From the * Department of Biology, University of Konstanz, D-78434 Konstanz, Germany; and Stazione Zoologica `Anton Dohrn,'
Villa Comunale, I-80121 Naples, Italy
Single-channel currents through calcium channels in muscle of a marine crustacean, the isopod Idotea baltica, were investigated in cell-attached patches. Inward barium currents were strongly voltage-dependent, and the channels were closed at the cell's resting membrane potential. The open probability (Po) increased e-fold for an 8.2 mV (±2.4, n = 13) depolarization. Channel openings were mainly brief (<0.3 ms) and evenly distributed throughout 100-ms pulses. Averaged, quasimacroscopic currents showed fast activation and deactivation and did not inactivate during 100-ms test pulses. Similarly, channel activity persisted at steadily depolarized holding potentials. With 200 mM Ba2+ as charge carrier, the average slope conductance from the unitary currents between +30 and +80 mV, was 20 pS (±2.6, n = 12). The proportion of long openings, which were very infrequent under control conditions, was greatly increased by preincubation of the muscle fibers with the calcium channel agonist, the dihydropyridine Bay K8644 (10-100 µM). Properties of these currents resemble those through the L-type calcium channels of mammalian nerve, smooth muscle, and cardiac muscle cells.
Key words: single channel; arthropodThe action potential in crustacean muscle fibers, originally shown by Fatt and Katz (1953) to be sodium independent, was later demonstrated to be carried by calcium ions (Fatt and Ginsborg, 1958
; Hagiwara and
Naka, 1964
; Hencek and Zachar, 1977
). Subsequently, it became clear that Ca action potentials are a general
feature of arthropod muscle (adult insect muscle: Washio,
1972
; larval insect muscle: Deitmer and Rathmayer,
1976
; scorpion muscle: Gilly and Scheuer, 1984
). Much
as in vertebrate cardiac muscle, the voltage-dependent
Ca2+ influx across the sarcolemmal membrane is necessary to elicit contractions in muscles of crustaceans (Zacharová and Zachar, 1967
; Gainer, 1968
; Hagiwara et
al., 1968
; Hidalgo et al., 1979
; Mounier and Goblet,
1987
) and other arthropods (Gilly and Scheuer, 1984
).
The influx of Ca2+ through the surface Ca channels
alone, however, is insufficient for excitation-contraction coupling (Mounier and Goblet, 1987
). As in vertebrate cardiac muscle, Ca2+ for the contraction of crustacean muscle comes primarily from a calcium-induced
Ca2+ release (CICR) (Fabiato, 1985
; Goblet and Mounier, 1986
; Lea and Ashley, 1989
; Györke and Palade,
1992
).
In contrast to vertebrate cardiac muscle, smooth
muscle and developing skeletal muscle, where the underlying single-channel Ca currents have been identified and characterized in great detail, crustacean muscle or, for that matter, arthropod muscle
is poorly
characterized with regard to the type and properties of single-channel Ca currents. Exceptions are a study on
single-channel Ca currents in cultured embryonic Drosophila skeletal muscle fibers (Leung and Byerly, 1991
),
and a report on a dihydropyridine-sensitive Ca channel
isolated from T-tubules of crayfish skeletal muscle and
incorporated into lipid bilayers (Hurnák et al., 1990
).
Also in crayfish skeletal muscle, the modulation of Ca
channels in the sarcolemmal membrane by the neuropeptide proctolin was described (Bishop et al., 1991
).
The fundamental importance of Ca channels for calcium-induced Ca2+ release in crustacean muscle and the channel's function as a possible target for modulation of contractions by peptides and biogenic amines is in contrast to the scarcity of information on the single-channel's properties. In the present study, we characterize single-channel Ba currents in the sarcolemmal membrane of a crustacean muscle and show that they resemble Ca channels in vertebrate cardiac muscle and other vertebrate L-type channels.
Preparation
Fast contracting fibers from abdominal extensor muscles of the
marine isopod Idotea baltica (Crustacea, Isopoda) are particularly well suited for single-channel recordings because they are largely free of connective tissue and accessible for patch-clamp recordings after modest enzymatic treatment. A detailed description of
the preparation and the arrangement of the muscles were published previously (Erxleben et al., 1995). Patch-clamp recordings
were performed in a small bath (0.5 ml vol) mounted on the
stage of an inverted microscope (ZEISS IM, Oberkochen, Germany) at 400× magnification. Experiments were done at room
temperature (20-25°C).
