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The Journal of Neuroscience, February 1, 1998, 18(3):878-886
The Ca2+ Channel 
3 Subunit
Differentially Modulates G-Protein Sensitivity of 
1A and
1B Ca2+ Channels
John P.
Roche and
Steven N.
Treistman
Department of Pharmacology and Molecular Toxicology, Program in
Neuroscience, University of Massachusetts Medical School, Worcester,
Massachusetts 01655
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ABSTRACT |
We have shown previously that the Ca2+ channel
3 subunit is capable of modulating tonic G-protein
inhibition of 
1A and 1B Ca2+ channels expressed in oocytes. Here we
determine the modulatory effect of the Ca2+ channel
3 subunit on M2 muscarinic
receptor-activated G-protein inhibition and whether the
3 subunit modulates the G-protein sensitivity of
1A and 1B currents equivalently. To
compare the relative inhibition by muscarinic activation, we have used
successive ACh applications to remove the large tonic inhibition of
these channels. We show that the resulting rebound potentiation results entirely from the loss of tonic G-protein inhibition; although the
currents are temporarily relieved of tonic inhibition, they are still
capable of undergoing inhibition through the muscarinic pathway. Using
this rebound protocol, we demonstrate that the inhibition of peak
current amplitude produced by M2 receptor activation is
similar for 1A and 1B calcium currents.
However, the contribution of the voltage-dependent component of
inhibition, characterized by reduced inhibition at very depolarized
voltage steps and the relief of inhibition by depolarizing prepulses,
was slightly greater for the 1B current than for the
1A current. After co-expression of the 3
subunit, the sensitivity to M2 receptor-induced G-protein inhibition was reduced for both 1A and 1B
currents; however, the reduction was significantly greater for
1A currents. Additionally, the difference in the voltage
dependence of inhibition of 1A and 1B
currents was heightened after co-expression of the
Ca2+ channel 3 subunit. Such
differential modulation of sensitivity to G-protein modulation may be
important for fine tuning release in neurons that contain both of these
Ca2+ channels.
Key words:
Ca2+ channels; G-proteins; 1A; 1B; Ca2+ channel subunit; voltage-dependent
inhibition; Xenopus oocyte; muscarinic M2
receptor; NEM
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INTRODUCTION |
Although the types of
Ca2+ channels present in neuronal synapses are
variable, there are numerous instances in which the N-type (1B) and P/Q-type (1A) are
colocalized presynaptically. The relative contribution of these channel
types to synaptic transmission is variable but often mediated by both,
acting in concert (Leubke et al., 1993
; Takahashi and Momiyama, 1993;
Castillo et al., 1994; Wheeler et al., 1994, 1996; Mintz et al., 1995;
Regehr and Mintz, 1996). Ca2+ has been shown to act
cooperatively in the presynaptic terminal to cause release of
neurotransmitter, and as a result, small changes in the amount of
Ca2+ entering the presynaptic neuron have large
effects on synaptic transmission (Dodge and Rahamimoff, 1967;
Heidleberger et al., 1994; Heinemann et al., 1994). Thus, even subtle
differences in modulation of the Ca2+ channel types
mediating neurotransmission would profoundly alter release.
Multiple varieties of Ca2+ channel inhibition by
heterotrimeric G-proteins have been documented. The most common form of
inhibition occurs in a voltage-dependent manner through activation of a
pertussis toxin (PTX)-sensitive G-protein. In voltage-clamp studies of
this type of inhibition, application of neurotransmitter causes a
slowing of the current activation kinetics, and current inhibition is greater at less depolarized voltages (Marchetti et al., 1986; Wanke et
al., 1987; Bean, 1989; Kasai and Aosaki, 1989). Other forms of
inhibition occur in a voltage-independent manner, involving activation
of a second messenger cascade (Beech et al., 1991; Bernheim et al.,
1991), or through a membrane-delimited pathway (Shapiro and Hille,
1993; Diverse-Pierluissi et al., 1995; Wollmuth et al., 1995).
Several Ca2+ channel 1 subunits as
well as a number of auxiliary subunits have been cloned recently
(Miller, 1992; Birnbaumer et al., 1994; Isom et al., 1994; Perez-Reyes
and Schneider, 1995; Catterall, 1996). The 1 subunit has
a putative structure similar to other channels in the voltage-gated ion
channel family, and expression of the 1 subunit alone is
sufficient for the formation of functional Ca2+
channels (Perez-Reyes et al., 1992). Current through 1
channels can be modulated by co-expression of auxiliary subunits. The
intracellularly situated subunit affects the amplitude and kinetics
of the Ca2+ currents of several cloned channels
(Lacerda et al., 1991; Mori et al., 1991; Wei et al., 1991; Williams et
al., 1992; Brust et al., 1993; Ellinor et al., 1993; Sather et al.,
1993; Soong et al., 1993; Stea et al., 1993).
