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Vol. 63, Issue 4, 832-843, April 2003
Modulation of CaV2.2 Calcium Channels: Probed by the Use of
Receptor-G
Tandems
Department of Pharmacology, University College London, London, United Kingdom (F.B., P.V., A.C.D.); and Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom (R.J.W., G.M.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The stable interaction of a G-protein coupled receptor and a particular
partner G-protein was made possible by creating tandems between the
2A adrenergic receptor (2A-R) and
pertussis toxin-resistant mutants of different G subunits of
heterotrimeric G-proteins. Both 2A-R-Go
and 2A-R-Gi proved able to reconstitute
agonist-induced voltage-dependent inhibition of N-type calcium channels
(CaV2.2) similar to the wild-type 2A-R when
expressed in COS-7 cells. The interaction of Gq with the
Gi/o signaling pathways was studied by expressing either
Gq or a chimeric construct based on Gq containing the last five amino acids of Gz, which is
activated by 2A-R. It was found that Gqz5
activated by the wild-type 2A-R inhibited
CaV2.2 currents in a voltage-independent fashion.
Furthermore, Gqz5 counteracted the voltage-dependent
inhibition resulting from 2A-R-Go
activation. We subsequently investigated the basis for the behavior of
Gqz5. Our evidence suggests that this occurs as a result
of a downstream effect of activation of Gqz5 because it
was blocked by C-terminal construct of phospholipase C
1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of
Gqz5 in
2A-R-Go-transfected cells. Conversely,
cells expressing both 2A-R-Go and
Gqz5 exhibited a partial restoration of
voltage-dependent inhibition in the presence of the PKC inhibitor
bisindolylmaleimide I (GF 109203X). The potential sites of
phosphorylation are discussed.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Calcium influx in any cell
requires fine tuning to guarantee the correct balance between
activation of calcium-dependent processes, such as muscle contraction
and neurotransmitter release, and calcium-induced cell damage.
G-protein-coupled receptors (GPCRs) play a role in negative feedback of
the activity of voltage-dependent calcium channels (Dolphin, 1995
).
Establishing the basis for the specificity of the relationships between
membrane receptors, G-proteins, and effectors has proven elusive, in
part because of the promiscuity of the partners involved when expressed
in heterologous systems. When different G-protein subunits are
over-expressed together with GPCRs and calcium channels, the degree of
specificity is rather low. For example, the
2A-adrenergic receptor
(2A-R) couples to all members of the
Gi/o family, including the pertussis toxin (PTX)-sensitive Go and Gi,
and the PTX-insensitive Gz (for review, see
Hille, 1994).
In native systems, however, receptors display a more selective
activation of endogenous G-proteins subtypes, with
Go being more important than
Gi in the inhibition of calcium currents in sensory neurons (Campbell et al., 1993). Furthermore, in sympathetic neurons, muscarinic activation of G-protein-activated inward-rectifier (GIRK) channels is mediated by Gi, whereas
muscarinic inhibition of N-type calcium channels is mediated by
GoA (Fernández-Fernández et al.,
2001). These results point to the importance of the cellular localization of each receptor and G-protein subtype.
For GPCRs that associate with PTX-sensitive G-proteins, production of
G
dimers seems to be responsible for the direct voltage-dependent inhibition of N- and P/Q-type channels (Herlitze et al., 1996; Stephens
et al., 1998), although it has also been proposed that in chick sensory
neurons, G results in activation of PKC, to mediate the
voltage-dependent inhibition caused by norepinephrine (Diversé-Pierluissi et al., 1995). Furthermore, G subunits
have also been implicated in mediating G-protein modulation
(Diversé-Pierluissi et al., 1995).
One way to identify the direct effects of a specific G-protein on
calcium channel activity is to link the G-protein subunit to the
receptor of choice to form a tandem construct. One of the advantages of
this approach is the elimination of one of the signal amplification
steps, occurring at the receptor/G-protein interaction level, because
the two components are constrained to work with a 1:1 stoichiometry.
Furthermore, there is increasing evidence against the established model
which sees G-proteins shuttling between receptor and effector, and
toward a view that there is a close localization of signal transduction
elements in distinct membrane domains (Seifert et al., 1999). We used
fusion proteins between the 2A-R and either
Gi1 or Go1, both of
which were rendered PTX-insensitive by means of a point mutation at
residue 351 (Bahia et al., 1998). The Ile351 G
mutants were chosen over other possible PTX-resistant mutants because
they resulted in the strongest activation by
2A-R (Bahia et al., 1998). Activation of these
tandems by the 2A-R agonist clonidine was
studied in COS-7 cells coexpressing N-type channels (CaV2.2) and comparing the response to that
produced by the activation of the wild-type
2A-R. These tandems have been found able to interact with endogenous G-proteins to a certain extent (Burt et al.,
1998). In the present study, treatment of cells with PTX before
recording allowed the receptor/G-protein tandems to be studied in
isolation, effectively removing the contribution of endogenous
Gi/o proteins.
The carboxyl terminus of the G subunit is not only a determinant of
its sensitivity to PTX-dependent ADP-ribosylation but is also essential
to confer specificity of coupling to GPCRs (Conklin et al.,
1993). To examine whether G dimers liberated from
Gq could also inhibit N-type
Ca2+ channels, we exploited a chimeric
Gq-protein. This construct was formed by a
Gq subunit in which the last 5 amino acids
were substituted for the corresponding amino acids from
Gz. The resulting Gqz5, unlike Gq itself,
is able both to couple to the 2A-R and to
activate effectors specific to the Gq family,
such as phospholipase C and the downstream protein kinase C (PKC)
(Conklin et al., 1996). We report the effects of such a construct in
isolation and when coexpressed with the
2A-R-Go fusion
protein and compare these effects with those of the wild type
Gq subunit. The involvement of downstream
effectors of Gqz5 is also examined.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Constructs.