Electrophysiological Techniques and Data Analysis
Standard electrophysiological techniques were used for potential
recording and current injection under current-clamp conditions (Erxleben et al., 1995). For two-electrode voltage-clamp experiments, an Axoclamp 2B (Axon Instruments Inc., Foster City, CA)
was used. These experiments were performed on fibers of the last
abdominal segment which are compact (270-450 µm long with a
diameter of 50-80 µm). Injection of current in the middle of the
fibers and recording at several points over the length of the fiber
showed that they are isopotential and thus suitable for two-electrode voltage-clamp. For isolation of Ca or Ba currents and suppression of K currents, a solution (TEA solution) consisting of
160 mM tetraethyl-ammonium, 2 mM 4-aminopyridine (4-AP),
320 mM NaCl, 8 mM KCl, 20 mM HEPES, pH 7.4, was used with
either 10 mM CaCl2 or BaCl2. In addition, the recording and current electrodes were filled with 3 M CsCl to suppress K currents.
Voltage-activated Ca or Ba currents were separated from linear
ionic and capacitive currents using a P/4 subtraction protocol.
Patch electrodes were fabricated from borosilicate glass (CLARK Electromedical Instruments, Reading, UK) coated with Sylgard 184 elastomer (Dow Corning Corp., Indianapolis, IN) to reduce noise. The electrodes were filled with high Ba2+ solution consisting of 200 mM BaCl2, 150 mM TEA, and 20 mM HEPES. The pH was adjusted to 7.4 with Ba(OH)2. The bath contained artificial sea water (ASW)1 consisting of 490 mM NaCl, 8 mM KCl, 10 mM CaCl2, 12 mM MgCl2, and 20 mM Tris, pH 7.4. Single-channel currents were recorded with an EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany) in the cell-attached configuration.
Pulse protocols and data acquisition were controlled by an interface (CED1401, CED, Cambridge, England for the patch-clamp experiments, and DigiData 1200, Axon Instruments for the current- and voltage-clamp experiments) connected to an IBM-compatible personal computer. Data were analyzed with software of
the CED patch and voltage-clamp suite or PCLAMP software (Axon
Instruments). Single-channel currents were low-pass filtered at 20 kHz, digitized by a modified PCM adapter (Sony) and stored on
video tape. For analysis and preparation of figures, the data were
re-filtered with a hardware 8-pole bessel filter with a cut-off frequency of 3 kHz (3 db). Alternatively, a variable software implementation of a Gaussian filter (PCLAMP software) was used. Data
were digitized at a sampling frequency of at least five times the filter frequency. Single-channel currents elicited by depolarization
up to 60 mV from the resting membrane potential were analyzed
after 3 kHz filtering. For larger depolarization 2 or 1 kHz filtering was used, depending on the signal-to-noise ratio of the recordings. The 3 kHz Gaussian filter, equivalent to a 10-90% rise
time (tr) of 100 µs, limits the detection of channel openings to
0.6 times tr, or 60 µs and currents larger than about two times tr,
or 200 µs, should be of unattenuated amplitude (Colquhoun and
Sigworth, 1983
). The choice of a 3 kHz filter is a compromise between acceptable time resolution and an acceptable proportion
of spurious events caused by excessive baseline noise. The frequency of channel openings at +50 mV was only in the order of
10/s and the rate of false openings due to noise should be at least
one to two orders of magnitude smaller than the opening rate
(Colquhoun and Sigworth, 1983
). For a recording at +50 mV
from the resting potential with current amplitudes of ~1 pA, this
implies a ratio of 5 between current amplitude and RMS baseline
noise, which, for a typical recording at +50 mV, was achieved
with a 3 kHz filter (between 0.15 and 0.2 pA RMS noise).
Steady-state open probabilities (Po) were determined from
patches with at most three active channels by summation of the
channel open times of all levels divided by the number of channels in the patch and the recording time. The number of channels in a patch (N) was judged by the maximum number of simultaneously open channels at depolarization of 80-100 mV
from rest. For voltage steps, Po values of 4-16 sweeps (100 ms)
were averaged, and Po values of channel activity during maintained depolarization were determined during 2-30 s periods,
depending on the opening frequency of the channels. Po values
were only calculated from periods of uniform channel activity,
while any times during which there was an obvious change to low
or zero activity were excluded (see Fig. 11).
For the preparation of the figures and calculation of averaged, quasimacroscopic currents, leak and capacitive currents were subtracted from the single-channel current records during voltage steps. This was achieved by adding averaged current records of negative pulses (which did not elicit any currents) to the test pulse current records. Since the leak-subtraction procedure introduces additional noise, Po values, single-channel current amplitudes and kinetics derived from voltage-step experiments were determined from the raw data.
Potentials are referred to as holding potential (HP) relative to
the membrane resting potential, or as membrane potential (Vm),
in which case a resting potential of 70 mV is assumed. This is
the average resting potential determined in earlier experiments with intracellular electrodes (Erxleben et al., 1995
). As for conventional intracellular voltage recordings, potentials are always
expressed relative to the exterior face of the membrane. Inward
currents are shown as downward deflections.
