Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
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ABSTRACT |
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Artim, Debra E. and
Stephen D. Meriney.
G-Protein-Modulated Ca2+ Current With Slowed
Activation Does Not Alter the Kinetics of Action Potential-Evoked
Ca2+ Current.
J. Neurophysiol. 84: 2417-2425, 2000.
We have studied voltage-dependent inhibition of
N-type calcium currents to investigate the effects of G-protein
modulation-induced alterations in channel gating on action
potential-evoked calcium current. In isolated chick ciliary ganglion
neurons, GTPS produced voltage-dependent inhibition that exhibited
slowed activation kinetics and was partially relieved by a conditioning
prepulse. Using step depolarizations to evoke calcium current, we
measured tail current amplitudes on abrupt repolarization to estimate
the time course of calcium channel activation from 1 to 30 ms. GTP
S prolonged significantly channel activation, consistent with the presence of kinetic slowing in the modulated whole cell current evoked
by 100-ms steps. Since kinetic slowing is caused by an altered voltage
dependence of channel activation (such that channels require stronger
or longer duration depolarization to open), we asked if GTP
S-induced
modulation would alter the time course of calcium channel activation
during an action potential. Using an action potential waveform as a
voltage command to evoke calcium current, we abruptly repolarized to
80 mV at various time points during the repolarization phase of the
action potential. The resulting tail current was used to estimate the
relative number of calcium channels that were open. Using action
potential waveforms of either 2.2- or 6-ms duration at half-amplitude,
there were no differences in the time course of calcium channel
activation, or in the percent activation at any time point tested
during the repolarization, when control and modulated currents were
compared. It is also possible that modulated channels might open
briefly and that these reluctant openings would effect the time course
of action potential-evoked calcium current. However, when control and
modulated currents were scaled to the same peak amplitude and
superimposed, there was no difference in the kinetics of the two
currents. Thus voltage-dependent inhibition did not alter the kinetics
of action potential-evoked current. These results suggest that
G-protein-modulated channels do not contribute significantly to calcium
current evoked by a single action potential.
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INTRODUCTION |
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High-voltage-activated, N-type
calcium channels are involved in many cellular processes including
neurotransmitter release. Inhibition of calcium current is a
well-documented mechanism of presynaptic modulation of neurotransmitter
release (Wu and Saggau 1997) and is induced by a variety
of neurotransmitters and peptides that exert their effects via
G-protein-coupled receptors (Dolphin 1998
; Hille
1994
). Many studies have investigated the mechanisms underlying
G-protein modulation of calcium channels and have revealed that a
membrane-delimited pathway, likely involving the direct interaction of
G-protein
subunits with the calcium channel, underlies many
instances of calcium channel inhibition (Herlitze et al.
1996
; Ikeda 1996
).
An intriguing characteristic of this inhibition is that it is often
voltage dependent (reviewed by Jones and Elmslie 1997). This voltage dependence is typically demonstrated by the ability of a
strong depolarizing step to relieve a large fraction of the inhibition
(Elmslie et al. 1990
). In whole cell recordings of calcium current, voltage-dependent inhibition is characterized by a
slowing of the activation kinetics, commonly referred to as kinetic
slowing (Boland and Bean 1993
; Elmslie and Jones
1994
; Golard and Siegelbaum 1993
). It is thought
that kinetic slowing is an example of relief of inhibition. The
sustained stimulus provided by a long step depolarization moves
modulated (reluctant) channels out of their reluctant mode into a
willing mode from which they open. This kinetic change is caused by a
positive shift in the voltage dependence of calcium channel activation
such that modulated channels require a stronger or longer
depolarization to move from a reluctant to willing state and then open
(Bean 1989
). So while unmodulated channels activate
normally, modulated channels will open more slowly, causing the
appearance of slowed calcium current activation during prolonged
depolarizations in whole cell recordings. Consistent with this
hypothesis, single-channel studies of calcium current inhibition have
demonstrated that voltage-dependent inhibition results in an increase
in the latency to first channel opening (Carabelli et al.
1996
; Patil et al. 1996
). This increase is
correlated with the presence of kinetic slowing in ensemble currents
and is relieved by a strong conditioning depolarization.