COS-7 cells were transiently transfected with
the following cDNAs: rabbit CaV2.2 (GenBank
accession no. D14157); rat 1b (GenBank accession no. X11394); and
mut-3 green fluorescent protein (GFP).
2A-R-G-protein fusion
proteins used throughout this study were prepared as described
previously (Cavalli et al., 2000). In brief,
Cys351 of rat Gi1 and
Go1 was mutated to Ile by site-directed
mutagenesis and then used to create the
2A-R-G fusion proteins using porcine 2A-R in pcDNA3. The
Ile19Ala, Glu20Ala (IE)
mutant of Go1 was constructed, based on
studies of an equivalent mutation (Ile25Ala,
Glu26Ala) of Gq (Evanko
et al., 2000), and this was then incorporated into the PTX-resistant
2A-R-Go fusion
protein. The wild-type Gq subunit
(Gq w.t.) and the
Gqz5 subunits described previously (Conklin et
al., 1993) were subcloned into pMT2. G-transducin (Gt) was in pcDNA3. The pEGFP-PLC-1ct
fusion construct of the C terminus of phospholipase C (PLC-1ct)
was described previously (Kammermeier and Ikeda, 1999).
Cell Culture and Transfections.
Cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% newborn
calf serum, penicillin (100 IU/ml) and streptomycin (100 µg/ml) (all
from Invitrogen, Paisley, UK) at 37°C, 5% CO2,
and passaged every 3 to 4 days. For transient transfections of the
different constructs, a cDNA mixture was made containing the
voltage-dependent calcium channel CaV2.2 subunit cDNA in a ratio of 3:1 with all the other constructs, 1b,
2A-R, 2A-R-G-protein
tandems, and/or the G subunits. Mut-3 GFP cDNA was also included at
a ratio of 0.2. For transfection, 10 µl of GenePORTER reagent
(Qbiogene, Harefield, UK) and 2 µl of cDNA mixture were preincubated
in 1 ml of Dulbecco's modified Eagle's medium at 20°C for 1 h
before addition to 35-mm Petri dishes containing approximately 2 × 106 cells. Cells were cultured at 37°C for
72 h, replated using a nonenzymatic cell dissociation medium
(Sigma, Poole, UK), and maintained at 27°C for 1 to 8 h, before
recording. PTX (Sigma) was used to inactivate the endogenous
Gi/o subunits by adding it to the culture
medium at a concentration of 40 to 100 ng/ml for 16 h before
replating the cells.
[3H]RS-79948-197 Binding.
To determine the
levels of expression of the various
2A-R-G-protein fusion proteins, the specific
binding of [3H]RS-79948-197 was measured as
described previously (Ward and Milligan, 2002).
[35S]GTPS Binding.
[35S]GTPS binding experiments were performed
essentially as described for receptor-G-protein tandems incorporating
G11 (Carrillo et al., 2002). These were
initiated by the addition of membranes containing 50 fmol of the fusion
constructs to an assay buffer [20 mM HEPES, pH 7.4, 3 mM
MgCl2, 100 mM NaCl, 1 µM guanosine 5'-diphosphate, 0.2 mM ascorbic acid, and 50 nCi of
[35S]GTPS] in the absence or presence of
clonidine (10 µM). Nonspecific binding was determined in the same
conditions but in the presence of 100 µM GTPS. Reactions were
incubated for 15 min at 30°C and were terminated by the addition of
0.5 ml of ice-cold buffer containing 20 mM HEPES, pH 7.4, 3 mM
MgCl2, and 100 mM NaCl. The samples were
centrifuged at 16,000g for 15 min at 4°C, and the
resulting pellets were resuspended in solubilization buffer (100 mM
Tris, 200 mM NaCl, 1 mM EDTA, and 1.25% Nonidet P-40) plus 0.2% SDS. Because all the 2A-R-G-protein tandems used in
these studies incorporated a hemagglutinin (HA) epitope tag at the N
terminus of the receptor, samples were precleared with Pansorbin
(Calbiochem, Nottingham, UK), followed by immunoprecipitation with the
anti-HA antiserum 12CA5 (Roche Diagnostics, Lewes, UK). Finally, the
immunocomplexes were washed twice with solubilization buffer, and bound
[35S]GTPS was measured by liquid
scintillation counting.
Immunoprecipitation and Immunodetection Studies.
To analyze
the interaction of
2A-R-Go with G
dimers, cells were transfected with
2A-R-Go or
2A-R-Ile19Ala,
Glu20Ala Go in the
absence or presence of plasmids encoding G-protein 1 and 2
subunits. Cells were washed once with ice-cold phosphate-buffered saline and immediately homogenized in a lysis medium containing 50 mM
HEPES, pH 7.4, 10 mM
Na4P2O7,
100 mM NaF, 10 mM EDTA, 0.1 mM
Na3VO4, 1% Triton X-100,
and a protease inhibitor cocktail (Complete; Roche). Cell lysates were
centrifuged (15 min, 13,000 rpm) and the supernatants precleared for
1 h with nonspecific serum and protein A. Next, samples were
incubated overnight with a polyclonal antiserum directed against the
C-terminal decapeptide of Go1 (Mullaney and
Milligan, 1990). The immunocomplexes were then captured with protein
A-agarose.