We have shown previously that 1A and 1B
Ca2+ channels expressed in Xenopus
oocytes are tonically inhibited by G-proteins, demonstrated by blockade
of a basally active G-protein population (Roche et al., 1995). Although
much useful information can be derived using this approach, further
characterization of the G-protein inhibition is better served by use of
an experimental paradigm in which the inhibition of
Ca2+ channels is controlled by activation of a
single receptor type, in a reversible manner. In the present study, we
use the muscarinic M2 receptor as a G-protein activation
pathway, incorporating an experimental paradigm in which the inhibition
results completely from muscarinic receptor activation, in the absence
of background tonic G-protein inhibition. We demonstrate that this
condition can be met and compare the G-protein inhibition of
1A and 1B Ca2+
currents before and after co-expression of the Ca2+
channel 3 subunit. Both the degree of inhibition and the
contributions of the voltage-dependent and voltage-independent
components of the inhibition were dependent on channel subunit
composition. Additionally, these parameters of 1A and
1B current inhibition were differentially modulated by
co-expression of the Ca2+ channel 3
subunit.
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MATERIALS AND METHODS |
Expression plasmids and oocyte preparation. Capped
RNA transcripts encoding full-length 1A (XbaI
linearized/SP6 RNA polymerase; gift of Dr. Y. Mori, University of
Cincinnati Medical Center), 1B (SalI/SP6;
gift of Dr. Y. Fujita, Kyoto University), and 3 (NotI/T7; gift of Dr. Edward Perez-Reyes, Loyola University
Medical Center) calcium channel subunits as well as the M2
muscarinic receptor (EcoRI, BglII/T7; gift of Dr.
Wolfgang Sadee, University of California, San Francisco) were
synthesized using a mMESSAGE mMACHINE in vitro transcription
kit (Ambion, Austin, TX). Xenopus laevis stage V-VI oocytes
were removed and treated with collagenase (Sigma type IV; Sigma, St.
Louis, MO) to remove the follicular layer. The oocytes were then
injected with cRNA encoding the M2 receptor along with
either 1A, or 1B alone (1:1) or in
combination with 3 (1:1:1). The concentration of all
individual RNAs before injection was 0.1 µg/µl, and 20-60 nl of
RNA mixed at the above ratios was injected. The oocytes were maintained
in culture at 18°C for at least 2 d in ND-96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM
CaCl2, 5 mM HEPES, pH 7.5) supplemented
with 2.5 mM sodium pyruvate and 2 mg/ml of gentamycin.
Electrophysiological recording and experimental treatments.
Two-electrode voltage-clamp currents were recorded using a Dagan CA-1
amplifier. Oocytes were clamped at a holding potential of 
80 mV, and
several different voltage protocols were used (specific protocols can
be found in the figure legends). Currents were filtered at 1 kHz, and a
p/2 or p/4 leak subtraction technique was used. Analysis was performed
off line with pClamp software version 6.0.2 (Axon Instruments, Foster
City, CA). Electrodes contained 3 M KCl and had resistances
of 0.5-2 M
. Oocytes were placed in a 1 ml chamber and perfused at a
rate of 0.5 ml/min. All recordings were made at room temperature using
bath solutions containing (in mM): BaOH 10, NaOH 50, CsOH
2, TEA-OH 20, N-methyl-D-glucamine 20, HEPES 5, titrated to pH 7.5 with methane sulfonic acid. In all experiments, 40 nl of a 50 mM stock solution of
K3-1,2-bis(aminophenoxy)ethane-N,N,N
,N-tetra-acetic acid (BAPTA) (Sigma) was injected at least 2 hr before the experiment. The final concentration of BAPTA inside the oocyte was estimated to be
between 2 and 5 mM, assuming an oocyte volume of 1 µl.
Acetylcholine chloride (Sigma) was dissolved in the external recording
solution from a 100 mM stock to a final concentration of 50 µM. For experiments using n-ethylmaleimide
(NEM) (Aldrich), NEM was dissolved in the external solution at a
concentration of 200 µM and applied to the oocyte for 2 min. In experiments using PTX, oocytes were incubated in 4 µg/ml of
PTX in ND-96 solution for 72 hr. All histograms are mean ± SEM.
The n for these experiments is represented above the
histograms.
