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The Journal of Neuroscience, August 1, 2000, 20(15):5623-5629
A Voltage-Independent Calcium Current Inhibitory Pathway
Activated by Muscarinic Agonists in Rat Sympathetic Neurons Requires
Both G q/11 and G 
Paul J.
Kammermeier,
Victor
Ruiz-Velasco, and
Stephen R.
Ikeda
Laboratory of Molecular Physiology, Guthrie Research Institute,
Sayre, Pennsylvania 18840
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ABSTRACT |
Calcium current modulation by the muscarinic cholinergic agonist
oxotremorine methiodide (oxo-M) was examined in sympathetic neurons
from the superior cervical ganglion of the rat. Oxo-M strongly
inhibited calcium currents via voltage-dependent (VD) and
voltage-independent (VI) pathways. These pathways could be separated
with the use of the specific M1 acetylcholine receptor antagonist M1-toxin and with pertussis toxin (PTX)
treatment. Expression by nuclear cDNA injection of the regulator of
G-protein signaling (RGS2) or a phospholipase C 1 C-terminal
construct (PLC-ct) selectively reduced VI oxo-M modulation in
PTX-treated and untreated cells. Expression of the G buffers
transducin (G tr) and a G-protein-coupled-receptor kinase (GRK3) construct (MAS-GRK3) eliminated oxo-M modulation. Activation of the heterologously expressed neurokinin type 1 receptor, a Gq/11-coupled receptor, resulted in VI calcium current
modulation. This modulation was eliminated with coexpression of
Gtr or MAS-GRK3. Cells expressing
G12 were tonically inhibited via the VD
pathway. Application of oxo-M to these cells produced VI modulation and reduced the amount of current inhibited via the VD pathway. Together, these results confirm the requirement for G in VD modulation and
implicate Gq-GTP and G as components in the
potentially novel VI pathway.
Key words:
N-type calcium channel; ion channel modulation; voltage
dependent; sympathetic neurons; SCG; G-protein
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INTRODUCTION |
Calcium current modulation by
G-protein-coupled receptors can proceed through many pathways. A well
studied and widespread mechanism in neurons is the voltage-dependent
(VD) inhibition seen in many systems (Hille, 1994 ). This modulation is
initiated by G-protein activation and mediated by the G-protein
subunit (Herlitze et al., 1996; Ikeda, 1996). G appears to
directly interact with N- and P/Q-type calcium channels (Zhang et al., 1996; DeWaard et al., 1997; Page et al., 1997; Zamponi et al., 1997), causing a shift in channel gating that results in smaller currents with slower activation kinetics at moderate voltages (Bean,
1989; Elmslie et al., 1990). When membrane potential is driven to
positive voltages, this shift can be at least partially reversed,
resulting in "facilitated" (relieved of inhibition) currents.
Therefore, kinetic slowing and facilitation can be used to identify
this mechanism. Conversely, other modulatory pathways inhibit calcium
channels by mechanisms insensitive to membrane potential. These forms
of inhibition are collectively referred to as voltage independent (VI)
and are often initiated by activation of PTX-insensitive G-proteins
(Hille, 1994).
Because of the expression of multiple muscarinic acetylcholine receptor
(mAchR) subtypes in superior cervical ganglion (SCG) neurons,
application of a nonspecific muscarinic agonist initiates multiple
modulatory pathways. The resulting calcium current inhibition has both
VD and VI components. Activation of M4 (Bernheim
et al., 1992) mAchRs, which couple to Gi/o
G-proteins, produces a VD, PTX-sensitive calcium current inhibition
(Bernheim et al., 1992; Delmas et al., 1998a,b). This inhibition
is mediated by the well defined pathway described above. Activation of
M1 mAchRs, which couple to
Gq/11 G-proteins, can produce a calcium current
inhibition that is insensitive to PTX and membrane potential. This
inhibition appears to have at least two components (Beech et al.,
1992). The first, termed "sman" (Beech et al., 1992), has a slow
onset, may use a diffusible second messenger because it does not appear to be membrane-delimited, and is sensitive to calcium buffering in rat
(Bernheim et al., 1992) but not mouse SCG (Shapiro et al., 1999). The
second, termed "fan," is faster in onset and insensitive to calcium
buffering (Beech et al., 1992; Delmas et al., 1998a,b). These
pathways may be used by other receptors, such as the angiotensin II and
substance P (SP) receptors (Shapiro and Hille, 1993; Shapiro et al.,
1994). Although some characterization of these pathways has been
completed, the underlying mechanisms have not been precisely defined.
In addition, experimental conditions (e.g., calcium buffering levels)
may influence whether these pathways are observed.