1 subunit (Green et al., 1990) and washed with TTBS. The PVDF membranes were incubated for 20 min with
horseradish peroxidase conjugated to anti-rabbit IgG (1:20,000) (Amersham Biosciences). Finally, they were washed with TTBS and developed by enhanced chemiluminescence.
Electrophysiology.
Fluorescent COS-7 cells expressing GFP
were chosen for whole-cell, patch-clamp recording. Borosilicate glass
electrodes were used with a resistance of 2 to 5 M
when filled with
a solution containing 140 mM cesium aspartate, 5 mM EGTA, 2 mM
MgCl2, 0.1 mM CaCl2, 2 mM
K2ATP, and 20 mM HEPES, pH adjusted to 7.2 with CsOH, 310 mOsM with sucrose. Cells were perfused with an
extracellular solution containing 160 mM tetraethylammonium-Br,
2 mM KCl, 1.0 NaHCO3, 1.0 MgCl2, 10 mM HEPES, 4 mM glucose, and 10 mM
BaCl2, pH 7.4, 320 mOsM with sucrose. Barium
currents were recorded using an Axopatch-1D amplifier (Axon
Instruments, Union City, CA). Data were filtered at 2 kHz, digitized at
5 to 10 kHz, and analyzed using pCLAMP 6 (Axon Instruments) and Origin
5.0 (Microcal, Northampton, MA). Cell capacitance compensation and
series resistance compensation between 65 and 80% were applied
electronically. Records are shown after leak subtraction (P/4 or P/8 protocol).
100 mV.
A strongly depolarizing prepulse PP of 30 to +100 mV was then delivered
before a second pulse (P2) to the same voltage as the first test pulse, to assess the voltage-dependence of current inhibition. The PP and the
second pulse were separated by a 10-ms repolarization time to 100 mV.
Pulses were delivered every 15 s. Currents were measured 10 ms
after the onset of both P1 and P2 and the average over a 2-ms period
was calculated and used for subsequent analysis. The 300-ms interval
between P1 and PP was sufficient to minimize the voltage-dependent
calcium channel inactivation caused by P1. The duration and amplitude
of the PP were chosen to produce maximal facilitation in the conditions
used (data not shown). Experiments were performed at room temperature
(20-24°C). Drugs were applied by the use of a gravity-fed,
electronically controlled, multibarrelled perfusion system. Current
density-voltage (I-V) relationships were fitted with a modified
Boltzmann equation as follows: I = Gmax (V Vrev)/(1 + exp((V V50,act)/k)), where
I is the current density (picoamperes per picofarad),
Gmax is the maximal conductance (nanosiemens per picofarad), Vrev is
the reversal potential, V50,act is the
mid-point voltage for current activation, and k is the slope
factor.
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act) was
calculated by fitting a single exponential to the current traces:
I = A × exp(t/act) + C, where
A is the amplitude of the component with time constant ,
and C is a constant. Data are expressed as mean ± S.E.M., and statistical significance between conditions was examined using Student's t test or paired t test, as appropriate.
Materials.
[3H]RS-79948-197 (90 Ci/mmol) was from Amersham Biosciences (Little Chalfont,
Buckinghamshire, UK), [35S]GTPS (1250 Ci/mmol) was from PerkinElmer Biosciences (Warrington, UK). Clonidine
hydrochloride (Calbiochem) was prepared as a
10
2 M stock in H2O. The
protein kinase C activator phorbol-12,13-dibutyrate (PDBu; Calbiochem)
and the PKC inhibitor bisindolylmaleimide I (GF 109203X, Calbiochem)
were prepared as 102 M stock in DMSO. All drugs
were diluted in the experimental solutions to the final concentrations indicated.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of the 2A Adrenergic
Receptor-Gi and -Go Tandems.
We
first expressed either 2A-R w.t. or the
PTX-insensitive receptor-G tandems
2A-R-Go1C351I
(2A-R-Go) or
2A-R-GiC351I
(2A-R-Gi) together
with the CaV2.2 calcium channel. The inhibition of the expressed Ba2+ currents
(IBa) by activation of the
2A-R w.t. was compared with the effect of the
receptor G-protein tandems (Fig. 1). Overall, the
2A-R agonist clonidine (10 µM) inhibited
N-type IBa via activation of both the free
2A-R and the tandem
2A-R-G constructs, as exemplified by the
current traces in Fig. 1, ac. The inhibition was rapid (< 15 s)
and reversible upon washing (data not shown). The extent of
IBa inhibition at 0 mV is given in Fig. 1e (
). In the absence of PTX, IBa was similarly reduced
by both the wild-type 2A-R (64.2 ± 6.6%, n = 9, Fig. 1a) and the tandems
2A-R-Go (77.6 ± 6.6%, n = 5, Fig. 1b) and
2A-R-Gi (64.1 ± 4.0%, n = 8, Fig. 1c). Thus, removal of the
amplification step between receptor and G-protein did not affect the
ability of Gi/o to produce inhibition of
CaV2.2 IBa.
constructs
are able to activate not only the tethered G subunit but also
endogenous subunits of the Gi/o family (Burt et
al., 1998). The use of PTX therefore allows isolation of the effects of
exogenous G subunits mutated to be PTX-resistant by rendering the
endogenous Gi/o subunits unable to couple to the
receptor. Preincubation of the cells with PTX greatly reduced the
inhibition produced by the 2A-R w.t. (see
traces in Fig. 1d and mean results in Fig. 1e). Conversely, PTX did not
significantly affect the functioning of the two PTX-insensitive
receptor G-protein tandems. The calcium channel currents at 0 mV were
still reduced by 74.1 ± 6.5% (n = 18, Fig. 1b)
and 62.9 ± 9.1% (n = 8, Fig. 1c) with the
Go and the Gi fusion
proteins, respectively, after pretreatment with the toxin (Fig. 1e).