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RESULTS |
Characterization of tonic G-protein inhibition after co-expression
of the unliganded muscarinic receptor
Co-expression of the M2 muscarinic acetylcholine
receptor with 1A or 1B
Ca2+ channels allowed us to activate a PTX-sensitive
G-protein population, presumably either endogenous G
i or
Go (Lechleiter et al., 1991). The presence of the
muscarinic receptor did not appear to affect the availability of the
endogenous G-protein pool for tonic inhibition of 1A and
1B channels. Figure 1
shows that 1A and 1B channels expressed
in the oocyte are subject to tonic voltage-dependent G-protein
inhibition, in a manner similar to that reported previously (Roche et
al., 1995), apparently unaffected by the presence of the unliganded
co-expressed muscarinic receptor. The level of tonic inhibition was
determined by comparing the current generated during a test voltage
step in the absence or presence of a strongly depolarizing voltage step
given before the test voltage step (Elmslie et al., 1990; Ikeda, 1991;
Lopez and Brown, 1991). At strongly depolarized voltages the channel is
able to overcome the G-protein inhibition, perhaps by temporary dissociation of the G-protein from the channel (Bean, 1989; Lopez and
Brown, 1991). In addition to showing the lack of effect of M2 receptor expression on this tonic inhibition, Figure
1C also illustrates the elimination of prepulse facilitation
by NEM and near-elimination by PTX, agents that are thought to bind to
and uncouple the Gi/Go classes of
G-protein subunit from receptor activation. Elimination of tonic
G-protein inhibition by these agents after co-expression of the
unliganded receptor occurs essentially as in the absence of the
unliganded receptor (Roche et al., 1995). Thus, the basic
characteristics of the tonic G-protein inhibition of 1A
and 1B Ca2+ channels appeared
unchanged by the presence of the unliganded receptor.
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Figure 1.
Co-expression of the M2 muscarinic
acetylcholine receptor does not modify the tonic inhibition of
1A and 1B Ca2+
currents by a basally active PTX-sensitive G-protein population. A, Depolarizing prepulse protocol used to relieve
voltage-dependent G-protein inhibition. B, Currents
elicited using this protocol. The protocol consists of a voltage step
to +10 mV both before (PP) and after
(+PP) a depolarizing prepulse to +100 mV for 75 msec.
C, Mean facilitation (±SEM) of current amplitude using
this prepulse protocol for 1A (left) and
1B (right) currents. Represented is the
facilitation seen in control conditions (Con), after
application of 200 µM NEM (NEM),
and after incubation with 4 µg/ml PTX (PTX) for
72 hr. The number for each experiment is represented
above the respective histogram. For these experiments RNA encoding
either 1A or 1B was coinjected with RNA
encoding the M2 muscarinic ACh receptor.
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Removal of tonic G-protein inhibition
The presence of tonic G-protein inhibition could complicate the
interpretation of results obtained with muscarinic receptor activation,
if the tonic inhibition differentially affects the two channel types or
in some way differentially occludes the receptor-induced G-protein-mediated inhibition of 1A and
1B Ca2+ channels. Therefore, it is
important to demonstrate that tonic inhibition is indeed not present
during subsequent experiments, and we use a number of approaches to
confirm this. It has been reported previously that after
neurotransmitter-induced G-protein inhibition of
Ca2+ channels there is a refractory period after the
removal of the transmitter during which tonic G-protein inhibition of
the channels is occluded (Kasai, 1991), and we examined whether this
protocol might allow us to observe muscarinic inhibition in the absence of the background of tonic inhibition.
Activation of the M2 receptor by ACh resulted in inhibition
of both 1A (Fig.
2B, trace 2)
and 1B (Fig. 2D, trace 2)
currents beyond the level of tonic inhibition. Additionally, after
removal of the ACh there was a large (two- to threefold) rebound of
current amplitude (Fig. 2B,D, trace 3).
Figure 2 also illustrates the similarity in current amplitude and
kinetics during the rebound phase (Fig. 2B,D, trace
3) as compared with currents after removal of tonic inhibition by
the application of NEM (Fig. 2B,D, trace 4).
These similarities were consistent with a temporary loss of tonic
inhibition, and we investigated this further, using 1B Ca2+ currents.
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Figure 2.
Removal of ACh results in rebound potentiation of
current amplitude for both 1A and 1B
Ca2+ currents. A, C, Time course of
muscarinic-mediated G-protein inhibition for both 1A
(left) and 1B (right)
currents. The oocyte was held at a potential of 80 mV, and the oocyte
was stepped to the test potential of +10 mV every 15 sec. The
circles represent the peak current amplitude at the test
potential of +10 mV. The black lines represent
application of 50 µM ACh, whereas the gray lines represent application of 200 µM NEM. Spaces
in which no circles are present indicate time periods in which other
protocols were instituted. B, D, Current traces for
various time points labeled on the graph in A and
C. Note the similarity between the current kinetics and
amplitude of the rebound current (3) and the
current after treatment with NEM (4).