The fact that voltage-dependent inhibition can be relieved by
strong depolarizations suggests that it may be sensitive to physiologically relevant voltage changes. In support of this idea, inhibition can be partially relieved by short trains of 1- to 2-ms step
depolarizations designed to mimic action potential (AP) stimulation (Williams et al. 1997; Womack and
McCleskey 1995
). Furthermore it has been shown that trains of
AP waveforms can relieve significantly inhibition of N-type channels in
chick dorsal root ganglion (DRG) neurons (Park and Dunlap
1998
) and of P/Q-type channels expressed in HEK 293 cells
(Brody et al. 1997
). Recently it has been shown that
relief of G-protein-mediated inhibition of calcium current contributes
to short-term synaptic facilitation in hippocampal autapses
(Brody and Yue 2000
). Thus voltage-dependent inhibition
may represent an activity-dependent form of modulation that fine tunes
the degree of transmitter release based on neuronal activity.
It is also possible that calcium channels open directly from the
modulated (reluctant) state. Recent studies have identified neurotransmitter-induced alterations in calcium channel gating that
provide evidence for calcium channel opening from the reluctant state
(Colecraft et al. 2000; Lee and Elmslie
2000
). These openings occurred without a delay in first latency
and with lower open probability and briefer open times than in the
normal gating mode. These reluctant openings are induced by
depolarizations within the range of membrane voltage typically reached
during an AP (equivalent to 0-10 mV in physiological calcium
concentration). Thus modulation-induced changes in channel gating may
alter the kinetics of calcium entry in two ways. The increase in first
latency of channel opening (due to the slow transition between the
reluctant and willing gating modes), and the presence of very brief,
direct reluctant openings may both be important determinants for the
time course of calcium entry.
The physiologic role of modulation-induced alterations in channel
gating kinetics remains unclear. While there are studies, discussed in
the preceding text, that address this issue with respect to
single-channel events, there are no studies that focus on potential
effects during a single AP. Modulated calcium channel gating could
alter the timing of peak current and thus the synaptic delay for
transmitter release. Alternatively if modulated channels gate too
slowly or briefly, they may not contribute significantly to AP-evoked
current. In this case, modulated channels would be effectively
eliminated from contributing to transmitter release. To examine this
issue, we have investigated the effects of voltage-dependent inhibition
on calcium current evoked by single AP waveforms. Using GTPS to
induce G protein-mediated, voltage-dependent inhibition of calcium
current and AP waveforms as voltage commands, we have recorded N-type
calcium current from the somata of chick ciliary ganglion neurons.
Analysis of the tail currents resulting from abrupt repolarization at
various times during the AP has enabled us to evaluate the time course
of calcium channel activation during a single AP. The results
demonstrate that the presence of voltage-dependent inhibition does not
alter the time course of calcium channel activation during a single AP,
suggesting that modulated channels do not contribute significantly to
AP-evoked calcium current.
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METHODS |
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Culture
Ciliary ganglia from White Leghorn chicken embryos at stage 40 (Hamburger and Hamilton 1951) were dissected in
oxygenated Tyrode containing (in mM) 134 NaCl, 3 KCl, 3 CaCl2, 1 MgCl2, 12 glucose,
and 20 NaH2CO3, pH 7.2. The
ganglia were then incubated at 37°C in collagenase (0.5 mg/ml)
followed by trypsin (0.08%) in Ca2+- and
Mg2+-free Tyrode for 20 min each. The ganglia
were mechanically dissociated by trituration through a fire-polished
Pasteur pipette in minimal essential media (MEM) with 10%
heat-inactivated horse serum and the suspension of cells was
centrifuged for 5 min at 500 g. The cell pellet was
resuspended in MEM plus 10% chick embryo extract, plated onto
poly-D-lysine-coated 35-mm plastic culture dishes and
incubated at 37°C. Cells were used for experiments 1-8 h after plating.
Electrophysiology
Calcium currents were recorded with an Axopatch 200B patch-clamp
amplifier in the gigaohm-seal whole cell configuration (Hamill et al. 1981). To isolate calcium currents, the cells were
bathed in a solution containing (in mM) 100 NaCl, 50 TEA-Cl, 10 HEPES, 5 glucose, 5 CaCl2, 2 MgCl2, 5 KCl, 5 3,4-diaminopyridine, and 500 nM
TTX. Pipettes were pulled on a Flaming/Brown Micropipette Puller
(Sutter Instruments; model P-97), coated with silicone elastomer
(Sylgard; Dow Corning, Midland, MI) and heat-polished. Electrode
resistance ranged from 0.5 to 2 M
. Pipettes were filled with an
internal solution containing (in mM) 120 CsCl, 10 HEPES, 11 EGTA, 5 TEA-Cl, 1 CaCl2, and 4 MgCl2 with 4 ATP-Mg, 0.3 GTP-Na, and 0.1 leupeptin added fresh daily to retard run down of calcium currents.
