Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont 05405
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ABSTRACT |
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Merriam, Laura A.,
Fabiana S. Scornik, and
Rodney L. Parsons.
Ca2+-Induced Ca2+ Release Activates
Spontaneous Miniature Outward Currents (SMOCs) in Parasympathetic
Cardiac Neurons.
J. Neurophysiol. 82: 540-550, 1999.
Mudpuppy parasympathetic cardiac neurons exhibit spontaneous
miniature outward currents (SMOCs) that are thought to be due to the
activation of clusters of large conductance Ca2+-activated
K+ channels (BK channels) by localized release of
Ca2+ from internal stores close to the plasma membrane.
Perforated-patch whole cell recordings were used to determine whether
Ca2+-induced Ca2+ release (CICR) is involved in
SMOC generation. We confirmed that BK channels are involved by showing
that SMOCs are inhibited by 100 nM iberiotoxin or 500 µM
tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is
decreased in solutions that contain 0 Ca2+/3.6 mM
Mg2+, and also in the presence of 1 µM nifedipine and 3 µM -conotoxin GVIA, suggesting that SMOC activation is dependent
on calcium influx. However, Ca2+ influx alone is not
sufficient; SMOC activation is also dependent on Ca2+
release from the caffeine- and ryanodine-sensitive Ca2+
store, because exposure to 2 mM caffeine consistently caused an
increase in SMOC frequency, and 10-100 µM ryanodine altered the
configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca2+ stores by the Ca-ATPase
inhibitor cyclopiazonic acid (10 µM) inhibited SMOC activity, even
when Ca2+ influx was not compromised. We also tested the
effects of the membrane-permeable Ca2+ chelators,
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 µM) caused no inhibition
of SMOC activation, whereas 10 µM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal
of caffeine and suggests that the source of Ca2+ that
triggers the internal Ca2+ release channel is different
from the source of Ca2+ that activates clusters of BK
channels. We propose that influx of Ca2+ through
voltage-dependent Ca2+ channels is required for SMOC
generation, but that the influx of Ca2+ triggers CICR from
intracellular stores, which then activates the BK channels responsible
for SMOC generation.
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INTRODUCTION |
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Neurons, like most cell types, contain
intracellular pools of Ca2+ that are important in
cellular signaling pathways (Berridge 1998; Henzi
and MacDermott 1992
; Kostyuk and Verkhratsky
1994
). The IP3-sensitive pools of
Ca2+ are known to be mobilized through activation
of a variety of G protein-coupled neurotransmitter receptors
(Berridge 1993
), but a role for the ryanodine-sensitive
Ca2+ stores in neurons is less clear. It has been
hypothesized that neuronal ryanodine-sensitive stores of
Ca2+ may amplify and prolong intracellular
Ca2+ signaling by
Ca2+-induced Ca2+ release
(CICR) using mechanisms similar to that demonstrated for cardiac and
smooth muscle cells (Bolton and Imaizumi 1996
; Fabiato and Fabiato 1984
; Kuba 1994
;
Verkhratsky and Shmigol 1996
). CICR in neurons was
initially well documented using pharmacological interventions
(Kuba 1994
; Verkhratsky and Shmigol
1996
). More recent results demonstrate physiological activation
of CICR in neurons (Cohen et al. 1997
; Hua et al.
1993
; Llano et al. 1994
; Moore et al.
1998
; Shmigol et al. 1995
; Yoshizaki et
al. 1995
).
A potential role of CICR in neurons could be to produce localized
elevations of internal Ca2+, which in turn would
activate Ca2+-dependent processes, thereby
modulating intracellular signaling. For instance, transient, localized
elevations of intracellular Ca2+, which are not
easily detected with traditional Ca2+ imaging
techniques, could activate Ca2+-dependent
channels in the plasma membrane, thus regulating membrane excitability
by altering the resting membrane potential. Indeed, the magnitude of
the resting membrane potential of many neurons is dependent on the
extent of the activation of large conductance Ca2+-activated potassium channels (BK channels)
(Marty 1989).
In some neurons, simultaneous activation of BK channels occurs
spontaneously under resting conditions (Fletcher and
Chiappinelli 1992; Hartzell et al. 1977
;
Mathers and Barker 1981
, 1984
;
Satin and Adams 1987
). This BK channel activation causes
spontaneous miniature hyperpolarizations (SMHs), or spontaneous
miniature outward currents (SMOCs) when recorded under voltage clamp.
These transient membrane events (SMHs or SMOCs) are proposed to be due to the activation of a cluster of BK channels by transient elevations of intracellular Ca2+ near the inner surface of
the plasma membrane (Satin and Adams 1987
). Thus
neuronal SMOCs may be similar to the spontaneous transient outward
currents (STOCs) measured in smooth muscle cells (Bolton and
Imaizumi 1996
; Nelson et al. 1995
) and could
potentially be involved in controlling neuronal excitability
(Berridge 1998
).