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If indeed the amplitude of the rebound current was larger because of
the loss of tonic G-protein inhibition, the facilitation of current
amplitude by depolarizing prepulses would be lost during this period.
When the depolarizing prepulse protocol was given either before
(Con.) or during (+ ACh.) ACh presentation,
significant prepulse facilitation was evident (Fig.
3A,B). However, after removal
of the ACh, during the period of potentiated current amplitude or
rebound (Reb.), very little or no prepulse facilitation was seen (Fig. 3A,B), providing support for the contention that
the tonic inhibition is lost during the period of rebound current potentiation.
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Figure 3.
Rebound current facilitation is a result of
temporary loss of tonic inhibition. A, 1B
currents elicited using the prepulse protocol illustrated in Figure
1A. Oocytes were stepped to +10 mV either with
(+PP) or without (PP) a depolarizing
voltage step to +100 mV for 75 msec. The prepulse was given 20 msec
before the test voltage step. Treatments: application of 50 µM ACh (+ ACH.); after the rebound of
current amplitude on removal of ACh (Reb.); and after
treatment of the oocyte with NEM (NEM). The representative currents were obtained from different oocytes. B, Summary of mean prepulse facilitation (±SEM) of
current amplitude after the various treatments (* denotes significant
difference from control; Student's t test;
p < 0.01). C, Summary of the mean potentiation (±SEM) of current by application of NEM
(hatched) and by removal of acetylcholine
(black). In all cases the control current was taken to
be the initial stable current amplitude (trace 1, Fig.
2A,C). The rebound potentiation
(black) was the peak current amplitude attained after
the removal of acetylcholine, both with (right) and
without (left) previous incubation with PTX. The mean percent potentiation of the peak current amplitude after application of
NEM (hatched) is also shown both with
(right) and without (left) PTX
pretreatment. D, Plot of current potentiation resulting
from NEM application versus potentiation resulting from removal of acetylcholine. The white squares represent oocytes in
which both rebound potentiation and NEM-induced potentiation were
measured. The black squares represent oocytes that were
pretreated with PTX and subsequently exposed to both treatments. The
correlation coefficient is 0.87 (p < 0.001). The slope of the linear fit = 1.04.
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Previously published data indicate that NEM relieves all of the tonic
inhibition, based on the loss of prepulse facilitation and the
occlusion of the actions of NEM by GDPS, which blocks all G-proteins
(Roche et al., 1995). Thus, for any given oocyte, if there is complete
loss of tonic inhibition during rebound, the degree of current
potentiation should be similar after NEM treatment and during the
rebound produced by withdrawal of transmitter. We measured the
potentiation after each of these treatments as the maximal current
attained after the treatment divided by the initial current (voltage
step to +10 mV). Frequently, as seen in Figure 2A,
the maximal rebound after ACh application was not attained after the
first application. Thus, repetitive ACh applications were given until a
maximal and stable level of rebound was attained. Figure 3C
shows that the mean potentiation observed in individual oocytes after
removal of acetylcholine was not significantly different from that
produced by exposure to NEM.
We also used this protocol with oocytes incubated with PTX for 72 hr
before the experiment to remove tonic inhibition. These oocytes were
still partially inhibited by ACh (36 ± 5.8%; n = 4), presumably through non-PTX-sensitive G-proteins. Little
potentiation was seen, however, either after removal of acetylcholine
or after application of NEM. The potentiation by NEM was plotted
against the potentiation resulting from rebound in individual oocytes. For these experiments, repetitive ACh applications were given until the
rebound potentiation reached a maximal steady state. The current was
then allowed to decay back to control values for a short period (~5
min), at which time NEM was applied to the oocyte. When the inhibition
from the two treatments was compared, the correlation coefficient was
0.87 and the slope of the linear fit was 1.04 (p < 0.0001), indicating a strong correlation (Fig. 3D). We
conclude from all of these data that the rebound of current amplitude
seen after the removal of ACh is a result of temporary loss of the
total tonic G-protein inhibition and that we can study receptor-induced
G-protein inhibition in the absence of tonic inhibition.
M2 receptor-induced inhibition of 1A and
1B currents in the absence of auxiliary subunits
Application of ACh and subsequent activation of the exogenously
expressed muscarinic M2 receptor during the rebound period caused a reduction of current amplitude (Fig.