Average values are given as mean ± standard deviation with n referring to the number of patches in the case of single-channel recordings, and the number of fibers in the current- and voltage-clamp experiments.
When abdominal extensor twitch muscle fibers of Idotea
baltica are depolarized by injection of constant current
under physiological conditions (i.e., in ASW), a graded
active electrical response is usually elicited, rather than
an action potential (Erxleben et al., 1995). After application of 10 µM Bay K8644, a dihydropyridine (DHP)
agonist of L-type Ca channels, this graded response was converted into an action potential (Fig. 1 A). Subsequent application of 10 µM nifedipine, an antagonist
of L-type Ca channels, reduces the action potential
again to a graded response (Fig. 1 A), (n = 2). If K currents were suppressed by including TEA and 4-AP in
the extracellular solution (TEA solution, see MATERIALS
AND METHODS), the fibers responded to suprathreshold
depolarizing current pulses with action potentials. This
response is well known from other crustacean muscle
fibers (Fatt and Katz, 1953
; Fatt and Ginsborg, 1958
).
The duration of the action potentials, defined as time
between maximal rate of rise and fall, ranged from
90-200 ms in Ca2+ solution (10 mM) and was greatly
increased (n = 4) to between 5.1 and 32 s in Ba2+ solution (Fig. 1, B and C). Since most voltage-dependent K
currents, in particular Ca2+-activated K currents, should
be blocked in the TEA solution (Araque and Buño,
1995
; Gielow et al., 1995
), the much longer action potentials in Ba2+ solution than in Ca2+ solution suggests
that the duration of the Ca spike is at least in part determined by calcium-dependent inactivation. Spikes in TEA solution were blocked by 20 µM nifedipine (data
not shown, n = 3).
The difference in inactivation between Ca and Ba currents (n = 4 and 5) is evident from the currents measured under voltage-clamp (Fig. 1 D). Whereas the decay of the Ca current includes a fast component of inactivation, the Ba current inactivates more slowly. The Ba current was reduced by nifedipine (n = 3), as expected from the block of the action potential, and completely blocked by 100 µM Cd2+ (n = 5), (Fig. 1, E and F). The most obvious effect of 10-50 µM Bay K8644 on the Ba current was a large increase in the tail currents (Fig. 1 F, n = 3), which were very brief and small in the absence of the drug (Fig. 1, D and E).
While the properties of the Ca and Ba currents in Idotea muscle fibers are merely illustrated here (a detailed analysis is to be published elsewhere), the converse effects of the L-type Ca channel modulators on Ca and Ba action potentials and currents clearly suggest the presence of L-type Ca channels in crustacean muscle and prompted us to characterize the single-channel currents underlying the active electrical responses in Idotea muscle fibers.
In cell-attached patches with Ba2+ solution in the pipette and normal saline in the bath, no channel activity
was seen at the cell's resting potential. Only when the
patch was depolarized by voltage steps of 20-30 mV
could very brief (100-300 µs) and infrequent inward
currents be observed. Inward currents became smaller
in amplitude but much more frequent with further depolarization (Fig. 2 B). Single-channel openings were
uniformly distributed throughout the pulses, indicating that there is little inactivation during the 100-ms
test pulses (Fig. 2 B).
To directly compare the single-channel Ba currents to macroscopic currents reported from this (Fig. 1) and other crustacean and arthropod preparations, we averaged single-channel currents that were elicited during voltage steps (see Fig. 2 B). The averaged and leak-subtracted, quasimacroscopic Ca currents showed fast activation and deactivation with no inactivation during the 100-ms pulses (Fig. 2 C). The averaged currents were highly voltage-dependent and reached a maximum at 90 mV depolarization (Fig. 2 D).
Single-channel Amplitude and Conductance
All-point histograms (Fig. 3 A) were used to estimate
the single-channel current amplitudes, and the 50%
value of the amplitude was used for setting the detection threshold of the analysis program. The single-channel conductance of the channel was determined
from distributions of the idealized single-channel current amplitudes as measured by the analysis program,
to which Gaussian distributions were fitted (Fig. 4 A).
To exclude data from attenuated openings, only amplitude values of openings longer than twice the rise time of the filter (tr) were included in these plots (see MATERIALS AND METHODS). A combined plot of all single-channel current-voltage relationships would have a lot
of scatter due to differences in the resting potential.