Experiments described in this study were designed to examine the
mechanisms of calcium current modulation via natively expressed mAchRs,
with emphasis on the observed VI inhibitory pathway. This pathway
differed from the voltage-independent inhibitory sman pathway described
above (Beech et al., 1992; Bernheim et al., 1992; Delmas et al.,
1998a,b). The experiments described below will show that the VI
and VD pathways can be separated and begin to elucidate the mechanism
of the potentially novel VI pathway.
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MATERIALS AND METHODS |
Cell isolation, cDNA injection, and plasmids. A
detailed description of the isolation and cDNA injection procedure is
available elsewhere (Ikeda, 1997). The methods used were approved by
the Institutional Animal Care and Use Committee. Briefly, the SCG were
removed from adult Wistar rats (175-225 gm) after decapitation and
incubated in Earle's balanced salt solution (Life Technologies, Rockville, MD) containing 0.4 mg/ml trypsin (Worthington Biochemicals, Freehold, NJ), 0.6 mg/ml collagenase D (Boehringer Mannheim,
Indianapolis, IN) and 0.05 mg/ml DNase I (Sigma, St. Louis, MO) for 1 hr at 35°C. Cells were then centrifuged (50 × g),
transferred to minimum essential medium (Fisher Scientific, Pittsburgh,
PA), plated on poly-L-lysine-coated 35 mm
polystyrene tissue culture dishes, and placed in an incubator (95% air
and 5% CO2; 100% humidity) at 37°C before DNA
injection. After injection, cells were incubated overnight at 37°C,
and patch-clamp experiments were performed the following day. Where
indicated, neurons were incubated overnight with PTX (0.5 µg/ml; List
Biological, Campbell, CA) in the culture media.
Injection of cDNA was performed with an Eppendorf 5246 microinjector
and 5171 micromanipulator (Eppendorf, Madison, WI) 4-6 hr after cell
isolation. Plasmids were stored at  20°C as a 1 µg/µl stock
solution in TE buffer (10 mM Tris, 1 mM EDTA,
pH 8). Transducin was injected at 0.1 µg/µl (in pcDNA3.1;
Invitrogen, Carlsbad, CA). RGS2 (pCI; Promega, Madison, WI), PLC--ct
(pEGFP-C1; Clontech, Palo Alto CA), and MAS-GRK3 (pcDNA3.1+;
Invitrogen) were injected at 0.1, 0.1, and 0.01 µg/µl,
respectively. Detailed descriptions of these constructs are available
in Kammermeier and Ikeda (1999). Type 1 neurokinin receptor
(NK1) (pCI; original plasmid from J. E. Krause, Neurogen Corporation) was injected at the concentrations noted
in the text. G1 and
G2 plasmids (pCI) were injected at 0.05 µg/µl. Neurons were coinjected with "enhanced" green
fluorescent protein (GFP) cDNA (0.005 µg/µl) (pEGFP-N1; Clontech)
to facilitate later identification of successfully injected cells.
All inserts were sequenced using an automated DNA sequencer (ABI 310, PE Applied Biosystems, Foster City, CA). PCR products were purified
with silica membrane spin columns (Qiagen, Valencia, CA) before
restriction digestion and ligation. Plasmids were propagated in
XL1-blue bacteria (Stratagene, La Jolla, CA), and minipreps were
prepared using Qiagen anion exchange columns.
Electrophysiology and data analysis. Patch pipettes were
made from 7052 glass (Garner Glass, Claremont, CA) and had resistances of 1-4 M . Series resistances were 2-7 M before electronic
compensation, which was typically 80%. Ruptured-patch whole-cell
recordings were made using an Axopatch 200 or 200A patch clamp
amplifier (Axon Instruments, Foster City, CA). Voltage protocol
generation and data acquisition were performed using custom software on
a Macintosh Quadra series computer (Apple Computer, Cupertino, CA) with
a MacADIOS II data acquisition board (G.W. Instruments, Somerville, MA). Currents were low-pass-filtered at 5 kHz using the four-pole Bessel filter in the patch-clamp amplifier, digitized at 2-5 kHz, and
stored on the computer for later analysis. Experiments were performed
at 21-24°C (room temperature). Data analysis was performed using
Igor software (Wavemetrics, Lake Oswego, OR).
