European Journal of Neuroscience, Vol. 14, pp. 283±292, 2001
ã Federation of European Neuroscience Societies
Multiple pertussis toxin-sensitive G-proteins can couple
receptors to GIRK channels in rat sympathetic neurons
when expressed heterologously, but only native
Gi-proteins do so in situ
Jose M. FernaÂndez-FernaÂndez,1* Fe C. Abogadie,1 Graeme Milligan,2 Patrick Delmas1 and David A. Brown1
1
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, UK
2
Keywords: a2-adrenergic receptors; GIRK channels, G-protein antisense-generating plasmids, M2 receptors, PTX-insensitive
G-protein mutants
Abstract
Although many G-protein-coupled neurotransmitter receptors are potentially capable of modulating both voltage-dependent Ca2+
channels (ICa) and G-protein-gated K+ channels (IGIRK), there is a substantial degree of selectivity in the coupling to one or other
of these channels in neurons. Thus, in rat superior cervical ganglion (SCG) neurons, M2 muscarinic acetylcholine receptors
(mAChRs) selectively activate IGIRK whereas M4 mAChRs selectively inhibit ICa. One source of selectivity might be that the two
receptors couple preferentially to different G-proteins. Using antisense depletion methods, we found that M2 mAChR-induced
activation of IGIRK is mediated by Gi whereas M4 mAChR-induced inhibition of ICa is mediated by GoA. Experiments with the bgsequestering peptides a-transducin and bARK1C-ter indicate that, although both effects are mediated by G-protein bg subunits, the
endogenous subunits involved in IGIRK inhibition differ from those involved in ICa inhibition. However, this pathway divergence
does not result from any fundamental selectivity in receptor±G-protein±channel coupling because both IGIRK and ICa modulation
can be rescued by heterologously expressed Gi or Go proteins after the endogenously coupled a-subunits have been inactivated
with Pertussis toxin (PTX). We suggest instead that the divergence in the pathways activated by the endogenous mAChRs
results from a differential topographical arrangement of receptor, G-protein and ion channel.
Introduction
Many G-protein-coupled neurotransmitter receptors (GPCRs) can
both inhibit voltage-dependent Ca2+ channels and activate G-proteingated inward recti®er K+ (GIRK/Kir3) channels. Both effects are
mediated through the receptor-triggered release of bg subunits from
heteromeric Pertussis toxin (PTX)-sensitive G-proteins and subsequent interaction with the ion channel (see Wickman & Clapham,
1995; Herlitze et al., 1996; Ikeda, 1996; Clapham & Neer, 1997;
Delmas et al., 1998a, b; Dolphin, 1998; Yamada et al., 1998).
Because both types of channel can be modulated by a wide range of
expressed bg subunit combinations (Wickman et al., 1994; Garcia
et al., 1998; Jeong & Ikeda, 2000; Zhou et al., 2000), it would be
expected that, in principal, individual GPCRs should affect both
channels. However, in normal nerve cells there appears to be a
substantial degree of selectivity in the coupling of individual
receptors to one or other of these channels. For example, a2adrenergic receptors inhibit Ca2+ channels but do not activate GIRK
channels in caudal raphe neurons (Li & Bayliss, 1998). Likewise, in
rat superior cervical ganglion (SCG) neurons, native M4 muscarinic
Correspondence: Professor David A. Brown, as above.
E-mail: d.a.brown@ucl.ac.uk
*Present address: Unitat de Senyalitzacio Cellular, Departament de CieÁncies
Experimentals i de la Salut, Universitat Pompeu Fabra, C/Dr Aiguader 80,
08003 Barcelona, Spain
Received 13 February 2001, revised 3 May 2001, accepted 18 May 2001
acetylcholine receptors (mAChRs) selectively inhibit N-type Ca2+
channels (Bernheim et al., 1992) but do not activate expressed GIRK
channels (Fernandez-Fernandez et al., 1999), whereas native M2
receptors show the opposite selectivity in channel coupling
(Fernandez-Fernandez et al., 1999). Because expressed M2 and M4
receptors couple to both types of channels in other systems, such as
oocytes and neuroblastoma cells (Higashida et al., 1990; Gadbut
et al., 1996), why the selectivity in SCG neurons?
One obvious source of selectivity might be that the two receptors
couple preferentially to different G-protein a subunits. Thus, ICa(N)
inhibition by M4 mAChRs in SCG neurons is mediated predominantly
(possibly exclusively) by GoA proteins (Delmas et al., 1998a), whereas
it is usually considered that activation of GIRK channels is normally
mediated by Gi proteins (Lledo et al., 1992). Therefore, one possibility
is that in rat SCG neurons M2 receptors selectively activate Gi proteins
whereas M4 receptors selectively activate Go proteins, and that GIRK
channels are preferentially activated by bg subunits freed from M2
mAChR-stimulated Gai whereas Ca2+ channels are preferentially
inhibited by bg subunits freed from M4 mAChR-activated Gao.
We have therefore assessed the contributions of different G-protein
a subunits (both endogenous and expressed) to the M2 mAChRinduced activation of expressed GIRK channels in rat SCG neurons.
We have also compared the results with those obtained on activating
native a2A-adrenoceptors, because these show a broader coupling
selectivity in these neurons; they can both activate GIRK channels
284 J. M. FernaÂndez-FernaÂndez et al.
and inhibit Ca2+ channels (Ruiz-Velasco & Ikeda, 1998), and both Go
and Gi contribute to noradrenergic Ca2+ channel inhibition (Delmas
et al., 1999). The results suggest that, while M2 mAChR-induced
GIRK activation is indeed mediated by Gi, this alone cannot explain
the discrimination of different mAChRS between GIRK and Ca2+
channels.