We therefore show a representative plot of single-channel current amplitude as a function of depolarization from one patch (Fig. 4 B). The mean conductance with
200 mM Ba2+ as charge carrier was 20 ± 2.6 pS (n = 12). A linear extrapolation from the single-channel amplitude plots to zero current gave an average apparent
reversal potential of +112 ± 15 mV (n = 12). With a
resting potential of 70 mV, the average value measured previously with intracellular microelectrodes
(Erxleben et al., 1995
), current reversal occurs at an absolute membrane potential of +42 mV. Because of the
highly asymmetric Ba2+ distribution under our recording conditions, a pronounced inward rectification is
predicted by the constant field theory (Hodgkin, 1951
).
Thus, a linear extrapolation will underestimate the true reversal potential. A probably more realistic value for
the reversal potential, +73 mV (absolute), was directly
obtained from averaged, leak-subtracted single-channel
currents that were recorded from multi-channel patches
during 150-mV voltage ramps (Fig. 5, A and B). Care
was taken to exclude traces with any contaminating outward potassium currents which would again bias the
current reversal potential towards more negative values.
Because all recordings of the single-channel currents
were done in the cell-attached configuration, the ion
selectivity of the channels could not be determined individually and for the same patch. Experiments with either artificial sea water or high potassium solution (Erxleben et al., 1995) in the patch pipette, however, never
showed any single-channel currents of comparable voltage range of activation, single-channel conductance, or
channel dwell times. Thus, it appears that the observed
Ba currents were through real Ca channels rather than
nonselective cation channels.
Evidence for the Presence of Just One Channel Type
Since previous studies of single-channel Ba currents in
the crayfish (Bishop et al., 1991) and Drosophila muscle
(Leung and Byerly, 1991
) and more recently of macroscopic Ba currents in Drosophila muscle (Gielow et al.,
1995
) suggest the presence of more than one type of
Ca channel in arthropod muscle, we had to establish
that there is really only one type of Ca channel in our
records. This was particularly important since many
channel openings were brief at threshold depolarization and, due to the frequency limitations, attenuated
in amplitude. Therefore, we analyzed channel openings by plotting the relationship between current amplitude and open time. Such plots show a single cluster of data points. As an example, data from currents elicited by maintained 50 mV depolarization are shown in
Fig. 6 A. Particularly for the open times in the first two
bins (50 and 100 µs) there is a tendency for larger currents to be of longer duration, which is what we expect
due to the frequency limitations. There is, however, no
indication of a second distinct cluster which would suggest the presence of another channel type. The same
plots were analyzed for 14 other patches that lasted
long enough to allow a quantitative evaluation. The summary (mean open time at 50 mV depolarization in relation to mean current amplitude) is plotted in Fig. 6 C.
Channel Open Times
At threshold depolarization (20-30 mV from the resting potential) channel openings were in the order of
100 µs, and many openings did not reach full amplitude with the bandwidth of 3 kHz that was required for
an adequate signal-to-noise ratio of our recordings. The
channel's gating was analyzed in continuous records during maintained (5-30 s) depolarization (Figs. 2 A and 7
A) during which there was no indication of a time-
dependent decrease in single-channel activity (see Modal
Gating). At depolarization of 40 mV and higher, longer
openings (milliseconds in duration) were occasionally
observed (Fig. 2 A), although most openings were still
in the 100-300 µs range. Consequently, open time distributions could be reasonably well fitted by a single exponential component (Fig. 7 B) since long openings, if
present at all, were much too infrequent to contribute
to the distributions. Channel open times increased with
depolarization (Fig. 7 C).
Voltage Dependence of the Channel's Open Probability
The dependence of the channel's opening probability
on the membrane potential (see Fig. 2) was investigated quantitatively in patches with, at the most, three
active channels, as judged from the maximum number
of simultaneously open channels at depolarization of
80-100 mV from rest. While the probability of finding
channels in any given patch was quite low (5-10%), the
open probability of the channel at large depolarization
was high, unless in the silent mode (see Modal Gating).
The average Po was 0.15 ± 0.08 (n = 12) at 80 mV,
0.34 ± 0.13 (n = 7) at 90 mV and 0.55 ± 0.07 (n = 5)
at 100 mV depolarization. Open probabilities (Po) were determined from two types of protocols: during voltage
steps (Fig. 2 B) and during maintained depolarization
(Figs. 2 A and 7 A). An example of a Po vs. holding potential plot is shown in Fig. 8, including data from
steady depolarization and voltage steps. With the exception of Po values at steady 20 and 30 mV depolarization, there is good agreement between values obtained
with the two methods. At 20 and 30 mV depolarization,
most channel openings are probably so brief that they
are filtered down to <50% amplitude by the 3 kHz upper frequency limitation. Consequently, these events escape the detection threshold and hence the apparent
Po value is lower than expected from the fitted Boltzmann
curve of the remaining data (Fig. 8). The average voltage
sensitivity, i.e., the depolarization necessary for an e-fold
change in Po, from this type of plot was +8.2 ± 2.4 mV
(n = 13). Half-maximal activation (V0.5) of the Ca channel required 89.3 ± 12.4 mV (n = 13) depolarization.
