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The Journal of Neuroscience, October 1, 2000, 20(19):7167-7173
Muscarinic Stimulation of
1E Ca Channels Is Selectively
Blocked by the Effector Antagonist Function of RGS2 and
Phospholipase C-
1
Karim
Melliti1,
Ulises
Meza2, and
Brett
Adams1
1 Department of Biology, Utah State University, Logan,
Utah 84322-5305, and 2 Department of Physiology and
Pharmacology, College of Medicine, Autonomous University of San Luis
Potosí, SLP 78210, México
 |
ABSTRACT |
Neuronal
1E Ca channel subunits are widely expressed in
mammalian brain, where they are thought to form R-type Ca channels. Recent studies have demonstrated that R-type channels contribute to
neurosecretion and dendritic Ca influx, but little is known concerning
their modulation. Here we show that
1E channels are strongly
stimulated, and only weakly inhibited, through M1 muscarinic acetylcholine receptors. Both forms of channel modulation are mediated
by pertussis toxin-insensitive G-proteins. Channel stimulation is
blocked by regulator of G-protein signaling 2 (RGS2) or the C-terminal region of phospholipase C-
1 (PLC
1ct), which have been
previously shown to function as GTPase-activating proteins for G
q.
In contrast, RGS2 and PLC
1ct do not block inhibition of
1E
through M1 receptors. Inhibition is prevented, however, by the
C-terminal region of
-adrenergic receptor kinase 1, which sequesters
G
dimers. Thus, stimulation of
1E is mediated by a pertussis
toxin-insensitive G
subunit (e.g., G
q), whereas inhibition
is mediated by G
. The ability of RGS2 and PLC
1ct to
selectively block stimulation indicates these proteins functioned primarily as effector antagonists. In support of this interpretation, RGS2 prevented stimulation of
1E with non-hydrolyzable guanosine 5'-0-(3-thiotriphosphate). We also report strong muscarinic
stimulation of rbE-II, a variant
1E Ca channel that is insensitive
to voltage-dependent inhibition. Our results predict that G
q-coupled
receptors predominantly stimulate native R-type Ca channels.
Receptor-mediated enhancement of R-type Ca currents may have important
consequences for neurosecretion, dendritic excitability, gene
expression, or other neuronal functions.
Key words:
CaV2.3; R-type calcium channel;
1E; RGS
protein; phospholipase C-
1; GAP; effector antagonist
 |
INTRODUCTION |
Native R-type Ca channels have been
defined by their resistance to selective antagonists of L-, N-, and
P/Q-type Ca channels (Randall and Tsien, 1995
). Only recently has a
selective antagonist of R-type channels been reported (Newcomb et al.,
1998
). Antisense depletion experiments suggest that neuronal R-type Ca
channels are formed by
1E subunits (Piedras-Rentería and
Tsien, 1998
; Tottene et al., 2000
).
1E subunits are widely expressed
in mammalian brain (Niidome et al., 1992
; Soong et al., 1993
; Wakamori
et al., 1994
; Williams et al., 1994
; Yokoyama et al., 1995
), and
several splice variants have been described (cf. Pereverzev et al.,
1998
). Although the physiological functions of R-type Ca channels are incompletely known, available evidence indicates that they contribute to dendritic Ca influx (Kavalali et al., 1997
) and neurosecretion by
some presynaptic terminals (Turner et al., 1995
; Wu et al., 1998
, 1999
;
Allen, 1999
; Wang et al., 1999
).
The G-protein-dependent modulation of N- and P/Q-type Ca channels has
been extensively studied (for review, see Hille, 1994
; Jones and
Elmslie, 1997
; Zamponi and Snutch, 1998
; Ikeda and Dunlap, 1999
; Bean,
2000
). In contrast, much less is known concerning the modulation of
R-type Ca channels. Previously, we reported that R-type channels formed
by rabbit
1E subunits are both inhibited and stimulated through M2
muscarinic acetylcholine receptors (Meza et al., 1999
). Our experiments
demonstrated that inhibition and stimulation of
1E are separate
processes that occur through distinct signaling pathways, both of which
couple to M2 receptors. Inhibition occurs through a fast, pertussis
toxin (PTX)-sensitive pathway, whereas stimulation occurs through a
slower, PTX-insensitive pathway (Meza et al., 1999
). It is intriguing
that R-type channels can be stimulated through muscarinic receptors,
because the closely-related N- and P/Q-type channels are typically
inhibited through G-protein-coupled receptors. Receptor-mediated
enhancement of R-type Ca currents may have important consequences for
dendritic Ca signaling, neurosecretion, or other neuronal functions.
In the present study, we have further examined the muscarinic
stimulation of recombinant R-type Ca channels.