The external (bath) solution contained (in mM): 155 Tris
hydroxymethyl aminomethane, 20 HEPES, 10 glucose, 10 CaCl2, and 0.0003 tetrodotoxin, pH 7.4, osmolality 320 mOsm/kg. The internal (pipette) solution contained (in
mM): 120 N-methyl-D-glucamine (NMG)
methanesulfonate, 20 TEA, 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl2, 4 MgATP, 0.3 Na2GTP, and 14 Tris creatine phosphate, pH 7.2, osmolality 300 mOsm/kg. M1-toxin was obtained from Peptides International
(Louisville, KY). Where indicated, external solutions contained 10 µM oxotremorine methiodide (oxo-M) or 10 µM norepinephrine (NE). All drugs and control
solutions were applied to cells using a custom, gravity-driven perfusion system positioned ~100 µm from the cell, which allowed rapid solution exchange (250 msec). M1-toxin
application via the perfusion system was begun before seal formation
and was maintained for the duration of the experiment. Solutions
containing the M1-toxin (30-100
nM) also contained 0.1 mg/ml cytochrome
c. U73122 (Sigma) was dissolved in dimethylsulfoxide and
applied to cells ~30 min before appropriate experiments. Percentage
inhibition of muscarinic responses was calculated as the decrease in
current of the third sweep in oxo-M (~30 sec, to allow steady state
to be reached) compared with the last sweep before application of the
drug. For NE responses, inhibition was calculated from the maximal
inhibition in the drug.
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RESULTS |
Calcium current inhibition by oxotremorine-M
The voltage protocol illustrated in Figure
1A was run every 10 sec
from a holding potential of 80 mV. This protocol (Elmslie et al.,
1990) consists of a test pulse to +10 mV (the "prepulse") followed
by a strong depolarizing step to +80 mV, a brief return to 80 mV, and
finally another test pulse to +10 mV (the "postpulse"), during
which currents inhibited by the VD pathway will be facilitated. Application of both 10 µM oxo-M and 10 µM NE potently inhibited the calcium current in
the prepulse, whereas inhibition by NE was much less potent in the
postpulse (Fig. 1A), indicating that NE modulation
was predominantly VD. The total oxo-M inhibition, in contrast, had a
large VI component (Fig. 1A,C).
However, a VD component of oxo-M inhibition was apparent because
postpulse currents were facilitated and kinetic slowing was routinely
observed in the prepulse currents (Fig. 1A). Although
the NE modulation was not completely reversed by the depolarizing
pulse, it was considered representative of the G-mediated, VD
pathway because it was PTX sensitive and nearly completely blocked by
G buffers (Kammermeier and Ikeda, 1999). Thus, the degree of
voltage dependence of NE inhibition was taken as an arbitrary standard
of VD inhibition. The effect of oxo-M was rapid, generally reaching
steady state in 20-30 sec (Fig. 1C). Figure
1B illustrates the average inhibition (+SEM)
of the prepulse and postpulse currents produced by application of 10 µM oxo-M and 10 µM NE.
Prepulse (filled bars) and postpulse (open
bars) inhibition by oxo-M was 76 ± 3 and 64 ± 3%
(n = 15). NE inhibited the prepulse and postpulse
currents by 56 ± 7 and 27 ± 4% (n = 13),
respectively. N-type calcium channels were assumed to be the
predominant channel modulated by oxo-M. In SCG neurons recorded under
these conditions, N-type current makes up the vast majority of the
total calcium current, and the contribution of L-type channels (which
can be modulated by Gq/11 activation in other
systems) is negligible (Zhu and Ikeda, 1994).
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Figure 1.
Oxo-M inhibits calcium currents in SCG neurons.
A, Sample current traces illustrating control
(con: before drug application) and inhibited currents
(oxo-M or NE: in the presence of the
drug). The holding potential for all experiments was 80 mV. The
voltage protocol, shown below the current traces, was repeated at 10 sec intervals. Calibration: 1 nA, 20 msec. B, Bar graph
indicating average (+SEM) prepulse inhibition (first test pulse to +10
mV in the illustrated voltage protocol: solid bars) and
postpulse inhibition (second test pulse to +10 mV: open
bars) by 10 µM oxo-M and 10 µM NE.
Numbers of cells for each drug are shown in parentheses.
C, Time course for cell shown in A.
Filled circles represent current measurements from the
prepulse; open circles are from the postpulse.
Measurements are taken 10 msec from the beginning of each step.
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VD and VI pathways can be separated using PTX and M1-toxin
To examine which mAchRs were responsible for each inhibitory
pathway, calcium current modulation was examined in the presence of
M1-toxin, a selective M1
mAchR antagonist (Max et al., 1993). Application of 10 µM
oxo-M to SCG neurons in the continuous presence of 30-100
nM M1-toxin produced predominantly VD
inhibition of the calcium currents as evidenced by kinetic slowing and
prepulse facilitation (Fig.
2A, top).
Interestingly, the magnitude of total inhibition in the prepulse was
similar to that of cells in the absence of
M1-toxin. However, the facilitation ratio
(postpulse current/prepulse current) of oxo-M-inhibited currents in the
presence of M1-toxin was 3.4 ± 0.2, compared with 1.9 ± 0.1 in control cells. In the presence of
M1-toxin, the average inhibition by oxo-M in the
prepulse and postpulse was 70 ± 6 and 36 ± 5%
(n = 9), respectively (Fig. 2B).
