1Sussex Centre for Neuroscience,
Yeoman, M. S.,
B. L. Brezden, and
P. R. Benjamin.
LVA and HVA Ca2+ Currents in Ventricular Muscle Cells
of the Lymnaea Heart.
J. Neurophysiol. 82: 2428-2440, 1999.
The single-electrode
voltage-clamp technique was used to characterize voltage-gated
Ca2+ currents in dissociated Lymnaea heart
ventricular cells. In the presence of 30 mM tetraethylammonium (TEA),
two distinct Ca2+ currents could be identified. The first
current activated between In the previous paper Yeoman and Benjamin
(1999) Voltage-gated calcium channels are a common feature of all invertebrate
and vertebrate muscles (McDonald et al. 1994 In vertebrates, the situation is more complex. Cardiac, skeletal, and
smooth muscle all possess both T- and L-type Ca2+
currents (McDonald et al. 1994 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 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 Solutions
To isolate either Ca2+ or
Ba2+ currents, the cells were perfused with 30 mM
tetraethylammonium (TEA). This was sufficient to block all the
K+ currents that normally masked the developing
inward currents (Yeoman and Benjamin 1999 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 Statistical analysis
All values presented in this paper represent means ± SE.
Unless stated otherwise in the text, tests for significance have been
performed using unpaired t-tests assuming unequal variances (Excel 97).
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
ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
70 and
60 mV. It was fully available for
activation at potentials more negative than
80 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
Ca2+ compared with Ba2+, although changing the
permeant ion had no observable effect on the kinetics of the evoked
currents. The current was blocked by Co2+ and
La3+ (1 mM) but was particularly sensitive to
Ni2+ ions (
50% block with 100 µM Ni2+)
and insensitive to low doses of the dihydropyridine Ca2+
channel antagonist, nifedipine. All these properties classify this
current as a member of the low-voltage-activated (LVA) T-type family
of Ca2+ currents. The activation threshold of the current
(
70 mV) suggests that it has a role in pacemaking and action
potential generation. Muscle contractions were first seen at
50 mV,
indicating that this current might supply some of the Ca2+
necessary for excitation-contraction coupling. The second, a high-voltage-activated (HVA) current, activated at potentials between
40 and
30 mV and was fully available for activation at potentials
more negative than
60 mV. This current was also fast to activate and
with Ca2+ as the permeant ion, inactivated completely
during the 200-ms voltage step. Substitution of Ba2+ for
Ca2+ 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 Co2+ and La3+ ions (1 mM) but was sensitive to micromolar concentrations of nifedipine
(
50% block 10 µM nifedipine) that were ineffective at blocking
the LVA current. These properties characterize this current as a L-type
Ca2+ 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 Ca2+ necessary for
excitation-contraction coupling. These data provide the first detailed
description of the voltage-dependent Ca2+ 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.
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ABSTRACT
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DISCUSSION
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described two voltage-gated K+
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 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.
). A variety of different Ca2+ currents have been
characterized (Kits and Mansvelder 1996
). In mollusks,
voltage-gated Ca2+ 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
40 mV) have been termed low-voltage activated
(LVA), whereas those that are activated at potentials more positive to
40 mV have been termed high-voltage activated (HVA). The majority of invertebrate muscles studied to date contain only the HVA type of
voltage-activated Ca2+ current. These HVA
currents vary slightly in their voltage sensitivity, but all could be
blocked with nifedipine. Substitution of Ba2+ for
Ca2+ 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
40 mV, is
fast to activate, and has rapid inactivation kinetics. These properties
are characteristic of vertebrate T-type Ca2+
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
Ca2+ current is the L-type current.
). Of the remaining
classes of Ca2+ 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 invertebrate hearts, very
little is known about the role of Ca2+ 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 Ca2+ (Hagiwara
and Byerly 1981
), but there are no studies where the currents responsible for action potential generation have been characterized. Two types of Ca2+ channel have previously
been characterized in dissociated 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
).
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). Ca2+ and
Ba2+ 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 Ca2+/Ba2+ currents
by substituting the
Ca2+/Ba2+ ions in the
saline with Co2+ ions. The remaining current was
classified as the leakage current. Unless otherwise stated, we have
used the latter pharmacological method.