Given that SMOCs might be caused by the simultaneous activation of clusters of BK channels, SMOCs provide a convenient model system to detect and analyze sources of localized Ca2+ involved in the activation of BK channels in neurons. Therefore, in the present study, we have analyzed SMOC generation in mudpuppy parasympathetic neurons to establish the relative roles of Ca2+ influx and Ca2+ release in activation of BK channels. In particular, our experiments test the hypothesis that the activation of SMOCs in mudpuppy cardiac neurons requires Ca2+-induced Ca2+ release from internal stores and further, that this pool of Ca2+ gates a closely localized cluster of BK channels. The experiments utilize voltage-clamp recordings from acutely isolated mudpuppy parasympathetic neurons and establish that SMOC generation requires Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs), which then triggers Ca2+ release from internal stores. As part of the study, we demonstrate that the internal Ca2+ released to generate SMOCs comes from a caffeine- and ryanodine-sensitive store.
Preliminary descriptions of initial aspects of this work have been
presented previously (Merriam et al. 1996;
Merriam and Parsons 1997
; Scornik et al.
1998
).
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METHODS |
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All experiments were performed on parasympathetic neurons
dissociated from mudpuppy (Necturus maculosus) cardiac
ganglia. Procedures for euthanasia were approved by the University of
Vermont Institutional Animal Care and Use Committee. The methods of
dissociation used a combination of collagenase, type I (Sigma, St.
Louis, MO) and neutral protease (Sigma) following methods described
previously (Merriam and Parsons 1995). All experiments
were completed at room temperature (21-22°C) with cells continuously
perfused by a gravity-fed bath solution (flow rate 5-10 ml/min) in a
recording chamber with a volume of 1 ml (Warner Instrument, Hamden, CT).
Electrophysiological methods
Recordings were made with the perforated-patch configuration of
the whole cell recording technique (Horn and Marty
1988). Either nystatin or amphotericin B were used to establish
electrical continuity between the patch pipette and cell interior. No
qualitative difference was noted between experiments with either of the
pore-forming compounds, but as the perforated-patch configuration was
more readily established and more stable with amphotericin B than
nystatin, most of the experiments used amphotericin B. Voltage-clamp
experiments were controlled using either Axopatch-1C/Labmaster DMA or
Axopatch 200/Digidata 1200 acquisition systems (Axon Instruments,
Foster City, CA). Currents were filtered at 2 kHz, stored on tape using a PCM recorder (A. R. Vetter , Rebersburg, PA), and then digitized (200-µS digitization rate) for further analysis using the SCAN program (Strathclyde Electrophysiology Software, John Dempster, University of Strathclyde, Glasgow, Scotland). Digitized files were
edited by eye, and SMOC frequency was determined by counting the number
of SMOCs in 60 s of digitized current traces. Peak amplitude and
50% decay time of individual SMOCs were analyzed using a threshold
detection utility that was set to detect events 0.5% of full scale.
This threshold, which equals 10 pA, was chosen to eliminate SMOCs that
were indistinguishable from baseline noise. For determination of
amplitudes and 50% decay times, 100-400 SMOCs were analyzed for each
condition; except for experiments in which the SMOC frequency
was greatly decreased, 20-150 SMOCs were analyzed. Reported voltages
were corrected for a junction potential of 10 mV; no corrections were
made for voltage-clamp error due to series resistance, except where
noted (Fig. 2, C-F). Data were normalized to control values
obtained in the same cell under the same conditions and are presented
as means ± SE. Statistical differences were determined by
Student's t-test, P < 0.05.
Solutions
The bath solution for SMOC recordings contained (in mM) 110 NaCl, 1.8 or 3.6 CaCl2, 2.5 KCl, 10 NaHEPES, and 0.0003 tetrodotoxin (TTX), pH 7.3. The pipette solution for patch recordings was (in mM) 80 Kaspartate, 40 KCl, 5 MgCl2, and 10 K-HEPES, pH 7.2. The patch pipettes were backfilled with 0.2 mg/ml amphotericin B or 0.33 mg/ml nystatin.
Drugs
All drugs used in the present study were obtained from
commercial sources: cyclopiazonic acid (CPA), iberiotoxin, and TTX from
Alomone Labs (Jerusalem, Israel); tetraethylammonium (TEA), caffeine,
3-isobutyl 1-methylxanthine (IBMX), and nifedipine from Sigma;
-conotoxin GVIA from Alexis Biochemicals (San Diego, CA; and Alomone
Labs); thapsigargin,
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) from Research Biochemicals International (Natick, MA); and EGTA-AM from Calbiochem (La Jolla, CA). All drugs were used at
concentrations consistent with those used in published studies and were
in excess of the established KD of
each drug. Thapsigargin, CPA, BAPTA-AM, and EGTA-AM were prepared as
1,000-10,000× concentrated stock solutions in DMSO and frozen until
use. Nifedipine was prepared in acetone as a 10-mM stock and stored at
20°C until used. Vehicle controls indicated that neither DMSO nor
acetone at the final concentrations had any measurable effects on SMOCs.
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RESULTS |
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SMOCs are potassium currents generated by the simultaneous activation of BK channnels
The initial experiments verified that mudpuppy neuronal SMOCs
resulted from the activation of large conductance
KCa channels (BK channels) but not small
conductance KCa channels (SK channels) by testing
whether 100 nM iberiotoxin or 100 nM apamin affected SMOC amplitude.