Very similar values for the voltage sensitivity (8.4 mV for an e-fold change in Ca current) and half-maximal activation (80 mV for V0.5) were obtained from averaged currents elicited during voltage ramps (Fig. 5, A and B) in the presence of the dihydropyridine Bay K8644 (see below). The voltage dependence of the averaged (leak-subtracted) ramp currents was calculated by fitting a Boltzmann-type equation to the ascending part of the I-V relationship (Fig. 5 B). The fit was restricted to the potential range where the single-channel I-V relationship was linear (Fig. 4 B), and a fit does not require that the constant-field rectification be taken into account.
Dihydropyridine Sensitivity
After preincubation of the preparation with the dihydropyridine Bay K8644, an agonist of L-type Ca channels in vertebrate muscle and neurons (see, e.g., Bechem
et al., 1988), long channel openings, which were very
infrequent in the absence of the drug, became apparent during either 100-ms voltage steps (Fig. 9 A) or continuous depolarization (Figs. 9 B and 10 A). As a consequence of the increased frequency of long openings in
the presence of Bay K8644, open time histograms show
a distinct second component with a time constant in
the millisecond range (Fig. 9 C).
Concentrations of 10-100 µM Bay K8644 induced long openings while 1 µM had no effect. No systematic difference was observed between concentrations of 10- 100 µM, possibly due to a limited solubility of Bay K8644. In experiments with Bay K8644 which showed a particularly high frequency of long openings, such as shown in Fig. 10 A, it became apparent that the single-channel current amplitudes of the long openings were larger than those of the short openings. While the all-point histograms and the measured amplitude distributions in the absence of Bay K8644 clearly contained only one class of amplitudes (Figs. 3 A and 4 A), the all-point histograms show a second component in the presence of Bay K8644 (Fig. 3 B). Likewise, the histograms of measured amplitudes showed a second distinct, larger peak (Fig. 10 B). Similarly, the relationship between open time and amplitude showed a second cluster in the presence of Bay K8644 (Fig. 6 B). In other experiments, where the frequency of long openings was lower, amplitude distributions only showed a slight skew towards larger amplitudes or a shoulder in the distribution (data not shown).
From the quasimacroscopic, averaged single-channel records it is obvious that the current deactivates very fast (Fig. 2 C). In the single-channel records this shows in the absence of tail currents, i.e., channel openings after the repolarizing voltage step. In the presence of Bay K8644, however, we observed tail currents in voltage step experiments (Fig. 9 D), presumably because the rate of deactivation is slowed in the presence of Bay K8644.
We did not see an effect of Bay K8644 on the voltage sensitivity of the Ca channel reported for L-type Ca currents and channels in other preparations (see DISCUSSION). There was no significant difference in the voltage sensitivity, which was 7.9 ± 2.9 mV (n = 7) without Bay K8644, and 8.5 ± 2.0 (n = 6) from patches with 25-100 µM Bay K8644. Similarly, no significant difference was found in the potential of half-maximal activation of the channel with or without Bay K8644 (85 ± 11 mV, n = 6 and 93 ± 13 mV, n = 7, respectively).
For a more complete pharmacological characterization of the channels it would of course be helpful to
study the effects not only of the DHP agonist Bay K8644
but also of DHP antagonists, such as nifedipine or nitrendipine and peptides of the conotoxin family. DHP
antagonists are presumably membrane permeant and
could thus be applied to the bath while recording in
the cell-attached configuration. The spontaneous switching of the Ca channels between silent and active mode
(see Modal Gating), however, combined with the long
time the DHPs require to exert their effects in this preparation, make the interpretation of changes in activity of the channels inherently difficult. Fast acting
toxins and channel blockers like Cd2+ require application to the external face of the membrane and cannot
be easily applied in the cell-attached configuration.
Preliminary attempts to obtain outside-out patches
were promising in that we could get channels in this
configuration. There was, however, rapid loss of activity
of the channels that resembled the Ca channels found
in the cell-attached mode. "Rundown" occurred even
with Mg-ATP in the pipette and with Bay K8644 in the
bath conditions which slow the rundown of whole-cell Ca currents and single-channel activity of L-type Ca
channels in many cells (see Bean, 1992
).
