This current was also fast to activate and with Ca21 as the
permeant ion, inactivated completely during the 200-ms voltage step.
Substitution of Ba21 for Ca21 increased the amplitude of the current
and significantly slowed the rate of inactivation. The inactivation of
this current appeared to be current rather than voltage dependent. This
current was blocked by Co21 and La31 ions (1 mM) but was sensitive
to micromolar concentrations of nifedipine ('50% block 10 mM
nifedipine) that were ineffective at blocking the LVA current. These
properties characterize this current as a L-type Ca21 current. The
voltage sensitivity of this current suggests that it is also important in
generating the spontaneous action potentials, and in providing some of
the Ca21 necessary for excitation-contraction coupling. These data
provide the first detailed description of the voltage-dependent Ca21
currents present in the heart muscle cells of an invertebrate and
indicate that pacemaking in the molluscan heart has some similarities
with that of the mammalian heart.
CA21 CURRENTS IN THE LYMNAEA HEART
ciated ventricle cells from the Lymnaea heart. These were
nonvoltage-gated calcium channels and were initially described by Brezden and Gardner (1990). They were subsequently shown to be activated following the application of the
peptide FMRFamide (Brezden et al. 1991). The P(open) time of
these channels was extremely low in the absence of the peptide
(0.008) (Brezden et al. 1999) and could therefore not be responsible for the generation of the action potentials recorded in
dissociated heart muscle cells (Yeoman and Benjamin 1999).
METHODS
Solutions
To isolate either Ca21 or Ba21 currents, the cells were perfused
with 30 mM tetraethylammonium (TEA). This was sufficient to block
all the K1 currents that normally masked the developing inward
currents (Yeoman and Benjamin 1999). These cells do not contain
voltage-gated Na1 currents (see RESULTS), so there was no need to
include the Na1 channel blocker, tetrodotoxin (TTX), in the bathing
solution. The standard solutions used were Ca21/, Ba21/, and Co21/
TEA and contained the following concentrations of ions (in mM): 20
Na1, 30 TEA, 1.7 K1, 3.5 Ca21 or Ba21 or Co21, 2 Mg21, and 10
N-(2-hydroxyethyl)piperazine-N9-(2-ethanesulfonic acid) (HEPES)
with the pH adjusted to 7.9 using NaOH. Occasionally, when the
Ba21 currents were difficult to resolve, the concentration of Ba21 in
the extracellular saline was increased to 10 mM. In these instances the
concentration of Na1 was reduced to compensate for the increased
concentration of Ba21 ions.
TEA, NiCl2, GdCl3, and LaCl3 were all obtained from Sigma
Chemical Co., Poole, U.K., and made up on the day of the experiment
by dissolving them in the bath solution. Nifedipine was also obtained
from Sigma. A stock solution was made by dissolving the nifedipine
in dimethyl sulfoxide (DMSO) and then diluting the solution in the
extracellular saline to yield a final concentration of 0.1% DMSO
vol/vol. Application of 0.1% DMSO alone was found to have no
effect on membrane ion currents. In the experiments that used v-conotoxin to test for the presence of N-type calcium currents, solutions of
the toxin were supplemented with 1 mg/ml of bovine serum albumin
to block putative binding sites on the perfusion tubing. All the
resulting solutions were adjusted to a pH of 7.9.
Statistical analysis
All values presented in this paper represent means 6 SE. Unless
stated otherwise in the text, tests for significance have been performed
using unpaired t-tests assuming unequal variances (Excel 97).
RESULTS
Initial identification of inward currents
Transient inward currents were observed in ;40% of cells
that had been subjected to voltage step protocols in normal
saline. These activated at around 270 mV but became masked
by the more slowly developing K1 currents at potentials more
positive than 240 mV. Ramp protocols showed two regions of
negative slope resistance (NSR) typical of the presence of two
distinct inward currents. In the example shown in Fig. 1A1, the
cell was held at 290 mV and then stepped briefly to 2120 mV
before being ramped to 140 mV over a 1.25-s period. Although the first phase of inward rectification is clear (I1; 262
mV) the second phase (I2) was almost completely masked by
the presence of the voltage-gated K1 currents that began to
activate between 250 and 240 mV. To resolve these inward
currents more clearly, the same cell was superfused with a bath
solution containing 30 mM TEA (Fig. 1A2). This had the effect
of blocking the majority of the outward currents and allowed
both phases of NSR to be seen more clearly. The first phase of
NSR activated around 267 mV and peaked at 260 mV and
FIG. 1. Isolation of voltage-dependent inward currents. A1: voltage ramp protocols (2120 to 140 mV, see
bottom) performed on dissociated ventricle cells bathed
in normal saline yielded whole cell currents that displayed 2 peaks of inward rectification (I1 and I2). A2:
addition of 30 mM tetraethylammonium (TEA) to the
bathing solution blocked a large proportion of the K1
currents allowing the 2 inward currents to be more
clearly visualized. A3: substitution of the Ca21 ions in
the bathing solution by Ba21 ions blocked a proportion
of the residual K1 currents and further enhanced the
amplitude of I2. A4: increasing the concentration of Ba21
ions to 10 mM blocked the majority of the outward K1
currents and further enhanced the amplitude of I2. B: 2
peaks of inward current were again visible when all the
extracellular Na1 ions had been replaced by Ba21 ions.
This indicated that these 2 inward currents are divalent
cationic currents. Recordings in A and B are from different cells.
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Methods describing the dissociation of heart ventricles and the
techniques used to stabilize the cells for intracellular recording are
detailed in the previous paper, as are the methods used for data
acquisition, analysis and presentation (Yeoman and Benjamin 1999).
Ca21 and Ba21 currents were isolated pharmacologically (see next
section) and leak-subtracted using one of two methods. The first used
pClamp’s on-line leak subtraction and was based on the resistance of
the membrane. The second method involved blocking the isolated
Ca21/Ba21 currents by substituting the Ca21/Ba21 ions in the saline
with Co21 ions. The remaining current was classified as the leakage
current. Unless otherwise stated, we have used the latter pharmacological method.
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M. S. YEOMAN, B. L. BREZDEN, AND P. R. BENJAMIN
eral other lines of evidence support the hypothesis that both the
LVA and HVA currents are Ca21 currents. First, in five experiments, application of TTX (1025 M) was unable to block
any component of the inward currents (data not shown). Second, in five experiments where all the K1 and Ca21 currents
were blocked pharmacologically by a mixture of TEA and
Co21, we failed to observe any inward current in response to
voltage ramp protocols (data not shown). Third, previous work
by Brezden and Gardner (1992) failed to show the presence of
any voltage-gated Na1 channels using the cell-attached patchclamp technique in ventricle cells isolated in an identical
manner to the present experiments. We therefore believe that
Lymnaea heart ventricle cells contain at least two voltage-gated
Ca21 currents.