Series resistance was measured by changes in the area and decay time
constant of the capacitive transient associated with a 10-mV
hyperpolarizaing voltage step and was 3.9 ± 1.0 M
(mean ± SD; n = 129). Currents were activated, acquired, and
leak-subtracted using a standard P/4 protocol by the software package
pClamp6 (Axon Instruments, Foster City, CA) running on a
Pentium-processor-based microcomputer. Currents were filtered using a
four-pole Bessel filter at 5 kHz and digitized at 25 kHz. Series
resistance was compensated by 80-90%, and a 5.8-mV liquid junction
potential was corrected before each recording. All cells were studied
at room temperature (22-25°C) and, with the exception of the series resistance value reported in this section, all values are expressed as
mean ± SE.
Action potential waveforms
Two different APs were used to construct the model AP waveforms
used here. The fast current-clamp mode of the Axopatch 200A (Magistretti et al. 1998), was used to evoke and record
APs at room temperature from small neurons (capacity = 6-7 pF),
presumed to be choroid neurons from the ciliary ganglion (see
Smith and O'Dowd 1994
). The perforated patch technique
was used with K2SO4 and KCl
replacing Cs2SO4 and CsCl
in the internal pipette solution and Tyrode saline as the external
solution. One of the choroid APs was used as a voltage command to evoke
calcium current. The chick DRG neuron AP was adapted from Park
and Dunlap (1998)
.
Pharmacological agents
Some experiments using GTPS were performed in the presence of
nifedipine (RBI, Natick, MA), which was solubilized in DMSO at 1 mM and
diluted into bath saline at 1 µM (0.1% DMSO final dilution). This
effectively isolates N-type calcium current in stage 40 ciliary
ganglion neurons (White et al. 1997
). There was no
significant difference between data obtained in the presence or absence
of nifedipine; thus the data were pooled. Somatostatin (SOM) and
GTP
S were obtained from Sigma (St. Louis, MO). SOM was dissolved in
deionized water to make a 10 µM stock solution, stored at
20°C,
and diluted into bath saline to a final concentration of 100 nM.
GTP
S was diluted into internal pipette saline to make 2 mM stock,
stored at
80°C, and used at a final concentration of 200 µM.
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RESULTS |
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GTPS-induced voltage-dependent inhibition of N-type calcium
current
Neurotransmitter-mediated modulation occurs via activation of G
proteins following receptor-ligand binding. To activate G proteins
directly, 200 µM GTPS was included in the intracellular pipette
solution. On obtaining the whole cell configuration, GTP
S caused a
rapid inhibition of calcium current that reached a steady state 5-10
min after obtaining whole cell access. Current was evoked every 10 s by a 100-ms voltage step from
80 to +10 mV. GTP
S inhibited peak
calcium current density by 54 ± 2.2% (Fig. 2A), and
the inhibition was partially voltage-dependent as measured by a
double-pulse protocol (Fig.
1A). During the first test
pulse, the current exhibited slowed activation kinetics. A conditioning step given 10 ms before the second test pulse increased the current magnitude and restored normal activation kinetics. The effect of the
conditioning step on relief of inhibition was expressed as the ratio of
the magnitude of current evoked by a step depolarization 10 ms after
the conditioning step to the magnitude of current evoked without a
preceding conditioning pulse. This ratio was calculated in all cells at
the beginning and end of each experiment (Fig. 1, B and
C). The voltage-dependent inhibition induced by GTP
S is
apparent by the significant increase in facilitation ratio (1.35 ± 0.05 for GTP
S vs. 0.90 ± 0.02 for control,
P < 0.001). A significant increase in facilitation
ratio persisted throughout the length of the recording and was still
present at the end of the experiment (1.22 ± 0.04 for GTP
S vs.
0.91 ± 0.03 for control, P < 0.01). Figure
2A shows
the current-voltage relationship for calcium currents both in the
control condition and with GTP
S included in the patch pipette. To
facilitate comparisons among cells of varying size, current is
expressed as current density (pA/pF). There was a robust inhibition of
calcium current at potentials at or near the potential that evokes peak
calcium current (0 mV) but little to no inhibition at more depolarized
potentials (Fig. 2B). GTP
S did not effect the potential
that evoked peak calcium current or the measured calcium reversal
potential. The voltage dependence of calcium channel activation was
determined by stepping to various potentials for 5 ms. The tail
currents that resulted from the repolarization to
80 mV were measured
and normalized to the maximum tail current from each cell (Fig.