Materials and methods
Cell culture
Sympathetic neurons were dissociated from superior cervical ganglia
(SCG) of 15- to 19-day-old male Sprague-Dawley rats (killed by CO2
asphyxiation) as described previously (Fernandez-Fernandez et al.,
1999). Cells were plated on laminin-coated glass coverslips. Cultured
neurons were maintained at 37 °C in an atmosphere of 5% CO2 in L15 medium supplemented with 10% fetal bovine serum, 2 mM
glutamine, 24 mM NaHCO3, 38 mM glucose, 50 U/ml penicillinstreptomycin and 25 ng/ml nerve growth factor. All culture reagents
were from Gibco except laminin, collagenase, trypsin (from Sigma)
and nerve growth factor (from Tocris).
DNA plasmids
The cloning and the speci®city of plasmids generating antisense RNA
to various G-protein a subunits has been described previously
(Abogadie et al., 1997; Delmas et al., 1998a). Antisense sequence of
rat GaoA (clone 207±8) was subcloned into pCR3 expression vector
(Invitrogen, NV Leek, The Netherlands). The antisense sequence of
rat Gaicommon(1±3) (clone 50±2) was subcloned into pCR3.1. This
clone corresponds to nucleotides 1045±1215 of Gai2 and shares
approximately 80% identity with Gai1 and Gai3. cDNA encoding the
C-terminus of b-adrenergic receptor kinase 1 (bARK1495±689) was
subcloned in pCIN1 as described previously (Delmas et al., 1998b).
The generation of PTX-insensitive Gai/o subunits (GaoA C351I, Gai1
C351I, Gai2 C352I and Gai3 C351I, mutated Gai/o subunits in which
the cysteine 351 or 352 residue was replaced with isoleucine) was as
detailed in Wise et al. (1997). cDNAs encoding these mutants were
subcloned into pCDNA3 (Invitrogen). Retinal Ga-transducin was
subcloned into pCDNA3. Plasmids expressing GIRK were generously donated by Dr F. Lesage. Plasmids were propagated in either
XL1-Blue or DH5 Escherichia coli and puri®ed using Qiagen
maxiprep columns (Hilden, Germany).
Transfection
SCG neurons cultured for 1 day were transfected using a biolistic
device (PDS-1000/He; Bio-Rad) or by microinjection. In the ®rst
method, neurons were bombarded with 1.6 mm gold particles (BioRad) precoated with the desired plasmid DNAs, as described
previously (Fernandez-Fernandez et al., 1999). For microinjection,
the plasmids were diluted to 100±500 mg/mL in calcium- and
glucose-free Krebs solution (290 mosm/L, pH 7.3) containing 0.5%
FITC-dextran (70 kDa) and pressure injected into the nucleus of SCG
neurons using an Eppendorf microinjector (Hamburg, Germany). A
green ¯uorescent protein (GFP)-encoding plasmid was used as a
marker to facilitate later identi®cation of neurons successfully
transfected. After transfection, neurons were returned immediately
to the incubator and maintained in culture for a further 1±2 days
before recording. When choosing a neuron for study, the neurons
were illuminated with 470±490 nm light to excite the GFP and
viewed through a 515 nm ®lter. Those cells with a medium level of
FIG. 1. Effect of bg-sequestering agents on the M2 muscarinic and a2adrenergic activation of expressed GIRK channels. (A±F) Representative
traces of GIRK currents (IGIRK) recorded from SCG neurons transfected
with Kir3.1- and Kir3.2-expressing plasmids alone (Control, A and B) or
together with either bARK1C-ter- (C and D) or a-transducin-expressing
plasmids (E and F), before (basal) and after application of either 10 mM
carbachol (CCh) or 10 mM norepinephrine (NE) (as indicated), with the
subsequent addition of 100 mM BaCl2 in the presence of the agonist
(+Ba2+). (G and H) Summary of GIRK current densities induced either by
10 mM CCh (G) or 10 mM NE (H), in the absence or presence of the
indicated bg-sequestering agent. The neurons were transfected using the
`biolistic' technique and recorded 1 day after transfection. GIRK currents
were evoked by 500 ms voltage ramps between ±140 mV and ±40 mV from
a holding potential of ±60 mV. Currents were recorded in 12 mM [K+]o.
The peak current amplitude was determined by averaging currents between
±125 mV and ±130 mV. Dashed lines in the current traces indicate the zero
current level. Data are presented as mean 6 SEM, and numbers indicate the
number of neurons tested. **P < 0.005; *P < 0.05
green ¯uorescence were selected for recording. For the PTX
experiments, transfected neurons were incubated in 0.5±1 mg/mL
PTX in culture medium at 37 °C for 18±24 h before recording.
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
G-proteins gating GIRK channels in SCG neurons
285
TABLE 1. Effect of bARK1C-ter and a-transducin on NE (10 mM)-induced ICa
inhibition
Transfection
ICa inhibition (%)
None
GFP
GFP +
GFP +
GFP +
GFP +
GFP +
49.2
48.9
33.8
18.4
24.4
8.5
9
bARK1C-ter
a-transducin
GIRKs
GIRKs + bARK1C-ter
GIRKs + a-transducin
6
6
6
6
6
6
6
2.9 (n = 20)
4 (n = 14)
4 (n = 15)*(vs. GFP)
3.6 (n = 6)*(vs. GFP)
4.5 (n = 9)*(vs. GFP)
1.6 (n = 6)*(vs. GFP + GIRKs)
1.7 (n = 6)*(vs. GFP + GIRKs)
Data are means 6SEM of the number of experiments indicated in parenthesis.
*P < 0.05.
Potassium current (IGIRK) recording
Potassium currents were measured at 32±35 °C from SCG neurons 1±
2 days after transfection, using the whole-cell patch-clamp technique.