Variation in the inward current size and the current
complement of heart ventricle cells
There were large variations in the size of the inward currents
that we recorded. This variation was found between cells taken
from the same or different batches of heart muscle cells. In
;60% of batches tested (n 5 32 batches), current recordings
had to be made using 10 mM Ba21 as the permeant ion to
visualize the currents. In the remaining 40% of the batches
(n 5 18 batches), the inward currents were large enough to be
recorded in normal Ca21 or Ba21 salines.
In the majority of fibers recorded (85%; n 5 60) two distinct
peaks of inward current could be seen (Fig. 2A3). However, a
significant proportion of the cells (15%, n 5 10) appeared to
contain either the LVA current (Fig. 2A1) or the HVA current
(Fig. 2A2).
Voltage dependence of LVA and HVA Ba21 currents
Figure 2, B1–B3, illustrates currents evoked by short (150
ms) voltage steps from 290 mV to potentials between 260 and
130 mV from the same cells as those illustrated in Fig. 3,
A1–A3, respectively. Figure 2B1 shows a typical example of
the currents evoked by a series of increasingly positive voltage
steps from a holding potential of 290 mV in a muscle cell that
contained only the LVA Ba21 current. The LVA current began
to activate between 260 and 250 mV (258 6 1 mV, mean 6
SE, n 5 10), peaked at potentials between 240 and 230 mV
and then decreased in amplitude at increasingly positive potentials, giving an extrapolated reversal potential of around
140 mV (Fig. 2C1). The HVA Ba21 current did not begin to
activate until 240 or 230 mV (234 6 2 mV, n 5 8) and
peaked at potentials between 210 and 0 mV. It then declined
in amplitude, yielding an extrapolated reversal potential of
140 mV (Fig. 2C2). An unpaired t-test showed the activation
thresholds of the two currents to be significantly different (P ,
0.001). Currents evoked from a cell containing both the LVA
and HVA currents are shown in Fig. 2B3. The current-voltage
(I-V) plot from this cell showed a characteristic shoulder on its
rising phase that corresponded to the peak of the LVA current.
Current amplitude continued to increase and peaked at between
210 and 0 mV (the peak of the HVA current). The current then
declined to give a predicted reversal potential of around 140
mV (Fig. 2C3).
Both inward currents had reversal potentials of around 140
mV. This was far from the theoretical reversal potential for
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was provisionally classified as the low-voltage–activated
(LVA) current. The second phase activated at around 245 mV
and peaked at 225 mV and is referred to as the high-voltage–
activated (HVA) current. Although the inclusion of 30 mM
TEA in the bathing media allowed us to resolve the inward
currents more clearly, there was still a significant outward
current present at the more depolarized potentials (more than
110 mV), indicating that not all the outward currents were
blocked by TEA.
To further characterize these inward currents, Ba21 ions
were substituted for Ca21 ions in the bathing solution. This had
a number of advantages. In the absence of Ca21 ions, Ba21 has
been shown to enhance the amplitude of a number of types of
voltage-gated Ca21 currents (Bean 1989a; Tsien et al. 1988), as
well as being able to block certain K1 currents (Armstrong and
Taylor 1980; Hille 1992; Latorre and Miller 1983). Its substitution for Ca21 in the extracellular solution would help block
any residual K1 currents not blocked by TEA and would also
prevent the activation of Ca21-dependent K1 currents also
known to be present in these cells. All of these effects would
allow the inward Ca21 currents to be more easily isolated.
Figure 1A3 is an example of a current record, from the same
cell as in A1 and A2, bathed in a 30-mM TEA/3.5-mM Ba21
saline with the current evoked using the same ramp protocol.
The whole cell current again showed two regions of NSR,
indicating the presence of two separate inward currents. As
predicted, substitution of Ca21 with Ba21 not only reduced the
residual outward current at potentials positive to 0 mV but also
caused a slight increase in the amplitudes of both inward
currents. These two effects were presumably responsible for
the shift in the reversal potential of the whole cell current to
more positive potentials (from between 210 and 0 mV in 3.5
mM Ca21 to between 120 and 130 mV in 3.5 mM Ba21).
Increasing the concentration of Ba21 ions in the saline to 10
mM (Fig. 1A4) further increased the amplitude of the HVA
current without significantly altering the amplitudes of either
the LVA current or the residual outward current. One unusual,
but consistent finding observed when Ba21 was substituted for
Ca21 in the bathing medium was an atypical shift in the
activation threshold of the two phases of inward rectification.
Differential effects of the two ions on surface charge screening
would normally predict a shift to the left as shown for the
voltage step protocols in Fig. 5, and thus we can only assume
that this is an artifact seen solely during voltage ramp protocols. The observation that the amplitude of the HVA current
alone was dependent on the extracellular concentration of Ba21
ions suggested that the current was due to the flow of divalent
ions and that Lymnaea ventricle cells contained two distinct
voltage-gated Ca21 currents. However, it was possible that the
LVA current as well as some component of the HVA current
were due to changes in the permeability of the cells to Na1
ions. To test for this, we replaced all the NaCl in the extracellular solution with BaCl2 giving a solution that contained 53.5
mM Ba21/30 mM TEA. Current records from cells ramped
from 2120 to 140 mV in this saline showed the same two
currents (Fig. 1B), suggesting that Na1 ions were not a significant charge carrier through these channels. The amplitude of
both the currents was greater than that seen using 10 mM Ba21.
This was presumably due to the increase in the driving force
for Ba21 entry and indicated that these currents were probably
carried by Ca21 ions in the normal physiological saline. Sev-
CA21 CURRENTS IN THE LYMNAEA HEART
2431
Ba21 (1175 mV). Similar anomalous reversal potentials for
Ba21 currents have been recorded in other molluscan and
vertebrate muscles (Bean 1989b; Brezina et al. 1994). In these
other preparations it was proposed that the channels were not
perfectly selective for Ba21 but instead could allow a significant efflux of K1 at depolarized potentials. Permeability to K1
would then tend to shift the reversal potential for the current
away from EBa toward Ek.