4A,B), which was roughly equivalent for 1A and 1B
Ca2+ channels (77 ± 2%, n = 15, for the 1A current vs 79 ± 1%,
n = 26, for the 1B current). Application
of ACh to oocytes that did not contain exogenous M2
receptor had no effect on either 1A or 1B
Ca2+ currents (data not shown). Also apparent is the
slowed activation kinetics of both 1A and
1B currents after application of ACh, a hallmark of
voltage-dependent G-protein-mediated inhibition (Marchetti et al.,
1986; Wanke et al., 1987; Bean, 1989; Kasai and Aosaki, 1989). The
time-to-peak current amplitude during a 250 msec voltage step to +10 mV
was shifted from 20 ± 1 msec for control to 115 ± 1 msec
(n = 13) after the application of ACh for the
1A currents, and from a value of 30 ± 4 msec for
control to 131 ± 10 msec (n = 19) for the
1B currents. From these data we conclude that muscarinic
M2 receptor-induced inhibition of 1A and
1B currents is equivalent at +10 mV. Additionally, the inhibition of both 1A and 1B currents
contains voltage-dependent components, which produce similarly slowed
activation kinetics.
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Figure 4.
Comparison of muscarinic M2
receptor-induced G-protein inhibition of 1A and
1B currents in the absence of tonic inhibition. A, B, Inhibition of current amplitude by reapplication
of acetylcholine to oocytes during the rebound phase of current
amplitude, which resulted from removal of previous application of
acetylcholine. The current during the rebound period is taken as
control (Control), whereas the current after
application of acetylcholine is represented as + ACh.
C, Slowing of the activation kinetics after application of acetylcholine. D, Inhibition of peak current
amplitude by activation of the M2 receptor in the absence
of tonic inhibition at various test voltages for 1A
(gray) and 1B
(black) currents. (* represents significant difference
in inhibition of 1A and 1B currents at a
given test pulse voltage; Student's independent t test;
p  0.01).
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We also tested the magnitude of inhibition at various voltages to
determine whether the inhibition of the two channels was differentially
voltage sensitive. This protocol revealed that M2
receptor-induced reduction in current amplitude at more depolarized voltage steps was greater for 1A than for
1B (Fig. 4D), suggesting that although
activation of the M2 pathway produces a similar magnitude
of inhibition for the two channel types, the inhibition of
1B contains a larger voltage-dependent component.
The M2-induced inhibition of both 1A and
1B currents was partially relieved by depolarizing
prepulses (Fig. 5A,B), another hallmark of voltage-dependent G-protein inhibition (Elmslie et al.,
1990; Ikeda, 1991; Lopez and Brown, 1991). Figure 5C,D shows that between 20 and 50% of the inhibition could be relieved by depolarizing voltage. Examination of the data in histogram form (Fig.
5E) demonstrates the divergence in the amount of inhibited current that was relieved by depolarizing prepulses, with
1B having a greater proportion of voltage-sensitive
inhibition, consistent with the greater inhibition of 1A
current at more depolarized voltage steps (Fig. 4C). We
conclude from these data that 1A and 1B
Ca2+ current amplitudes are equivalently sensitive
to G-protein inhibition; however, the inhibition of 1B
currents is more voltage sensitive than the inhibition of
1A currents.
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Figure 5.
Voltage-dependent component of
M2-mediated inhibition of 1A and
1B in the absence of the Ca2+ channel
3 subunit. A, B, Facilitation of
1A and 1B Ca2+
currents using the prepulse protocol illustrated in Figure
1A. C, D, Normalized current
versus voltage plots of rebound current ( ), subsequent inhibition by
application of 50 µM ACh ( ), and facilitation of the
inhibited current by a depolarizing prepulse to +100 mV for 75 msec
( ). E, Facilitation of current amplitude by the
prepulse voltage protocol illustrated previously, for 1A (gray) and 1B
(black) currents. Facilitation is measured as the percentage of current inhibited by application of ACh, which is subsequently relieved by the prepulse voltage protocol.
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The Ca2+ channel 3 subunit
differentially modulates the magnitude of M2-mediated
G-protein inhibition of 1A and 1B
Ca2+ currents
We reported previously that co-expression of the
Ca2+ channel 3 subunit significantly
reduced tonic inhibition of 1A and 1B Ca2+ currents (Roche et al., 1995). In the presence
of the 3 subunit, 1A and
1B currents show a relatively small amount of rebound after removal of ACh and little or no prepulse facilitation (data not
shown), indicating that only a small degree of tonic inhibition remains. Using the rebound protocol to eliminate the already reduced background tonic inhibition, we determined the ability of the 3 subunit to modulate muscarinic receptor-induced
inhibition and furthermore whether the Ca2+ channel
3 subunit differentially modifies the muscarinic
receptor-induced inhibition of 1A and 1B
Ca2+ currents.