2C). Each plot was fit by a Boltzmann function. As expected
for voltage-dependent inhibition, GTP
S caused a shift in the voltage
dependence of the channels to more depolarized potentials and a
decrease in the steepness of the curve.
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Time course of calcium channel activation during a step depolarization
To investigate the effects of calcium channel inhibition on the
time course of channel activation, we used square pulses to +30 mV of
varying duration (0.5-30 ms) to evoke calcium current. Steps to +30 mV
were used here to facilitate comparison of these data with those
obtained by AP waveforms that peaked at +30 mV. Tail currents resulting
from the repolarization to 80 mV were measured and used as an
estimate of the relative number of calcium channels open after that
duration of depolarization. This protocol revealed a difference in
calcium channel activation between control and GTP
S-modulated
conditions (Fig. 3, A-C).
Control calcium current activated rapidly with depolarization and
reached peak activation after approximately 2-3 ms of depolarization.
In contrast, modulated current activated more slowly, requiring at
least 15 ms of depolarization to be activated maximally. The percent
activation of calcium current differed between control and GTP
S
conditions at each time point tested from 1 to 10 ms of depolarization
(Fig. 3C). Thus GTP
S slowed significantly the time course
of calcium channel activation.
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Time course of calcium channel activation during an AP
To investigate calcium channel activation during an AP, we used a
series of modified AP waveforms as voltage commands to evoke calcium
current (see Pattillo et al. 1999). The AP was recorded from a chick ciliary ganglion neuron (see METHODS), scaled
to a peak of +30 mV and a resting potential of
60 mV, and was 2.2 ms
in duration at half-amplitude (Fig.
4A). The rising phase was modeled by a series of ramps that faithfully followed the actual AP
waveform and was followed by a brief plateau phase. The AP falling
phase was simplified to a single ramp to allow for consistent alterations. The AP waveform was repolarized abruptly to
80 mV at
various time points during the repolarizing phase. We measured the tail
current resulting from this step to
80 mV and used this measurement
as an estimate of the relative number of calcium channels open at any
given point of the AP (Fig. 4B). To correct for rundown during the course of the experiment, each tail current evoked by a
modified AP was compared with a control recording taken within 30 s of the experimental recording. Additionally, the order in which the
modified AP waveforms were applied was varied among experiments. These
voltage commands were given either under normal whole cell conditions
or with GTP
S included in the patch pipette to induce
voltage-dependent inhibition of calcium current. Figure 5A shows the summary data
obtained using this protocol. Each tail current ratio (experimental
tail current/control current) was normalized to the maximum ratio
recorded from each cell (defined as 100%). The ratios were then
plotted as a function of percent AP repolarization. Plotting the data
in this manner allows one to determine the time course of calcium
current activation during the falling phase of an AP. GTP
S did not
alter the time course of channel activation during the AP as peak
activation occurred at the same point in the AP in both control and
inhibited conditions. Furthermore there were no significant differences
between control and GTP
S in the percent activation at any time
during the repolarization. Since the proportion of current that
displays kinetic slowing has been shown dependent on the amplitude of
the test potential (Golard and Siegelbaum 1993
), calcium
current activation was also studied during AP waveforms that peaked at
+10 mV. As with the AP waveforms to +30 mV, there were no significant
differences in percent activation between control and GTP
S-modulated
currents (Fig. 5B).
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Increasing AP duration fails to reveal a change in activation time course
To test the hypothesis that the choroid AP used in the preceding
text did not provide sufficient stimulus to convert reluctant channels
to a willing gating mode, we tested the effects of an AP of longer
duration. A chick DRG AP adapted from Park and Dunlap (1998) (
80-mV resting potential, +24-mV peak amplitude, and
6-ms duration at half-amplitude) was used to create a series of
modified AP waveforms as described in the preceding text (see Fig.
6A). Again, we measured the
tail currents that resulted from abrupt repolarization to
80 mV at
various times during the repolarizing phase of the AP and used this
measure to estimate the proportion of calcium channels open at that
time. We hypothesized that this AP waveform would provide sufficient
depolarization to recruit kinetically slowed channels and thus slow the
time course of whole cell current activation when channels were
modulated. However, as with the choroid AP waveforms, GTP
S did not
change the time course of channel activation during this long AP (Fig.
6B).