21
FIG. 3. A: variations in the extracellular Ba
concentration increases the
amplitude of the recorded LVA Ba21 current but has no effect on its rate of
inactivation. B: increasing the concentration of extracellular Ba21 ions increases the amplitude of the recorded HVA Ba21 current as well as increasing
its inactivation rate. The scaled currents represent the currents obtained in 3.5
mM Ba21 scaled to the peak of the current obtained in 10 mM Ba21.
Activation and deactivation of the LVA and HVA currents
In this and the following section we have quantified the time
courses of the activation and inactivation of the two voltagegated Ba21 currents.
The LVA current was fast to activate with the time taken for
the current to reach its peak decreasing as the potential to
which the cell was stepped became more positive. Values
ranged from 56.7 6 8.7 ms with voltage steps to 260 mV
(threshold potential) to 17.1 6 1.5 ms with steps to 240 mV,
which activated the peak current (n 5 6; Fig. 2B1). Deactivation of the LVA current following the termination of the
voltage step was extremely rapid, making a detailed analysis of
the tail currents impossible. The time for the HVA current to
reach its peak was also rapid and decreased with increasingly
more positive voltage steps (Fig. 2B2). The time taken for the
current to reach its peak decreased from 44.9 6 6.7 ms with
voltage steps to 240 mV (threshold potential) to 29 6 2.2 ms
with voltage steps to 210 mV, which activated the peak HVA
current (n 5 6; Fig. 2B2). Like the LVA current, deactivation
of the HVA current was again extremely rapid, and the time
course of the tail currents could not be resolved with sufficient
clarity to perform a detailed analysis. A t-test analysis performed on the time-to-peak for both currents showed them to
be significantly different (P , 0.001).
Inactivation of LVA and HVA currents during short
voltage steps
As we described earlier, at potentials close to or more
positive than those that evoked the largest LVA currents (more
positive than 250 mV), the majority of the LVA current
(.95%) inactivated completely during a 150-ms voltage step
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21
FIG. 2. Ventricle cells contain a mixture of 2 types of voltage-gated Ba
currents. Voltage ramp protocols (2120 to 150 mV)
demonstrated that the majority of Lymnaea ventricle cells contained a mixture of both high-voltage–activated (HVA) and
21
low-voltage–activated (LVA) Ba currents (A3). In a minority of cells, only LVA Ba21 currents (A1), or HVA Ba21 currents (A2)
could be recorded. B1–B3: voltage step protocols were performed on the same cells as A1–A3 to demonstrate the voltage sensitivity
and time-dependent kinetics of these currents. C1–C3: I-V plots of the currents recorded in cells B1–B3.
2432
M. S. YEOMAN, B. L. BREZDEN, AND P. R. BENJAMIN
inactivation of the LVA Ba21 currents recorded in the same
experiments was not significantly affected by changes in the
extracellular concentrations of divalent ions (Fig. 3A), confirming that inactivation of the LVA current is not current dependent.
The values of the time constants detailed above provide
further information confirming the distinctness of the two
currents. However, because of our inability to completely
isolate either of the two currents, these values may not represent the true kinetics of the pure currents.
Ca21 and Ba21 permeability of the LVA and HVA currents
Based on the voltage sensitivity and time-dependent kinetics
of the LVA and HVA currents, these two currents appeared to
be members of the T-type and L-type family of Ca21 currents,
respectively. Further evidence in support of this classification
would be obtained by examining the relative permeabilites of
the two currents to Ca21 or Ba21 ions. T-type channels have
previously been shown to have no preference for Ca21 ions
over Ba21 ions (Alvarez and Vassort 1992; Bean 1985;
Chesnoy-Marchais 1985; Coyne et al. 1987). L-type channels
on the other hand have been shown, in a variety of invertebrate
and vertebrate muscles, to conduct Ba21 ions much better than
Ca21 (Bean 1989b; Tsien et al. 1988). We have therefore
examined the relative amplitudes of the LVA and HVA currents in Lymnaea ventricle cells in the presence of equimolar
(3.5 mM) physiological concentrations of either Ca21 or Ba21.
Figure 4 shows examples from three different cells of both
LVA (A1–A3) and HVA currents (B1–B3) recorded in the two
different salines.
LVA currents were evoked by a 200-ms voltage step from
290 to 250 mV. In 3.5 mM Ca21 they were fast to activate
and in the majority of cases (12 of 13 cells tested) inactivated
completely during the 200-ms voltage step (Fig. 4, A1 and A3).
In the remaining cell tested, a sustained inactivating component of the LVA Ca21 current was present (Fig. 4A2). Due to
the infrequent appearance of this current, we have not been
able to determine whether or not this current is different from
the more typical LVA current recorded in the majority (92%)
of cells. Substitution of Ba21 for Ca21 caused a marked
reduction in the amplitude of the LVA current in the majority
of fibers tested 50.6 6 10.6% (mean 6 SE; in 12 of the 13
fibers tested, Fig. 4, A1 and A3). This demonstrated, that on
average, LVA channels were more permeable to Ca21 than
Ba21 ions. In one cell only, LVA currents were recorded that
showed equal permeability to Ca21 and Ba21 (Fig. 4A2). It is
interesting to note that this was seen in the cell that displayed
the noninactivating current.
HVA currents were evoked by voltage steps from 290 to
210 mV. The HVA Ba21 currents were typically larger than
the corresponding LVA currents recorded from the same
cells. They were all fast to activate but showed varying rates
of inactivation (compare Fig. 4, B1 and B3 with B2). Compared to the Ca21 currents recorded from the same cells,
HVA Ba21 currents were noticeably larger (48.6 6 6.7%
mean 6 SE; 10 of 13 cells tested). This indicated that these
channels conduct Ba21 better than Ca21. In the other three
cells tested, one showed no increase, whereas the others
showed decreases of 156 and 242%, respectively. HVA
Ca21 currents like the Ba21 currents were fast to activate
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(Fig. 2B1). However, at potentials close to the activation
threshold (270 mV), a component of the LVA current failed to
inactivate, leaving a small residual inward current (the possible
relevance of this sustained current is detailed further in DISCUSSION). The rate of inactivation of the LVA current was fitted by
a single exponential. The rate increased as the membrane was
stepped to more depolarized potentials between 260 and 220
mV, indicating a strong voltage dependence (260 mV; t 5
59.4 6 7.6 ms, n 5 6; 220 mV, t 5 15.6 6 1.6 ms, n 5 6;
Fig. 2B1). However, at potentials more positive than 220 mV
(220 to 130 mV), the rate of inactivation began to slow (e.g.,
0 mV, t 5 28.9 6 6.3 ms; n 5 6). This indicated that some
other mechanism might be important for determining inactivation rates over this potential range. It was possible that this
represented some form of current-dependent inactivation.