). These cells
do not contain voltage-gated Na+ currents (see
RESULTS), so there was no need to include the
Na+ channel blocker, tetrodotoxin (TTX), in the
bathing solution. The standard solutions used were
Ca2+/, Ba2+/, and
Co2+/TEA and contained the following
concentrations of ions (in mM): 20 Na+, 30 TEA,
1.7 K+, 3.5 Ca2+ or
Ba2+ or Co2+, 2 Mg2+, and 10 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic
acid) (HEPES) with the pH adjusted to 7.9 using NaOH. Occasionally, when the Ba2+ currents were difficult to resolve,
the concentration of Ba2+ in the extracellular
saline was increased to 10 mM. In these instances the concentration of
Na+ was reduced to compensate for the increased
concentration of Ba2+ ions.
-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.
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ABSTRACT
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70 mV but became masked by the more slowly
developing K+ currents at potentials more
positive than
40 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
90
mV and then stepped briefly to
120 mV before being ramped to +40 mV
over a 1.25-s period. Although the first phase of inward rectification
is clear (I1;
62 mV) the second
phase (I2) was almost completely
masked by the presence of the voltage-gated K+
currents that began to activate between
50 and
40 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
67 mV and peaked at
60 mV and was provisionally
classified as the low-voltage-activated (LVA) current. The second
phase activated at around
45 mV and peaked at
25 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 +10 mV),
indicating that not all the outward currents were blocked by TEA.
View larger version (27K):
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Fig. 1.
Isolation of voltage-dependent inward currents. A1:
voltage ramp protocols ( 120 to +40 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 K+ currents allowing the 2 inward
currents to be more clearly visualized. A3: substitution
of the Ca2+ ions in the bathing solution by
Ba2+ ions blocked a proportion of the residual
K+ currents and further enhanced the amplitude of
I2. A4: increasing the
concentration of Ba2+ ions to 10 mM blocked the majority of
the outward K+ currents and further enhanced the amplitude
of I2. B: 2 peaks of inward
current were again visible when all the extracellular Na+
ions had been replaced by Ba2+ ions. This indicated that
these 2 inward currents are divalent cationic currents. Recordings in
A and B are from different cells.
To further characterize these inward currents,
Ba2+ ions were substituted for
Ca2+ ions in the bathing solution. This had a
number of advantages. In the absence of Ca2+
ions, Ba2+ has been shown to enhance the
amplitude of a number of types of voltage-gated
Ca2+ currents (Bean 1989a;
Tsien et al. 1988
), as well as being able to
block certain K+ currents (Armstrong and Taylor
1980
; Hille 1992
; Latorre and Miller 1983
). Its substitution for Ca2+ in the
extracellular solution would help block any residual K+
currents not blocked by TEA and would also prevent the activation of
Ca2+-dependent K+ currents also known to be
present in these cells. All of these effects would allow the inward
Ca2+ 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 Ba2+ 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 Ca2+ with Ba2+ 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
10 and 0 mV in 3.5 mM Ca2+ to
between +20 and +30 mV in 3.5 mM Ba2+). Increasing the
concentration of Ba2+ 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 Ba2+ was substituted for Ca2+ 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 Ba2+
ions suggested that the current was due to the flow of divalent ions
and that Lymnaea ventricle cells contained two distinct
voltage-gated Ca2+ 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 Na+ ions. To
test for this, we replaced all the NaCl in the extracellular solution
with BaCl2 giving a solution that contained 53.5 mM
Ba2+/30 mM TEA. Current records from cells ramped from
120 to +40 mV in this saline showed the same two currents (Fig.
1B), suggesting that Na+ ions were not a
significant charge carrier through these channels. The amplitude of
both the currents was greater than that seen using 10 mM
Ba2+. This was presumably due to the increase in the
driving force for Ba2+ entry and indicated that these
currents were probably carried by Ca2+ ions in the normal
physiological saline. Several other lines of evidence support the
hypothesis that both the LVA and HVA currents are Ca2+
currents. First, in five experiments, application of TTX
(10
5 M) was unable to block any component of the inward
currents (data not shown). Second, in five experiments where all the
K+ and Ca2+ currents were blocked
pharmacologically by a mixture of TEA and Co2+, 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 Na+ channels using the cell-attached patch-clamp 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 Ca2+ 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 = 32 batches), current recordings had to be made using 10 mM Ba2+ as the permeant ion to visualize the currents. In the remaining 40% of the batches (n = 18 batches), the inward currents were large enough to be recorded in normal Ca2+ or Ba2+ salines.