1E subunits were
expressed in HEK293 cells with M1 muscarinic acetylcholine receptors,
which preferentially couple to heterotrimeric G-proteins of the G
q
subfamily (Felder, 1995
). We find that M1 receptors predominantly
stimulate, rather than inhibit,
1E channels. Additionally, we find
that stimulation of
1E is selectively blocked by regulator of
G-protein signaling 2 (RGS2) and the C-terminal region of phospholipase C-
1 (PLC
1ct), two proteins known to function as GTPase-activating proteins (GAPs) for G
q. Interestingly, the effects of RGS2 and PLC
1ct in our experiments can only be explained if these proteins functioned primarily as effector antagonists. Altogether, our data
suggest that
1E is stimulated through a G
q-coupled signaling pathway. These observations predict that G
q-coupled receptors stimulate native R-type Ca channels in vivo. Our results
also provide new insight into mechanisms by which RGS proteins can influence the receptor-mediated modulation of voltage-gated Ca channels.
 |
MATERIALS AND METHODS |
Cell culture and transfection. Human embryonic kidney
(HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA) and propagated in culture medium containing 90% DMEM,
10% fetal bovine serum, and 50 µg/ml gentamycin. The cells were trypsinized weekly and replated onto 60 mm culture dishes at 20%
confluence. CaPO4 precipitation was used to
transfect these cells within 3-5 d of plating. The transfection
mixture contained expression plasmids encoding
1E,
2-
, and
3 Ca channel subunits at 1.25 µg of each cDNA per dish, plus an
expression plasmid encoding the M1 muscarinic acetylcholine receptor at
0.25 µg of cDNA per dish. In selected experiments, the transfection
mixture also included expression plasmids encoding RGS2, RGS8, or the
C-terminal region (Thr903-Leu1216)
of PLC
1 at 0.625 µg of cDNA per dish. RGS2, RGS8, and PLC
1ct were expressed as fusions to the C terminus of enhanced green fluorescence protein (EGFP). In specific experiments, an expression plasmid encoding
Gly495-Leu689
of
-adrenergic receptor kinase 1 (
ARK1; denoted
ARK1ct) was transfected at 1.25 µg of cDNA per dish. For the experiments
illustrated in Figure 7, cells were transfected at 1.25 µg/dish with
an expression plasmid encoding a variant
1E subunit (denoted rbE-II)
cloned from rat hippocampus by Soong et al. (1993)
. These cells were cotransfected with the M1 receptor as described above or alternatively with a plasmid encoding the M2 muscarinic acetylcholine receptor at
0.0625 µg/dish. For all transfections that did not include EGFP
fusion proteins, a separate plasmid encoding EGFP was included at 0.125 µg/dish. The day after transfection, cells were briefly trypsinized
and replated onto 12 mm round glass coverslips. Electrophysiological experiments were performed 16-24 hr later. Successfully transfected cells were visually identified by their green fluorescence under ultraviolet illumination; only green cells were used for experiments.
Expression plasmids. cDNA encoding rabbit
1E (GenBank
accession number X67856) was in pcDNA3.1+ (Invitrogen, San Diego, CA).
rbE-II (accession number L15453) and rat
2-
(M86621) were in pMT2
(Genetics Institute, Cambridge, MA). Rabbit
3 (X64300) was in pcDNA3
(Invitrogen). Human M1 muscarinic acetylcholine receptor (X52068) was
in pCD. Human M2 muscarinic receptor (X15264) was in pRK5 (Genentech,
South San Francisco, CA). Jellyfish enhanced green fluorescent protein
(U55763) was in pEGFP (Clontech, Cambridge, UK). Human RGS2 (L13463)
and rat RGS8 (AB006013) were in pCI (Promega, Madison, WI). EGFP-RGS2 and EGFP-RGS8 were in pEGFP-C2 and pEGFP-C1 (Clontech), respectively. A
deletion mutant of RGS8 (denoted
RGS8) was in pEGFP-C1; this construct encodes EGFP fused to an RGS8 protein lacking
Phe57-Pro160.
cDNA encoding
Thr903-Leu1216
of rat PLC
1 (M20636) was in pEGFP-C1 (Clontech). cDNA encoding Gly495-Leu689
of
ARK1 (M34019) was in pRK5 (Koch et al., 1994
).
Patch-clamp recordings. Large-bore patch pipettes were
pulled from 100 µl borosilicate glass micropipettes (VWR 53432-921) and filled with a solution containing (in mM): 155 CsCl, 10 Cs2-EGTA, 4 Mg-ATP, 0.32 Li-GTP, and 10 HEPES, pH
7.4, with CsOH. For the experiments illustrated in Figure 6, equimolar
guanosine 5'-0-(3-thiotriphosphate) (GTP-
-S) was substituted for GTP
in the pipette solution. Aliquots of the pipette solution were stored
at
80°C, kept on ice after thawing, and filtered at 0.22 µm
immediately before use. Pipette tips were coated with paraffin to
reduce capacitance and then fire-polished. Filled patch pipettes had DC
resistances of 1.0-1.5 M
. The bath solution contained (in
mM): 145 NaCl, 40 CaCl2, 2 KCl, and
10 HEPES, pH 7.4, with NaOH. Ca currents were recorded in the
whole-cell configuration. After forming a gigaohm seal in the
cell-attached configuration, residual pipette capacitance was
compensated using the negative capacitance circuit of the amplifier.
The DC resistance of the whole-cell configuration was routinely >1
G
. The steady holding potential was
90 mV. No corrections were
made for liquid junction potentials. Depolarizations to potentials near
the peak of the current-voltage relationship (+30 mV) were delivered
every 1-10 sec; the stimulation rate was adjusted for each cell to
maximize sampling resolution and to minimize cumulative inactivation.