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Relationship between kinetic slowing and voltage dependence of inhibition
Many groups have reported the presence of slowed activation
kinetics together with voltage-dependent calcium channel inhibition (reviewed by Jones and Elmslie 1997). Although it is not
necessarily apparent in all cases of voltage-dependent inhibition,
kinetic slowing is thought to be a marker of voltage-dependent
inhibition. Thus we sought to look for a correlation between kinetic
slowing and the degree of voltage-dependent inhibition to determine the extent to which these two phenomena are coincident. Data for this experiment were obtained from 47 cells to which 100 nM SOM was applied
to produce voltage-dependent inhibition. In these cells, SOM has been
shown to inhibit preferentially N-type calcium channels in a
voltage-dependent manner, exhibiting kinetic slowing when measured
using traditional whole cell recording techniques (Meriney et
al. 1994
; White et al. 1997
). Measures of
kinetic slowing and of prepulse relief of inhibition were taken from
each cell. Kinetic slowing was quantified as follows. Calcium current
was evoked by a 100-ms step depolarization to +10 mV in the presence
and absence of 100 nM SOM. The percent SOM-induced inhibition was measured at the time of peak control calcium current and after 95 ms of
depolarization. Kinetic slowing was defined as the ratio of percent
inhibition at the peak to the percent inhibition late in the
depolarization (Fig. 7A). The
voltage dependence of inhibition was determined with a standard
double-pulse protocol (see Fig. 1A). A 30-ms depolarizing
prepulse to +100 mV was applied 10 ms before a second test pulse. This
prepulse relieved partially the SOM-induced inhibition, providing a
robust measure of the voltage dependence of inhibition. There was a
significant correlation (R = 0.51, P < 0.0003) between kinetic slowing and the voltage dependence of
inhibition (Fig. 7C).
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We also investigated the relationship between kinetic slowing and relief of inhibition produced by an AP train. Kinetic slowing was quantified as described in the preceding text, but now voltage dependence was defined as the inhibition relieved by a train of eight choroid AP waveforms delivered at 100 Hz (Fig. 7B). The percent inhibition of current evoked by the eighth AP in the train was compared with the percent inhibition of current evoked by the first AP in the train to produce a measure of inhibition relief, thus an indication of the voltage dependence of inhibition. Again, there was a significant correlation (R = 0.63, P < 0.0001) between kinetic slowing and the degree of inhibition relief produced by the AP train (Fig. 7D). Thus kinetic slowing reliably predicted the extent of activity-dependent relief induced by this AP train.
Time course of action potential-evoked calcium current
To examine the effect of SOM-induced inhibition on the activation time course of AP-evoked currents, we compared control and SOM-modulated calcium currents evoked by the first and the eighth APs in a 100-Hz train. To facilitate a comparison of the activation of these currents, all traces were normalized to the maximum current evoked from each cell. Thus control and modulated currents were scaled to be of identical amplitude. Data were pooled from 18 cells in which the kinetic slowing ratio was 1.53 or greater and expressed as the mean normalized current ± SE (Fig. 8A). There was no difference in the time course of current activation between control and modulated currents. Examination of the calcium current evoked from the cell displaying the greatest degree of kinetic slowing (kinetic slowing ratio = 2.93; Fig. 8B), also showed that the time course of current activation was identical for control and modulated currents when the currents were normalized to the same peak amplitude (Fig. 8C).
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DISCUSSION |
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Calcium current inhibition is a well-documented mechanism
underlying presynaptic inhibition (Wu and Saggau 1997).
Thus the voltage dependence of inhibition may provide a mechanism
whereby transmitter release is modulated based on the level of neuronal activity. Our investigation has focused on the effects of
G-protein-mediated, voltage-dependent inhibition on AP-evoked calcium
current. Using traditional whole cell voltage-clamp methods, we have
used GTP
S to induce voltage-dependent inhibition of N-type calcium
current in chick ciliary ganglion neurons. The inhibition displayed the characteristics common to voltage-dependent inhibition: altered voltage
dependence of steady-state activation, relief by a conditioning prepulse, and kinetic slowing.
To address the physiologic relevance of calcium current inhibition, we
have looked for functional consequences of G-protein-mediated modulation that occur in a voltage-dependent manner in response to
single AP stimulation. These are hypothesized to occur if significant numbers of modulated channels either open directly from the reluctant state (very brief opening), or convert to the willing gating mode (delayed opening) during a single AP depolarization. A significant current contribution from either of these types of openings would be
expected to alter the time course of calcium entry during an AP and
thus affect synaptic delay and/or the magnitude of transmitter released
(Sabatini and Regehr 1996). The absence of a significant contribution argues that voltage-dependent modulation is relevant only
during trains of APs as has been suggested previously (Brody and
Yue 2000
; Brody et al. 1997
; Park and
Dunlap 1998
).