In the majority of fibers recorded (85%; n = 60) two distinct peaks of inward current could be seen (Fig. 2A3). However, a significant proportion of the cells (15%, n = 10) appeared to contain either the LVA current (Fig. 2A1) or the HVA current (Fig. 2A2).
|
Voltage dependence of LVA and HVA Ba2+ currents
Figure 2, B1-B3, illustrates currents evoked by short
(150 ms) voltage steps from 90 mV to potentials between
60 and +30 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
90 mV in a muscle cell that contained only the LVA
Ba2+ current. The LVA current began to activate
between
60 and
50 mV (
58 ± 1 mV, mean ± SE,
n = 10), peaked at potentials between
40 and
30 mV
and then decreased in amplitude at increasingly positive potentials,
giving an extrapolated reversal potential of around +40 mV (Fig.
2C1). The HVA Ba2+ current did not
begin to activate until
40 or
30 mV (
34 ± 2 mV,
n = 8) and peaked at potentials between
10 and 0 mV.
It then declined in amplitude, yielding an extrapolated reversal potential of +40 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
10 and 0 mV (the peak of
the HVA current). The current then declined to give a predicted
reversal potential of around +40 mV (Fig. 2C3).
|
Both inward currents had reversal potentials of around +40 mV. This was
far from the theoretical reversal potential for
Ba2+ (+175 mV). Similar anomalous reversal
potentials for Ba2+ 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
Ba2+ but instead could allow a significant efflux
of K+ at depolarized potentials. Permeability to
K+ would then tend to shift the reversal
potential for the current away from
EBa toward
Ek.
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 voltage-gated Ba2+ 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 ± 8.7 ms with voltage steps to 60 mV (threshold potential) to 17.1 ± 1.5 ms with steps to
40 mV, which activated the peak current
(n = 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 ± 6.7 ms with voltage steps to
40 mV (threshold potential)
to 29 ± 2.2 ms with voltage steps to
10 mV, which activated the peak HVA current (n = 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
50 mV), the majority of the LVA current (>95%) inactivated completely during a 150-ms voltage step (Fig. 2B1). However,
at potentials close to the activation threshold (
70 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
60 and
20 mV, indicating a strong voltage dependence (
60
mV;
= 59.4 ± 7.6 ms, n = 6;
20 mV,
= 15.6 ± 1.6 ms, n = 6; Fig. 2B1). However, at potentials more positive than
20 mV
(
20 to +30 mV), the rate of inactivation began to slow (e.g., 0 mV,
= 28.9 ± 6.3 ms; n = 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
10 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 Ba2+
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 1 was
present in the highest proportion (
100 times more than
2) 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 (
20 mV),
inactivation was weak (
1 = 320 ± 46.3 ms,
2 = 15.6 ± 1.6 ms; n = 6). The rate of inactivation increased with more positive voltage
steps and became maximal around the peak of the HVA current (0 mV;
1 = 116 ± 21.6 ms,
2 = 16.5 ± 1.6 ms; n = 6). At potentials more positive than +20 mV, inactivation of the HVA
current again became weaker (
1 = 280 ± 36.3 ms,
2 = 16.1 ± 1.2 ms;
n = 6). Using an ANOVA followed by a paired
t-test, the values for
1 recorded at
20 and +20 mV were found to be significantly different to those at
0 mV. The inactivation of the Ba2+ 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
Ba2+ current was recorded in salines where the
extracellular concentration of Ba2+ 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
+10 mV from a holding potential of 90 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
1 was the only time constant to be significantly altered.
1 for currents recorded in 3.5 mM
Ba2+ was 235 ± 19.5 ms (n = 6). This value decreased to 130.3 ± 4.7 ms for currents recorded
in 10 mM Ba2+ (paired t-test,
P < 0.001). In contrast the rate of inactivation of
the LVA Ba2+ 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.
Ca2+ and Ba2+ 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 Ca2+ currents,
respectively. Further evidence in support of this classification would
be obtained by examining the relative permeabilites of the two currents
to Ca2+ or Ba2+ ions.
T-type channels have previously been shown to have no preference for
Ca2+ ions over Ba2+ 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
Ba2+ ions much better than
Ca2+ (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 Ca2+ or Ba2+. 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 90 to
50 mV.