Experiments repeated with a lower concentration of clonidine (100 nM)
gave comparable results in terms of degree of inhibition, demonstrating
that maximal receptor activation was achieved at the concentration of
agonist used (data not shown).
Inhibition of N-type currents by the
receptor-Gi/o tandems was largely
voltage-dependent, as seen by using a double pulse voltage-clamp
protocol (Fig. 1, a
-d). The PP was able to reverse the
agonist-induced inhibition induced by either
2A-R w.t. (Fig. 1a) or the
2A-R-Go (Fig. 1b) and
2A-R-Gi (Fig. 1c)
tandems, whereas incubation with PTX eliminated the voltage-dependent
effects of the 2A-R w.t. (Fig. 1d). The amount
of inhibition by clonidine before and after the PP is summarized in
Fig. 1e. The resultant "facilitation" (determined as the P2 current
amplitude divided by P1 current amplitude) was substantial for all
three receptor constructs. In all cases, however, removal of inhibition
during P2 by the PP to +100 mV was never complete, indicating a
voltage-independent inhibitory component.
As a corollary of the voltage-dependence of the inhibition of
IBa by clonidine, it should also be abolished at
large step potentials. The voltage-clamp protocol used to examine this
was similar to that shown in Fig. 1 with the exception that both test pulses (P1 and P2) were varied from 40 to +70 mV in 10-mV increments. Example traces are shown in Fig. 2a,
whereas the mean I-V plots for values measured in P1, before and during
application of clonidine, for cells expressing the
2A-R-Go
(n = 8) are shown in Fig. 2b. With all receptor
constructs, the agonist caused both a reduction in
IBa and a depolarizing shift in the I-V
relationship. The V50,act during P1 was
significantly depolarized for cells expressing
2A-R-Go, from
6.4 ± 3.1 to +9.0 ± 4.0 mV (p < 0.05, n = 6, Fig. 2b) and, for cells expressing
2A-R-Gi, from
+2.5 ± 2.4 to +9.7 ± 0.7 mV (p < 0.05, n = 6). No significant differences in the
Vrev or in the
Gmax were detected (Fig. 2b; data not
shown). The P2/P1 facilitation ratios for the different test potentials
are reported in Fig. 2, c-d. The PP revealed some tonic facilitation
in the absence of the agonist (), which was more marked when
expressing the 2A-R w.t., where P2/P1 was
2.3 ± 0.3 at 0 mV (Fig. 2c). Clonidine enhanced the
voltage-dependent facilitation, although the effects were much greater
for the 2A-R-Go
tandem than for the 2A-R w.t. (Fig. 2d).
Maximal facilitation was obtained at 10 or 0 mV and it was absent
above +20 mV.
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2A-R-G
tandems cause a reduction in current amplitude but the activation phase
of the current was typically slowed during P1; this effect was reversed
by the PP (e.g., Fig. 1, a-c). For example, for those cells
transfected with the
2A-R-Go tandem, the
act at 0 mV during P1 was 3.7 ± 0.5 ms
in control and 6.1 ± 1.1 ms during clonidine application (n = 10, p < 0.05, see Fig. 1b). This
slowed activation was reversed by Gt, which
acts as a G sink to sequester free G subunits but does not
couple to the 2A-R. Example traces are shown
in Fig. 3a (top). After cotransfection of
Gt with
2A-R-Go, there was no
longer a difference in the act values measured
in control and clonidine during P1 (2.9 ± 0.6 ms and 3.4 ± 0.5 ms, respectively, n = 9). Along with this effect,
Gt was able significantly to reduce inhibition
by clonidine at 0 mV from 74.1 ± 6.5 to 43.0 ± 6.7%
(p < 0.001; Fig. 3b) and to reduce the P2/P1
facilitation ratio in the presence of clonidine to 1.54 ± 0.24 at
0 mV, although this was still significantly greater than the P2/P1
ratio under control conditions (Fig. 3c).
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2A-R tandems are able to reconstitute
inhibitory effects on CaV2.2 calcium channel
currents by means of the tethered Gi/o that
are almost identical to the wild-type receptor coupling to endogenous
G-proteins and that such effects are very likely to be mediated purely
by G dimers. It has been found previously that mutation of both Ile25 and Glu26 of
Gq to Ala severely limits interaction with the
G complex (Evanko et al., 2000). These residues are highly
conserved in other G-protein subunits. We thus constructed a form
of the PTX-resistant
2A-R-Go tandem (IE)
that also incorporated the equivalent mutations of
Ile19Ala and Glu20Ala in
Go. Application of clonidine to cells
expressing the IE form of the
2A-R-Go tandem
produced no inhibition of IBa, and no effect on
facilitation (Fig. 3, a, bottom, and b-c). It is also evident that
these CaV2.2 currents show some tonic modulation, being slowly activating and facilitated by a prepulse, although this is
no greater than for the free 2A-R (Fig. 2c).