Currents were filtered at 2-10 kHz using the built-in Bessel filter
(four-pole low-pass) of an Axopatch 200B amplifier (Axon Instruments,
Foster City, CA) and sampled at 10-50 kHz using a Digidata 1200 analog-to-digital board installed in a Gateway Pentium computer. The
pCLAMP 7.0 software programs Clampex and Clampfit were used for data
acquisition and analysis, respectively. Figures were made using the
software program Origin (version 6.0).
Linear cell capacitance (C) was determined by
integrating the area under the whole-cell capacity transient, which was
evoked by a voltage-clamp step from
90 to
80 mV; the whole-cell
capacitance compensation circuit of the amplifier was turned off during
this measurement. The average value of C was 18 ± 1 pF
(mean ± SEM; n = 160 cells). To minimize voltage
errors, the time constant for decay of the whole-cell capacity
transient (t) was reduced as much as possible using the
analog series resistance compensation circuit of the amplifier. Series
resistance (RS) was calculated as
t × (1/C), where t was the time
constant for decay of the whole-cell capacity transient. The average
values of t and RS,
measured before electronic compensation, were 54 ± 2 µsec and
3.2 ± 0.1 M
, respectively (n = 160). Maximal
Ca current amplitude was 1790 ± 140 pA (n = 160;
test potential, +30 mV). After electronic compensation of t
and RS, the average maximum voltage
error was 3.9 ± 0.3 mV (n = 160).
All currents were corrected for linear capacitance, and leakage
currents using
P/6 or
P/4 subtraction. Ca current amplitudes were measured at the time of peak inward current. Comparisons were by
ANOVA or by unpaired, two-tailed t tests, with
p < 0.05 considered significant. Application of
carbachol (CCh) was by bath exchange or local superfusion through
macropipette positioning close to the cell under study. CCh was
dissolved directly in the bath solution. Temperature (20-24°C) was
continuously monitored using a miniature thermocouple placed in the
recording chamber.
 |
RESULTS |
1E Ca channels are predominantly stimulated through M1
muscarinic acetylcholine receptors
Figure 1 illustrates Ca currents
recorded from an HEK293 cell coexpressing rabbit
1E subunits and
human M1 muscarinic acetylcholine receptors. As seen in the plot of
current amplitudes versus time (Fig. 1A), application
of CCh initially produced a small, rapid decrease (inhibition) of
1E
current amplitude (point b). This initial inhibition
was soon followed (point c) by a substantial increase
in the current amplitude (stimulation). After CCh washout, current
amplitude again transiently increased (point d) by an amount comparable with that of the initial inhibition. This secondary increase at washout apparently corresponds to relief of the initial inhibition (see below). The observed modulation of
1E Ca channels was attributable to coexpressed M1 receptors, because it was absent from cells not cotransfected with muscarinic receptors and was completely blocked by atropine (Meza et al., 1999
).

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Figure 1.
1E Ca channels are predominantly stimulated
through M1 muscarinic acetylcholine receptors. A,
Whole-cell Ca currents were evoked every 5 sec by step depolarizations
from 90 to + 30 mV. Ca current amplitudes are plotted as a function
of time during a representative experiment. Application of CCh (1 mM) is indicated by a horizontal bar. Linear
cell capacitance (C) = 13 pF; series
resistance (RS) = 2.1 M .
B, Selected Ca currents recorded at the times indicated in
A. C, Summary data for inhibition and stimulation of
1E Ca currents through coexpressed M1 receptors. For each cell,
inhibition was measured as the difference between the amplitudes of
currents a and b, normalized with respect
to the amplitude of current a. Stimulation was measured
as the difference between the amplitudes of currents b
and c, normalized with respect to the amplitude of
current a. Error bars represent ±SEM.
|
|
1E current amplitudes were inhibited by only 6 ± 2%
(n = 17) through M1 receptors. In contrast, the
magnitude of stimulation was substantially larger (40 ± 6%;
n = 17). Thus, M1 receptors predominantly stimulate
1E Ca channels (Fig. 1C). Previously, we found that
1E
currents were inhibited by ~40% and stimulated by ~20% through
the M2 subtype of muscarinic acetylcholine receptor (Meza et al.,
1999
). This comparison indicates that M1 receptors produce smaller
inhibition and larger stimulation of
1E Ca channels than M2 receptors.
We next examined the dose dependency for stimulation of
1E Ca
channels through M1 receptors (Fig. 2).
Application of 10 µM CCh produced approximately
half-maximal stimulation, and 100 µM CCh generated
maximal stimulation. The concentration of CCh used throughout the
remainder of this study (1 mM) was therefore clearly saturating. These dose-response data are in general agreement with
previously reported agonist binding affinities of cloned M1 receptors
(Peralta et al., 1987
).

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Figure 2.
Dose-response data for modulation of 1E Ca
channels through M1 receptors. Top, Representative Ca
currents recorded before the application of CCh and during maximal
stimulation by various concentrations of CCh. The control currents are
aligned with the horizontal dotted line.
Bottom, Average inhibition and stimulation of 1E
currents by various CCh concentrations. Inhibition and stimulation are
expressed relative to the control Ca current in each cell, recorded
immediately before CCh application. Each cell was exposed once to a
single concentration of CCh. The voltage protocol is as in Figure 1. In
selected experiments, cells were exposed to 100 nM
staurosporine before, during, and after the application of CCh.