For these experiments, SMOCs were recorded from neurons that were
bathed in a solution containing 1.8 mM Ca2+ and
voltage clamped to depolarized potentials. SMOC amplitudes, 50% decay
times, and frequency were unaffected by the presence of apamin
(n = 3, Fig. 1,
A1 and B, Table
1); in contrast, SMOCs were eliminated
during exposure to iberiotoxin (n = 4, Fig.
1A3). We also tested the effect of TEA on SMOC amplitude to
further demonstrate that BK channel activation was responsible for SMOC generation. Example results obtained with 500 µM TEA, which
illustrate the TEA-induced decrease in SMOC amplitude, are shown in
Fig. 1A2. In five cells exposed to 500 µM TEA, SMOC
amplitude was reduced to 56.2 ± 3.6% (mean ± SE) of
control amplitudes measured in the same cells (Fig. 1B,
Table 1). Results of other experiments with 100 µM 2 mM TEA
demonstrated that the TEA-induced decrease in SMOC amplitude was
concentration dependent and reversible (data not shown).
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SMOC activation characteristics are voltage dependent
Satin and Adams (1987) reported previously that
SMOCs could be recorded from cultured bullfrog sympathetic neurons and
from mudpuppy parasympathetic neurons in solutions containing cadmium (Cd2+). These authors characterized the
voltage-dependent characteristics of SMOC activity in bullfrog
sympathetic neurons, and found that SMOC frequency, amplitude, and
decay time all increased with depolarization in the presence of 100 µM Cd2+. They also suggested, from experiments
with iontophoretically applied Ca2+, that
the SMOC decay characteristics were determined by the closing behavior
of BK channels. Fewer experiments involved mudpuppy parasympathetic neurons, but Satin and Adams (1987)
did observe SMOC
activity in the presence of 200 µM Cd2+ in
these neurons but saw no evidence that SMOC frequency increased on
membrane depolarization.
We tested whether the characteristics of SMOC activation were altered
in the dissociated mudpuppy neurons when 200 µM
Cd2+ was added to the bath solution. In these
neurons, the addition of 200 µM Cd2+ decreased
the SMOC frequency markedly and, in some cells, eliminated SMOC
activity completely. With 3.6 mM Ca2+ and 200 µM Cd2+ in the bath solution, SMOC activity
decreased quickly to a reduced frequency and was maintained at this
reduced frequency throughout the rest of the recording period (Fig.
2A). Under these conditions with SMOC frequency reduced, it was possible to resolve characteristics of individual events. Thus we used cells maintained in 3.6 mM Ca2+ and 200 µM Cd2+ to
measure the voltage dependence of the frequency, amplitude, and decay
of mudpuppy SMOCs. The cells were voltage clamped to potentials between
50 and +20 mV. Figure 2B shows examples of averaged SMOCs
recorded at two different voltages. Under these recording conditions,
the SMOC frequency-membrane voltage relationship was bell-shaped with
the maximum frequency occurring near
20 mV (Fig. 2C). SMOC
amplitude and 50% decay also were voltage dependent; both increased as
the holding potential was made more positive (Fig. 2, D and
E). Even though SMOC frequency was decreased in the presence
of 200 µM Cd2+, SMOC amplitudes or decay times
were not significantly different from control (see Table 1).
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Caffeine stimulates SMOC activity
The possibility that Ca2+ released from an
internal store was involved in SMOC generation was tested using 2 mM
caffeine. Caffeine stimulates Ca2+ release from
neuronal ryanodine-sensitive Ca2+ stores by
sensitizing the release channel to Ca2+
(McPherson et al. 1991). For these experiments, the
cells were voltage clamped to membrane potentials between
20 and 0 mV
and bathed in a solution containing 1.8 mM Ca2+
and 200 µM Cd2+.
The frequency of SMOCs was consistently increased in the presence of 2 mM caffeine (Fig. 3A and
D). Caffeine did not affect SMOC amplitude, but in the
presence of caffeine, SMOC 50% decay time was increased significantly,
to 186 ± 9% of control values (n = 3; Fig. 3,
B and C, see Table 1). Also, a decrease in the outward current was consistently observed when caffeine was applied (Fig. 3A). Caffeine has previously been shown to directly
block delayed rectifier K+ channels
(Reiser et al. 1996), which could account for the
observed effect. All of the effects of caffeine reversed quickly on
removal of caffeine.
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Control experiments were done to test whether the prolongation of SMOC
50% decay time in caffeine was related to the ability of caffeine to
inhibit cellular phosphodiesterases (Ashcroft et al.
1972) rather than to its ability to induce
Ca2+ release from internal stores. We tested
whether 2 mM caffeine increased SMOC 50% decay time in cells that had
been pretreated with the potent phosphodiesterase inhibitor IBMX. In
three cells, following pretreatment with 500 µM IBMX, 2 mM caffeine
significantly increased 50% decay time (200 ± 21% of the value
in IBMX alone), but SMOC amplitude was not affected by caffeine in the
IBMX-treated cells (92 ± 2%). Thus we suggest that the increase
in 50% decay time by caffeine was not related to a caffeine-induced
inhibition of phosphodiesterases, but rather represented a direct
effect of caffeine on the Ca2+ release channel.