Modal Gating
The complete absence of inactivation of either single-channel currents (Figs. 2 B and 9 A) or quasimacroscopic currents (Fig. 2 C) during 100-ms voltage steps prompted us to look at channel activity during extended periods. Even during several seconds of depolarization, there is no inactivation of the single-channel Ba currents. This is demonstrated in a plot of open probability versus time (Fig. 11 A). If the activity is observed over even longer times (minutes), spontaneous changes between periods of activity and periods with no or very few openings become apparent (Fig. 11 B), indicating different modes of gating.
If we look at the open probability over time in the absence of Bay K8644 we find that, with the exception of
periods during which the channel seems unavailable
for opening, Po values averaged over a 100-ms "sweep"
are fairly homogeneous (Fig. 12, A and B) and appear
as a single, approximately Gaussian distribution in Po
histograms (Fig. 12, D and E). The effect of stronger
depolarization, i.e., from +60 to +70 mV leads to a
shift of this distribution, towards higher Po values, as expected due to the voltage sensitivity of the channel
(Fig. 12 E). In the presence of Bay K8644, two changes in the plots of Po over time become evident. The distribution of Po values becomes broader, and there is a
number of high Po sweeps which are clearly from a different population. These high Po sweeps consist mainly
of long openings like the sample traces shown in Figs.
9, A and B, and 10 A. In addition there is a tendency for
clusters of long openings to occur. For the recording
shown in Fig. 12, C and F, for example, the total probability of high Po sweeps (defined as those with a Po
0.5) is 5%, and it is therefore unlikely that clusters of
four or five consecutive high Po sweeps, as can be seen
in Fig. 12 C, will be observed by chance. Likewise, null
Po sweeps, which correspond to the first bins in the distributions (Fig. 12, D-F) appear in clusters, as can be seen in Fig. 12 C (between 15 and 19 s of the record).
Evidence for a Single Ca Channel Type
A detailed biophysical analysis of the Ca channels in
Idotea muscle fibers presented here is hindered by the
rapid kinetics and small amplitude of the Ca channel
currents. At depolarization up to +50 mV (relative to
the resting membrane potential) a significant proportion of openings is either not detected at all on the basis of the 50% threshold criterion, or remains poorly resolved. Therefore, we cannot exclude the possibility that
some of these openings are from other than the DHP-sensitive Ca channel. The evidence for the presence of
just one channel type, however, is as follows. (a) Plots
of open time vs. single-channel amplitude show only one cluster of data points and a distinct second cluster
appears only in the presence of Bay K8644 (Fig. 6, A
and B). (b) The combined distribution of open time vs.
single-channel amplitude from all patches (Fig. 6 C) is
fairly homogeneous, and provides no evidence for another population of channels. (c) Neither the quasimacroscopic I-V curves from voltage-jump experiments
(Fig. 2 D) nor those obtained with voltage-ramps (Fig. 5
B) show a "shoulder" or "bump" which would indicate
the presence of another population of channels. If,
however, different channel-types were to activate within
the same range of potentials, as is the case for two Ca
current components in Drosophila larval muscle (Gielow
et al., 1995), this would not necessarily show in the I-V
curves. (d) It could be argued that the long-duration
openings represent another type of Ca channel that is
rarely active in the absence of Bay K8644 and more frequently open in the presence of the drug. The fact is,
however, that we recorded currents with brief and long
openings (i.e., Figs. 9 B and 10 A) in patches that
clearly showed no double openings even with strong
depolarization (100 mV) where the average Po was 0.55. This shows that the larger amplitude, long-duration openings actually represent another conformation of the
same channel rather than a second channel type.
Finding two conductance levels in the presence of
Bay K8644 (Fig. 10) is not unprecedented: multiple
conductance levels in the presence of Bay K8644 have
been reported for L-type Ca channels in cardiac muscle
(Lacerda and Brown, 1989) and GH3 cells (Kunze and
Ritchie, 1990
).
Comparison to Ca Channels in Other Arthropod Muscles
The only other arthropod muscle single-channel Ca
currents described so far that seem similar to the currents we found in the sarcolemmal membrane of Idotea
muscle fibers are those from cultured Drosophila myotubes (Leung and Byerly, 1991). These channels have a
similar conductance (18.5 pS with 100 mM Ba2+ solution), show no inactivation during 90-ms test pulses,
and, like the Idotea Ca channels, exhibit rapid kinetics
(Leung and Byerly, 1991
). Drosophila muscle Ca channels,
however, resemble more closely the N-type Ca channels
of vertebrate cells (Leung and Byerly, 1991
), based on
their similarity to Ca channels in Drosophila neurons,
which were shown to be insensitive to dihydropyridines (Byerly and Leung, 1988
). In crayfish slow contracting
skeletal muscle fibers, two Ca channels have been described with conductances of 14 and 38 pS (Bishop et
al., 1991
). Gating properties and voltage dependence
of these channels have not been determined, but both are activated by depolarization. The mean open times
of these Ca channels with about 40 ms for the 14 pS
and 10 ms (judging from the published records) for
the 38 pS channel, are, however, much longer than of
the Ca channel reported here for fast contracting fibers. A Ca channel isolated from T-tubular membrane
of crayfish skeletal muscle (of unknown type) and incorporated into lipid bilayers (Hurnák et al., 1990
) has
the DHP sensitivity of our Ca channel and a similar
conductance, 16 pS, compared to 20 pS in our study.