To examine the binding of G to the IE mutant of
2A-R-Go, either
2A-R-Go or the IE
form of this construct was cotransfected together with plasmids
encoding the G1 and G2 subunits. Cell lysates were subsequently
immunoprecipitated with an antiserum (OC) that identifies the
C-terminal decapeptide of Go1. Such samples
were then resolved by SDS-PAGE, transferred to PVDF membranes, and
immunoblotted with an antiserum (BN) that identifies the N-terminal decapeptide of G1. Although the 2A-R-G
o tandem allowed coimmunoprecipitation of 1
subunit (Fig. 3d, lane 2), this was not observed for the IE form of the
tandem receptor (Fig. 3d, lane 3).
Investigation of 2A-R-Gq and
2A-R-Gqz5 Chimeras.
Because the
expression of the receptor/G-protein tandems indicated that the release
of activated G subunits, Gi and
Go, does not play any direct role in
G-protein-effector coupling for calcium channel inhibition, we were
interested in studying whether G released from another class of
G-protein, Gq, could also participate in the
inhibitory process. However Gq is known not to
couple efficiently to the 2A-R (Dorn et al.,
1997). To use the same receptor for activation of both
Gi/o and Gq pathways, we
therefore employed the chimeric construct
Gqz5. This subunit conserved the main structure of Gq but the last five amino acids
were substituted for those of Gz, a
PTX-resistant member of the Gi/o family that does
couple to the 2A-R (Conklin et al.,
1993). Tandem
2A-R-Gq and
2A-R-Gqz5 constructs
were assembled, and their functionality was assessed biochemically.
2A-R-G tandems by clonidine was obtained by
monitoring agonist-induced binding of
[35S]GTPS. Expression levels of the
2A-R-containing fusion proteins in membranes
of PTX-treated cells were quantified by saturation ligand binding
studies employing the high-affinity
2-adrenoceptor antagonist
[3H]RS-79948-197.
[35S]GTPS binding studies were performed in
the presence and absence of clonidine (10 µM) on membrane fractions
expressing equal amounts of the various fusion proteins. After this,
the anti-HA antibody 12CA5 was used to immunoprecipitate the samples,
because all of these constructs contained an N-terminal HA epitope tag.
Significant levels of [35S]GTPS binding were
observed for both the Go- and
Gi-containing fusion proteins; this was
stimulated markedly by the presence of clonidine (Fig.
4). In contrast, little binding of
[35S]GTPS was observed to the
2A-R-Gq and
2A-R-Gqz5 constructs, even in the presence of clonidine, consistent with a lack of activation of these G-proteins by the associated 2A-R.
The inability of clonidine to promote binding of
[35S]GTPS to the fusion proteins containing
Gq does not reflect the well appreciated
difficulty in monitoring nucleotide exchange for such G-proteins in
standard [35S]GTPS binding assays. We have
recently shown that combination of use of receptor-G-protein tandems
and selective immunoprecipitation allows a 30-fold stimulation of
binding in the presence of agonist when such G-proteins are linked in
tandem with appropriate receptors (Carrillo et al., 2002). A
preliminary investigation also failed to show any clonidine-mediated
inhibition of CaV2.2 via the
2A-R-Gqz5 tandem, but
because these receptor-Gq tandems were
nonfunctional biochemically, their coupling to
CaV2.2 was not further examined.
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q and
Gqz5 to examine whether G released from
Gq or Gqz5 can signal to
N-type calcium channels (Fig. 5a). We
confirmed, by coexpressing the 2A-R w.t. with
Gq w.t. in cells treated with PTX, that
Gq did not couple directly to the
2A-R. Perfusion of clonidine induced only
7.1 ± 1.1% reduction in the current (n = 5, Fig.
5b). In contrast, expression of Gqz5 with the
2A-R w.t. resulted in significantly greater
inhibition of CaV2.2 currents by clonidine
(35.8 ± 8.6%, n = 9, Fig. 5, a and b).
Surprisingly however, this was not removed by a PP to +100 mV, the
inhibition in P2 being 34.5 ± 5.4% (n = 9, Fig.
5b). Thus, the inhibition elicited by Gqz5 was
much greater than that elicited by Gq w.t.
(p < 0.001) but was not voltage-dependent. The P2/P1
facilitation ratio in the presence of Gqz5 was
around unity and was unaffected by the presence of agonist (0.98 ± 0.07 in control, 1.20 ± 0.23 in clonidine, p > 0.05, Fig. 5c). Current traces in the presence of
Gqz5 showed no evidence of slowing of the
kinetics of activation in response to clonidine (e.g., traces in Fig.
5a and data not shown). To determine whether voltage-dependent inhibition was completely absent for Gqz5, we
also examined the voltage-dependence of inhibition over a range of
potentials. However, no obvious facilitation was evident at any test
potential (data not shown). These results demonstrate that the
C-terminal modification of Gq allowed
Gqz5 to couple to the
2A-R, causing a reduction in
IBa, although the inhibition was
voltage-independent and smaller than that elicited by the tandems
2A-R-Go and
2A-R-Gi or the wild
type 2A-R coupling to endogenous G-proteins.
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Interaction between 2A-R-Go and
Gqz5.