Borosilicate glass electrodes (2±4 MW) were ®lled with a solution
containing (in mM): KCl, 60; potassium acetate, 60; MgCl2, 2.5;
Hepes, 30; BAPTA, 10; Na2 ATP, 2; and Na3GTP, 0.1 (adjusted to
pH 7.2 with KOH and 290 mosmol/L). Cells were superfused
initially with a Krebs solution containing (in mM): NaCl, 110;
NaHCO3, 23; KCl, 3; MgCl2, 1.2; CaCl2, 2.5; Hepes, 5; glucose, 11;
tetrodotoxin (TTX), 0.0005 (bubbled with a 95% O2±5% CO2
mixture, pH 7.4). The cells were then superfused with a solution
containing (in mM): NaCl, 101; NaHCO3, 23; KCl, 12; MgCl2, 5;
Hepes, 5; glucose, 11; TTX, 0.0005 (bubbled with a 95% O2±5% CO2
mixture, pH 7.4). Membrane currents were recorded with an
Axoclamp 2B ampli®er (discontinuous single-electrode voltage
clamp, sample rate 6.6 kHz; Axon Instruments, Foster City, CA,
USA). K+ currents were typically evoked by 500 ms voltage ramp
between ±140 mV and ±40 mV (holding potential ±60 mV), low-pass
®ltered at 1 kHz and sampled at 6.67 kHz. The peak amplitude of the
current was acquired by averaging currents between ±125 mV and
±130 mV. Activation of IGIRK was measured 1 min after perfusion
with a 12 mM K+ solution containing the agonist [carbachol (CCh) or
norepinephrine (NE)], using a gravity-fed perfusion system (10 mL/
min). Muscarinic antagonists (tripitramine and pirenzepine), when
used, were present 2 min before and during CCh application.
Calcium current (ICa) recording
Voltage-gated Ca2+ currents (ICa) were recorded in whole-cell mode
(32±35 °C) from neurons cultured for 1±3 days. The solution in the
patch electrodes (2±4 MW) contained (in mM): CsCl, 13; caesium
acetate, 120; MgCl2, 2.5; Hepes, 10; BAPTA, 10; Na2ATP, 2 and
Na3GTP, 0.1 (adjusted to pH 7.2 with CsOH and 290 mosmol/L).
Neurons were bathed in Krebs solution and voltage clamped at ±80 mV
using an Axoclamp 2B ampli®er (continuous single-electrode voltage
clamp). Ca2+ currents were evoked by a double-pulse voltage protocol
consisting of a 10 ms test pulse to +10 mV applied before and after a
50 ms conditioning pulse to +100 mV. Currents were low-pass ®ltered
at 1 kHz and sampled at 33.3 kHz. The amplitude of ICa was estimated
by digitally subtracting the outward current remaining during the same
voltage protocol in the presence of Krebs solution containing CdCl2
100 mM. Inhibition of ICa was measured 1 min after the normal
perfusion solution was changed to one containing either NE or CCh.
Antagonists, when used, were perfused (10 mL/min) in the bath for at
least 2 min before testing with CCh.
FIG. 2. Basal GIRK currents in transfected cells and effect of Gb1g2
subunits on expressed GIRK currents. (A) Current traces obtained from a
nontransfected SCG neuron (left) and from a neuron transfected with
Kir3.1- and Kir3.2-expressing plasmids (right), before and after application
of 100 mM Ba2+ in the absence of agonists. Note that N-ethylmaleimide
(NEM) 50 mM blocked the tonic activation of GIRK currents. (B)
Representative current records obtained from SCG neurons injected with
Kir3.1- and Kir3.2-expressing plasmids alone (Control) or together with
Gb1- and Gg2-expressing plasmids (b1g2), before (basal) and after
application of 10 mM NE. (C) Summary of basal, NE-induced and total
(basal plus NE) current densities in the absence (white bars) and the
presence (black bars) of Gb1g2 overexpression. After nuclear injection of
cDNAs (Kir3.1- and Kir3.2-expressing plasmids 100 mg/mL; Gb1- and Gg2expressing plasmids 200 mg/mL), SCG neurons were maintained in culture
for 1 day before electrophysiological recording. GIRK currents were
recorded and measured as described in Fig. 1. Data are presented as
mean 6 SEM, and numbers indicate the number of neurons tested.
***P < 0.0001; **P < 0.005
Immunocytochemistry
Immunocytochemistry was performed as described previously
(Abogadie et al., 1997). Brie¯y, SCG neurons were acetone-®xed
and stained using rabbit polyclonal anti-Gao (sc-387) and anti-Gai1±3
(sc-262; Santa Cruz Biotechnology, CA, USA) diluted 1 : 1000.
Bound antibodies were detected using biotinylated Fab2 swine antirabbit IgG antibody (Dako, Denmark) conjugated with alkaline
phosphatase (1 : 500 dilution). The speci®city of the staining was
assessed by preabsorbing the antibody with a 10-fold excess of the
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
286 J. M. FernaÂndez-FernaÂndez et al.
FIG. 3. Effect of Gai1±3 and GaoA-antisense
expression on receptor-mediated modulation of
expressed GIRK channels and native Ca2+
channels. (Left and central panels) Typical
GIRK current records from SCG neurons
injected with Kir3.1- and Kir3.2-expressing
plasmids alone (Control, A and B) or along
with either GaoA (C and D) or Gai1±3
antisense plasmids (E and F), before (basal)
and after application of either 3 mM carbachol
(CCh) or 1 mM NE as indicated. The records
shown for each transfection group were
obtained from the same neurons. (Right panel)
Superimposed Ca2+ current traces before and
after application of 1 mM NE obtained from a
noninjected SCG neuron (H) and from neurons
injected with either GaoA (I) or Gai1±3
antisense plasmids (J). Dashed lines indicate
the zero current level. (G) Summary of GIRK
current densities induced either by 3 mM CCh
or 1 mM NE, in the absence or presence of the
indicated Ga antisense plasmid. (K) Summary
of calcium current inhibition induced by 1 mM
NE in the absence or presence of the indicated
Ga antisense plasmid. After nuclear injection
of cDNAs (Kir3.1- and Kir3.2-expressing
plasmids 100 mg/mL; Ga antisense plasmids
500 mg/mL), SCG neurons were maintained in
culture for a further 2 days before
electrophysiological recording. GIRK currents
were recorded as in Fig. 1. Calcium currents
were generated by a double-pulse voltage
protocol consisting of a 10 ms test pulse to
+10 mV applied before and after a 50 ms
conditioning pulse to +100 mV (see Materials
and methods); the current response to the
conditioning pulse is omitted for clarity. Data
are presented as mean 6 SEM, and numbers
indicate the number of neurons tested.