We also analyzed the effects of high concentrations of caffeine on
Ca2+ release from internal stores in mudpuppy
neurons. The results presented in Fig. 3E demonstrate that
repeated 30-s applications of 30 mM caffeine initiated a large outward
current typical of a caffeine-induced outward current caused by release
of Ca2+ from internal stores (Marrion and
Adams 1992). We found that with repeated caffeine applications,
the large transient outward currents were comparable if the interval
between caffeine challenges was >2 min. This result demonstrates that
the caffeine-sensitive stores in the mudpuppy neurons readily refill
with Ca2+ after caffeine challenges. The
caffeine-induced outward currents recorded when 200 µM
Cd2+ was present were similar to those recorded
in cells not exposed to Cd2+ (not shown).
SMOC activity is depressed by removal of external Ca2+ and by blockers of VDCCs
The observation that SMOC frequency was decreased in the presence
of Cd2+ suggested that Ca2+
influx through surface membrane Ca2+ channels was
involved in SMOC generation. To test the role of external
Ca2+, we substituted Mg2+
for Ca2+ in the external solution and examined
SMOC frequency. SMOCs initially were recorded at holding voltages
between 20 and 0 mV in a control solution that contained 3.6 mM
Ca2+ and 200 µM Cd2+. The
bath solution was then changed to a
Ca2+-deficient solution containing 3.6 mM
Mg2+ and 200 µM CdCl2. In
six cells, SMOCs disappeared quickly after switching to the
Ca2+-deficient solution (Fig.
4A). SMOC activity recovered
if the cells were returned to the Ca2+-containing
solution (n = 3, Fig. 4, A and
B3). Alternatively, when SMOC frequency had decreased to
zero in the Ca2+-deficient solution, the addition
of 3 mM caffeine to the Ca2+-deficient solution
reinitiated SMOC activity (n = 3, Fig. 4A2). To ensure that results observed were not related to the presence of 200 µM Cd2+, we repeated the experiments without
Cd2+. In these experiments (n = 5), SMOC activity also disappeared when the 3.6 mM
Ca2+ was replaced by 3.6 mM
Mg2+, and the time course of SMOC disappearance
was closely dependent on the speed of the gravity flow system. In three
experiments in which we adjusted the the gravity flow to a fast flow
rate of 10 ml/min, we observed that SMOC frequency began to decrease quickly after switching to the Ca2+-deficient
solution, so that the frequency was decreased to 3% of the control
frequency within 30 s of the solution change (Fig. 4B1).
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We also observed that in four cells in which SMOCs disappeared in the Ca2+-deficient solution, application of 30 mM caffeine initiated a large transient outward current, which demonstrated that the intracellular stores still contained Ca2+ (Fig. 4B2). However, once Ca2+ stores were emptied by caffeine in the Ca2+-deficient solution, subsequent applications of high caffeine did not activate SMOCs or any outward current (n = 3, data not shown).
The results of these experiments demonstrated that SMOC activity was inhibited by exposure to the Ca2+-deficient solution even though caffeine-sensitive internal Ca2+ stores still contained Ca2+. SMOC activity could be transiently activated by application of caffeine, but once the internal stores of Ca2+ were depleted, SMOC activity could not be reinitiated.
The observation that SMOC frequency was decreased in
Ca2+-deficient or
Cd2+-containing solutions suggested that
Ca2+ influx through surface membrane
Ca2+ channels was important for SMOC generation.
Additional experiments were done to test whether the channels involved
were VDCCs. Previously, Merriam and Parsons (1995)
demonstrated that mudpuppy parasympathetic neurons express both
dihydropyridine- and
-conotoxin GVIA (ctx-GVIA)-sensitive Ca2+ channels, but that most of the whole cell
Ca2+ current flowed through ctx-GVIA-sensitive
channels. We tested whether inhibition of VDCCs affected SMOC
generation and, further, whether a specific class of VDCC was
preferentially involved. The Ca2+ concentration
in the bathing solution was 1.8 mM, and the cells were voltage clamped
to
10 or 0 mV. The addition of 1 µM nifedipine decreased SMOC
frequency by 18.5 ± 4.3%, whereas 3 µM ctx-GVIA plus 1 µM
nifedipine decreased SMOC frequency by 93.1 ± 2.3% relative to
the control frequency (n = 5, Fig.
5A). The reverse order of drug
application was also tested in an additional experiment. In this cell,
3 µM ctx-GVIA decreased SMOC frequency by 83.3% relative to control,
and the addition of 1 µM nifedipine to ctx-GVIA blocked SMOC
frequency further, so that the frequency was decreased by 96.7% of
control (data not shown). SMOC frequencies were counted 0.5-2 min
after exposure to each drug. Although SMOC frequency was consistently
decreased by exposure to the combination of ctx-GVIA and nifedipine
(Fig. 5A), SMOC amplitudes were not decreased (see Table 1).
Thus the decrease in SMOC frequency was not due to a decrease in
amplitude of individual SMOCs. SMOCs measured in the presence of the
VDCC inhibitors exhibited a small but statistically significant
increase in 50% decay time (Table 1), but the explanation for this
observed increase is unclear.