These data confirm that activation of M1 mAchRs
is involved in VI inhibition and suggest that
M1-toxin is an effective M1
mAchR antagonist.
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Figure 2.
VD and VI oxo-M pathways can be separated using
M1-toxin and PTX treatment. A, Sample
current traces illustrating control (con: before drug
application) and oxo-M-inhibited currents (oxo-M)
in cells treated with M1-toxin (100 nM in the
bath for the duration of the experiment; top) and cells
treated overnight with PTX (bottom). The voltage
protocol is the same as in Figure 1A.
Calibration: 1 nA, 20 msec. B, Bar graph depicting
average (+SEM) prepulse and postpulse inhibition of calcium currents by
10 µM oxo-M in control cells (Con), in the
presence of 30-100 nM M1-toxin
(M1Tx), and in cells pretreated
with 0.5 µg/ml PTX (PTX).
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After overnight exposure to 0.5 µg/ml PTX, calcium current inhibition
by 10 µM oxo-M was less potent (Fig.
2A, bottom). Additionally, the inhibition
appeared to be VI, because there was little facilitation, and kinetic
slowing was rarely observed. Inhibition of prepulse and postpulse
currents by oxo-M in PTX-treated cells was 37 ± 3 and 33 ± 3%, respectively (n = 28) (Fig. 2B).
As a control for the effectiveness of PTX, inhibition by NE, an
2-adrenergic receptor-mediated pathway
(Schofield, 1990), was examined. Prepulse and postpulse NE inhibition
was 15 ± 2 and 7 ± 1% (n = 10) in
PTX-treated cells, respectively, compared with 56 ± 7 and 27 ± 4% in untreated cells. These data confirm that VI modulation of
calcium currents in SCG neurons proceeds through a PTX-insensitive
pathway and that VD modulation is PTX sensitive.
Expression of RGS2 and PLC-ct selectively reduces VI
oxo-M inhibition
To test whether oxo-M-mediated, VI calcium current inhibition
proceeds through a Gq/11-mediated pathway,
calcium current inhibition was examined in the presence of RGS2 and a
GFP-tagged, C-terminal construct of phospholipase C1 (PLC-ct)
(Kammermeier and Ikeda, 1999). Both of these constructs can
selectively bind Gq/11-GTP (Wu et al., 1993;
Heximer et al., 1997). In contrast to uninjected cells (Fig.
3A), cells expressing RGS2 or
PLC-ct exhibited predominantly VD inhibition on exposure to oxo-M
(Fig. 3B,C). Under these
conditions, prepulse inhibition was similar to control cells, but
postpulse inhibition was reduced to 47 ± 5% in cells expressing
RGS2 (n = 5) and 35 ± 6% in cells expressing PLC-ct (n = 5) (Fig. 3D).
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Figure 3.
Expression of RGS2 or PLC-ct reduces VI
modulation. A-C, Sample current traces
(left) and corresponding time courses
(right) for uninjected cells and cells expressing RGS2
or PLC-ct, respectively. The voltage protocol is the same as in
Figure 1A. Calibration: 1 nA, 20 msec for all
current traces. D, E, Bar graphs
indicating average (+SEM) prepulse (filled bars)
and postpulse (open bars) inhibition for uninjected
(control) cells and cells expressing RGS2 and
PLC-ct for untreated cells (D) and cells
treated with 0.5 µg/ml PTX (E).
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This effect was also apparent when the VI pathway was examined in
isolation. Figure 3E illustrates averaged data from
PTX-treated, uninjected cells (Fig. 3E,
control) and those expressing either RGS2 or
PLC-ct. The average prepulse and postpulse inhibition for these
groups was 37 ± 3 and 33 ± 3% for uninjected cells
(n = 28), 41 ± 13 and 15 ± 8% for
RGS2-expressing cells (n = 3), and 29 ± 3 and
9 ± 3% for PLC-ct-expressing cells (n = 6)
after overnight PTX treatment. Therefore, the VI inhibition that was exhibited in PTX-treated cells appeared to be converted to VD inhibition when Gq/11-GTP was buffered.
Apparently, the G that mediated the VD inhibition in this
experiment was released on Gq/11 activation,
indicating that G from either Gi/o or
Gq/11 has the ability to produce VD inhibition
under certain circumstances. Together, these data indicate that the VI
component of oxo-M modulation requires
Gq/11-GTP. However, activation of
phospholipase C did not appear to be involved in this pathway, because
the phospholipase C inhibitor U73122 had no effect on calcium
current modulation by oxo-M in PTX-treated cells (data not shown).