However, there was no correlation between the inactivation
rate and the peak amplitude of the current or the amount of
current flowing across the membrane (determined by the integral of the current waveform). The most likely explanation was
that the inactivation of the LVA current was generally voltage
dependent but that the slowing in the inactivation rate at
potentials more positive to 210 mV was due to contamination
of the LVA current by a small amount of the slower inactivating HVA current. This had previously shown to be maximally
active at these potentials.
In contrast to the LVA currents, the HVA Ba21 currents
showed only partial inactivation during the 150-ms voltage
step (Fig. 2B2). A double exponential was found to be the best
fit for the rate of inactivation. The exponential was fitted to
declining current from its peak at the start of the depolarizing
voltage step to its final level at the end of the step. Of the two
components of the double exponential fit t1 was present in the
highest proportion ('100 times more than t2) and was the only
component to vary significantly with alterations in the membrane potential of the cell. At potentials just positive to the
activation threshold (220 mV), inactivation was weak (t1 5
320 6 46.3 ms, t2 5 15.6 6 1.6 ms; n 5 6). The rate of
inactivation increased with more positive voltage steps and
became maximal around the peak of the HVA current (0 mV;
t1 5 116 6 21.6 ms, t2 5 16.5 6 1.6 ms; n 5 6). At potentials
more positive than 120 mV, inactivation of the HVA current
again became weaker (t1 5 280 6 36.3 ms, t2 5 16.1 6 1.2
ms; n 5 6). Using an ANOVA followed by a paired t-test, the
values for t1 recorded at 220 and 120 mV were found to be
significantly different to those at 0 mV. The inactivation of the
Ba21 HVA current therefore does not appear to be only voltage
dependent but may also be current dependent.
This was examined in experiments where inactivation of the
HVA Ba21 current was recorded in salines where the extracellular concentration of Ba21 ions had been increased from
3.5 to 10 mM to increase the current flow through the HVA
channels. Figure 3B shows an example of one such experiment
where HVA currents were evoked by 150-ms voltage steps to
110 mV from a holding potential of 290 mV. Increases in the
concentration of divalent ions bathing the cells clearly increased the rate of inactivation of the HVA current confirming
a current-dependent mechanism of inactivation. Again t1 was
the only time constant to be significantly altered. t1 for currents
recorded in 3.5 mM Ba21 was 235 6 19.5 ms (n 5 6). This
value decreased to 130.3 6 4.7 ms for currents recorded in 10
mM Ba21 (paired t-test, P , 0.001). In contrast the rate of
CA21 CURRENTS IN THE LYMNAEA HEART
2433
Comparison of the voltage dependence of LVA and HVA
currents using normal concentrations (3.5 mM) of Ca21 or
Ba21 as the permeant ions
but showed complete inactivation during the 200-ms voltage
step. The inactivation rates of the HVA Ca21 currents were
fitted best with a double exponential and were therefore
similar to the HVA Ba21 currents described earlier. However, unlike the HVA Ba21 currents the proportions of the
two components comprising the inactivation phase of the
HVA Ca21 currents are approximately equal. Changing the
permeant ion caused significant changes in the first time
constant (t1) from 81.6 6 11.3 ms (Ca21) to 364.2 6 46.4
ms (Ba21; n 5 8, P , 0.001), whereas there were no
changes in t2 5 16.6 6 1.5 ms (Ca21) to 15.5 6 1.8 ms
(Ba21). The faster inactivation rate seen in cells bathed in
the Ca21-containing saline could be due to the activation of
a Ca21-dependent K1 current, that has been shown to be
present in other molluscan muscles (Brezina and Weiss
1995; Laurienti and Blankenship 1996b), but this has not
been examined in the present experiments.
The changes in conductance that we have recorded for both
LVA and HVA currents in the Ca21 and Ba21 salines are
consistent with these two currents being members of the T- and
L-type calcium channel families, respectively.
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21
FIG. 4. Permeability of LVA and HVA currents to Ca
and Ba21 ions.
A1–A3: LVA currents evoked from 3 separate cells by voltage steps to 250
mV from holding potentials of 290 mV in salines containing either 3.5 mM
Ca21 or 3.5 mM Ba21 ions. B1–B3: HVA currents evoked from the same 3
cells as A1–A3, by voltage steps to 210 mV from a holding potential of 290
mV. Currents were again recorded with either 3.5 mM Ca21 or 3.5 mM Ba21
ions as the permeant ion.
It was important to generate I-V curves for the HVA and LVA
currents at concentrations of Ca21 resembling those present in
normal blood (3.5 mM) and with similar concentrations of Ba21
ions, for comparison. The function of the currents could then be
discussed in relation to the known operating range of voltages
seen in previous recordings of beating activity in the isolated cells
(Yeoman and Benjamin 1999) carried out in normal saline. Figure
5 shows a series of current recordings from a single cell in normal
saline with either Ca21 (Fig. 5A) or Ba21 (Fig. 5B) as the permeant ion. In Fig. 5A1 Ca21 currents were evoked by holding the
cell at 290 mV and stepping it to a range of potentials between
280 and 140 mV. An I-V plot of the total inward currents are
shown in Fig. 5A4 (■). The whole cell inward current was seen to
activate between 275 and 265 mV and showed an initial peak
around 250 mV (LVA current) and a second larger peak of
inward current between 210 and 0 mV (HVA current). By
holding the cell at more depolarized potentials (250 mV; Fig.