In contrast to the results obtained in the absence of the
3 subunit, the inhibition of current amplitude induced
by the M2 receptor is different for the two channel types.
Co-expression of the Ca2+ channel 3
subunit reduced the sensitivity of both the 1A and 1B Ca2+ currents to
M2-induced G-protein inhibition. However, the
3 subunit blocked the muscarinic M2
inhibition of the 1A current to a much greater extent
than the 1B current. This is illustrated in Figure
6A,B, which shows
representative 1A3 and
1B3 currents before and after application
of ACh. These current traces also demonstrate the greater slowing of
1B3 current activation kinetics compared
with that of 1A3 current during
M2 receptor activation, in contrast to the results obtained
in the absence of the Ca2+ channel 3
subunit. The time-to-peak current is quantitated in Figure
6C. The slowed activation kinetics of the
1B3 current is similar to the slowing
seen when 1B is expressed alone.
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Figure 6.
Co-expression of the Ca2+
channel 3 subunit differentially modulates the
inhibition induced by activation of the muscarinic M2
receptor. A, B, Currents elicited by a voltage step to
+10 mV from a holding potential of 80 mV before
(Control) and after (+ACh)
application of 50 µM acetylcholine. C,
Slowing of activation kinetics by application of acetylcholine after
co-expression of the Ca2+ channel 3
subunit (voltage step to +10 mV). D, Inhibition of current amplitude at various test potentials for both
1A3 (gray) and
1B3 (black) currents. (*
represents significant difference between inhibition of
1A3 and 1B3
currents at a given test pulse voltage; Student's independent
t test; p 0.01)
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Slowing of the current activation kinetics is frequently associated
with voltage-dependent inhibition, and the Ca2+
channel 3 subunit differentially affects this aspect of
inhibition for the two channels, suggesting that the 3
subunit may differentially affect the voltage dependence of G-protein
inhibition. This interpretation is strengthened by analysis of the
inhibition at various test pulse voltages, which reveals a pronounced
difference in the magnitude of the inhibition at relatively negative
voltage steps (Fig. 6D), again in contrast to the
results seen without the Ca2+ channel
3 subunit. In addition, the relief of G-protein
inhibition by depolarizing prepulses was significantly greater for
1B3 currents than for
1A3 currents (Fig.
7). Figure 7A,B illustrates the effect of a depolarizing prepulse to +100 mV, demonstrating clearly
the difference in current facilitation for these two cloned channels
after co-expression of the 3 subunit. The relief of G-protein inhibition using this protocol is 46.8 ± 0.03% for
1B3 currents, whereas there is no
significant relief of inhibition for 1A3
currents (Fig. 7C). These data indicate that not only does
the Ca2+ channel 3 subunit
differentially modulate the magnitude of M2 receptor-induced G-protein inhibition of these two cloned channels, but
it also heightens the inherent differences in the voltage dependence of
M2 receptor-induced inhibition.
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Figure 7.
Co-expression of the Ca2+
channel 3 subunit differentially modulates the
voltage-dependent inhibitory characteristics associated with
M2-induced G-protein inhibition. A, B,
Facilitation of 1A3 and
1B3 Ca2+ currents
using the prepulse protocol illustrated in Figure
1A. C, D, Normalized current
versus voltage plots of rebound current (), subsequent inhibition by
application of 50 µM ACh (), and facilitation of the
inhibited current by a depolarizing prepulse to +100 mV for 75 msec
(). E, Facilitation of
1A3 (gray) and 1B3 (black) current
amplitude. Facilitation was measured as the percentage of inhibited
current, which was reversed by the depolarizing prepulse (* represents
significant difference between facilitation of
1A3 and 1B3
currents at a given test pulse voltage; Student's t
test; p 0.01).
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Comparison of rate of prepulse facilitation of
1A3 and 1B3
Ca2+ currents
The voltage dependence of G-protein inhibition is thought to arise
from the voltage-dependent dissociation of the G-protein from the
Ca2+ channel (Bean, 1989; Lopez and Brown, 1991). We
have demonstrated that the Ca2+ channel
3 subunit differentially modifies the magnitude as well as the voltage dependence of G-protein inhibition of 1A
and 1B Ca2+ channels, raising the
possibility that G-protein dissociation rates for the two channel types
are dissimilar. Figure
8A,B shows the rate of
facilitation of Ca2+ currents when the prepulse
duration is varied and the voltage is held constant. The time constants
derived from these data were similar [7.12 ± 1.24 msec for
1A3 (n = 4) and 6.91 ± 0.95 msec for 1B3 (n = 5)], suggesting that the rate of relief of G-protein inhibition does
not explain the differences in inhibitory characteristics of these two
channel types. However, it should be noted that only a small portion of
the inhibition of 1A3 currents is
relieved using these prepulse protocols (~6% facilitation for
1A3 vs ~80% for
1B3), and caution should be used
in interpreting this result as a definitive indicator of G-protein
affinity for the 1A3 channel.