In 3.5 mM Ca2+ 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 Ca2+ 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 Ba2+ for
Ca2+ caused a marked reduction in the amplitude
of the LVA current in the majority of fibers tested 50.6 ± 10.6%
(mean ± SE; in 12 of the 13 fibers tested, Fig. 4, A1
and A3). This demonstrated, that on average, LVA channels
were more permeable to Ca2+ than
Ba2+ ions. In one cell only, LVA currents were
recorded that showed equal permeability to Ca2+
and Ba2+ (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 90 to
10 mV.
The HVA Ba2+ 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 Ca2+ currents recorded from the same
cells, HVA Ba2+ currents were noticeably larger
(48.6 ± 6.7% mean ± SE; 10 of 13 cells tested). This
indicated that these channels conduct Ba2+ better
than Ca2+. In the other three cells tested, one
showed no increase, whereas the others showed decreases of 156 and
242%, respectively. HVA Ca2+ currents like the
Ba2+ currents were fast to activate but showed
complete inactivation during the 200-ms voltage step. The inactivation
rates of the HVA Ca2+ currents were fitted best
with a double exponential and were therefore similar to the HVA
Ba2+ currents described earlier. However, unlike
the HVA Ba2+ currents the proportions of the two
components comprising the inactivation phase of the HVA
Ca2+ currents are approximately equal. Changing
the permeant ion caused significant changes in the first time constant
(
1) from 81.6 ± 11.3 ms
(Ca2+) to 364.2 ± 46.4 ms
(Ba2+; n = 8, P < 0.001), whereas there were no changes in
2 = 16.6 ± 1.5 ms (Ca2+) to 15.5 ± 1.8 ms (Ba2+). The faster inactivation rate seen in
cells bathed in the Ca2+-containing saline could
be due to the activation of a Ca2+-dependent
K+ 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 Ca2+ and Ba2+ salines are consistent with these two currents being members of the T- and L-type calcium channel families, respectively.
Comparison of the voltage dependence of LVA and HVA currents using normal concentrations (3.5 mM) of Ca2+ or Ba2+ as the permeant ions
It was important to generate I-V curves for the
HVA and LVA currents at concentrations of Ca2+
resembling those present in normal blood (3.5 mM) and with similar concentrations of Ba2+ 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
Ca2+ (Fig. 5A) or
Ba2+ (Fig. 5B) as the permeant ion. In
Fig. 5A1 Ca2+ currents were evoked by
holding the cell at
90 mV and stepping it to a range of potentials
between
80 and +40 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
75 and
65 mV and showed an
initial peak around
50 mV (LVA current) and a second larger peak of
inward current between
10 and 0 mV (HVA current). By holding the cell
at more depolarized potentials (
50 mV; Fig. 5A2), it was
possible to inactivate all the LVA current so that the same series of
voltage steps (
80 to +40 mV) now evoked an almost pure HVA current.
This current activated around
40 mV (
42 ± 2 mV;
n = 8) and peaked between
10 and 0 mV before
declining in amplitude and yielding an extrapolated reversal potential
of around +50 mV (
, 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
75 and
65 mV
(
72.0 ± 12 mV; n = 10) and peaked at between
50 and
40 mV before declining in amplitude and showing an apparent
reversal potential around +50 mV. The small inflection seen at
20 mV
probably represented a small proportion of the HVA current that is also blocked by holding the cell at
50 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
Ba2+ saline. Currents were again evoked from
holding potentials of
90 mV (Fig. 5B1) or
50 mV (Fig.
5B2) with LVA currents again isolated by subtracting
currents recorded from a holding potential of
50 mV from those evoked
from a holding potential of
90 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
Ba2+ were extremely small and difficult to
resolve due to the relatively low permeability of the channels to
Ba2+ (see Fig. 4). At potentials more positive
than
30 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
50 mV (
, Fig.
5B4). Because of their small size we were unable to obtain
reliable data for the voltage dependence of the LVA
Ba2+ current recorded under these conditions. HVA
currents on the other hand activated between
50 and
40 mV, peaked
at potentials between
20 and
10 mV, and reversed around +30 mV.