**P < 0.005; *P < 0.05
relevant immunogenic peptides (also
Biotechnology) (Delmas et al., 1998a).
from
Santa
Cruz
Chemicals and drugs
Tripitramine was generously donated by Professor Carlo Melchiorre
(Universita di Bologna, Italy). Other chemicals and drugs were
obtained from Sigma.
Statistics
All data are expressed as means 6 SEM. Student's t-test (unpaired)
or ANOVA followed by a posthoc test, as appropiate, was performed to
examine statistical signi®cance. P < 0.05 was considered signi®cant.
Results
Sequestration of bg subunits by a-transducin, but not by
bARK1C-ter, depresses agonist activation of Kir3.1/3.2 currents
It has been shown that Gbg subunits can activate GIRK channels in
native tissues and in heterologous expression systems (Logothetis
et al., 1987; Wickman & Clapham, 1995; Yamada et al., 1998). We
aimed to test whether endogenous Gbg subunits are involved in the
activation of expressed GIRK channels induced by M2 muscarinic
and a2-adrenergic receptors in SCG neurons, and, if so, whether the
activation is mediated by the same endogenous Gbg subunits as those
apparently responsible for ICa inhibition (Delmas et al., 1999). For
this we expressed two peptides containing Gbg-binding domains,
thereby competing with GIRK channels for free Gbg dimers: the
carboxyl terminal domain of bARK1 (bARK1C-ter) and a-transducin.
These peptides differ in their ef®ciency in binding different Gbg
subunits (Daaka et al., 1997) and can discriminate between different
modes of ICa inhibition mediated by different G-proteins in rat SCG
neurons (Delmas et al., 1998a, b, 1999).
Expression of bARK1C-ter had no effect on the activation of GIRK
currents (IGIRK) by the mAChR stimulant CCh (19.7 6 5.6 pA/pF,
n = 8 vs. 18.1 6 2.8 pA/pF, n = 18) (Fig. 1A, C and G). Similar
results were obtained when studying the NE-induced activation of
IGIRK (21.3 6 2.1 pA/pF, n = 11 vs. 27.1 6 4.5 pA/pF, n = 21)
(Fig. 1B, D and H). In order to test if bARK1C-ter was functionally
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
G-proteins gating GIRK channels in SCG neurons
287
FIG. 4. Inef®cient reconstitution of M2
muscarinic receptor coupling to expressed
GIRK channels by expression of PTXinsensitive Gai/o mutants. (A) Current traces in
the absence (basal) or presence of 10 mM
carbachol (CCh), recorded from SCG neurons
transfected with Kir3.1- and Kir3.2-expressing
plasmids alone or along with different PTXinsensitive Gai/o mutant subunits (GaoA, Gai1,
Gai2 or Gai3) and treated (+) or not treated (±)
with PTX as indicated. Dashed lines indicate
the zero current level. (B) Summary of basal
(white bars) and CCh-induced (black bars)
current densities, under conditions described
above, in SCG neurons untreated (left) or
treated (right) with PTX. After nuclear
injection of cDNAs (Kir3.1- and Kir3.2expressing plasmids, 100 mg/mL; Gai/o
mutant-expressing plasmids, 100 mg/mL), SCG
neurons were maintained in culture for 1 day
before electrophysiological recording. PTXtreated neurons were incubated overnight (18±
24 h) with 0.5 mg ml±1 PTX. GIRK currents
were recorded as in Fig. 1. Data are presented
as mean 6 SEM, and numbers indicate the
number of neurons tested. *P < 0.05
expressed we performed parallel experiments investigating the
adrenergic inhibition of calcium currents, as it has been shown
previously that such inhibition was partially prevented by bARK1C-ter
(Delmas et al., 1999). The results are summarized in Table 1. Thus,
bARK1C-ter expressed alone signi®cantly reduced the adrenergic
inhibition of ICa when compared to control untransfected cells or cells
transfected with a GFP-expressing plasmid. As previously described
by Ruiz-Velasco & Ikeda (1998), such inhibition was also reduced in
cells transfected with Kir3.1/3.2-expressing plasmids. Nevertheless,
in neurons cotransfected with both plasmids expressing Kir3.1/3.2
and bARK1C-ter, the inhibition of ICa induced by NE was attenuated
signi®cantly when compared to neurons expressing GIRK channels
alone. Thus, bARK1C-ter was functionally expressed and reduced ICa
inhibition by NE, but was unable to affect the activation of GIRK
currents by either NE or CCh.
By contrast, expression of the other bg-sequestering agent, atransducin, substantially reduced the activation of expressed GIRK
currents by both muscarinic (1.5 6 0.4 pA/pF, n = 10 vs.
18.1 6 2.8 pA/pF, n = 18) (Fig. 1A, E and G) and adrenergic
(6.9 6 1.2 pA/pF, n = 10 vs. 27.1 6 4.5 pA/pF, n = 21) receptors
(Fig. 1B, F and H).
We have previously reported that cotransfection of SCG neurons
with Kir3.1- and Kir3.2-expressing plasmids increased basal GIRK
currents seen in the absence of applied agonist (Fernandez-Fernandez
et al., 1999). Thus, the percentage of inwardly rectifying current
sensitive to 100 mM Ba2+ in transfected SCG neurons, in the absence
of any agonist, was nearly double that in nontransfected cells (see
Fig. 2A). Although in neurons coexpressing a-transducin, basal
(agonist-independent) currents appeared to be slightly smaller
(12.8 6 2.4 pA/pF, n = 10) than those in cells expressing GIRKs
alone (18.5 6 1.8 pA/pF, n = 21) or GIRKs along with bARK1C-ter
(18.4 6 2 pA/pF, n = 11), the difference was not statistically
signi®cant.