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We also tested whether the decreased SMOC frequency during exposure to Ca2+ channel blockers was due to depletion of intracellular Ca2+ stores. After SMOC activity had decreased to very low frequencies, application of 3 mM caffeine stimulated additional SMOC activity (n = 2, data not shown). In five inhibitor-treated cells, application of 30 mM caffeine, given when SMOC activity had declined to very low frequencies, elicited a large outward current (Fig. 5B). Thus, when SMOC activity was depressed during exposure to the Ca2+ channel blockers, internal stores had not been depleted of Ca2+. We also noted that even though SMOC activity decreased to very low frequencies after treatment with the Ca2+ channel blockers, repeated caffeine applications initiated large-amplitude, transient outward currents. This result differed from that obtained with cells kept in the Ca2+-deficient solution. In Ca2+-deficient solutions, once stores were depleted of Ca2 by caffeine, subsequent caffeine applications could not induce an outward current because intracellular Ca2+ stores were not replenished.
SMOC activity is progressively diminished during exposure to CPA
Our results with caffeine demonstrated that SMOC activity could be
initiated by release of Ca2+ from internal
stores. Furthermore, our results suggested that if internal stores
became depleted of Ca2+, then SMOC activity was
depressed. In another series of experiments, we tested directly the
effect of depletion of internal Ca2+ stores on
SMOC generation. For these experiments, we analyzed the effect of 10 µM CPA on SMOC frequency in cells voltage clamped to 10 mV and
bathed in 1.8 mM Ca2+ solution. CPA inhibits the
sarco/endoplasmic reticulum Ca2+ ATPase (SERCA),
which sequesters Ca2+ into the endoplasmic
reticulum (ER); during continued exposure to 10 µM CPA, internal
Ca2+ stores gradually become depleted as
Ca2+ leaks out of the ER (for review, see
Thomas and Hanley 1994
).
In the presence of 10 µM CPA, SMOC activity declined gradually, and, by ~15 min, SMOC frequency was decreased to very low levels (n = 3, Fig. 6A and B). SMOC activity returned once CPA was removed (Fig. 6C). At a time when the SMOC frequency in CPA had declined >60% (9 min), analysis of individual events indicated that there was no CPA-induced change in amplitude or half decay time (n = 3; see Table 1). SMOCs also disappeared in two additional cells that were treated with the irreversible SERCA inhibitor, thapsigargin (100 nM, data not shown).
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Additional experiments were done to verify that after internal
Ca2+ stores were depleted by exposure to 10 µM
CPA, SMOCs could not be activated. For these experiments, 30 mM
caffeine was applied repeatedly to control cells and cells treated with
10 µM CPA. The bathing solution contained 1.8 mM
Ca2+, and the cells were voltage clamped to 10
mV. As illustrated in Fig. 3D, repeated applications of 30 mM caffeine to control cells produced large transient outward currents
even when the interval between the caffeine applications was only
90 s, because the internal stores readily refill. In contrast,
during exposure to CPA, a single 30-s application of caffeine induced a
large transient outward current, but subsequent applications of
caffeine could not, because stores were depleted of
Ca2+ (Fig. 7,
A-C). After the first application of caffeine, no SMOCs were recorded, even though Ca2+ influx was not
compromised. When CPA was removed, SMOC activity resumed and the
application of 30 mM caffeine induced a large outward current (Fig. 7,
D and E). The transient outward current could
also be initiated by subsequent applications of 30 mM caffeine. Similar
results were obtained in three cells.
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SMOC activity and configuration are affected by ryanodine
The effects of caffeine suggested Ca2+
release from ryanodine-sensitive stores was involved in SMOC
generation. We tested the involvement of ryanodine-sensitive stores in
experiments in which the effect of ryanodine (10, 50, or 100 µM) on
SMOC frequency was analyzed in cells voltage clamped between 20 and 0 mV. For most experiments, the bath solution contained 1.8 or 3.6 mM
Ca2+, and 200 µM Cd2+ was
present to allow resolution of individual SMOCs.
In 10 cells, SMOC frequency declined during ryanodine application (10 µM, 3 cells; 50 µM, 3 cells; 100 µM, 4 cells). We did not observe any obvious concentration-dependent effects, presumably because ryanodine had been added by bath superfusion, and the actual concentration that reached the ryanodine receptor was unknown. In some cells at each concentration, there was an initial increase in SMOC frequency following the ryanodine application (Fig. 8A), but in all cells, with continued exposure to ryanodine, the SMOC frequency and amplitudes decreased progressively until the events became indistinguishable from noise. As the SMOCs became smaller, quantification of individual events was impossible, but in all cells, SMOC activity had disappeared within 30 min. In addition, in 60% of the cells, ryanodine caused an obvious alteration in SMOC configuration (Fig. 8). Before ryanodine exposure, individual SMOCs exhibited a fast rise to peak and then decayed more slowly back to the baseline (Fig. 8B1). In ryanodine-treated cells, the rising phase was not noticeably different from that in control cells, but the decay phase in some cells was greatly exaggerated. In these instances, the SMOCs decayed only part way to a plateau level that remained for many tens of milliseconds (Fig. 8, B2 and B3). This plateau phase appeared similar to what would be expected if the ryanodine had blocked the release channel in an open subconductance state, and the Ca2+ release had been prolonged (see DISCUSSION).
|
To verify that SMOC disappearance in the presence of ryanodine was not
simply related to the presence of 200 µM Cd2+,
we recorded SMOCs at 10 mV in three additional cells without Cd2+ present. In these cells, 100 µM ryanodine
also decreased SMOC frequency in a time-dependent manner. As observed
in the ryanodine- and Cd2+-treated cells, SMOC
activity initially increased, then became more difficult to distinguish
as SMOCs merged with the noise. All SMOC activity had disappeared
within 30 min.