It has been observed previously that chimeric
receptor-G constructs are able to activate not only the tethered
G subunit but also endogenous subunits of the
Gi/o family (Burt et al., 1998). Accordingly,
Gqz5 might be expected also to interact with, and to be activated by, the
2A-R-Go tandem used
in this part of the study. We investigated this potential interaction
by coexpressing the tandem
2A-R-Go with the
Gqz5 subunit, and treating all cells with PTX.
2A-R-Go with
Gqz5 was significantly smaller than in cells
expressing 2A-R-Go
alone (Fig. 6a). Inhibition was 21.0 ± 12.4% in P1 (n = 16, p < 0.01, Fig. 6b). Interestingly, the presence of Gqz5
also almost abolished facilitation by the PP at all potentials examined
(Fig. 6c). For example the P2/P1 facilitation ratio in clonidine was
1.24 ± 0.38 at 0 mV and 0.87 ± 0.11 at +10 mV
(n = 11, both p < 0.01 compared with
the much greater facilitation shown by the
2A-R-Go alone). As a
corollary of this, no agonist-induced depolarizing shift of the I-V
relationship for IBa was detected (data not
shown). Furthermore, no slowing of the activation kinetics was evident
during P1 (e.g., Fig. 6a). In summary, coexpression of
Gqz5 with
2A-R-Go reduced the
inhibition and reversed the P2/P1 facilitation observed upon activation
of the 2A-R-Go alone.
|
Mechanism of Action of Gqz5.
We addressed the
possibility that the effects produced by Gqz5
on CaV2.2 channel modulation might be mediated by
a signaling pathway downstream from Gq rather
than directly by the Gqz5 subunit. It has been
proposed that overexpression of any G subunit could abolish calcium
channel inhibition by sequestering G subunits, which would
therefore become unavailable for receptor activation (Jeong and Ikeda,
1999). However, this will depend on the balance between G activation
to form free G-GTP and G interaction with the G-GDP
species. In such a scenario, coexpression of
Gqz5 could buffer the effect of the G
released upon activation of 2A-R-Go, in a similar
way to transducin; as we have shown, however, Gqz5 is able to be activated. Once activated,
it would then lead to stimulation of phospholipase C (Conklin et al.,
1993), causing breakdown of phosphatidylinositol
4,5-bisphosphate (PIP2) into inositol
1,4,5-trisphosphate and diacylglycerol, the latter stimulating PKC.
Activation of PKC has been reported to counter G-protein modulation of
rat CaV2.2 (Zamponi et al., 1997; Hamid et al., 1999). However, elevation of PIP2 has also been
shown to modulate CaV2.1, mimicking that by
G (Wu et al., 2002). To investigate whether the reduction in
inhibition and loss of facilitation in our coexpression studies with
Gqz5 were caused by a G buffering effect
or by a specific downstream effect of activated
Gqz5 protein, we first chose to block the
downstream action of activated Gqz5 by
coexpressing the C-terminal peptide of phospholipase C-1
(PLC-1ct), which binds activated Gq and
acts as a GTPase-activating protein (Kammermeier and Ikeda, 1999).
Inhibition by clonidine in cells coexpressing
2A-R-Go and
Gqz5 together with PLC-1ct returned to
levels comparable with when
2A-R-Go was expressed
alone (Fig. 6b). Furthermore the P2/P1 facilitation ratio in the
presence of clonidine was increased relative to that in the presence of 2A-R-Go and
Gqz5 at all potentials between 0 and +20 mV,
being 4.88 ± 2.48 at 0 mV and 2.25 ± 0.66 at +10 mV (Fig.
6d, n = 6, p < 0.05 relative to
facilitation in clonidine for
2A-R-Go and Gqz5 alone at 0 and +10 mV).
1ct only binds activated Gq
species and was able to reverse the effect of
Gqz5, this must occur via its GTP-bound form.
We therefore examined the role of downstream effectors of Gq. We investigated the effect of activating PKC
to mimic the presence of Gq as a signal
transduction component, and simultaneously removed its presence as a
potential G buffering agent. We used PDBu, an activator of PKC,
on cells expressing the
2A-R-Go fusion protein. After assessing the inhibition of CaV2.2
currents and the voltage-dependent facilitation elicited by clonidine
alone, cells were perfused with PDBu (500 nM) in the presence of
clonidine (Fig. 7, a and b). Within 5 min
after the start of PDBu application, IBa
partially recovered from inhibition by clonidine. During P1, inhibition
by clonidine was reduced from 77.8 ± 6.1 to 56.1 ± 9.4% in
the additional presence of PDBu (Fig. 7c, n = 7, p < 0.001). Application of PDBu also resulted in
reduced current facilitation (Fig. 7d, n = 7). After
application of PDBu and clonidine, the current during P1 showed a rapid
activation phase, further evidence for the loss of voltage-dependent
inhibition (e.g., Fig. 7a, traces). Both the loss of inhibition and the
reduction of facilitation are similar to the effect of
Gqz5. Application of PDBu (500 nM) in the
absence of receptor activation did not cause any increase of
CaV2.2 IBa, rather reducing
it by 37 ± 9% after application for 3 min, with a loss of
control facilitation (n = 6, data not shown).
|
qz5, we observed that the PKC inhibitor
GF109203X partially restored the voltage-dependence of G-protein
modulation in the presence of Gqz5. After a
30-min preincubation with 1 µM GF 109203X, application of clonidine
to cells expressing 2A-R-Go and
Gqz5 produced a 54 ± 15% inhibition of
IBa at 0 mV (n = 9), and the
P2/P1 facilitation ratio approached that in the absence of
Gqz5 [2.4 ± 0.5 (n = 9)]. These two pieces of data indicate that PKC activation is at least
in part responsible for the effects of Gqz5.
| |
Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The Advantage of Using GPCR-G Protein Tandems.