5A2), it was possible to inactivate all the LVA current so that the
same series of voltage steps (280 to 140 mV) now evoked an
almost pure HVA current. This current activated around 240 mV
(242 6 2 mV; n 5 8) and peaked between 210 and 0 mV before
declining in amplitude and yielding an extrapolated reversal potential of around 150 mV (F, Fig. 5A4). By subtracting the
currents recorded in Fig. 5A2 from those recorded in Fig. 5A1 we
were able to isolate the LVA current (Fig. 5A3). This current
activated between 275 and 265 mV (272.0 6 12 mV; n 5 10)
and peaked at between 250 and 240 mV before declining in
amplitude and showing an apparent reversal potential around 150
mV. The small inflection seen at 220 mV probably represented a
small proportion of the HVA current that is also blocked by
holding the cell at 250 mV. Figure 5B shows a series of current
records from the same cell as that recorded in Fig. 5A, but this
time superfused with a 3.5 mM Ba21 saline. Currents were again
evoked from holding potentials of 290 mV (Fig. 5B1) or 250
mV (Fig. 5B2) with LVA currents again isolated by subtracting
currents recorded from a holding potential of 250 mV from those
evoked from a holding potential of 290 mV (Fig. 5B3). LVA
currents recorded under these conditions were again fast to inactivate (Fig. 5B3). The HVA currents on the other hand showed the
similar slow inactivation rates that we had observed previously
(see Fig. 4). The I-V plots of the isolated currents are shown in
Fig. 5B4. LVA currents recorded in 3.5 mM Ba21 were extremely
small and difficult to resolve due to the relatively low permeability
of the channels to Ba21 (see Fig. 4). At potentials more positive
than 230 mV, their small size meant that there was significant
contamination of this current from the small proportion of HVA
current that was also blocked by holding the cell at 250 mV (},
Fig. 5B4). Because of their small size we were unable to obtain
reliable data for the voltage dependence of the LVA Ba21 current
recorded under these conditions. HVA currents on the other hand
activated between 250 and 240 mV, peaked at potentials between 220 and 210 mV, and reversed around 130 mV. This
negative shift in the I-V curve for the HVA Ba21 current compared with the HVA Ca21 current is probably due to the fact that
Ca21 is a better screen of the surface membrane charge than
equimolar concentrations of Ba21 (Hille 1992).
2434
M. S. YEOMAN, B. L. BREZDEN, AND P. R. BENJAMIN
Steady-state inactivation of HVA and LVA currents
The resting membrane potential of Lymnaea heart ventricle
cells was previously shown to be about 255 mV (Brezden and
Gardner 1986; Buckett et al. 1990; Yeoman and Benjamin
1999). An analysis of the I-V relationship of the LVA current
detailed in the previous section indicated that from holding
potentials of 290 mV there was a significant activation of this
current between 270 and 260 mV, suggesting that this current
could contribute significantly to the depolarization that drives
spontaneous spiking activity in the cells (Yeoman and Benjamin 1999). However, steady-state inactivation at normal
membrane potentials might make the contribution of the LVA
current ineffective. This was examined by holding cells at a
variety of membrane potentials (between 2110 and 220 mV)
and stepping to 250 mV to activate the LVA current. The
experiments used 3.5 mM Ca21 as the permeant ion. The
amplitude of LVA currents was unaffected by holding potentials more negative than 270 mV. As the holding potential was
made more positive than 270 mV, the amplitude of the evoked
current declined and was abolished completely by holding
potentials more positive than 240 mV (Fig. 6A1). The data
from three cells showing the effect of holding potential on the
amplitude of the evoked current is plotted in Fig. 6A2. The
three sets of data could be fitted with a Boltzmann curve that
showed that the LVA current was half inactivated at 258 mV
and had a slope factor of 4.1 6 0.3 mV. Due to the steep slope
of the inactivation curve, only about one-third of the LVA
current would be available at 255 mV, the resting membrane
potential of the cell, but could still contribute to the pacemaker
activity of the isolated heart muscle cells (see DISCUSSION).
HVA currents were recorded using 3.5 mM Ba21 as the
permeant ion and were activated by short 150-ms voltage steps
from a variety of holding potentials (2110 to 220 mV) to 0
mV. We chose not to use Ca21 as the permeant ion because of
the risk of contaminating the evoked Ba21 current with unblocked K1 currents that would be significantly activated at
this potential. Unlike the LVA current, the HVA current was
relatively unaffected by holding potentials as positive as 260
mV. At potentials more positive than this, the current again
declined in amplitude and was abolished completely by holding potentials of around 220 mV. Sample current traces of
HVA currents evoked from a variety of holding potentials are
shown in Fig. 6B1. The combined data from three such experiments is plotted in Fig. 6B2 and again has been fitted with a
Boltzmann curve, indicating that the HVA current is half
inactivated at a holding potential of 248 mV and had a slope
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21
FIG. 5. Comparison of the voltage sensitivities of LVA and HVA currents using 3.5 mM Ca
or 3.5 mM Ba21 as the permeant
ion. A1: whole cell LVA and HVA Ca21 currents evoked by voltage steps from a holding potential of 290 mV. A2: whole cell
HVA Ca21 currents evoked by voltage steps from a depolarized holding potential (250 mV) designed to inactivate the majority
of the LVA current. A3: currents recorded in A1 minus A2 yield a net LVA current. A4: I-V plots of whole cell (■), LVA (}), and
HVA Ca21 currents (●). B1: HVA and LVA Ba21 currents evoked from holding potentials of 290 mV. B2: HVA Ba21 currents
evoked from a holding potential of 250 mV that inactivates the majority of the LVA current. B3: subtraction of currents recorded
in B1 minus B2 to yield pure LVA Ba21 current. B4: I-V plot of the whole cell (■), LVA (}), and HVA Ba21 currents (●).
Recordings in A and B are all from the same cell.
CA21 CURRENTS IN THE LYMNAEA HEART
factor of 4.1 6 0.3 mV. Thus, although a significant proportion
of this current is available for activation at the RMP of the cell,
its relatively positive activation threshold (240 mV) means
that this current cannot contribute to the initiation of pacemaking in these cells, but could contribute to the action potentials
once they had been triggered.
Pharmacological profile of LVA and HVA currents
As in other systems La31, Gd31, and Co21 produced 100%
block of both LVA and HVA currents at 3.5 mM, whereas a
similar block could be obtained using 1 mM Cd21 (data not
shown). These data are in agreement with previous reports and
provide further evidence that both the LVA and HVA currents
are carried predominantly by Ca21 ions. Here we were mainly
interested in characterizing compounds that were capable of
selectively blocking one or other of the two Ca21 currents.
Previous work has shown that the dihydropyridine compounds
such as nifedipine are capable of selectively blocking L-type
calcium currents (Bean 1989a; Brezina et al. 1994; Tsien et al.