This negative shift in the I-V curve for the HVA
Ba2+ current compared with the HVA
Ca2+ current is probably due to the fact that
Ca2+ is a better screen of the surface membrane
charge than equimolar concentrations of Ba2+
(Hille 1992
).
|
Steady-state inactivation of HVA and LVA currents
The resting membrane potential of Lymnaea heart
ventricle cells was previously shown to be about 55 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
90 mV there was a significant activation of this current between
70 and
60 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
110 and
20 mV) and stepping to
50 mV to activate the LVA current. The
experiments used 3.5 mM Ca2+ as the permeant ion. The
amplitude of LVA currents was unaffected by holding potentials more
negative than
70 mV. As the holding potential was made more positive
than
70 mV, the amplitude of the evoked current declined and was
abolished completely by holding potentials more positive than
40 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
58 mV and had a slope factor of 4.1 ± 0.3 mV. Due to the steep slope of the inactivation
curve, only about one-third of the LVA current would be available at
55 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 Ba2+ as the
permeant ion and were activated by short 150-ms voltage steps from a
variety of holding potentials (110 to
20 mV) to 0 mV. We chose not
to use Ca2+ as the permeant ion because of the risk of
contaminating the evoked Ba2+ current with unblocked
K+ 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
60 mV. At potentials
more positive than this, the current again declined in amplitude and was abolished completely by holding potentials of around
20 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
48 mV and had a slope
factor of 4.1 ± 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 (
40 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 La3+,
Gd3+, and Co2+ produced
100% block of both LVA and HVA currents at 3.5 mM, whereas a similar
block could be obtained using 1 mM Cd2+ (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 Ca2+ ions. Here we were mainly
interested in characterizing compounds that were capable of selectively
blocking one or other of the two Ca2+ 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 µM nifedipine were examined. In this
experiment, currents were recorded in fibers bathed in the elevated
Ba2+ solution (10 mM). LVA and HVA currents were
evoked by holding the fiber at
90 mV and stepping it to
40 and 0 mV, respectively (these were the peak potentials for these currents in
the elevated Ba2+ saline). Figure 7A1
shows a record of an HVA current evoked in normal saline (control) or
in the presence of 10 µM 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 ± 5.6%; n = 8; P < 0.01) compared with the instantaneous block (32.4 ± 4.74%; n = 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 +50 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 µM nifedipine using the
standard protocol (Fig. 7A) versus a protocol where the cell
was prestepped to +50 mV. Prepulsing the cell to +50 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 ± 7.8% at the end of the voltage pulse during the standard protocol
compared with the 86.2 ± 9.3% (mean ± SE) seen with the
prepulse. T-type calcium channels, on the other hand 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
30 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
-conotoxin GVIA (Hille 1992
). In four
experiments of the sort described above, application of 10 µM
-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 Ni2+
(Bonvallett 1987; Hagiwara et al. 1988
).
We therefore examined the ability of Ni2+ to
selectively block the LVA channel. In Fig.
8A1 application of 100 µM
Ni2+ produced a significant block of the LVA
channel (38.9 ± 2.07%; n = 8; P < 0.01). At these concentrations Ni2+ 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 µM Ni2+ is shown in
Fig. 8B. In the presence of the Ni2+
ions, there is a marked reduction in the current flowing between
60
and
40 mV, where the LVA current is maximally active. Increasing the
concentration of Ni2+ to 1 mM completely 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).
|
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DISCUSSION |
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Lymnaea heart ventricle cells contain two types of voltage-gated Ca2+ 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 voltage-gated
K+ 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 Ca2+ 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 Ca2+ current activated
at potentials between 75 and
65 mV, peaked between
50 and
40
mV, and reversed between +30 and +40 mV. This low threshold for
activation is characteristic of LVA and T-type
Ca2+ 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
50 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
58 mV and completely inactivated at
holding potentials more positive than
40 mV. These values were
consistent with those obtained for T-type 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 Ca2+ or Ba2+ (3.5 mM) showed
that, in the majority of cells tested, LVA channels were more permeable
to Ca2+ than Ba2+ ions. The majority of T-type
channels in muscle cells appear to show little or no preference for
either Ca2+ or Ba2+ 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 Ni2+ ions (100 µM;
50% block). Sensitivity to Ni2+ 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 µM
Ni2+ (Hagiwara et al. 1988
) and are
therefore much more sensitive than the Lymnaea current.
However, the Ni2+ sensitivity of the Lymnaea
LVA current is comparable to other invertebrate LVA currents, which
were also relatively insensitive to Ni2+ (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 Ca2+ currents.