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Figure 8.
Prepulse facilitation of
1A3 and 1B3
currents as a function of prepulse duration. A, B,
Facilitation of current with a prepulse to +100 mV for varying
durations, in a representative experiment. The data were fit with a
single exponential, and the time constant of this fit is shown. In both
cases, facilitation of current was normalized to the values of
facilitation seen in the absence of G-protein inhibition, to eliminate
any contribution of desensitization produced by these protocols.
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DISCUSSION |
Our results demonstrate that co-expression of the calcium channel
3 subunit differentially modulates both the magnitude
and voltage dependence of M2 receptor-induced G-protein
inhibition of 1A and 1B
Ca2+ currents. The magnitude of inhibition,
previously equivalent for 1A and 1B
Ca2+ currents at moderate depolarizations, becomes
greater for 1B3 currents compared with
1A3 currents. In addition, G-protein inhibition of 1A3 channels becomes less
voltage dependent, whereas inhibition of
1B3 channels becomes more voltage
dependent than 1A and 1B channels were in
the absence of the subunit. However, we found no difference in the
rate of prepulse relief of G-protein inhibition between
1A3 and 1B3
currents, suggesting no difference in the rate at which the G-proteins
dissociate from these channels at equivalent voltages.
Frequently, voltage-dependent and voltage-independent forms of
inhibition are separable components (Beech et al., 1991, 1992; Bernheim
et al., 1991; Leubke and Dunlap, 1994; Diverse-Pierluissi et al., 1995;
Wollmuth et al., 1995), which is attributable to the activation of
distinct biochemical pathways by G-protein-coupled receptor(s). Thus, a
shift in the contribution of voltage-dependent versus
voltage-independent components to overall inhibition could be explained
by a blockade of one of these pathways by the subunit. However,
this is not likely to explain our results, because the Ca2+ channel 3 subunit would have to
selectively eliminate the voltage-dependent inhibitory pathway for the
1A channel and the voltage-independent pathway for the
1B channel.
An alternative explanation is that changes in the voltage dependence
may reflect inherent differences in the rate of dissociation of the
G-protein from the Ca2+ channel 1
subunit, which is accentuated by the Ca2+ channel
3 subunit. Differences in the rate of voltage-dependent relief of somatostatin receptor-induced inhibition have been documented previously and proposed to explain differences in the magnitude of
inhibition of 1A1 and
1B1 currents (Zhang et al., 1996). In
these studies, the rate of G-protein dissociation was twofold faster
for the 1A1 Ca2+
channel, and this was suggested to be responsible for the smaller magnitude of G-protein inhibition of this channel type, compared with
1B3 channels. However, a similar
comparison of G-protein inhibition of N- and Q-type
Ca2+ currents in adrenal chromaffin cells (Currie
and Fox, 1997) uncovered no differences in the rate of relief of
inhibition by depolarizing prepulses, although the N- and Q-type
currents displayed differences in the magnitude of G-protein inhibition
similar to the differences seen in 1A and
1B channels expressed in oocytes. Our results show no
difference in G-protein dissociation rate, probed using prepulse
protocols, between 1A3 and
1B3, similar to the results of
Currie and Fox (1997); however, caution should be exercised in the
interpretation of these results. If G-protein dissociation occurs at
extremely fast rates, a significant amount of dissociation will occur
during the test pulse step to +10 mV. Subsequent attempts to cause
further G-protein dissociation using depolarizing prepulses would cause
little additional dissociation and might not accurately reflect the
overall state of G-protein dissociation. Such extremely fast
dissociation is consistent with the voltage-dependent characteristics of the G-protein inhibition of 1A3
current. Our results show decreased slowing of activation kinetics and
decreased prepulse facilitation of 1A3
current, consistent with an increased rate of G-protein dissociation
from 1A3 channels. Biochemical
determination of relative binding affinities for G-protein subunits for
both the 1A and 1B
Ca2+ channels might shed definitive light on this
subject. However, voltage-dependent channel conformations may be
necessary to see differences in affinity of the G-proteins for these
two channels. Whatever the mechanism, it remains that the inhibition of
1B3 current is more easily reversed by
voltage and is thus more voltage dependent.