1988); so if the HVA current in Lymnaea ventricle cells was
sensitive to nifedipine, it would provide further evidence that
this current was a member of the L-type calcium channel
family. Figure 7 shows an example of such an experiment in
which the sensitivities of both LVA and HVA currents to 10
mM nifedipine were examined. In this experiment, currents
were recorded in fibers bathed in the elevated Ba21 solution
(10 mM). LVA and HVA currents were evoked by holding the
fiber at 290 mV and stepping it to 240 and 0 mV, respectively
(these were the peak potentials for these currents in the elevated Ba21 saline). Figure 7A1 shows a record of an HVA
current evoked in normal saline (control) or in the presence of
10 mM nifedipine. The current in nifedipine is markedly reduced, indicating that a significant proportion of the current is
sensitive to this dihydropyridine compound. Interestingly, the
block was more potent toward the end of the voltage step
(57.3 6 5.6%; n 5 8; P , 0.01) compared with the instantaneous block (32.4 6 4.74%; n 5 8; P , 0.01). This is in
agreement with previous studies that showed that nifedipine
antagonized the channel by means of an open channel block
(Bean et al. 1986; Carbone and Swandulla 1989). We investigated this possibility by prepulsing the fibers to 150 mV to
maximally activate the HVA channels and then tested the block
by nifedipine. Figure 7C shows a comparison of the current
blocked by 10 mM nifedipine using the standard protocol (Fig.
7A) versus a protocol where the cell was prestepped to 150
mV. Prepulsing the cell to 150 mV can clearly be seen to
facilitate the block of the HVA current by nifedipine. In three
preparations the instantaneous current was blocked by 68.8 6
7.8% at the end of the voltage pulse during the standard
protocol compared with the 86.2 6 9.3% (mean 6 SE) seen
with the prepulse. T-type calcium channels, on the other hand
21
FIG. 7. Nifedipine sensitivity of LVA and HVA Ba
currents. A1: HVA Ba21 currents evoked by voltage steps
from 290 to 0 mV were significantly reduced following
the application of 10 mM nifedipine. A2: LVA Ba21
currents evoked by voltage steps from 290 to 240 mV
were unaffected by 10 mM nifedipine. B: I-V plot of
whole cell Ba21 currents in the presence (●) and absence
(■) of 10 mM nifedipine. C: blockade of the HVA Ba21
current is enhanced in cells that were prepulsed to 150
mV compared with control currents evoked in the absence
of a prepulse.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on May 2, 2017
21
FIG. 6. Holding potential dependence of LVA and HVA Ca
currents. A1:
LVA currents evoked from a variety of different holding potentials (2110 to
230 mV, see marked potentials) by test potentials to 250 mV. A2: plot of
holding potential dependence of LVA Ca21 currents from 3 different cells
(different symbols). Solid line is a Boltzmann fit of the 3 sets of data. Dotted
line represents the potential at which 50% of the current is inactivated. B1:
HVA currents evoked from a variety of different holding potentials (2110 to
220 mV, see marked potentials) by test potentials to 210 mV. B2: plot of
holding potential dependence of HVA Ba21 currents from 3 different cells
(different symbols). Solid line is a Boltzmann fit of the 3 sets of data. Dotted
line represents the potential at which 50% of the current is inactivated.
2435
2436
M. S. YEOMAN, B. L. BREZDEN, AND P. R. BENJAMIN
21
FIG. 8. Ni
sensitivity of LVA and HVA Ba21 currents.
A1: LVA Ba21 currents evoked by voltage steps from 290 to
240 mV were markedly reduced following the superfusion of
100 mM Ni21. A2: HVA Ba21 currents evoked by voltage
steps from 290 to 0 mV were not significantly affected
following superfusion with 100 mM Ni21. B: I-V plot of
whole cell Ba21 currents in the presence (●) and absence (■)
of 100 mM Ni21. Arrow indicates the region of the I-V curve
where Ni21 causes a marked reduction of the whole cell
current. Recordings in A1 and A2 and data plotted in B are
from the same cell.
blocked the LVA current but also blocked over 80% of the
HVA current (data not shown). Other potential blockers of
LVA currents such as amiloride (Tytgat et al. 1990) showed no
selectivity for the LVA over the HVA current (data not
shown).
DISCUSSION
Lymnaea heart ventricle cells contain two types of voltagegated Ca21 currents
Ramp protocols, performed on voltage-clamped Lymnaea
heart ventricle cells bathed in normal saline, indicated the
presence of two distinct inward currents. These currents have
been termed LVA and HVA currents based on a classification
by Skeer et al. (1996). These currents could be isolated by
perfusing the ventricle cells with TEA to block the voltagegated K1 currents (Yeoman and Benjamin 1999). Further more
detailed electrophysiological and pharmacological analysis of
the two currents, mainly using step voltage clamp protocols,
led us to identify them as members of the T- and L-type
families of Ca21 currents, which have been extensively studied
in vertebrates and are present in a wide variety of different
muscle types (McDonald et al. 1994).
The Lymnaea LVA Ca21 current activated at potentials
between 275 and 265 mV, peaked between 250 and 240
mV, and reversed between 130 and 140 mV. This low threshold for activation is characteristic of LVA and T-type Ca21
currents in both invertebrates (Kits and Mansvelder 1996) and
vertebrates (Alvarez and Vassort 1992; Hagiwara et al. 1988;
McDonald et al. 1994). The current was fast to activate and at
potentials above 250 mV showed complete inactivation during
short (200 ms) voltage steps. The time constant for inactivation
appeared to be voltage dependent, which was again consistent
with data obtained from T-type currents in vertebrate atrial
(Bean 1989a), smooth (Akaike et al. 1989), and skeletal muscles (Cognard et al. 1986). The LVA current also showed
progressive steady-state inactivation as the holding potential
from which it was evoked was made more positive. The LVA
current was half inactivated at 258 mV and completely inactivated at holding potentials more positive than 240 mV.
These values were consistent with those obtained for T-type
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have previously been shown to be insensitive to concentrations
of dihydropyridines that produce a significant block of L-type
channels (Bean 1985; Hagiwara et al. 1988). Consistent with
this was our observation that nifedipine had no significant
effects on the amplitude of the LVA current (P . 0.05; Fig.
7A2). The I-V relationship of peak current evoked from the cell
recorded in Fig. 7A in the presence and absence of nifedipine
is illustrated in Fig. 7B. The figure clearly shows that nifedipine blocks a current that activates between 230 and 0 mV, the
voltage range where the HVA current is maximally active. The
lack of block at potentials more positive than 0 mV indicates
that the block may show some voltage dependence.
The HVA current in Lymnaea heart ventricle cells required
approximately a 10-fold higher concentration of nifedipine to
produce the same %block when compared with the L-type
current in another invertebrate muscle (ARC muscle) (Brezina
et al. 1994). This raised the possibility that the HVA current in
Lymnaea ventricle cells may be made up of more than one
current type. One possibility was that the HVA current we
were recording consisted of a mixture of N- and L-type currents. N-type channels like L-type are another example of
HVA currents. However, they can be specifically blocked by
v-conotoxin GVIA (Hille 1992). In four experiments of the
sort described above, application of 10 mM v-conotoxin GVIA
failed to cause any significant block of the HVA current. This
indicated that the HVA current in Lymnaea ventricle cells
appeared to consist of a homogenous population of L-type
channels that was relatively insensitive to nifedipine.