We also tested the effect of overexpressing exogenous b1g2
subunits (Fig. 2B and C). This signi®cantly increased basal current
density, and reduced GIRK current activation by the adrenergic
agonist NE. However, the total current activation (b1g2 plus NE) was
no greater than that activated by NE alone. Therefore, an excess of bg
subunits mimicked and occluded GIRK channel activation by
agonists.
Depletion of Gai subunits, but not GaoA, reduces agonist
activation of Kir3.1/3.2 currents
The above results suggest that in SCG neurons a2-adrenergic and M2
muscarinic receptors activate heterologously expressed Kir3.1/3.2
channels by endogenous bg subunits derived from PTX-sensitive Gproteins. However, comparison with previous results obtained on the
GPCR-induced ICa inhibition with Gbg-sequestering agents (Delmas
et al., 1999) suggests that the bg-subunits responsible for GIRKactivation might differ from those responsible for ICa inhibition,
implying perhaps an association with, and release from, different asubunits. To test this more directly, we expressed antisense RNAs
against individual Ga subunits (Fig. 3). In these experiments we used
a test concentration of 3 mM for CCh and 1 mM for NE. These are
submaximal concentrations for IGIRK activation and ICa inhibition
(see Ruiz-Velasco & Ikeda, 1998; Delmas et al., 1999; FernandezFernandez et al., 1999) and so provide a suitably sensitive assay for
any change in receptor±G-protein coupling ef®ciency.
Intranuclear injection of antisense-encoding plasmids against GaoA
and Gai1±3 resulted in a speci®c reduction in the immunoreactivity
for the relevant Ga protein (Delmas et al., 1998a; Haley et al., 1998).
Activation of IGIRK by 1 mM NE or 3 mM CCh was then examined
48 h after injection when the effects of the antisense RNAs plateaued
(Haley et al., 1998; Fig. 3A±G). In SCG neurons injected with
Kir3.1- and Kir3.2-expressing plasmids, current densities activated by
NE or CCh were 20.1 6 3.1 pA/pF (n = 17) and 14.9 6 2.3 pA/pF
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
288 J. M. FernaÂndez-FernaÂndez et al.
FIG. 5. Reconstitution of a2-adrenergic
receptor coupling to expressed GIRK channels
by expression of PTX-insensitive Gai/o
mutants. (A) Current traces in the absence
(basal) or presence of 10 mM NE, recorded
from SCG neurons transfected with Kir3.1and Kir3.2-expressing plasmids alone or along
with different PTX-insensitive Gai/o mutant
subunits (GaoA, Gai1, Gai2 or Gai3) and
treated (+) or not (±) with PTX (as indicated).
The records shown for each experimental
condition in this ®gure and in Fig. 4 were
obtained from the same neurons Dashed lines
indicate the zero current level. (B) Summary
of NE-induced current densities, under
conditions described above, in SCG neurons
untreated (left) or treated (right) with PTX.
After nuclear injection of cDNAs (Kir3.1- and
Kir3.2-expressing plasmids, 100 mg/mL; Gai/o
mutants-expressing plasmids, 100 mg/mL),
SCG neurons were maintained in culture for 1
day before electrophysiological recording.
PTX-treated neurons were incubated overnight
(18±24 h) with 0.5 mg/mL PTX. GIRK
currents were recorded as in Fig. 1. Data are
presented as mean 6 SEM, and numbers
indicate the number of neurons tested.
*P < 0.05
(n = 23), respectively. Depletion of GaoA subunits did not alter the
adrenergic (23.9 6 2.3 pA/pF, n = 24) or the muscarinic
(14.8 6 2.8 pA/pF, n = 23) activation of IGIRK (Fig. 3C, D and G).
By contrast, activation by NE and CCh was reduced signi®cantly in
cells coinjected with the Gai1±3 antisense-expressing plasmid
(9.9 6 1.3 pA/pF, n = 24 and 5.71 6 1.3 pA/pF, n = 21, respectively; Fig. 3E±G).
Gai1±3 antisense also produced a signi®cant reduction in the basal
(agonist-independent) current density (11.3 6 0.6 pA/pF, n = 25;
Fig. 3E and F), when compared to cells injected with GIRK channel
subunits alone (18.9 6 1.25 pA/pF, n = 23) or together with GaoAantisense (19.3 6 1.9 pA/pF, n = 24).
In order to test if GaoA depletion was suf®cient to have an effect on
cellular responses mediated by this G-protein, we made parallel
measurements of the effect of GaoA antisense on calcium current
inhibition by NE (Fig. 3H±K). As reported previously (Delmas et al.,
1999), expression of GaoA antisense depressed calcium current
inhibition by 1 mM NE from 44.7 6 4.4% (n = 10) to 26.5 6 4.2%
(n = 9). Also, basal ICa facilitation produced by a depolarizing
prepulse was higher in neurons expressing this antisense (Fig. 3I),
probably due to Gbg subunits released from GoA heterotrimers
following GaoA depletion (see Delmas et al., 1998a). However,
calcium current inhibition was not signi®cantly affected by expression of Gai1±3 antisense (46.2 6 5.4%, n = 10) (Fig. 3J).
Reconstitution of M2 muscarinic and a2-adrenergic receptor
coupling to Kir3.1/3.2 channels by PTX-insensitive Gai/o
mutants
The above experiments show that Gbg subunits released from Gi
rather than Go proteins are responsible for both M2 muscarinic and
a2-adrenergic receptor-mediated activation of Kir3.1/3.2 channels in
rat SCG neurons. To assess whether this coupling speci®city in situ is
due to a speci®c functional interaction between receptors, Gi/o
proteins and GIRK channels, we investigated the potential ability of
different members of the PTX-sensitive family of G-proteins to drive
receptor-induced activation of GIRK channels in these neurons. For
these experiments, SCG neurons were injected, along with Kir3.1and Kir3.2-expressing plasmids, with cDNAs encoding mutated Gai/o
subunits insensitive to PTX (see Materials and methods) and then
treated for 18±24 h with PTX to annul any response mediated through
endogenous Ga subunits. We then studied the degree of reconstitution of the muscarinic and adrenergic responses for each Ga subunit
mutant.