Staurosporine (STAURO.) was dissolved in DMSO to make a
stock solution of 1 mM. The final concentration of DMSO in
the bath was 0.01%, which alone had no effects on 1E
currents.
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|
In our previous study (Meza et al., 1999
), we found that the M2
receptor-mediated stimulation of
1E was prevented by staurosporine, a broad-spectrum inhibitor of serine-threonine kinases. As shown in
Figure 2, staurosporine also prevented stimulation of
1E through M1
receptors, suggesting that stimulation results from a pathway that
couples to both receptor subtypes. Because M1 receptors produce larger
stimulation than M2 receptors, the responsible signaling pathway
apparently couples more efficiently to M1 receptors. The effect of
staurosporine is consistent with a previous report that
1E Ca
channels are stimulated through a protein kinase C-dependent pathway in
Xenopus oocytes (Stea et al., 1995
).
As shown in Figure 2, the average magnitude of inhibition was somewhat
larger in staurosporine-treated cells (18 ± 10%) than in control
cells (6 ± 2%), although this difference was not statistically significant (p > 0.05). Our impression is that
stimulation causes a slight underestimation in the measurement of
inhibition; however, this underestimation does not affect the
conclusions of this study (see below).
Stimulation of
1E is mediated by a PTX-insensitive
G
subunit
PTX catalyzes the ADP ribosylation of G
i subfamily proteins at
a cysteine residue near the C terminus, thereby decoupling these G
subunits from receptors (West et al., 1985
; Avigan et al., 1992
). We
used PTX to investigate which G-proteins are responsible for modulation
of
1E through M1 receptors. Figure
3B shows currents recorded
from a cell exposed to PTX (500 ng/ml) overnight, and Figure
3A illustrates currents recorded from an untreated control cell. It was clear that PTX had no appreciable effects on inhibition or
stimulation of
1E. Averaged results are presented in Figure 5. These
data suggest that, in the case of M1 receptors, inhibition and
stimulation of
1E are both mediated by PTX-insensitive
G-proteins.

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Figure 3.
Stimulation of 1E is mediated by a
PTX-insensitive G subunit. Left, 1E current amplitudes
are plotted as a function of time during representative experiments.
Right, Whole-cell Ca currents recorded at times
indicated in the corresponding plots. A, Modulation of
1E currents by M1 receptors in a control cell (same cell as in Fig.
1A). C = 13 pF;
RS = 2.1 M . B, PTX does not
affect inhibition or stimulation of 1E through M1 receptors. Cells
were preincubated with PTX; 200-500 ng/ml) for at least 20 hr.
C = 9 pF; RS = 3.6 M . C, Coexpression of ARK1ct selectively blocks
inhibition of 1E through M1 receptors. C = 11 pF; RS = 3.2 M . Other details are as in
Figure 1.
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|
To determine which subunits (G
or G
) of the
PTX-insensitive G-protein are responsible for modulation of
1E, we
expressed
ARK1ct. This region
(Gly495-Leu689)
of
ARK1 sequesters G
dimers (Koch et al., 1994
), including G
released through activation of G
q-coupled M3 receptors
(Stehno-Bittel et al., 1995
). As shown in Figure 3C,
coexpression of
ARK1ct blocked the initial inhibition of
1E as
well as the secondary increase in current amplitude after CCh washout.
Overall,
1E currents were inhibited by only 0.7 ± 0.7%
(n = 8) in
ARK1ct-expressing cells, compared with
6.4 ± 1.8% (n = 17) inhibition in control cells
(p < 0.05; see Fig. 5). These results with
ARK1ct suggest that inhibition of
1E is mediated by G
dimers released as a consequence of M1 receptor activation.
In contrast, stimulation of
1E was unaffected by
ARK1ct (see Fig.
5). Thus,
1E currents were stimulated by 40 ± 6% in control cells (n = 17) and by 43 ± 7% in
ARK1ct-expressing cells (n = 7). Altogether, these
data indicate that stimulation involves signaling by a PTX-insensitive
G
subunit.
PLC
1ct, RGS2, and RGS8 selectively block stimulation
of
1E
M1 receptors preferentially couple to G-proteins of the G
q
subfamily (Felder, 1995
), suggesting that G
q mediates stimulation of
1E. To test this hypothesis, we took advantage of recent work showing that PLC
1 and RGS2 can modify interactions of G
q with its
downstream effectors. PLC
1 is the principal effector enzyme of
G
q; interestingly, this phopholipase also functions as a powerful GAP for G
q (Berstein et al., 1992
; Biddlecome et al., 1996
). For our
purposes, we chose to express only the C terminus of PLC
1 (Thr903-Leu1216;
denoted PLC
1ct), because this portion of the protein contains intrinsic GAP activity (Paulssen et al., 1996
), but it completely lacks
phospholipase activity (Wu et al., 1993
).
As illustrated in Figure
4B, coexpression of
PLC
1ct greatly reduced stimulation of
1E. Thus, stimulation was
only ~4% in cells expressing PLC
1ct, compared with ~40%
stimulation in control cells (Fig. 5).