Buffers of G block inhibition by oxo-M
Because VD inhibition is mediated by G, and VI inhibition
appears to require Gq/11-GTP, oxo-M modulation
was examined in cells expressing proteins that bind G. When
transducin, a G subunit from retina (Gtr),
or a membrane-associating C-terminal construct of a G-protein-coupled
receptor kinase (termed MAS-GRK3), two G-binding proteins, were
expressed in SCG neurons, the total oxo-M inhibition was nearly
eliminated (Fig. 4A).
Prepulse and postpulse oxo-M inhibition of
Gtr-expressing cells was 11 ± 5 and
13 ± 4%, respectively (n = 8). In
MAS-GRK3-expressing cells, prepulse and postpulse inhibition was 7 ± 3 and 11 ± 4%, respectively (n = 9) (Fig.
4B). Finally, this experiment was repeated in cells pretreated with PTX. The predominantly VI modulation normally seen in
these cells was eliminated by expression of
Gtr or MAS-GRK3 (Fig. 4C).
PTX-treated cells expressing Gtr had prepulse
and postpulse inhibition of 6 ± 2 and 11 ± 2%,
respectively (n = 17). Prepulse and postpulse
inhibition in MAS-GRK3-expressing cells was 2 ± 2 and 6 ± 3%, respectively (n = 16) (Fig. 4D).
It seems unlikely that the effects of G buffers on oxo-M
modulation resulted from nonspecific actions because neither of these
agents altered oxo-M-mediated M-type potassium current modulation.
M-current was inhibited 77 ± 1% in control cells, 84 ± 7%
in Gtr-expressing cells, and 77 ± 6% in
MAS-GRK3-expressing cells; n = 3 for each group. These
data confirm that both the VD and VI pathways require G. It
should be noted that in early attempts in this study, this experiment
appeared to produce selective elimination of the VD pathway in
PTX-untreated cells. The apparent VI inhibition that remained was
determined to have resulted from inefficient Gtr and MAS-GRK3 expression or inefficient
buffering attributable to the large amount of G released on oxo-M
application. To circumvent this problem, NE-induced inhibition was used
as a paired positive control for expression of the G buffers.
Only cells exhibiting <15% inhibition by NE were included in the
final analysis shown in Figure 4, A and B.
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Figure 4.
Expression of MAS-GRK3 or Gtr
eliminates oxo-M modulation. A, Sample current traces
illustrating currents before (con) and during oxo-M
application (oxo-M) in a MAS-GRK3-expressing
cell. B, Bar graph depicting average (+SEM)
prepulse and postpulse inhibition of calcium currents in
uninjected cells and cells expressing MAS-GRK3 or Gtr.
C, D, Same as A and
B, but for cells pretreated overnight with 0.5 µg/ml
PTX. Calibration: 1 nA, 20 msec. The voltage protocol is the same as in
Figure 1A.
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Neurokinin type 1 receptor-mediated VI inhibition is inhibited by
G buffering
The experiments above indicate that oxo-M-mediated VI calcium
current inhibition requires G in addition to
Gq/11-GTP. To test the generality of these
effects, modulation of the calcium current was examined after
activation of an independent Gq/11-coupled receptor. For this experiment, NK1 was chosen.
cDNA encoding NK1 was injected at several
concentrations ranging from 0.006 to 120 ng/µl to determine an
optimal level for NK1 expression (Fig.
5A). SP was used as the
NK1 agonist (Krause et al., 1994). After nuclear injection of 6 ng/µl NK1 cDNA, maximal calcium
current inhibition was obtained. Concentrations below 0.6 ng/µl
produced no detectable changes in calcium current amplitude (but see
Shapiro and Hille, 1993). Subsequent experiments were therefore
performed after injection of 6 ng/µl NK1 cDNA.
In addition, experiments with NK1 were performed using -neurokinin (-NK) as the NK1 agonist,
because the action of -NK was more readily reversible than that of
SP (data not shown).
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Figure 5.
-NK application to NK1-expressing
cells produces a Gtr-sensitive, VI inhibition.
A, Bar graph indicating the average (+SEM) maximal
calcium current inhibition by 1 µM SP (in the prepulse)
in cells after nuclear injection of the indicated concentration of
NK1 cDNA (see Materials and Methods for description of
injection procedure). B, Sample current traces
illustrating 1 µM -NK inhibition in cells expressing
NK1 alone (top) or with Gtr
(bottom). Calibration: B,
top, 1 nA, 20 msec; B,
bottom, 0.5 nA, 20 msec. C, Bar graph
depicting average prepulse and postpulse inhibition by 1 µM -NK for NK1-expressing cells (injected
with 6 ng/µl NK1 cDNA).