BAPTA eliminates SMOC activity
We have presented results that support the hypothesis that SMOCs
are initiated by Ca2+-induced
Ca2+ release from caffeine- and
ryanodine-sensitive internal stores. Evidence has been presented
demonstrating an involvement of both Ca2+ influx
and Ca2+ release from internal stores in SMOC
generation. Additional experiments were done to test the effects of the
membrane-permeable Ca2+ chelators, BAPTA-AM and
EGTA-AM, on SMOC activity. Although the affinity of BAPTA and EGTA for
Ca2+ are similar, the association rate for
Ca2+ is ~150 times greater for BAPTA than EGTA
(Naraghi 1997). Thus it is possible to distinguish
between fast and slow Ca2+-stimulated events that
occur over short distances by use of these two chelators. For these
experiments, the cells were bathed in 1.8 mM
Ca2+ and voltage clamped to
10 mV.
Eight cells were loaded with 10 µM BAPTA-AM for time periods ranging from 10 to 37 min, at room temperature in the dark. Whole cell recordings were initiated at different times. In three cells, the recordings were begun before BAPTA-AM exposure and continued during the incubation period. In the remaining cells, the recordings were initiated after the BAPTA-AM loading had ended with the longest time interval being ~90 min. In all cases, SMOC frequency decreased progressively after application of BAPTA-AM. In those cells in which the recording was started before BAPTA-AM application, SMOCs were present, but during the exposure to BAPTA-AM the frequency gradually declined until by ~20 min, no SMOCs were recorded (Fig. 9, A1 and A2). In the remaining cells that were pretreated with BAPTA-AM, SMOC frequency (if SMOCS were present) was very low and continued to decline further over time.
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In four BAPTA-AM-loaded cells, after SMOC activity had ceased, 3 mM caffeine was applied. SMOC frequency increased quickly in these cells (Fig. 9A3). The SMOC activity also quickly declined on removal of caffeine (not shown). The induction and reversal of SMOC activity by caffeine could be repeated throughout the length of the recording, which in the case of one cell was longer than 90 min.
We also tested whether loading cells with EGTA-AM had a comparable effect to that of BAPTA on SMOC generation. Four cells were loaded with 10 µM EGTA-AM for ~25 min before initiation of SMOC recording. Recordings were then continued for 40-120 min after loading. It was possible to record SMOCs from EGTA-loaded cells for the duration of recording without any decrease in frequency (Fig. 9B).
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DISCUSSION |
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SMOCs in mudpuppy neurons are spontaneous, transient outward
K+ currents that are abolished by iberiotoxin and
TEA, but not by apamin; thus confirming the role of BK channels
(Satin and Adams 1987) and demonstrating that activation
of SK channels did not contribute to SMOC generation.
SMOC frequency was greatly diminished by conditions that eliminated or
reduced Ca2+ influx, such as removal of
external Ca2+, or addition of
Ca2+ channel blockers. Mathers and Barker
(1984) also found that spontaneous hyperpolarizations could not
be recorded in cultured dorsal root ganglion (DRG) cells
exposed to a Ca2+-deficient solution. However, in
the DRG cells, the recordings were made after a 15- to 30-min
pretreatment in Ca2+-deficient solution, during
which time the stores could have been depleted of
Ca2+. In our experiments, the decrease in SMOC
frequency occurred at times when the Ca2+ stores
still contained sufficient Ca2+ to support SMOC
activity induced by caffeine. Thus depletion of internal stores was not
the cause of the decrease in SMOC activation in our experiments.
Also, only SMOC frequency, but not SMOC amplitude, was affected by conditions that decreased Ca2+ influx through VDCC. If Ca2+ influx directly gated the BK channels contributing to SMOC generation, SMOC amplitude should have fallen as fewer and fewer BK channels were activated. This was not the case, suggesting instead that Ca2+ from VDCC did not activate BK channels directly, but triggered an intermediate step such as Ca2+ release from internal stores that then gated BK channel activation.
SMOC frequency exhibited a bell-shaped dependence on membrane voltage
when 200 µM CdCl2 was included in the bath
solution (Fig. 2C). A similar voltage dependence was noted
by Satin and Adams (1987), who suggested that there
might be coupling between the internal
Ca2+ release sites and the surface
membrane so that the voltage dependence of SMOC frequency reflected a
voltage dependence of the Ca2+ release channels
themselves. However, the voltage dependence of mudpuppy SMOC frequency
is very similar to the voltage dependence of Ca2+
influx through VDCCs in mudpuppy neurons (Merriam and Parsons 1995
) and mimics the relationship between
Ca2+ influx through VDCCs and
Ca2+ release probability in cardiac myocytes
(Santana et al. 1996
). Even though 200 µM
Cd2+ was present in our experiments,
Ca2+ influx very likely was not completely
blocked, because removal of external Ca2+
resulted in more effective block of SMOC activity than did
Cd2+ alone. As described by Santana et al.