Surprisingly, PLC
1ct did not also reduce inhibition of
1E through
M1 receptors. Overall,
1E currents were inhibited by 10 ± 2%
(n = 10) in PLC
1ct-expressing cells, compared with 6 ± 2% (n = 17) inhibition in control cells
(p > 0.1). The lack of effect on inhibition was
unexpected, given the previously demonstrated ability of PLC
1 to act
as a powerful GAP for G
q (Berstein et al., 1992
).

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Figure 4.
Stimulation of 1E is selectively blocked by
PLC 1ct, RGS2, and RGS8. A, Modulation of 1E by M1
receptors in a control cell. C = 16 pF;
RS = 2.8 M . B, Modulation of 1E
by M1 receptors in a cell coexpressing PLC 1ct. C = 24 pF; RS = 1.3 M . C,
Modulation of 1E by M1 receptors in a cell coexpressing RGS2.
C = 11 pF; RS = 5.1 M . D, Modulation of 1E by M1 receptors in a cell
coexpressing RGS8. C = 13 pF;
RS = 2.9 M . Other details are as in Figure
1.
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Figure 5.
Modulation of 1E Ca currents through M1
receptors. Inhibition and stimulation are expressed relative to the
control Ca current in each cell, recorded immediately before CCh
application. Each cell was exposed once to a single concentration of
CCh. Means were compared using one-way ANOVA or by an unpaired,
two-tailed t test. Asterisks indicate
significant differences from the control mean (*p < 0.05; **p < 0.01; ***p < 0.001). The average Ca current densities (measured in response to a
test pulse to +30 mV) in each group were 106 ± 17 pA/pF
(n = 17) in control, 104 ± 23 pA/pF
(n = 10) in PTX-treated, 80 ± 21 pA/pF
(n = 10) in PLC 1ct-expressing, 196 ± 88 pA/pF (n = 9) in RGS2-expressing, 68 ± 10 pA/pF (n = 8) in ARK1ct-expressing, 124 ± 19 pA/pF (n = 18) in RGS8-expressing, and 149 ± 38 pA/pF (n = 7) in RGS8-expressing
cells.
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To further test the hypothesis that G
q mediates stimulation of
1E, we used RGS2, because previous studies have found that RGS2
preferentially interacts with G
q in vitro (Chen et al., 1997
; Heximer et al., 1997
; but see Ingi et al., 1998
). Stimulation of
1E was also significantly reduced by coexpression of RGS2 (Fig.
4C). Altogether, stimulation was 7 ± 4% in
RGS2-expressing cells (n = 9), compared with 40 ± 6% (n = 17) stimulation in control cells
(p < 0.01). In contrast, inhibition of
1E
was unaffected by RGS2 (Fig. 5). Thus, the effects of RGS2 were
basically identical to those of PLC
1ct; that is, both proteins
strongly reduced stimulation of
1E without also reducing inhibition.
Because PLC
1ct and RGS2 have been previously demonstrated to
interact with G
q, these findings support the hypothesis that
stimulation is mediated through a G
q-coupled signaling pathway.
For comparison with PLC
1ct and RGS2, we tested RGS8, which has been
previously shown to act as a GAP for G
i subfamily proteins (Saitoh
et al., 1997
; Melliti et al., 1999
). Coexpression of RGS8 significantly
reduced stimulation of
1E (Fig. 4D) but to a
lesser extent than either PLC
1ct or RGS2 (Fig. 5). Inhibition of
1E was unaffected by RGS8 (Fig. 5). The ability of RGS8 to reduce stimulation suggests that it can interact with G
q, although less effectively than either PLC
1ct or RGS2.
As a control for expression of PLC
1ct, RGS2, and RGS8, we used the
deletion mutant
RGS8 (Melliti et al., 1999
). This mutant lacks amino
acids
Phe57-Pro160,
which constitute the major portion of the conserved RGS core domain
(Berman and Gilman, 1998
). The RGS core domain mediates binding of RGS
proteins to the switch regions of G
(Tesmer et al., 1997
). We have
previously shown that
RGS8 does not act as a GAP to attenuate N-type
Ca channel inhibition by G
i subfamily proteins (Melliti et al.,
1999
). As summarized in Figure 5, coexpression of
RGS8 had no effect
on the modulation of
1E through M1 receptors. Thus, the RGS core
domain is apparently necessary for the observed effects of RGS2 and
RGS8 in our experiments.
In summary, only stimulation of
1E was attenuated by PLC
1ct,
RGS2, and RGS8. Notably, these proteins failed to reduce inhibition of
1E through M1 receptors (Fig. 5). The selective block of stimulation by PLC
1ct, RGS2, and RGS8 cannot be explained by the GAP activity of
these proteins, because simply accelerating GTP hydrolysis should have
attenuated signaling by both G
and G
and should have reduced
both stimulation and inhibition of
1E. However, the selective block
of stimulation is consistent with the interpretation that PLC
1ct,
RGS2, and RGS8 functioned primarily as effector antagonists in our experiments.