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Application of 1 µM -NK to cells expressing
NK1 produced a VI calcium current inhibition in
which little kinetic slowing or prepulse facilitation was observed
(Fig. 5B, top). -NK produced inhibition of
48 ± 6% in the prepulse and 41 ± 4% in the postpulse in
control NK1-expressing cells. Cells expressing
Gtr in addition to NK1
exhibited little calcium current inhibition on exposure to 1 µM -NK (Fig. 5B,
bottom). Prepulse and postpulse inhibition in these cells
were 3 ± 2 and 10 ± 3%, respectively (n = 10) (Fig. 5C). This result indicates that the VI inhibition
mediated by NK1 is sensitive to
Gtr and thus requires activated G.
Therefore, the interpretation that VI inhibition requires G is
supported. Finally, cells coexpressing NK1 and
PLC-ct exhibited VD inhibition on -NK application (data not
shown). In these cells, prepulse inhibition was 24 ± 3% and
postpulse inhibition was 4 ± 2% (n = 4). These
data indicate that NK1 activation can activate
the same VI pathway as M1 mAchR activation and
support the finding that this pathway requires
Gq/11-GTP.
Cells expressing G exhibit oxo-M inhibition in the postpulse
but not prepulse
The data above indicate that the VI oxo-M-mediated pathway
requires not only Gq/11-GTP but G as
well. As a test for independence of the two pathways, oxo-M modulation
of calcium current was examined in cells expressing
G12. These cells
exhibited strong basal facilitation (basal facilitation ratio = 3.2 ± 0.3) and kinetic slowing in the prepulse (Fig.
6A, con), as
expected (Herlitze et al., 1996; Ikeda, 1996). On exposure to oxo-M,
the calcium currents were inhibited potently in the postpulse but
remained largely unchanged in the prepulse (Fig. 6B).
In addition, oxo-M application resulted in some relief of kinetic
slowing in the prepulse (Fig. 6A,
oxo-M). Oxo-M produced a 5 ± 7% inhibition in
the prepulse and a 40 ± 5% inhibition in the postpulse, which reduced the facilitation ratio to 1.9 ± 0.2 (n = 11) (Fig. 6C).
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Figure 6.
Cells expressing G12
exhibit postpulse but not prepulse inhibition on exposure to oxo-M.
A, Sample current traces illustrating prepulse and
postpulse currents before and during application of 10 µM
oxo-M. Calibration: 0.3 nA, 20 msec. B, Time course of
oxo-M effect for cell shown in A. C,
Average (+SEM) oxo-M inhibition in the prepulse (filled
bars) and postpulse (open bars) for untreated
cells and cells treated overnight with 0.5 µg/ml PTX.
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Finally, this experiment was repeated in cells treated overnight with
PTX (Fig. 6C). In
G12-expressing cells
pretreated with PTX, prepulse inhibition was 2 ± 6% and
postpulse inhibition was 36 ± 3%, which reduced the facilitation
ratio to 1.9 ± 0.2 (n = 9) (Fig. 6C),
a value indistinguishable from the corresponding value obtained from
PTX-untreated cells. These results lend further support to the
hypothesis that the VI pathway shares a component with the VD pathway
(i.e., G). If these two mechanisms operated independently, the
prepulse and postpulse currents would be expected to be inhibited
similarly in
G12-expressing cells.
Therefore, these data support the conclusion that G (in addition
to Gq/11) is required for VI inhibition.
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DISCUSSION |
This study presents the finding that muscarinic receptor
activation can initiate a potentially novel VI calcium current
inhibitory pathway that requires Gq/11-GTP and
G. In addition, evidence has been presented to confirm that in
sympathetic neurons from the rat SCG, activation of mAchRs by oxo-M
produces VD and VI calcium current inhibition, mediated by
M4 and M1 muscarinic
receptors, respectively. Evidence is also presented that suggests that
the VD component is mediated by G-protein subunits, in agreement with previous studies (Herlitze et al., 1996; Ikeda, 1996).
Previous studies (Beech et al., 1991, 1992; Bernheim et al., 1991,
1992; Delmas et al., 1998a,b) have characterized a VI calcium channel inhibitory pathway in SCG neurons activated by muscarinic receptors, termed sman by Beech et al. (1992). This pathway appeared to
require a diffusible intracellular second messenger and was sensitive
to high concentrations of intracellular BAPTA (Bernheim et al., 1992).