(1996)
, even when Ca2+ channel open
probability is greatly reduced, the Ca2+ flux
through an individual open channel is not compromised and is sufficient
to activate ryanodine receptors, particularly at more hyperpolarized
potentials where there is a large driving force for
Ca2+. Therefore we propose that the SMOC
frequency-voltage relationship reflects voltage dependence of
Ca2+ influx, and not voltage dependence
of the Ca2+ release channel.
Previously, Ca2+ influx through specific classes
of VDCC has been correlated with activation of particular
Ca2+-activated K+ channels
(Davies et al. 1996; Marrion and Tavalin
1998
; Wisgirda and Dryer 1994
). However, in our
study, Ca2+ influx through a specific VDCC type
did not selectively activate SMOCs. Preferential blockade of VDCC types
decreased SMOC frequency by the same proportion as the inhibition of
specific voltage-gated Ca2+ channels that
contribute to the whole cell Ca2+ current
(Merriam and Parsons 1995
). The lack of any specific role of a particular VDCC type suggests that the source of
Ca2+ that is involved in triggering release may
not be critical; any Ca2+ source that contributes
free Ca2+ to the microenvironment surrounding the
release channel may be sufficient to stimulate CICR (McPherson
and Campbell 1993
; Sutko and Airey 1996
;
Zucchi and Ronca-Testoni 1997
). This suggestion is
supported by the observation that ryanodine (Fig. 8) can transiently increase SMOC frequency as Ca2+ begins to leak
out of intracellular stores. The converse is true also; any
experimental perturbation that reduces the free
[Ca2+]i in the
microenvironment would decrease the probability of
Ca2+ release and therefore decrease SMOC frequency.
Low concentrations of caffeine increased SMOC frequency, suggesting
that Ca2+ release from caffeine-sensitive
Ca2+ stores were involved in SMOC generation in
mudpuppy neurons. Results of earlier studies also indicated that
caffeine increased the frequency of spontaneous miniature
hyperpolarizations in neurons (Fletcher and Chiapinelli
1992; Mathers and Barker 1984
). Marrion and Adams (1992)
suggested, based on an analysis of current
variance, that the caffeine-induced (10 mM) outward current recorded in cultured bullfrog sympathetic neurons arose from the summation of
individual SMOCs. We report here, that 2 mM caffeine did not change
SMOC amplitude but did increase SMOC 50% decay time. Caffeine increases the open probability (Po) of
the release channels by decreasing the closed state dwell time of the
channel, thus resulting in more frequent openings. The open time and
conductance of the release channel are not altered under these
conditions (Sitsapesan and Williams 1990
). We propose
that the caffeine-induced prolongation of the 50% decay time most
likely reflected multiple activations of BK channels in the vicinity of
the release channel (DiChiara and Reinhart
1995
). However, SMOC amplitude was not increased; thus
either Ca2+ buffering is sufficiently fast to
prevent accumulation of Ca2+ in the vicinity of
the BK channel, or the concentration of Ca2+
released is normally high enough so that BK channel activation is
maximal, thus any additional increase in Ca2+
would not affect SMOC amplitude.
Ryanodine binds with high affinity to the caffeine-sensitive
Ca2+ release channel and alters its
gating behavior and conductance. At lower concentrations, ryanodine can
lock the release channel in an open, subconductance state, whereas
higher concentrations of ryanodine will block the release channel in a
use-dependent manner (Lai et al. 1992; Meissner
1986
, 1994
; Sutko and Airey 1996
;
Zucchi and Ronca-Testoni 1997
). In all mudpuppy neurons tested, ryanodine decreased SMOC frequency in a time-dependent manner.
Also, in some cells, SMOC configuration was markedly altered. In these
cells, in the presence of ryanodine, SMOC decay sometimes exhibited a
flat and prolonged phase, similar to what would be expected if the
Ca2+ level remained elevated, as the
release channel were locked in an open, subconductance state. This
altered configuration was remarkably similar to the altered
configuration of Ca2+ sparks observed in rat
cardiac myocytes in the presence of ryanodine (Cheng et al.
1993
). The results with ryanodine add further support for the
hypothesis that Ca2+ release from
ryanodine-sensitive intracellular stores controls activation of the BK
channels involved in SMOC generation.
SMOC activity was eliminated in the presence of the SERCA inhibitor,
CPA. The decrease in SMOC frequency was time dependent and occurred
when Ca2+ influx through VDCC was not
compromised. In addition, after internal Ca2+
stores were depleted by 30 mM caffeine in the presence of CPA, SMOCs
could not be activated. SMOCs quickly returned when CPA was removed and
the internal stores were allowed to replenish. These results provided
additional support for the view that Ca2+
released from internal stores initiated SMOC activity, and that Ca2+ influx through VDCCs, by itself, was not
sufficient for SMOC activation. The observation that the SMOC frequency
decreased as the stores became more and more depleted suggested that
lumenal concentration of Ca2+ also is a critical
factor in determining SMOC frequency. Interestingly, although the
Ca2+ stores contained less
Ca2+ over time, SMOC amplitude did not change
over the same time period. We propose two alternative mechanisms that
could potentially account for these observations. First, the
Ca2+ release process might be quantized
(Cheek et al. 1993), so that a fixed amount of
Ca2+ is always released even though total levels
in the store diminish. Alternatively, the amount of
Ca2+ released exceeds that needed to activate the
BK channels located very close to the release channels. Thus the local
Ca2+ levels are normally well above threshold
concentrations so that the amount released from the stores could
decrease greatly before there was any noticeable change in number of
activated BK channels. The Ca2+ concentration in
the local domain must reach quite high levels given that SMOCs occur at
voltages as negative as
50 to
60 mV; potentials at which BK
channels require relatively high concentrations of
Ca2+ for activation (Barrett et al.