RGS2 prevents GTP-
-S-mediated stimulation of
1E
Previous studies have found that RGS2 and RGS4 reduce signaling by
G
q activated with GTP-
-S (Hepler et al., 1997
; Heximer et al.,
1997
). GTP-
-S is nonhydrolyzable; thus, these effects of RGS2 and
RGS4 cannot be attributed to their GAP activities. Rather, it is
thought that RGS2 and RGS4 can function as effector antagonists by
binding to the switch regions of G
q, thereby blocking its
interactions with downstream effectors. To further examine whether
PLC
1ct, RGS2, and RGS8 could have functioned as effector antagonists
in our experiments, we used GTP-
-S to produce stimulation of
1E.
These experiments were performed on cells coexpressing
1E Ca
channels and M1 receptors and incubated with PTX (200-500 ng/ml)
overnight to inactivate G
i/o proteins. Equimolar GTP-
-S was
substituted for GTP in the pipette solution, and
1E current amplitudes were monitored as a function of time after establishing the
whole-cell configuration (i.e., break-in). As shown in Figure 6, intracellular dialysis with GTP-
-S
significantly increased
1E currents in control cells. The magnitude
of this increase was comparable (~40%) with that produced by
coexpressed M1 receptors (compare Fig. 1). By contrast, GTP-
-S
failed to stimulate
1E currents in cells coexpressing RGS2 (Fig. 6).
Apparently, GTP-
-S activated the stimulatory pathway in control
cells but not in cells coexpressing RGS2. Because GTP-
-S is
nonhydrolyzable, RGS2 cannot have blocked the stimulatory pathway by
functioning as a GAP; it must have functioned as an effector
antagonist. These results support our hypothesis that PLC
1ct, RGS2,
and RGS8 selectively blocked stimulation of
1E by acting as effector
antagonists.

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Figure 6.
RGS2 prevents stimulation of 1E by
intracellular GTP- -S. Whole-cell Ca currents were recorded from
HEK293 cells expressing rabbit 1E Ca channels and human M1
receptors; some cells also expressed RGS2. The pipette solution
contained 0.32 mM GTP- -S in place of GTP; aliquots of
this solution were thawed from 80°C, kept on ice during
experiments, and discarded within 3 hr of thawing. Recordings from
control and RGS2-expressing cells were alternated. The cells were
incubated with PTX (200-500 ng/ml) overnight before experiments.
Immediately after establishment of the whole-cell configuration
(break-in), Ca currents were evoked by 10 msec depolarizations to +30
mV, delivered every 10 sec from a steady holding potential of 90 mV.
Top, Representative whole-cell Ca currents recorded at
break-in and 5 min later from a control (left) and an
RGS2-expressing (right) cell. RS = 2.5 M (control) and 2.4 M (RGS2). Bottom,
Average ± SEM Ca current amplitudes at various times after
break-in. Current amplitudes are expressed relative to the initial Ca
current amplitude recorded in each cell at break-in. For times between
0 and 2 min, symbols represent data from 15 control and
10 RGS2-expressing cells; beyond 2 min, symbols
represent data from 8 control and 7 RGS2-coexpressing cells. Initial Ca
current densities were 99 ± 17 pA/pF (control) and 124 ± 22 pA/pF (RGS2).
|
|
rbE-II is stimulated through muscarinic receptors
Previous studies have demonstrated that rbE-II, an
1E subunit
cloned from rat hippocampus (Soong et al., 1993
), is insensitive to
inhibition through µ-opioid and dopamine receptors (Bourinet et al.,
1996
; Page et al., 1998
). It was recently shown by Page et al. (1998)
that an N-terminal domain within
1B and
1E subunits is essential
for their voltage-dependent inhibition by G
dimers. rbE-II lacks
this domain, which accounts for its insensitivity to voltage-dependent
inhibition (Page et al., 1998
).
To determine whether rbE-II can undergo muscarinic stimulation, we
coexpressed it with M1 or M2 receptors. In agreement with Bourinet et
al. (1996)
and Page et al. (1998)
, we found that rbE-II displayed no
appreciable voltage-dependent inhibition through either muscarinic
receptor. However, rbE-II was prominently stimulated through both
receptors. In response to M2 receptors, rbE-II exhibited stimulation
that simply reversed after CCh washout (Fig.
7A). However, with M1
receptors rbE-II currents often exhibited a secondary increase after
CCh washout (Fig. 7B). The mechanism of this secondary increase is presently unclear; further work is being done.

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Figure 7.
rbE-II Ca channels are strongly stimulated through
muscarinic receptors. Left, rbE-II current amplitudes
are plotted as a function of time during representative experiments.
Right, Whole-cell Ca currents recorded at times
indicated in the corresponding plot. A, Stimulation of
rbE-II current through M2 receptors (M2R). Ca currents
were evoked every 3 sec by step depolarizations from 90 to +30 mV.
C = 50 pF; RS = 1.1 M .
The application of CCh (50 µM) is indicated by a
horizontal bar; this CCh concentration produces maximal
activation of M2 receptors (Melliti et al., 1999 ; Meza et al., 1999 ).