We sought to
recreate proximity between a GPCR, the 2AR,
and a specific G-protein by using tandem constructs. Both the chimeric receptors 2A-R-Gi and
2A-R-Go reconstituted
N-type current inhibition, comparable with the
2AR w.t. Similarly, it has been found that a
tandem between the muscarinic m2 receptor and
Gz was able to modulate GIRK channels by
release of G (Vorobiov et al., 2000). This is in contrast to
their inability to activate downstream effectors via the G moiety
(Sautel and Milligan, 1998; Burt et al., 1998), presumably because the
G-subunits are not amplified and also because they are constrained.
The conclusion of these results is that the release of G from
both the activated GPCR tandems is completely sufficient to produce
typical voltage-dependent inhibition of N-type calcium channels. This
is confirmed by the inability of the IE mutant of
2A-R-Go, which does
not bind G, to mediate inhibition of CaV2.2
by clonidine. Although tonic facilitation was seen with this mutant in
the absence of agonist (Fig. 3c), this was no greater than for the
nontandem 2A-R (Fig. 2c), where inhibition by
clonidine was observed (Fig. 1e).
). However, we did not find a clear correlation
between the G-subunit in the tandem and the voltage-dependence of
the inhibition, although there was a slightly greater voltage-dependent effect with the
2A-R-Go fusion
protein. This may relate to the endogenous G dimers with which
the G subunits preferentially associate. Indeed, the kinetics and
voltage-dependence of G dissociation and reassociation are
dependent on the nature of the G dimers (Stephens et al., 1998).
Effects of Gq on G-Protein Modulation of Calcium
Channels.
The role of Gq in G-protein
modulation of calcium currents remains unclear. It has been shown that
Gq is not involved in modulation by the
2A-R of the (largely N type) calcium currents
in mouse sympathetic neurons (Haley et al., 2000). In the present
study, expression of Gq produced negligible
inhibition of N-type channels, consistent with its very low ability to
couple to the 2A-R (Chabre et al., 1994). In
contrast, the chimeric counterpart, Gqz5,
allowed significant inhibition of CaV2.2,
indicating that substitution of the C terminus of
Gz enhanced the coupling to the
2A-R (Conklin et al., 1993). However,
Gqz5 showed a reduced ability to inhibit IBa compared with Gi/o. The
inhibition also showed a lack of voltage-dependence; together, these
results suggested that Gqz5 acts via a different or modified signaling mechanism compared with
Gi/o. A similar voltage-independent inhibition of
Ca2+ channels by the
Gq-coupled muscarinic m1 receptor was shown to involve both the Gq and G subunits
(Kammermeier et al., 2000). Furthermore, the voltage-independent
inhibition was converted into voltage-dependent inhibition by
sequestering activated Gq (Kammermeier and
Ikeda, 1999).
qz5
with 2A-R-Go caused
first a reduction of clonidine-induced inhibition of
CaV2.2 and second a loss of voltage-dependent
facilitation. This action of Gqz5 could result
from a number of mechanisms: 1) G buffering, as suggested for
Gq (Jeong and Ikeda, 1999), or 2)
Gqz5 might interact with, and be activated by,
the 2A-R-Go tandem.
It has been observed previously that chimeric receptor-G constructs are able to activate not only the tethered G subunit but also endogenous subunits of the Gi/o family (Burt et
al., 1998). In the case of Gqz5 this would
result in downstream activation of phospholipase C, resulting in
elevation of inositol 1,4,5-trisphosphate and diacylglycerol and
concomitant reduction of PIP2. One potential downstream pathway would be PKC activation and subsequent
phosphorylation of either the calcium channel or the
2A-R to suppress G-protein modulation. Another
potential downstream pathway would be via reduction of
PIP2, because elevation of
PIP2 mimics and may play an essential role in
G-protein modulation (Wu et al., 2002).
We have addressed these possibilities in turn. If the mechanism were
G sequestration, Gqz5 should act
identically to Gt. However
Gt reduced inhibition of
CaV2.2 via
2A-R-Go from 75 to
43% but did not abolish facilitation in the presence of clonidine (Fig. 3a, traces). In contrast, Gqz5 reduced
inhibition by clonidine to 36% but completely abolished facilitation
(Fig. 5a, traces). Furthermore, in cells coexpressing
2A-R-Go and
Gqz5, it was possible to restore typical
Go-mediated facilitation by enhancing the GTPase
activity of activated Gqz5 with PLC-1ct.
This suggests that the effect of Gqz5 is
downstream from its activation.
Two pieces of evidence indicate that PKC activation is involved in the
response to Gqz5, although it may not
represent the entire story. Firstly, application of a PKC agonist to
cells expressing 2A-R-Go mimicked the
effects of Gqz5, resulting in reduced inhibition by clonidine and loss of PP facilitation. Secondly, in cells
coexpressing 2A-R-Go
and Gqz5, Go-mediated
inhibition and facilitation were restored with a PKC inhibitor. Taken
together, these results suggest that activation of the PLC-
signaling pathway by the 2A-R coupling to
Gqz5 can oppose G-protein-mediated inhibition of
CaV2.2.
Potential Targets for PKC Phosphorylation.