1982
).
The experiments with Ca2+ chelators provided
important insight into the characteristics of the
Ca2+ pathways involved in SMOC generation. These
conclusions are based on the fact that BAPTA, under the conditions of
these experiments, has the same binding affinity, but faster
association kinetics than EGTA (Naraghi 1997). First,
the results with BAPTA demonstrated that two distinct
Ca2+ pathways are involved: 1) a
Ca2+ domain coupling Ca2+
influx to the activation of the ryanodine-sensitive ER
Ca2+ release channels and 2) a
Ca2+ domain coupling Ca2+
release from the ER release channel to plasma membrane BK channels. BAPTA appeared to chelate the transient increases of intracellular Ca2+ due to Ca2+ influx
through plasma membrane VDCCs, but could not effectively buffer the
transient increase of Ca2+ in the microdomains
coupling the ER release channels to BK channels, as caffeine could
reinitiate SMOC activity in BAPTA-loaded cells. EGTA was totally
ineffective, indicating that the Ca2+ domain for
both pathways are restricted to <1-2 µm (Deisseroth et al.
1996
; Neher 1998
). In addition, the inability of
BAPTA to block SMOC initiation by caffeine indicates that the increase in intracellular Ca2+ concentration following
release from the ER release channel must be quite high as well as very
localized. Thus we propose that the cytoplasmic face of the plasma
membrane BK channels is very likely within 10-20 nm of the ER release
channel, and furthermore, that the Ca2+
concentration within this local domain conceivably may reach 100 µM.
SMOCs are short lived, with a half decay time of ~6-7 ms at 10 mV.
Ca2+ removal from the vicinity of the BK channels
must be very efficient and rapid if the SMOC decay time primarily
reflects BK channel closing (Satin and Adams 1987
).
Furthermore, the fact that SMOC time course was readily altered by
conditions, i.e., the presence of caffeine or ryanodine, that change
the Po of the release channel suggest
that the Ca2+ release event normally is very
brief and transient. In neurons, like other cell types,
Ca2+ homeostasis is highly regulated, with
numerous mechanisms to restore resting Ca2+
levels after a transient rise. Active uptake of
Ca2+ into the ER by the SERCA pump might be a
critical mechanism (Gomez et al. 1996
;
Steenbergen and Fay 1996
). However, SMOC decay time was
not altered in the presence of CPA, suggesting that removal of
Ca2+ by the SERCA pump does not determine SMOC
time course. Recently Cseresnyes et al. (1997)
described
a novel, fast Ca2+ uptake mechanism in frog
sympathetic neurons, activated by Ca2+ released
from internal stores. A similar mechanism could potentially be involved
in rapidly removing the Ca2+ that gated SMOC
activity, although more study is required to establish determinants of
SMOC decay.
Neuronal SMOCs are quite similar to spontaneous transient outward
currents (STOCs) recorded from smooth muscle cells (Bolton and
Imaizumi 1996; Nelson et al. 1995
). The STOCs
are thought to represent synchronous activation of BK channels by
transient, highly localized elevations of Ca2+
released from ryanodine-sensitive internal stores (Nelson et al.
1995
). Nelson and colleagues (1995)
have
provided evidence that rapid, transient Ca2+
signals, termed "Ca2+ sparks," are
responsible for STOC generation in arterial smooth muscle cells. We
postulate that a similar highly localized, rapid Ca2+ transient (i.e., neuronal sparks) could be
responsible for SMOC activation in mudpuppy neurons. However, given
that mudpuppy SMOCs have a faster time course than smooth muscle STOCs,
the intracellular Ca2+ transient may be faster
and more effectively buffered in neurons than in smooth muscle. A
recent study in PC12 cells and hippocampal neurons (Koizumi et
al. 1999
) has demonstrated Ca2+ release
signals that may be important in neuronal signaling pathways. However,
these Ca2+ signals have durations >200
ms and thus are unlikely to be similar to the
Ca2+ release events that underlie SMOC activity
in mudpuppy parasympathetic neurons.
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ACKNOWLEDGMENTS |
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The authors thank Dr. John Dempster for providing the SCAN program. The authors also thank Drs. Mark Nelson and Joseph Patlak for helpful discussions during the course of this study and Dr. Patlak for critical comments during the preparation of this manuscript.
This work was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-23978 to R. L. Parsons and American Heart Association Grant 9820031T to F. S. Scornik.
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FOOTNOTES |
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Address reprint requests to R. L. Parsons.
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 3 November 1998; accepted in final form 1 April 1999.
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REFERENCES |
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