In contrast, T-type channels in muscles have been shown to
be sensitive to micromolar concentrations of Ni21 (Bonvallett
1987; Hagiwara et al. 1988). We therefore examined the ability
of Ni21 to selectively block the LVA channel. In Fig. 8A1
application of 100 mM Ni21 produced a significant block of the
LVA channel (38.9 6 2.07%; n 5 8; P , 0.01). At these
concentrations Ni21 did not affect the amplitude of the HVA
current (Fig. 8A2). An example of the current-voltage relationship of the same cell in the presence and absence of 100 mM
Ni21 is shown in Fig. 8B. In the presence of the Ni21 ions,
there is a marked reduction in the current flowing between 260
and 240 mV, where the LVA current is maximally active.
Increasing the concentration of Ni21 to 1 mM completely
CA21 CURRENTS IN THE LYMNAEA HEART
FIG. 9. Schematic representation of the voltage sensitivities of the voltagegated K1 and Ca21 currents and their relationship to the proposed voltage
operating range of Lymnaea heart ventricle cells. The operating range was
defined at the lower end as the potential (263 mV) that allowed spontaneous
action potential generation and the upper level by the most positive voltage
reached at the peak of the action potential (211 mV).
manipulations designed to alter the size of the current that
flowed through HVA channels (increasing the extracellular
concentration of divalent ions) had the appropriate effects on
the inactivation rates. Equally, the increased inactivation
observed with Ca21 as the permeant ion could be an artifact
due to residual unblocked Ca21-dependent K1 currents that
would not be activated in Ba21-containing solutions (Hermann
and Gorman 1979). Similar findings have been described in
other invertebrate preparations (Brezina et al. 1994). The Lymnaea HVA current also showed marked steady-state inactivation when the voltage from which the current was evoked was
made more positive. Typically the HVA current was half
inactivated at 248 mV and completely inactivated at holding
potentials more positive than 230 mV. Again these data are
consistent with those obtained in other invertebrate and vertebrate preparations (Brezina et al. 1994) (half inactivation 245
mV). Further confirmation that the HVA current we had characterized was a member of the L-type Ca21 current family was
its block by the dihydropyridine (DHP), nifedipine. The DHP
binding site has previously been shown to be a part of the
L-type Ca21 channel, and therefore the block of our HVA
current by nifedipine confirms that it is a member of the L-type
family of Ca21 channels.
What is the possible role of the two voltage-dependent Ca21
currents in regulating heart beat in the snail Lymnaea?
We have identified and characterized four major currents in
Lymnaea heart ventricle cells, two voltage-dependent K1 currents [IK(A) and IK(V)] (Yeoman and Benjamin 1999) and two
voltage-gated Ca21 currents (LVA and HVA) whose voltage
sensitivities are shown in Fig. 9. Yeoman and Benjamin (1999)
described the properties of small spikelike action potentials
that could be evoked in dissociated heart muscle cells following hyperpolarization of cells from their resting membrane
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on May 2, 2017
currents recorded from a variety of muscles types and neurons
(McDonald et al. 1994). However, direct comparisons were
extremely difficult as the majority of T-type currents were
extremely small and were therefore recorded using elevated,
rather than normal concentrations of the divalent ions.
An examination of the relative permeabilities of the LVA
channel to normal levels of Ca21 or Ba21 (3.5 mM) showed
that, in the majority of cells tested, LVA channels were more
permeable to Ca21 than Ba21 ions. The majority of T-type
channels in muscle cells appear to show little or no preference
for either Ca21 or Ba21 as a permeant ion (Alvarez and Vassort
1992; Bean 1985; Chesnoy-Marchais 1985; Coyne et al. 1987),
and therefore in this respect the LVA channel was different
from those previously described in the vertebrate literature.
Lymnaea ventricle LVA currents were also sensitive to block
by extracellular Ni21 ions (100 mM; '50% block). Sensitivity
to Ni21 ions is another characteristic of T-type calcium currents (Bonvallett 1987; Hagiwara et al. 1988). On average
vertebrate T-type currents are completely blocked by 40 mM
Ni21 (Hagiwara et al. 1988) and are therefore much more
sensitive than the Lymnaea current. However, the Ni21 sensitivity of the Lymnaea LVA current is comparable to other
invertebrate LVA currents, which were also relatively insensitive to Ni21 (Kiss and Osipenko 1991). These data therefore
provide convincing evidence that the LVA current in Lymnaea
ventricle cells is a member of the T-type family of Ca21
currents.
The HVA current that we have characterized has a number
of properties that resemble those of previously described Ltype Ca21 currents. With normal levels of extracellular Ca21
(3.5 mM) the activation threshold was ;30 mV more positive
than that for the LVA current (240 to 230 mV) and peaked at
potentials between 210 and 0 mV. This was comparable to
values in invertebrate neurons (Byerly and Hagiwara 1982;
Eckert and Ewald 1983) and vertebrate muscle and neurons
(reviewed by Bean 1989b). In extracellular solutions containing physiological levels of Ca21, the HVA current was fast to
activate and appeared to inactivate completely during standard
200-ms voltage steps. In equimolar Ba21 solutions, HVA currents were again fast to activate but exhibited much slower
rates of inactivation during standard 200-ms voltage steps.
Previous work on both HVA and L-type currents in other
systems has demonstrated similar differential effects of Ca21
and Ba21 ions on inactivation (Eckert and Chad 1984). This
change in the inactivation rate could be due to a number of
factors. First, under physiological conditions the inactivation of
the HVA and L-type currents has been shown to be strongly
dependent on the intracellular concentration of Ca21 ions and
only weakly voltage dependent (i.e., that portion of the inactivation remaining in currents recorded in Ba21-containing
solutions). In a given extracellular solution the rate of inactivation of the HVA currents recorded at a variety of different
membrane potentials showed no correlation to the voltage at
which the cell was being held but was more closely related to
the size of the evoked current. Thus at physiological concentrations of extracellular ions, this would be related to the
amount of Ca21 entering the cell. However, current-dependent
inactivation is also seen with Ba21 as the charge carrier,
although the responses of the cell are greatly attenuated, indicating that the channels are more sensitive to Ca21. This
conclusion was further substantiated by the observation that
2437
2438
M. S. YEOMAN, B. L. BREZDEN, AND P. R. BENJAMIN
basal relay neurons of vertebrates (Huguenard and Prince
1994) and invertebrates (Adams and Benson 1985). We are
currently performing similar experiments in snail muscle cells
to determine the role of the LVA current.