PKC-mediated
phosphorylation might occur at several sites, either separately or in
combination. PKC activation has been shown to cause
phosphorylation-dependent desensitization of the
2A-R in COS-7 and Chinese hamster ovary cells
(Liang et al., 1998). It is possible that this process may play a role
in the effect of Gqz5, although it is unclear
how this could result in a selective loss of facilitation while
substantial clonidine-mediated inhibition remains. Alternatively, PKC
could phosphorylate one or more calcium channel subunits, thus
rendering the channel less responsive to G-protein mediated inhibition
and abolishing facilitation. There are a number of possible mechanisms
by which this might occur. Phosphorylation of
CaV2.2 or an accessory subunit may result in
the loss of its ability to be modulated by G. Residues in the
I-II linker of rat CaV2.2 have been proposed to
be a target of phosphorylation by PKC and to be responsible for PKC
antagonism of G-protein modulation (Zamponi et al., 1997). Evidence has
been presented recently that this process involves binding of
Gq and PKC to the C terminus of
CaV2.2 (Simen et al., 2001). Direct activation of
PKC has been shown to counteract inhibition of N-type calcium channels
by norepinephrine (Shapiro et al., 1996). Subsequently, the importance
was examined of phosphorylation sites on the I-II linker of rat
CaV2.2, including Thr422
and Ser425, in the modulation by PKC (Zamponi et
al., 1997). An increase in calcium current was observed when mimicking
channel phosphorylation on either of these residues by mutation to Glu,
and a reduction of G-protein modulation by somatostatin when
Thr422 was mutated to Glu (Hamid et al., 1999).
However, this effect was subsequently observed only with G1 and not
with other G subunits, calling into question its general relevance
(Cooper et al., 2000). Indeed, we have shown that G-protein modulation of CaV2.2 is not dependent on the presence of the
I-II linker of a modulatable calcium channel, whereas the N terminus is
essential (Canti et al., 1999).
).
The mechanism of the reduction of G-protein modulation and loss of
P2/P1 facilitation that is characteristic of coexpression of
Gqz5 thus seems to involve its activation, and
at least in part involves downstream activation of PKC, but the main
target site(s) for phosphorylation may not be the calcium channel 1 subunit. Facilitation involves the unbinding of G subunits from the channel during the depolarizing PP (Stephens et al., 1998); this
requires the functional interaction of CaV
subunits (Canti et al., 2000, 2001; Meir et al., 2000). In the absence
of coexpressed CaV subunit, we observed
previously that activation of the D2 dopamine receptor produced only a
small voltage-independent inhibition of CaV2.2
calcium channels, whereas in the presence of
CaV subunits, the inhibition was much larger
and voltage-dependent (Meir et al., 2000). It is therefore possible
that phosphorylation of the CaV subunit might
mediate the loss of facilitation resulting from
Gqz5 coexpression. Indeed, another
CaV subunit, 2a, is phosphorylated
stoichiometrically by PKC (Puri et al., 1997). We will examine in a
future study whether phosphorylation of CaV subunits by PKC is responsible for the effects of
Gqz5.
| |
Acknowledgments |
|---|
We are grateful to N. Balaguero, W. S. Pratt, M. Nieto-Rostro, and K. Chaggar for technical assistance, to Drs. K. M. Page and N. S. Berrow for molecular biology expertise, and to
A. Meir for helpful discussion. We thank the following for gifts of
cDNA: Dr. L. E. Limbird (Vanderbilt University, Nashville, TN)
(2A-R); Dr/B. Conklin (J. David Gladstone
Institutes, San Francisco, CA) (Gq and
Gqz5); and Dr. S. Ikeda (National Institutes
of Health, Bethesda, MD) (PLC-1ct).
| |
Footnotes |
|---|
Received September 11, 2002; Accepted January 14, 2003
Address correspondence to: A.C. Dolphin, Department of Pharmacology, Medawar Building, University College London, Gower Street, London WC1E 6BT, UK. E-mail: a.dolphin{at}ucl.ac.uk
| |
Abbreviations |
|---|
GPCR, G-protein-coupled receptor;
2A-R, 2A-adrenergic receptor;
PTX, pertussis toxin;
GIRK, G-protein-coupled inward rectifier K channel;
GFP, green fluorescent protein;
w.t., wild type;
RS-79948-197, [8aR,12aS,13aS]5,6,8a,9,10,11,12,12a,13,13a-decahydro-12-ethanesulfonyl-3-methoxy-6H-isoquino[2,1-g]-[1,6]naphthyridine hydrochloride);
GTPS, guanosine
5'-O-(3-thio)triphosphate;
HA, hemagglutinin;
PAGE, polyacrylamide gel electrophoresis;
PVDF, polyvinylidene fluoride;
TTBS, Tween 20/Tris buffered saline;
PDBu, phorbol dibutyrate;
GF 109203X, bisindolylmaleimide I;
PKC, protein kinase C;
PLC-1ct, phospholipase C-1 C terminus;
PP, prepulse;
Gt, G-transducin;
PIP2, phosphatidylinositol
4,5-bisphosphate;
IE, Ile19Ala, Glu20Ala.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) the depolarizing PP. Values are reported without
and with pretreatment with PTX for the 
) and after (
) application of clonidine (n = 8). I-V plots were fitted with a modified Boltzmann equation (see
Materials and Methods). c and d, values of
IBa facilitation ratios (P2/P1) in control (
), for the 
)
the depolarizing PP. d, voltage-dependent facilitation for the same
cells as in c in control (