The HVA current that we have characterized has a number of properties
that resemble those of previously described L-type Ca2+
currents. With normal levels of extracellular Ca2+ (3.5 mM)
the activation threshold was ~30 mV more positive than that for the
LVA current (40 to
30 mV) and peaked at potentials between
10 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 Ca2+, the HVA current was fast to
activate and appeared to inactivate completely during standard 200-ms
voltage steps. In equimolar Ba2+ 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 Ca2+ and Ba2+ 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 Ca2+ ions and only weakly voltage
dependent (i.e., that portion of the inactivation remaining in currents
recorded in Ba2+-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 Ca2+ entering the cell. However,
current-dependent inactivation is also seen with Ba2+ as
the charge carrier, although the responses of the cell are greatly
attenuated, indicating that the channels are more sensitive to
Ca2+. This conclusion was further substantiated by the
observation that 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 Ca2+ as the permeant ion could be an artifact due to
residual unblocked Ca2+-dependent K+ currents
that would not be activated in Ba2+-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
48 mV and completely inactivated at holding
potentials more positive than
30 mV. Again these data are consistent
with those obtained in other invertebrate and vertebrate preparations
(Brezina et al. 1994
) (half inactivation
45 mV).
Further confirmation that the HVA current we had characterized was a
member of the L-type Ca2+ 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 Ca2+
channel, and therefore the block of our HVA current by nifedipine confirms that it is a member of the L-type family of Ca2+ channels.
What is the possible role of the two voltage-dependent Ca2+ 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
K+ currents
[IK(A) and
IK(V)] (Yeoman and Benjamin
1999) and two voltage-gated Ca2+
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
potential (
55 mV) to potentials around
63 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
Cd2+. Based on this data and the voltage
sensitivity of the Ca2+ 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 (
75 to
65
mV) and the relatively depolarized resting membrane potential of these
cells (
55 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 M
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
K+ 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
K+ channel blocker (Yeoman and Benjamin
1999
).
|
The activity of the LVA current is typically seen as a fast activating,
rapidly decaying current near 55 mV, the resting membrane potential
of the cell. However, at more negative potentials (
70 to
60 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 (
55
mV) to potentials near
63 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
K+ 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 Ni2+ 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 ventrobasal 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 (50 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 Ba2+ over
Ca2+ (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 (
72 mV)
currents compared with those for the two voltage-dependent
K+ currents (Yeoman and Benjamin
1999
). This would allow a significant depolarization (fast
spike) via the influx of Ca2+ before it could be
checked by a significant activation of the K+
currents (Fig. 9). The second is the faster activation rates of the
Ca2+ currents compared with the two
K+ currents that would also favor spiking.
Role of extracellular Ca2+ 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
50 mV, with contractions increasing in strength as the potential of
the cell was increased to
20 mV. Based on our recordings of
ventricular action potentials, this probably represents the upper limit
of the voltages reached by the cells under normal physiological
conditions. Our observation that contractions start at potentials as
negative as
50 mV is indicative that the first source of
Ca2+ is via an influx through LVA
Ca2+ channels, which are the only
Ca2+ channels active at this potential (Fig. 9).
Significant increases in intracellular Ca2+
levels due to influx via LVA channels have been described in a variety
of different types of neurons (Markram and Sakmann
1994
; Wang et al. 1997
). However, recordable
changes in intracellular Ca2+ levels due to influx via LVA
channels in muscle are unusual with HVA channels providing the main
entry pathway for this ion (Benham and Tsien 1988
;
Nelson et al. 1988
). In the sinoatrial node the maximum
currents that flow through T-type (LVA) channels are relatively small
compared with those that flow through HVA channels (Hagiwara et
al. 1988
). In Lymnaea the situation appears to
be different. Here the magnitude of the LVA current appears remarkably
similar to that of the HVA current, although some caution should be
taken when interpreting these data because it is not clear how the
amplitude or the inactivation rate of the HVA current has been
contaminated by the possible presence of unblocked K+
currents. Their presence would tend to reduce the peak amplitude and
increase the apparent rate of inactivation. Despite this, we can see
that there is a significant current flow through the LVA channels that
would act to increase intracellular Ca2+ levels. Further
depolarization of the muscle cell to potentials more positive than
50
mV would tend to activate HVA currents allowing these channels to
contribute to the influx of Ca2+ (Fig. 9). The relative
contributions of the LVA and HVA currents to elevating intracellular
Ca2+ levels are at present unclear. However, current
investigations involving selectively blocking of one or the other of
the two Ca2+ currents should provide us with a clearer idea
of the relative roles of these two currents.
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ACKNOWLEDGMENTS |
---|
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.
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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 July 1999.
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REFERENCES |
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