But again, the available data on the T-tubular channel is insufficient to allow a more detailed comparison, and
different types of fibers may have different sets of Ca
channels.
Comparison to Macroscopic Ca Currents
In the absence of Bay K8644, the macroscopic Ca current (Fig. 1, D and E) and quasimacroscopic Ba current
(Fig. 2 C) is of the fast activating, fast deactivating type,
as originally described for the L-type macroscopic Ca
currents in a pituitary cell line (Matteson and Armstrong, 1986). In the single-channel records, fast deactivation shows in the absence of tail currents, i.e., channel openings after repolarization. The observation that
there is no decline in single-channel activity either during voltage steps (Figs. 2 B and 9 A) or steady depolarization (Figs. 2 A, 7 A, and 10 A) nor any inactivation of
the quasimacroscopic currents (Fig. 2 C) indicates that
the sarcolemmal DHP-sensitive Ca channel in Idotea
muscle does not inactivate voltage dependently. Based on voltage-clamp studies of Ca current inactivation in
crayfish muscle, Ca channels in arthropod skeletal muscle were initially thought to inactivate voltage dependently, analogous to the classical Hodgkin-Huxley Na
current (Hencek and Zachar, 1977
). Later studies concluded that inactivation was either primarily Ca2+-
dependent (in insect muscle: Ashcroft and Stanfield,
1982
; Salkoff and Wyman, 1983
) or found a combined
calcium-induced and voltage-dependent inactivation
(in crab muscle: Mounier et al., 1988
). Under physiological conditions, i.e. with Ca2+ instead of Ba2+ as
charge carrier, our single channel data would only allow calcium-dependent inactivation for the macroscopic current through the sarcolemmal Ca channels.
This view is supported by the large difference between the duration of Ca and Ba action potentials and the
more rapid inactivation of the Ca over the Ba current
(Fig. 1, B-D). Further investigation of the macroscopic
Ca and Ba currents will have to show if the slow but
clearly present inactivation of the macroscopic Ba current reflects a different population of Ca channels located in the T-system, or if other factors like ion depletion from the T-tubules (Almers et al., 1981
; Ashcroft
and Stanfield, 1982
) can account for the difference between inactivation of the macroscopic and single-channel currents.
Dihydropyridine Sensitivity
The ability of the dihydropyridine (±) Bay K8644 to increase the frequency of long openings, a characteristic
feature unique to L-type Ca channels, is the main argument for classification of the crustacean muscle Ca
channel as L-type. Compared to mammalian cardiac
muscle or nerve L-type channels, the sensitivity is quite low: at least 10 µM Bay K8644 was needed for a significant effect. However, a similarly high concentration of
Bay K8644 (5 µM) seems to be required for the "slow"
L-type Ca channel of vertebrate skeletal muscle, that
was only recently characterized in its native membrane
(Dirksen and Beam, 1995).
The sensitivity of mammalian L-type channels to the
agonist S ()-Bay K8644 is much higher than to the antagonist R (+)-Bay K8644, both of which constitute the
racemic compound (±) Bay K8644 which is usually
used. We also used the racemic compound and do not
know the crustacean Ca channel's sensitivity ratio for
agonist/antagonist. Sensitivity to the pure agonist
could be higher.
Since we were unable to record from Ca channels in
outside-out patches, we could not readily test the effects of DHP antagonists and peptide blockers on the
sarcolemmal Ca channels. The current- and voltage-clamp experiments (Fig. 1), however, show that the macroscopic current is sensitive to DHP agonists and
antagonists. The view that at least part of the Ca current in arthropod muscle is indeed through L-type Ca
channels is also supported by a recent investigation on
macroscopic currents in Drosophila larval muscle. Here,
two types of current, resembling those through mammalian T- and L-type channels, have been resolved and
pharmacologically analyzed (Gielow et al., 1995).