Expression of the mutants alone (without PTX treatment) did not
enhance membrane currents, and in fact reduced (signi®cantly in
some cases) both basal and agonist-activated GIRK currents
(Figs 4B and 5B). A similar result has been observed by others
(Leaney & Tinker, 2000; Leaney et al. 2000), and is most likely to be
attributed to sequestration of free Gbg subunits. As expected, PTX
treatment abolished the activation of IGIRK by 10 mM CCh (Fig. 4)
and produced a signi®cant attenuation of GIRK channel activation by
10 mM NE (3 6 0.3 pA/pF, n = 16 vs. 30.7 6 3.9 pA/pF, n = 25;
Fig. 5). Basal currents were signi®cantly greater in PTX-treated cells
(28.3 6 3 pA/pF, n = 18) than those in PTX-untreated neurons
(19.3 6 1.6 pA/pF, n = 26). In order to test if PTX-induced
enhancement of basal currents was due to uncoupling of an inhibitory
pathway following PTX treatment, we studied the effect on basal
currents of N-ethylmaleimide (NEM), which prevents receptor±Gi/o
protein interaction (Shapiro et al., 1994). The application of NEM
(50 mM) for at least 2 min did not increase basal currents, but
produced a decrease of 8.8 6 1.1 pA/pF (n = 13) similar to that
induced by 100 mM Ba2+ in the absence of agonist (10 6 1 pA/pF;
n = 17; see, e.g. Fig. 2A, right panel). The increase in basal currents
induced by PTX was reduced by expression of PTX-insensitive Gai/o
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
G-proteins gating GIRK channels in SCG neurons
FIG. 6. Ef®cient reconstitution of CCh-induced activation of expressed
GIRK channels by coexpression of PTX-insensitive GaoA and Gai3 mutant
subunits with Gb1g2. (A) Current traces in the absence (basal) or presence
of 10 mM CCh, recorded from SCG neurons transfected with Kir3.1- and
Kir3.2-expressing plasmids along with different PTX-insensitive Gai/o
mutant subunits (GaoA, Gai1, Gai2 or Gai3) plus Gb1g2, and treated with
PTX. Dashed lines indicate the zero current level. (B) Summary of basal
(white bars) and CCh-induced (black bars) current densities in SCG neurons
expressing Kir3.1/Kir3.2 channels alone or along with Gb1g2 and the
indicated Gai/o mutant, untreated (left) or treated (right) with PTX. After
nuclear injection of cDNAs (Kir3.1- and Kir3.2-expressing plasmids,
100 mg/mL; Gai/o mutants-expressing plasmids, 100 mg/mL; Gb1- and Gg2expressing plasmids, 200 mg/mL), SCG neurons were maintained in culture
for 1 day before electrophysiological recording. PTX-treated neurons were
incubated overnight (18±24 h) with 0.5 mg/mL PTX. GIRK currents were
recorded as in Fig. 1. Data are presented as mean 6 SEM, and numbers
indicate the number of neurons tested. *P < 0.05
mutants until they attained similar amplitudes to the basal currents in
PTX-untreated cells.
In PTX-treated neurons, a small reconstitution of CCh-induced
IGIRK activation could be detected on expressing either PTXinsensitive GaoAC351I or Gai3C351I, whereas expression of
Gai1C351I or Gai2C351I mutants had no effect (Fig. 4). By contrast,
in the same neurons, NE-activation of GIRK current was reconstituted substantially, and to a similar extent, by expression of any of the
above mentioned Gai/o mutants (Fig. 5).
The lack of effective reconstitution of the M2 muscarinic-mediated
GIRK current activation by the Gai/o mutants could be explained by
an inability of these Ga subunits to interact with this particular
receptor after mutation (C®I). Another possible explanation relates
to the above-mentioned capability of these mutants to sequester free
Gbg subunits, masking any possible reconstitution (as the muscarinic
response is smaller, on average, than that induced by adrenergic
receptors). To overcome this inhibitory effect of the mutant asubunits, we coexpressed these together with Gb1g2 subunits. This
289
FIG. 7. Reconstitution of a2-adrenergic receptor coupling to expressed
GIRK channels by coexpression of PTX-insensitive Gai/o mutants with
Gb1g2. Summary of NE-induced current densities in SCG neurons
expressing Kir3.1/Kir3.2 channels alone or together with different PTXinsensitive Gai/o mutant subunits (GaoA, Gai1, Gai2 or Gai3) and Gb1g2,
untreated (A) or treated (B) with PTX. After nuclear injection of cDNAs
(Kir3.1- and Kir3.2-expressing plasmids, 100 mg/mL; Gai/o mutantsexpressing plasmids, 100 mg/mL; Gb1- and Gg2-expressing plasmids,
200 mg/mL), SCG neurons were maintained in culture for 1 day before
electrophysiological recording. GIRK currents were recorded as in Fig. 1.
PTX-treated neurons were incubated overnight (18±24 h) with 0.5 mg/mL
PTX. Data shown for each experimental condition in this ®gure and in
Fig. 6 were obtained from the same neurons. Data are presented as
mean 6 SEM, and numbers indicate the number of neurons tested.