B, Stimulation of rbE-II current through M1 receptors
(M1R). Ca currents were evoked every 5 sec by step
depolarizations from 90 to +30 mV. C = 28 pF;
RS = 1.5 M . The concentration of applied CCh
was 1 mM. C, Average stimulation of rbE-II
currents through M1 and M2 receptors.
|
|
Results obtained with rbE-II Ca channels are summarized in Figure
7C. Altogether, rbE-II currents were stimulated by 35 ± 11% (n = 10) through M2 receptors and by 55 ± 8% (n = 13) through M1 receptors (only the primary
phase of stimulation is included in the latter measurement). The larger
stimulation of rbE-II through the M1 receptor is consistent with the
hypothesis that stimulation is mediated by G
q, because M1 receptors
couple preferentially to this G
subunit (Felder, 1995
). Stimulation
of rbE-II was statistically indistinguishable (p > 0.1) from stimulation of the rabbit
1E Ca channel [19 ± 3% through M2 receptors (n = 11) and 40 ± 6% through M1 receptors (n = 17)]. In summary, these
experiments demonstrate that rbE-II Ca channels are significantly
stimulated through M1 and M2 muscarinic receptors. Because rbE-II lacks
the N-terminal domain essential for voltage-dependent, G
-mediated inhibition (Page et al., 1998
), this domain apparently is not involved
in the muscarinic stimulation of
1E Ca channels.
 |
DISCUSSION |
We have shown that
1E Ca channels are strongly stimulated and
only weakly inhibited through M1 muscarinic acetylcholine receptors. Both forms of channel modulation are insensitive to PTX. Stimulation of
1E is blocked by PLC
1ct and RGS2, two proteins previously demonstrated to function as GAPs for G
q (Berstein et al., 1992
; Paulssen et al., 1996
; Heximer et al., 1997
). In contrast, stimulation is unaffected by
ARK1ct, which sequesters G
subunits and which blocks inhibition of
1E through M1 receptors (Figs. 4C,
5). Together these results indicate that stimulation of
1E involves
signaling by a PTX-insensitive G
subunit. Because M1 receptors
preferentially couple to heterotrimeric G-proteins of the G
q
subfamily (Felder, 1995
), we hypothesize that stimulation is mediated
through G
q. Our results predict that native R-type Ca channels are
predominantly stimulated through endogenous muscarinic and possibly
other G
q-coupled receptors. This possibility is supported by the
strong muscarinic stimulation of rbE-II Ca channels (Fig. 7).
The finding that
ARK1ct blocks inhibition of
1E suggests that
inhibition is mediated by G
dimers (Figs. 3C, 5).
Notably, M1 receptors produce relatively weak inhibition of
1E
(compare Figs. 1, 5). Thus, even in staurosporine-treated cells where
stimulation was prevented and inhibition was consequently not
underestimated,
1E currents were inhibited by only ~18% (Fig. 2).
In comparison, the M2 subtype of muscarinic receptor produces ~40%
inhibition of
1E Ca currents under identical experimental conditions
(Meza et al., 1999
). The weak inhibition of
1E through M1 receptors may reflect the type of G
dimer involved. Previously, Fletcher et
al. (1998)
found that G
5 preferentially associates with G
q, suggesting that G
q-coupled receptors such as the M1 receptor will
liberate G
dimers containing G
5 (assuming that G
5 is present in HEK293 cells). Because G
5 produces relatively weak voltage-dependent inhibition of native N-type Ca channels
(García et al., 1998
; Ruiz-Velasco and Ikeda, 2000
), it may
also produce relatively weak inhibition of R-type Ca channels formed by
1E.
PLC
1ct, RGS2, and RGS8 functioned primarily as
effector antagonists
PLC
1ct, RGS2, and RGS8 blocked stimulation of
1E without
also reducing its inhibition through M1 receptors (Fig. 5). The selective block of stimulation is surprising, given that PLC
1 and
RGS2 have been previously shown to act as powerful GAPs for G
q
in vitro (Berstein et al., 1992
; Biddlecome et al., 1996
; Ingi et al., 1998
). If PLC
1ct and RGS2 had behaved mainly as GAPs,
they would have accelerated conversion of G
-GTP into G
-GDP, thereby promoting reassociation of G
with its G
dimer (Berman and Gilman, 1998
). In this event, both inhibition and stimulation of
1E would have been attenuated. That only stimulation was reduced argues that PLC
1ct, RGS2, and RGS8 functioned primarily as effector antagonists in our experiments. This interpretation is supported by
results obtained using GTP-
-S. We found that GTP-
-S produced stimulation of
1E in control cells but not in cells coexpressing RGS2 (Fig. 6). Because GTP-
-S cannot be hydrolyzed by G
subunits, RGS2 must have functioned exclusively as an effector antagonist to
prevent stimulation of
1E in these experiments (Fig. 6). However, these experiments with GTP-
-S do not preclude the possibility that
RGS2, RGS8, and PLC
1ct also functioned as GAPs, in addition to
functioning as effector antagonists, in experiments in which M1
receptors and hydrolyzable GTP produced stimulation of
1E (e.g.,
Fig. 4).
Previous studies have demonstrated that RGS2 and RGS4 can act as
effector antagonists under certain conditions (Hepler et al., 1997
;
Heximer et al., 1997
; Yan et al., 1997
). In contrast, effector
antagonism by PLC
1 or RGS8 has not been previously reported. However, Kammermeier and Ikeda (1999)
found that PLC
1ct blocked the
voltage-independent, G
q-mediated component of N-type Ca channel inhibition in superior cervical ganglion neurons but left the voltage-dependent, G
-mediated component of inhibition intact. Their experiments suggest that PLC
1ct interfered with signaling by
G
q but not with signaling by its G
dimer. These results of
Kammermeier and Ikeda (1999)
are also consistent with the idea that
PLC
1ct can function as an effector antagonist for G
q.