Voltage Sensitivity and Ion Selectivity
The average voltage sensitivity of the crustacean Ca
channel of 8.2 mV depolarization required for an e-fold
change in open probability is similar to the values reported, for example, for single L-type Ca channels in
chick dorsal root ganglion neurons (5.5 mV) (Fox et
al., 1987), whole-cell L-type currents in atrial myocytes
(9.7 mV) (Bechem and Hoffmann, 1993
), and DHP-sensitive skeletal muscle Ca channels in bilayers (6.9 mV) (Ma et al., 1991
) or in the intact muscle (5.3 mV)
(Dirksen and Beam, 1995
). With L-type Ca channels of
smooth muscle from cerebral arteries, the crustacean
Ca channel not only shares the voltage-sensitivity but
also the range of half-maximal activation as well as single channel conductance (5-7 mV for e-fold change in
Po; V0.5 = +13.5 mV and
= 19.4 pS in 90 mM barium;
Worley et al., 1991
; Quayle et al., 1993
).
Under our experimental conditions we did not see
the shift in the activation curve (Fig. 8) to more negative potentials in the presence of Bay K8644 reported
previously for macroscopic L-type Ca currents (Bechem
and Hoffmann, 1993; Sanguinetti et al., 1986
) or single-channel currents (Quayle et al., 1993
). Since we recorded channel activity in cell-attached patches with
unknown resting potential (see RESULTS), we would expect any such shift in the activation curve to be lost due
to differences in the resting potentials of muscle fibers.
Ca Channel Gating Modes
During long-term recordings (Fig. 11) of the Ca channels,
spontaneous shifts occurred between periods with openings and periods with no openings. Similarly, patches
that initially did not seem to contain any channels often showed channel activity only after several minutes
of recording. This switching between periods when the
channel can be opened by depolarization and periods
during which depolarization fails to elicit channel openings resembles the gating modes 1 and 0 described for
L-type Ca channels of vertebrate ventricular cells (Hess
et al., 1984). Similarly, the long openings, particularly
evident in the presence of Bay K8644, might correspond
at least qualitatively
to mode 2 openings of
Hess et al. (1984)
.
Physiological Consequences
What is the physiological function of the described sarcolemmal Ca channel? Since Ca2+ influx through the
surface membrane (i.e., sarcolemma and transverse tubules) alone is probably unable to initiate contractions
in crustacean muscle fibers (Mounier and Goblet,
1987), the sarcolemmal Ca channels cannot be directly
responsible for muscle contraction. Furthermore, they
are unlikely to even contribute directly to the CICR
necessary for contraction since, in a large diameter muscle fiber like that of the crustacean, the sarcolemmal
membrane is too far away from the sarcoplasmatic
reticulum to allow for activation by Ca2+ diffusion. It
seems instead that the sarcolemmal Ca channels are
mainly for the electrogenic response which, in Idotea
muscle fibers, can be either graded or regenerative in
the form of action potentials (Erxleben et al., 1995
). It
seems likely, however, that the same L-type channel
found in the sarcolemma by the single-channel recordings also exists in the T-tubules, since a large fraction of
the macroscopic current is blocked by nifedipine (Fig.
1 E) and the activation and deactivation kinetics of
quasimacroscopic single-channel and macroscopic currents are comparable (Figs. 1 and 2).
As discussed above, the data on the crustacean Ca
channel presented here argue for it being L-type. One
of the hallmarks of vertebrate L-type Ca channels is
their susceptibility to modulation. Especially modulation that is mediated by phosphorylation through a
cAMP/protein kinase A pathway is well established for
vertebrate cardiac L-type channels (see, e.g., Hofmann
et al., 1994; Campbell and Strauss, 1995
) and neuronal
L-type channels (see, e.g., Armstrong et al., 1991
). The
suggested identity of the crustacean Ca channel with
L-type channels also makes it a likely target for modulation. Modulation leading to an increase in Ca current
has in fact been observed in lobster skeletal muscle with
octopamine and serotonin (Kravitz et al., 1980
), and on
the single-channel level, two calcium-permeable channels in flexor muscle fibers of the crayfish were shown
to be up-regulated by proctolin (Bishop et al., 1991
).
In conclusion, we have shown a voltage-dependent, dihydropyridine-sensitive L-type Ca channel in the sarcolemmal membrane of a crustacean muscle. Thus it appears that both vertebrate cardiac muscle and crustacean skeletal muscle may not only have a common mechanism of excitation-contraction coupling (a calcium- induced Ca2+ release) but also share the Ca channel type through which Ca influx occurs.
Original version received 23 July 1996 and accepted version received 29 November 1996.
Address correspondence to Christian Erxleben, Stazione Zoologica `Anton Dohrn,' Villa Comunale, I-80121 Naples, Italy. Fax: 039-81-7641-355; E-mail: erxleben{at}alpha.szn.it
1 Abbreviations used in this paper: ASW, artificial sea water; DHP, dihydropyridine.We thank Dr. David L. Armstrong for critically reading an early version of the manuscript and Mary A. Cahill for editorial assistance.
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 156.