*P < 0.05
effectively prevented the reduction in basal current, and hence
prevented the inhibitory effect of the mutants (Figs 6 and 7). Under
these conditions, GaoAC351I (six of 12 cells) and Gai3C351I (six of
11 cells) effectively reconstituted the CCh-activation of IGIRK in
transfected neurons treated with PTX, whereas coupling between
muscarinic receptors and Kir3.1/3.2 channels through Gai1C351I or
Gai2C352I remained negligible (Fig. 6). As expected from previous
results, any of the Gai/o mutants reconstituted the NE-activation of
expressed GIRK currents in the same neurons (Fig. 7). As previously
reported for the voltage-dependent inhibition of N-type Ca2+ channels
via a2-adrenergic receptors in SCG neurons (Jeong & Ikeda, 2000),
successful rescue of receptor-mediated IGIRK activation by PTXinsensitive Gai/o mutants was dependent on the right stoichiometry
between expressed Ga and Gbg subunits. Thus, if expression of Gbg
exceeded that of Ga, signi®cant basal tonic activation of GIRK
currents was observed and, consequently, agonist-activated currents
were occluded (as in Fig. 2); those cells were not included in the
analysis. By contrast, if the expression of Ga greatly surpassed that of
Gbg, similar results to those obtained in the absence of Gbg
expression (as in Figs 4 and 5) were observed.
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
290 J. M. FernaÂndez-FernaÂndez et al.
(n = 8), giving a ratio S2 : S1 of 0.72 6 0.07 (n = 8). When
tripitramine was present 2 min before and during S2 a signi®cant
decrease of activated GIRK currents could be observed (ratio S2 : S1
of 0.15 6 0.04, n = 8; Fig. 8A). By contrast, no signi®cant difference could be detected when pirenzepine was used (ratio S2 : S1 of
0.65 6 0.04, n = 10; Fig. 8B). Similar results were observed on
PTX-treated neurons expressing either GaoAC351 or Ga13C351I
along with Gb1g2, and Kir3.1/3.2 channels. Thus, the ratios S2 : 1
were 0.17 6 0.01 (n = 6) and 0.21 6 0.04 (n = 3) for 6 nM
tripitramine, and 0.67 6 0.06 (n = 6) and 0.79 6 0.08 (n = 3) for
100 nM pirenzepine in cells expressing GaoAC351I or Gai3C351I,
respectively (Fig. 8A and B). In order to con®rm the selectivity for
M2- and M4-mediated response of these antagonists, we tested the
effect on the M4 muscarinic inhibition of ICa in nontransfected SCG
neurons. As anticipated, 6 nM tripitramine did not alter the S2 : S1
ratio for ICa inhibition (0.78 6 0.06, n = 6 vs. 0.77 6 0.07, n = 10;
Fig. 8C), whereas 100 nM pirenzepine reduced it to 0.51 6 0.05
(n = 10; Fig. 8D).
FIG. 8. Muscarinic pharmacology of GaoA- and Gai3-mediated activation of
expressed GIRK channels. Effect of 6 nM tripitramine (A) and 100 nM
pirenzepine (B) on CCh-induced activation of GIRK currents (IGIRK) in
SCG neurons expressing Kir3.1/Kir3.2 channels alone (PTX-untreated; two
left-hand blocks) or together with Gb1g2 plus either PTX-insensitive GaoA
or Gai3 mutant subunit (PTX-treated; two right-hand blocks). GIRK
currents were activated by two successive exposures to 10 mM CCh (S1 and
S2) in the absence (white bars) or presence during S2 (black bars) of the
indicated antagonist. S2 : S1 current ratios were calculated using peak IGIRK
amplitudes (acquired by averaging currents between ±125 mV and
±130 mV). (C and D) Effect of 6 nM tripitramine (C) and 100 nM
pirenzepine (D) on carbachol-induced inhibition of calcium currents in
nontransfected SCG neurons, determined using the same protocol. GIRK
currents were recorded as in Fig. 1. Data are presented as mean 6 SEM,
and numbers indicate the number of neurons tested. **P < 0.005;
*P < 0.05.
The reason why the NE response was ef®ciently reconstituted by
PTX-insensitive Gai/o mutants without a requirement of Gbg
expression whereas the CCh response was not could be related to
the sensitivity of the channels to Gbg and the relative `ef®cacy' with
which the receptors generate free Gbg. Thus, up to four molecules of
Gbg subunits can bind to GIRK channels (Corey & Clapham, 2001),
and the apparent cooperativity of binding means that large GIRK
currents can only be generated at relatively high Gbg concentrations
(Ivanova-Nikolova & Breitweiser, 1997). So, if the density of M2
receptors or the `ef®cacy' for free Gbg formation by activated M2
receptors was lower than that of a2 receptors (as suggested by the
smaller currents induced by CCh), responses to NE might be
reconstituted more readily by Gai/o mutants using the (limited) pool
of Gbg already available without the need to add extra Gbg.
To de®ne the muscarinic receptor (M2 vs. M4) responsible for the
activation of IGIRK via the PTX-insensitive GaoAC351I and
Gai3C351I mutants, we used two muscarinic antagonists with inverse
af®nities (differing on average by at least 10-fold) for M2 and M4
receptors: tripitramine (6 nM) and pirenzepine (100 nM; see Caul®eld
& Birdsall, 1998). We tested these concentrations as they selectively
antagonized M2 muscarinic activation of IGIRK and M4 muscarinic
inhibition of ICa, respectively (Fernandez-Fernandez et al., 1999). In
control neurons only expressing Kir3.1/3.2 channels and not treated
with PTX, the magnitude of IGIRK activated by CCh (10 mM) was
19.3 6 2.5 pA/pF (n = 8) (S1). A second exposure to 10 mM CCh
(S2) 2±3 min later produced GIRK currents of 14 6 2.1 pA/pF
Discussion
The present results show that the involvement of G-protein subunits
in the coupling of M2 mAChRs to expressed GIRK channels in rat
SCG neurons can be distinguished from their role in the voltagedependent component of Ca2+ current inhibition mediated by M4
mAChRs in two respects. First, the antisense depletion experiments
show that activation of GIRK channels is preferentially mediated by
endogenous Gai whereas inhibition of ICa is mediated primarily or
exclusively by endogenous Gao (see Delmas et al., 1998a). This is in
accordance with previous observations on rat anterior pituitary cells,
where Go and Gi3 selectively link dopamine receptors to Ca2+
channels and GIRK channels, respectively (Lledo et al., 1992).