Our observation that sustained LVA currents could occur at
relatively depolarized potentials (250 mV) in a few cells was
an interesting one. Previous work has shown that LVA currents
present in neurons have extremely heterogeneous kinetics.
LVA currents have been typically classified into two classes
according to their rates of inactivation. Thus currents are either
fast inactivating transient currents or slow inactivating sustained currents (Adams and Levitan 1985; Eckert and Lux
1975). A number of these sustained LVA currents have been
shown to be sensitive to DHP blockers and show a marked
selectivity for Ba21 over Ca21 (Eckert and Lux 1976), suggesting that they are atypical L-type channels. In dissociated
Lymnaea ventricle cells their appearance was extremely infrequent and as such precluded a study of their pharmacology and
ion selectivity. However, their presence is interesting because
they may contribute significantly to the pacemaker potential in
the cells where they occur.
How might the voltage-gated currents present in Lymnaea
ventricle cells contribute to action potential generation?
In a myogenic tissue such as the Lymnaea heart, action
potential generation is of fundamental importance because it
provides a mechanism that allows each muscle fiber to depolarize in the absence of neuronal input. In all excitable cells,
whether an action potential is generated or not is determined by
the balance of inward and outward currents flowing across the
cell membrane. In some molluscan and vertebrate smooth
muscles the ability of the cells to fire action potentials is
prevented by the presence of large fast outward currents whose
voltage sensitivity and kinetics are such that they are capable of
counteracting the depolarizing drive due to the cationic inward
currents, thereby blocking action potential generation. We have
demonstrated that all dissociated ventricular muscle cells can
generate action potential–like events, whose properties were a
hybrid of those recorded from pacemaker cells in different
regions of the mammalian heart (Irisawa et al. 1993). Two
observations probably underlie our ability to record spikelike
action potentials. The first is the relatively negative activation
threshold of the LVA (272 mV) currents compared with those
for the two voltage-dependent K1 currents (Yeoman and Benjamin 1999). This would allow a significant depolarization
(fast spike) via the influx of Ca21 before it could be checked by
a significant activation of the K1 currents (Fig. 9). The second
is the faster activation rates of the Ca21 currents compared
with the two K1 currents that would also favor spiking.
Role of extracellular Ca21 in excitation-contraction
coupling?
In experiments described in Yeoman and Benjamin (1999)
we determined that the threshold potential for activation of
contractions in Lymnaea ventricle cells was 250 mV, with
contractions increasing in strength as the potential of the cell
was increased to 220 mV. Based on our recordings of ventricular action potentials, this probably represents the upper
limit of the voltages reached by the cells under normal phys-
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on May 2, 2017
potential (255 mV) to potentials around 263 mV (the lower
operating range of the muscle cell, Fig. 9). Spikes were completely inhibited following superfusion of the cells with high
concentrations (3.5 mM) of Cd21. Based on this data and the
voltage sensitivity of the Ca21 currents obtained in this paper,
we propose that the rising phase of ventricular action potentials
is due to the sequential opening of LVA followed by HVA
channels. The low activation threshold of the LVA current
(275 to 265 mV) and the relatively depolarized resting membrane potential of these cells (255 mV) makes it ideally suited
to provide the initial depolarizing drive to the cell. As mentioned in the previous paper (Yeoman and Benjamin 1999) the
high-input resistance of these cells '900 MV means that a
small current flow across the membrane will lead to a substantial depolarization of the cell. This depolarization would then
activate the HVA current, which in turn would lead to a further
depolarization of the cell (Fig. 9). Similar roles for T- and
L-type calcium currents have been proposed for mammalian
sinoatrial node cells (Hagiwara et al. 1988). The peak of the
action potential (upper operating range of the muscle cell, Fig.
9) would occur when there was no net current flow across the
muscle cell. This would be determined by the inactivation of
the LVA and HVA currents and the relatively slow activation
of IK(A) and IK(V). Further inactivation of the LVA and HVA
currents and the continuing development of the K1 currents
would lead to the repolarization of the muscle cell (Fig. 9).
This is consistent with the observation that the falling phase of
the action potential was sensitive to TEA, a K1 channel
blocker (Yeoman and Benjamin 1999).
The activity of the LVA current is typically seen as a fast
activating, rapidly decaying current near 255 mV, the resting
membrane potential of the cell. However, at more negative
potentials (270 to 260 mV), evoked currents are long-lasting
and might therefore be able to provide a sustained depolarizing
drive that could underlie repetitive spiking in these muscle
cells as well as initiating individual beats. This observation
may help explain why we found it necessary to hyperpolarize
dissociated muscle cells from their resting membrane potential
(255 mV) to potentials near 263 mV, to obtain repetitive
spiking in current-clamp experiments (Yeoman and Benjamin
1999). Hyperpolarization would also make more of the LVA
current available for activation as well as helping to inactivate
any residual K1 currents [IK(V)] thus causing the net current
flow to become inward (pacemaker current). It was possible
that by hyperpolarizing the muscle cells we were activating a
different type of current such as Ih/If (hyperpolarization-activated current) that has been shown to drive pacemaker activity
in the sinoatrial node (Difrancesco 1986). However, in a detailed analysis of .20 cells, we failed to show the presence of
any hyperpolarization-activated currents. Thus in the absence
of any other current, it appeared that the low-threshold voltage
for the LVA channel made it a suitable candidate for providing
the main current flow necessary for driving repetitive action
potentials in these ventricle cells. Indeed, selective blocking of
the LVA current by Ni21 has been shown to cause a marked
slowing of repetitive action potentials in rabbit sinoatrial node
cells, indicating that this current does have a significant role in
pacemaking in vertebrate heart cells (Hagiwara et al. 1988).
Similarly, T-type currents have also been shown to underlie
pacemaking and rhythmic activity in a number of neuronal
structures such as the thalamic reticular nucleus and ventro-
CA21 CURRENTS IN THE LYMNAEA HEART
M. S. Yeoman and P. R. Benjamin received financial support from United
Kingdom Biotechnology and Biological Sciences Research Council Grant
IR3521-1. B. L. Brezden was supported by a grant from the BBSRC Travelling
Fellowship.
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.
Received 1 April 1999; accepted in final form July 1999.
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