It has been suggested recently that the Xenopus oocyte
contains an endogenous subunit (3XO)
(Tareilus et al., 1997), which is suggested to increase expression at
low expression levels and modulate channel activity at higher
expression levels, possibly by binding to multiple sites on the
Ca2+ channel 1 subunit. It is clear
that G-protein inhibition of Ca2+ channels in our
experiments is significantly altered by co-expression of
3. We do not believe that the endogenous subunit
significantly couples with exogenous 1 subunits, on the
basis of the following data. (1) The endogenous Ca2+
current is very small compared with the currents seen with the 1 subunits expressed alone (~10 nA compared with
~1000 nA for 1B), and therefore the endogenous
3 subunit would have to be expressed in excess of the
endogenous 1 subunit to have any effect on exogenously
expressed Ca2+ channels (Mori et al., 1991; Bourinet
et al., 1992), but (2) injection of exogenous subunit increases the
current amplitude of the endogenous Ca2+ currents,
indicating that the endogenous subunit is not likely expressed in
overabundance (Lacerda et al., 1994); (3) certain 1B
clones, incapable of expressing without exogenous subunit, can be
expressed in high densities when placed in a Xenopus
expression vector, suggesting not only that endogenous subunit is
insufficient for expression of these clones, but also that the subunit is not a requirement for high density expression (Lin et al.,
1997). However, the potential role of endogenous subunits can be
fully determined only by quantitating expression levels.
We used the refractory period that follows the removal of transmitter,
during which tonic inhibition is absent (rebound), to examine the
actions of G-proteins activated by M2 activation. The
mechanism behind this rebound is unclear at this time, but the
phenomenon may have significant physiological implications. For
example, a cell that has been inhibited by the action of two or more
transmitters, on removal of one of these transmitters might be
refractory to the actions of the second transmitter. Tonic G-protein
inhibition may influence transmission at some synapses where
presynaptic elements might exhibit heightened release as a result of
temporary removal of tonic inhibition for a period of time after
removal of a neurotransmitter.
The influence of calcium channel subunit composition on G-protein
modulation is also likely to have significant functional consequences.
When transmitter release is triggered by lengthy, high-frequency trains
of action potentials, the voltage-dependent form of G-protein
inhibition will be minimized (Elmslie et al., 1990; Ikeda, 1991). This
would lead to a greater amount of Ca2+ entry from
channels that are inhibited in a voltage-dependent manner and in turn
lead to a greater amount of transmitter release. It is apparent from
our experiments that the ability of release to become facilitated will
depend on both the expression of particular 1 subunits
as well as on the degree of subunit association with these
1 subunits. Transmitter release at many CNS synapses is
initiated by Ca2+ permeating both N-type
(1B) and P/Q-type (1A)
Ca2+ channels. The relative contribution of these
channel types to synaptic transmission is variable but often mediated
by both acting in concert (Leubke et al., 1993; Takahashi and Momiyama,
1993; Castillo et al., 1994; Wheeler et al., 1994; Mintz et al., 1995; Regehr and Mintz, 1996; Wheeler et al., 1996). At these synapses, regulation of both the expression of the Ca2+
channel subunit and the relative abundance of a particular 1 subunit will finely regulate the overall sensitivity
of the synapse to G-protein modulation, as well as its response to
trains of depolarizing action potentials. Regulation of inhibitory
modulation at the level of the target channel would allow more precise
tuning than would regulation of inhibitory modulation at the level of the G-protein, in which a larger range of downstream targets would be
affected.
In conclusion, these data indicate that the both the magnitude as well
as the voltage dependence of G-protein-mediated inhibition of the
1A and 1B Ca2+
currents is differentially modulated by the Ca2+
channel 3 subunit. This may play an important role in
the CNS to regulate the plasticity of synapses.
| |
FOOTNOTES |
Received July 3, 1997; revised Nov. 14, 1997; accepted Nov. 17, 1997.
This work was supported by National Institutes of Health (NIH) Grants
AA05542 and AA08003 to S.N.T. and NIH predoctoral fellowship to J.P.R.
We thank Dr. Ann Rittenhouse for careful reading of this manuscript,
and Andy Wilson and Lynda Zorn for expert technical assistance.
Correspondence should be addressed to Dr. Steven N. Treistman,
Department of Pharmacology and Molecular Toxicology, University of
Massachusetts Medical Center, Worcester, MA 01655.
Dr. Roche's present address: Department of Physiology and Biophysics,
University of Washington, Seattle, WA 98195.
| |
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Abstract
Introduction