Second, whereas the inhibitory effects of transducin expression
indicate that both effects are mediated the bg subunits freed from the
respective endogenous Gabg trimers, the active subunits appear not
to be identical, since inhibition of ICa was readily suppressed by the
bARK-1C-ter peptide (Delmas et al., 1998a), whereas activation of
IGIRK was not. The same differences apply to the activation of IGIRK
and the voltage-dependent component of ICa inhibition by a2adrenoceptor stimulation (compare the present results with those of
Delmas et al., 1998b, 1999).
This selectivity appears not to result from a fundamental difference
in the selectivity with which the receptors interact with G-protein a
subunits. Thus, when the coupling to endogenous Ga subunits was
prevented with Pertussis toxin, M2 mAChR-triggered activation of
IGIRK could be `rescued' by either GaoA or Gai3 when heterologously
expressed with bg subunits; and IGIRK activation by NE could be
rescued indiscriminately by GaoA, Gai1, Gai2 or Gai3.
Likewise, it seems unlikely that the ICa/IGIRK selectivity resulted
from a fundamental difference in the ability of the bg subunits freed
from the respective G-protein trimers to inhibit Ca2+ channels or
activate GIRK channels, because both channel types can be
modulated by a wide and substantially overlapping range of bg
subunits (Wickman et al., 1994; Garcia et al., 1998; Jeong & Ikeda,
2000; Zhou et al., 2000).
Finally, it is clear from experiments on other cells that the M2
IGIRK/M4 ICa selectivity pattern observed in these SCG neurons is not
a rigid or predetermined relationship. Thus, M4 receptors can readily
activate GIRK channels when expressed in oocytes (Gadbut et al.,
1996) or HEK cells (Leaney & Tinker, 2000) whereas expressed M2
receptors can inhibit Ca2+ currents in neuroblastoma cells (Higashida
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
G-proteins gating GIRK channels in SCG neurons
291
FIG. 9. Comparison of ion channels modulatory pathways using (A) endogenous and (B) heterologously expressed Gi/o proteins in rat SCG neurons. M4, M4
muscarinic receptors; a2, a2-adrenergic receptors; M2, M2 muscarinic receptors; N-ICa, N-type Ca2+ channels; GIRK1/2, GIRK1/GIRK2 channels;
±, inhibition; +, activation. The G-protein subunits responsible for the indicated modulation are shown in grey. Data for coupling between receptors and
endogenous G-proteins are from Delmas et al. (1998a, b; 1999) and from this study. Data for coupling between receptors and heterologously expressed Gi/o
proteins are from this study and from Jeong & Ikeda (2000).
et al., 1990). Further, the muscarinic inhibition of ICa in basal
forebrain neurons is mediated by endogenous M2 receptors, not M4
receptors (Allen & Brown, 1993).
Instead, we suggest that the coupling speci®city observed in SCG
neurons in situ might arise from topographical constraints resulting
from physical proximity between receptors, G-proteins and ion
channels, as suggested from the work of Huang et al. (1995),
Slesinger et al. (1995) and Stanley & Mirotznik (1997). Thus, in SCG
neurons, M2 muscarinic receptors might be tightly coupled to Gi
proteins and expressed GIRK channels, precluding access to Ca2+
channels (Fig. 9). Conversely, M4 muscarinic receptors might be in
close association with Go proteins and Ca2+ channels and preclude
coupling to GIRK channels. By contrast, a2-adrenergic receptors
would appear to participate in both `microdomains'.
One other feature of interest concerns the basal (agonist-independent) activation of GIRK channels in transfected neurons. Basal
activation of GIRK currents in transfected SCG neurons has been
previously described (Fernandez-Fernandez et al., 1999) but not
studied in detail. Here we show that the basal GIRK currents were
reduced by NEM treatment, suggesting that these were generated by
resting G-protein turnover. Furthermore, because the currents were
reduced selectively by expression of Gai antisense RNA and were
reduced signi®cantly by some bg-sequestering agents (in particular,
GaoAC351I and Gai3C351I mutants), basal activation appears to
result from the action of bg subunits freed from Gi trimers.
Collectively, these observations suggest that basal activation is a
consequence of the activation of Gi by agonist-free receptors, as
initially shown for the cardiac inward recti®er channels linked to M2
muscarinic receptors (Soejima & Noda, 1984; Ito et al., 1991). We
also observed an enhancement of basal GIRK currents in transfected
cells treated with PTX. This increase is unlikely to have been due to
uncoupling of an inhibitory pathway (cf. Schreibmayer et al., 1996)
because the effect of PTX was not replicated by NEM. Instead, this
enhanced response might be a consequence of an increase in free
Gbg, as it was attenuated by expression of different PTX-insensitive
Gai/o mutants.
Acknowledgements
We are grateful to Dr Florian Lesage (Institut de Pharmacologie Moleculaire
et Cellulaire, Sophia Antipolis, Valbonne, France) who provided the GIRK1and 2-expressing plasmids and to Professor Carlo Melchiorre (Dipartimento di
Scienze Farmaceutiche, Universita di Bologna, Italy) for the muscarinic
antagonist tripitramine. This work was supported by The Wellcome Trust.
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 283±292
292 J. M. FernaÂndez-FernaÂndez et al.
Abbreviations
bARK1C-ter, C-terminus of b-adrenoceptor kinase 1; CCh, carbachol; GIRK,
G-protein-activated inwardly rectifying K+ GPCR, G-protein-coupled receptor; ICa, Ca2+ current; mAChR, muscarinic acetylcholine receptor; NE,
norepinephrine; NEM, N-ethylmaleimide; PTX, Pertussis toxin; SCG, superior cervical ganglion.
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