Sman also activated with a much slower time course than the VD
inhibition. It is unlikely that the VI pathway observed in the present
study corresponds to the sman pathway for the following reasons. First,
the present study was conducted with 11 mM intracellular EGTA. In addition, results obtained with 10 mM BAPTA in the
pipette were similar to those reported in this study (data not shown). These levels (10-11 mM) of EGTA or BAPTA would be expected
to produce similar calcium buffering as the 20 mM BAPTA
used by the Hille and Brown groups (Beech et al., 1991, 1992; Bernheim
et al., 1991, 1992; Delmas et al., 1998a,b). Indeed, a
gonadotropin-releasing hormone-mediated pathway akin to the sman
pathway was shown to be sensitive to 10 mM EGTA under
conditions similar to those presented here (Lewis and Ikeda, 1997).
Therefore, with this level of calcium buffering, activation of the sman
pathway would be unlikely. Second, the VI inhibition observed in this
study appeared to activate somewhat more quickly than sman (Fig. 1).
Many factors of this study differed from previous studies that
characterized muscarinic, VI calcium current modulation in SCG neurons
(Beech et al., 1991; Delmas et al., 1998a), namely the ionic
composition of the recording solutions and temperature. Factors such as
these may influence which pathways can be activated during whole-cell
patch-clamp experiments as well as rates of onset for each pathway.
Separate from the sman pathway, another VI component, termed fan, was
observed by Beech et al. (1992), which activated more rapidly than sman and was not sensitive to intracellular BAPTA. The VI pathway presented here may correspond to the fan pathway. However, this pathway was not as well characterized as sman and did not appear to contribute a great deal to the total inhibition, perhaps because of the recording conditions, so direct comparison is difficult. There are other examples
of G-protein-coupled receptors producing VD or VI inhibition of N-type
calcium current (Hille, 1994). Some receptors, by activating multiple
classes of G-proteins, can initiate VD and VI pathways simultaneously
(Filippov et al., 1998; Delmas et al., 1999; Kammermeier and Ikeda,
1999). It should also be noted that results from mammalian SCG neurons
may differ significantly from those obtained in avian neurons
(Diverse-Pierluissi et al., 1997).
The requirement for G in the VD pathway has been well established
(Herlitze et al., 1996; Ikeda, 1996). However, a recent study (Delmas
et al., 1999) has presented evidence that Go- and Gi-associated subunits mediating
2-adrenergic calcium current modulation in SCG
neurons can produce relatively voltage-dependent and
voltage-independent inhibition, respectively. Our results indicate that
a VI pathway mediated by M1 mAchRs requires
Gq/11-GTP in addition to G. This pathway
does not appear to require G from a specific source. It is
unlikely, however, that any component of adrenergic modulation in SCG
neurons is akin to the VI, muscarinic pathway in the present study,
because of the lack of Gq/11 involvement. Under conditions similar to those presented here, RGS2 or PLC-ct expression failed to alter the voltage dependence of NE-mediated calcium current inhibition (Kammermeier and Ikeda, 1999).
The strategies of the experiments performed in this study are
summarized schematically in Figure 7.
Each diagram illustrates the available components (arrows)
of the M1 and M4 mAchR
signaling pathways under each experimental condition and the type(s) of modulation observed (VD, VI, or both). These experiments were designed
to eliminate one or more of these components while the calcium current
modulation was observed, thereby allowing identification of the
contributing components. Figure 7 (inset) also summarizes the available components under each condition in which VD
(top) or VI (bottom) modulation was observed.
(Note that G from either source, M1 or
M4, was considered functionally equivalent
because each has been shown to produce VD modulation; see Figs. 2,
3E). Under all conditions in which VD modulation was
observed, the only component available in every case was G,
confirming that G mediates this pathway. In contrast, VI
modulation was observed in only two experiments, uninjected control
(Fig. 7, top row, first column) and uninjected,
PTX-treated (Fig. 7, top row, second column). The
common components in these experiments were Gq
and G. This indicates that one or both of these components are
necessary for the VI pathway. Closer inspection reveals that when
either of these components was present in isolation, VI modulation was not observed (Fig. 7, RGS2/PLC-ct +PTX, center row,
second column, and Gtr/MAS-GRK3
+PTX, bottom row, second column). This confirms that both Gq and G are necessary to
activate the VI pathway.
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|
Figure 7.
Schematic summary of experiments. Each diagram
illustrates the two G-protein-signaling components associated with each
receptor (M1 or M4 mAchR) and the type of
modulation observed under each condition (VD,
VI, or No inhibition).
Arrows indicate that a component is free to be activated
and unhindered in its interaction with its effectors. X
indicates that a component is unavailable for activation or effector
interaction. Note that the smaller, lightened
arrow associated with Gi/o indicates only that
this component was not found to be involved in either of the pathways
of interest in this study and does not imply degree of signal strength.
Top row, Schematics for uninjected, untreated cells
(Control, first column), uninjected,
PTX-treated cells (+PTX, second column),
and uninjected cells exposed to M1-toxin
(+M1-toxin, third
column). Center row, Schematics for RGS2 or
PLC-ct-expressing, untreated cells (RGS/PLCb-ct,
first column) and RGS2 or PLC-ct-expressing,
PTX-treated cells (+PTX, second column).