Physical interactions between G
subunits and RGS proteins take place
at the switch regions of G
, which are also involved in binding
G
dimers and downstream effectors such as PLC
1 (Wall et al.,
1995
; Lambright et al., 1996
; Tesmer et al., 1997
; Berman and Gilman,
1998
). It is therefore unlikely that G
can associate with G
while an RGS protein (or effector) is bound. Prolonged association
between G
and an RGS protein would be expected to delay heterotrimer
formation and would be predicted to block signaling by G
but to
allow continued signaling by its G
dimer. This mechanism was
recently proposed by Bünemann and Hosey (1998)
to explain the
apparently increased availability of G
dimers in cells
overexpressing RGS4. Prolonged association between G
and an RGS
protein might result if GTP hydrolysis was relatively slow or if the
RGS protein dissociated slowly from G
after GTP hydrolysis (Berman
and Gilman, 1998
). Alternatively, an RGS protein might remain
associated with G
over multiple GTPase cycles, as proposed for
PLC
1 and G
q (Biddlecome et al., 1996
). In this latter example, an
RGS protein (or PLC
1) could function simultaneously, or alternately,
as an effector antagonist and as a GAP.
Previously, we demonstrated that RGS proteins shift the dose-response
curve for Ca channel inhibition to higher agonist concentrations (Melliti et al., 1999
). RGS proteins have also been shown to accelerate recovery of Ca channels from inhibition after agonist washout (Jeong
and Ikeda, 1998
; Melliti et al., 1999
). These effects of RGS proteins
can be adequately explained by their GAP activity. In these two
previous studies, Ca channels were inhibited by PTX-sensitive G-proteins belonging to the G
i subfamily (Jeong and Ikeda, 1998
; Melliti et al., 1999
). In the present study, channel modulation was
mediated by PTX-insensitive G-proteins (probably G
q), and the
coexpressed RGS proteins (and PLC
1ct) appeared to function primarily
as effector antagonists. Thus, whether G
-interacting proteins such
as RGS and PLC
1 behave mainly as GAPs or as effector antagonists
might depend on the G
subunit involved. Consistent with this idea,
most previous studies have used G
q to reveal the effector antagonist
function of RGS proteins (Hepler et al., 1997
; Heximer et al., 1997
;
Yan et al., 1997
; Kammermeier and Ikeda, 1999
).
Our present results are the first demonstration that RGS proteins (and
PLC
1ct) can influence receptor-mediated stimulation, as opposed to
inhibition, of voltage-gated Ca channels. Our findings contribute to a
growing body of evidence that RGS and other G
-interacting proteins
play important roles in ion channel modulation (cf. Doupnik et al.,
1997
; Saitoh et al., 1997
; Bünemann and Hosey, 1998
; Jeong and
Ikeda, 1998
; Herlitze et al., 1999
; Kammermeier and Ikeda, 1999
;
Melliti et al., 1999
).
Physiological significance of
1E Ca channel stimulation
Muscarinic receptors are widely expressed in mammalian brain and
are prevalent in hippocampus, dentate gyrus, amygdala, and cortex
(Buckley et al., 1988
). These same regions of the brain also express
1E subunits (Niidome et al., 1992
; Soong et al., 1993
; Wakamori et
al., 1994
; Williams et al., 1994
; Yokoyama et al., 1995
). Additionally,
muscarinic receptors and
1E subunits are both found on neuronal
somata and dendrites (Hersch et al., 1994
; Yokoyama et al., 1995
;
Westenbroek et al., 1998
). Thus, we speculate that native R-type Ca
channels are modulated through muscarinic receptors in central neurons.
Only a few studies have examined receptor-mediated modulation of native
R-type Ca channels. Jeong and Wurster (1997)
observed muscarinic
inhibition of R-type currents in intracardiac neurons, and Overholt and
Prabhakar (1999)
found adrenergic inhibition of R-type currents in
carotid body glomus cells. To our knowledge, receptor-mediated
stimulation of native R-type Ca channels has not been reported.
However, our present results demonstrate that
1E Ca channels are
strongly stimulated through G
q-coupled M1 receptors. It seems likely
that native R-type Ca channels are also stimulated through muscarinic
and perhaps other G
q-coupled receptors in vivo.
Stimulation of native R-type currents may have important consequences
for dendritic Ca signaling, neurosecretion, gene expression, or other
neuronal functions.
 |
FOOTNOTES |
Received April 14, 2000; revised July 11, 2000; accepted July 14, 2000.
This work was supported by National Institutes of Health Grant NS34423
to B.A., American Heart Association Established Investigator Award
0040067N to B.A., and Consejo Nacional de Ciencia y Tecnologia Grant
31391-N to U.M. K.M. was the recipient of a fellowship from the
Philippe Foundation.
Correspondence should be addressed to Brett Adams, Department of
Biology, 5305 Old Main Hill, Utah State University, Logan, UT
84322-5305. E-mail: brett{at}biology.usu.edu.
 |
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