Using APs of different duration and amplitudes, we did not find any
evidence that voltage-dependent modulation altered the time course for
calcium current entry during a single AP. Taken together, these results
suggest that modulated N-type channels do not contribute significantly
to calcium current evoked by a single AP in chick ciliary ganglion
neurons. The absence of measurable effects could be due to several
issues. Studies that have focused on kinetic slowing, both at the whole
cell and single channel levels, have evoked calcium current using
sustained step depolarizations (Bean 1989; Grassi
and Lux 1989
; Patil et al. 1996
). This protocol reveals a robust change in activation kinetics associated with voltage-dependent inhibition of calcium current. However, comparably less is known about how channels behave when stimulated with APs. A
ramp depolarization (as occurs during the AP rising phase) and a step
depolarization may produce a different time course of channel activation. Additionally, an AP may not depolarize a cell to the same
degree as a step depolarization of the same duration. In our recordings
of modulated calcium current, there is evidence that modulated channels
are partially recruited with a 2- or 6-ms step depolarization but not
with an AP of 2- or 6-ms duration at half-amplitude. However, the AP is
not an equivalent stimulus, producing much less depolarization than a
step pulse of similar duration. Given the dramatic increase in latency
to first channel opening (10- to 20-fold) reported for modulated N-type
calcium channels (Carabelli et al. 1996
), modulated
channels may not be recruited to convert to a willing gating mode by a
single AP, even of relatively long duration.
Another issue to consider when interpreting these data is that if
direct channel openings from the reluctant mode occur, they may in fact
contribute a very small percentage of the total AP-evoked current.
Single-channel evidence of reluctant channel openings has shown them to
be infrequent and to have a very brief mean open time (Colecraft
et al. 2000). Thus direct reluctant openings, if they occur,
may contribute a very small amount of current that is not detectable in
our whole cell recordings.
Our results suggest that activated G proteins may result in the
modulation of a proportion of the calcium channels such that during an
AP, they are effectively closed and prevented from contributing to
calcium influx. This could have both temporal and spatial consequences on transmitter release. Given the nonlinear relationship between calcium influx and transmitter release (see Augustine and
Charlton 1986), even small increases in calcium influx caused
by AP train-induced relief of inhibition could produce significant
increases in the magnitude of transmitter released. In a temporal
sense, relief of voltage-dependent inhibition has been shown to occur
with a train or burst of APs (Brody et al. 1997
;
Park and Dunlap 1998
), and this may contribute to
short-term synaptic facilitation (Brody and Yue 2000
).
Activation of G proteins has also been shown to enhance paired-pulse
synaptic facilitation (Dittman and Regehr 1997
;
Dunwiddie and Hass 1985
). If modulated calcium channels are effectively not contributing to single AP-evoked release but can be
recruited with bursts of APs, this suggests that release sites
positioned near modulated channels may become functional only near the
end of a burst of APs. Thus G-protein modulation of calcium current
could influence not only the degree of transmitter released with a
single AP but may also influence when transmitter release is
facilitated during a burst of activity.
The spatial arrangement of modulated channels could also have profound
effects on transmitter release. Models of calcium current inhibition
and transmitter release have suggested that both the degree of
inhibition and facilitation of release are greatly influenced by the
location and distribution of modulated and unmodulated channels
(Bertram and Behan 1999). For example, if modulated
channels reside near release sites, there would be increased
facilitation of release during bursts of APs. This could be
particularly relevant in nerve terminals if the calcium channels
coupled to release sites (through interaction with syntaxin) are most
susceptible to regulation by G proteins (see Stanley and
Mirotznik 1997
).
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ACKNOWLEDGMENTS |
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We thank J. Simples, R. Poage, J. Pattillo, D. King, and J. Dilmore for many helpful discussions and critical evaluation of the manuscript.
This work was supported by National Institutes of Health Grants NS-32345 (S. D. Meriney) and MH-18273 (D. E. Artim) and by a Grant-in-Aid from the American Heart Association (S. D. Meriney).
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FOOTNOTES |
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Address for reprint requests: S. D. Meriney, Dept. of Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260 (E-mail: Meriney{at}imap.pitt.edu).
Received 13 March 2000; accepted in final form 31 July 2000.
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REFERENCES |
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