Bottom row, Schematics for Gtr or
MAS-GRK3-expressing, untreated cells
(Gtr/MAS-GRK3,
first column), and for Gtr or
MAS-GRK3-expressing, PTX-treated cells (+PTX,
second column). Inset indicates which one
of the components was available (+) or unavailable () under each
condition in which VD (top) or VI
(bottom) inhibition was observed.
|
|
Because these separate pathways use a common molecule (G), it can
be assumed that the inhibition produced by each pathway will not be
strictly additive. This fact can explain the apparent discrepancy that
arises when each pathway is compared in isolation using
M1-toxin or PTX treatment (Fig. 2). Clearly, the
inhibition produced in control cells is less than the sum of that
produced by each pathway in isolation, at least in the prepulse.
However, because little is known about the molecular mechanism of the
VI pathway, it is difficult to speculate on the degree of inhibition expected when the two are activated together. For example, the role of
Gq/11-GTP and G in VI modulation is not
known. One may speculate that Gq/11-GTP can
bind to (or activate a secondary molecule that can bind to) the
G-bound calcium channel. This interaction may simply render the
inhibition of G voltage independent by preventing G release
in response to strong depolarizations. On the contrary, this
interaction may produce further inhibition unrelated to the VD
mechanism beyond its requirement for G. The data in Figure 6
indicate that the prepulse inhibition produced by each pathway is
similar (when at least some of the VD-modulated channels become VI
modulated), but this information alone does not allow either
possibility to be ruled out. Still, these data raise the question of
whether other G-GTPs can induce effects similar to that of
Gq/11-GTP, but with varying potencies. This possibility may explain why in some systems VD inhibition is completely reversed, or nearly so, by depolarizing voltage steps, whereas in
others (where other forms of G-GTP may be involved) the facilitation is only partial (but see Jones and Elmslie, 1997; Delmas et al., 1998b).
To definitively confirm that the VI pathway of calcium current
modulation required both Gq/11-GTP and
G, we attempted to overexpress a constitutively active
Gq (Gq Q209L) (Qian
et al., 1993) and introduce G (for example by application of NE)
to mimic VI inhibition. However, in attempting this experiment we encountered several problems that made interpretation difficult. First,
cells expressing constitutively active Gq
exhibited greatly reduced calcium currents. Unfortunately, the
remaining current possessed a strongly inactivating time course unlike
that seen in control cells inhibited by oxo-M (Delmas et al.,
1998a,b). This led to concerns that long-term exposure to
constitutively active Gq may induce changes in
the cells (such as the initiation of the phospholipase C pathway and
even possible activation of small G-protein pathways) unrelated to the
pathways that we wished to study. These changes may not be observable
in the short-term under patch-clamp conditions. Another concern was
that the VI inhibitory pathway would be saturated by the presence of
constitutively active Gq and basally activated
G.
In conclusion, we have confirmed that muscarinic calcium current
inhibition in sympathetic neurons from the rat SCG possesses both a VD
and a VI component. The VD pathway is PTX sensitive, mediated by
G, and appears to be activated by M4 mAchRs
because it is insensitive to the specific M1
mAchR antagonist, M1-toxin. The VI pathway is PTX
insensitive and is initiated by M1 mAchRs. In
addition, this VI pathway is sensitive to the presence of RGS2 and
PLC-ct, indicating that it requires
Gq/11-GTP. Finally, this pathway also appears
to require activation of G, as is apparent by its sensitivity to
MAS-GRK3 and Gtr. An apparently similar VI
modulation initiated by NK1 activation was
sensitive to buffering of G. Further studies are necessary to
elucidate the details of the mechanism of VI inhibition in SCG neurons
and to determine whether this mechanism is used in other systems.
| |
FOOTNOTES |
Received Feb. 24, 2000; revised May 5, 2000; accepted May 11, 2000.
This work was supported by National Institutes of Health Grants GM56180
and NS37615 (S.R.I.) and NS10943 (P.J.K.). We thank Marina King for
valuable technical assistance and Dr. Seong-Woo Jeong for helpful
critique of this work. We also thank M. I. Simon, R. J. Lefkowitz, D. R. Forsdyke, J. E. Krause, and S. G. Rhee for generously providing plasmids.
Correspondence should be addressed to Stephen R. Ikeda, Guthrie
Research Institute, Guthrie Foundation for Education and Research, One
Guthrie Square, Sayre, PA 18840. E-mail:
sikeda{at}inet.guthrie.org.
| |
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TOP
ABSTRACT
INTRODUCTION
