From the Centre for Clinical Pharmacology, The BHF
Laboratories, Department of Medicine, University College London, Rm.
420, 5 University St., London WC1E 6JJ and the
Division of
Biochemistry and Molecular Biology, University of Glasgow, Glasgow,
G12 8QQ, United Kingdom
Received for publication, December 3, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Traditionally the consequences of activation of
G-protein-coupled receptors (GPCRs) by an agonist are studied using
biochemical assays. In this study we use live cells and take advantage
of a G-protein-gated inwardly rectifying potassium channel
(Kir3.1+3.2A) that is activated by the direct binding of G The activation of G-protein-coupled receptors
(GPCRs)1 by extracellular
ligands is an important mechanism involved in a multitude of
physiological responses and is of central importance in drug development and therapeutics (1). The activated receptor couples to
G-proteins of various subtypes that then activates effector pathways
either directly or indirectly. This combination of agonist, receptor,
and G-protein is referred to as the "ternary complex" and is
thought to be the key essential determinant of the magnitude of the
downstream response (2, 3). The most recent formulations propose a
cubic ternary complex model with a large number of equilibrium constants between various states governing efficacy (2, 4, 5). The
important species is the activated receptor/agonist/G-protein complex.
It is proposed that for any combination of these three elements the
particular active conformation (or conformational space) is unique and
can thus have distinctive signaling consequences (2, 4-6). An agonist
binds more favorably to the active receptor species and thus at
equilibrium favors its' formation. Recently this model has been
extended to also incorporate the kinetics of G-protein activation and
deactivation and indicate that a kinetic model, as opposed to an
equilibrium model, may potentially have quite different properties
(7).
Generally these phenomena have been studied by the use of biochemical
assays, using cell homogenates or fractions, or by measuring the
behavior of a physiological response many steps downstream from the
G-protein cycle. It is apparent that there is a gap in our
understanding about how these signaling pathways behave dynamically in
intact cells. This is important, because, in reality, the release of
hormones and neurotransmitters varies over the second time scale.
Agonist binding to receptor is generally agreed to be diffusion-limited and much faster than the activation of downstream signaling events. However, there are a number of more controversial issues regarding models of receptor activation of G-proteins. Is there kinetic evidence
for the unique conformation of the ternary complex? What is the role of
both the isoform and concentration of G-protein in dictating the
dynamic behavior? Is the encounter of receptor with the G-protein
rate-limiting, and do receptor and G-protein exist in a pre-coupled
complex? To address these questions we have used members of the Kir3.0
family of inwardly rectifying K+ channels that are gated by
G-proteins as a reporter. G-protein-gated inwardly rectifying
K+ channels were first identified in atrial myocytes where
they are activated by acetylcholine at muscarinic M2
receptors (8-10). It was subsequently shown that this activation was
membrane-delimited (11), mimicked by non-hydrolysable GTP analogues
(12), and sensitive to pertussis toxin (PTx), implicating the
inhibitory family of G-proteins (Gi/o) (13). It is now
apparent that G-protein-gated inwardly rectifying K+
currents are also present in many neuronal cell types (14-16). Cloning
efforts have revealed the molecular counterparts of these currents, and
the channel is a heteromultimer of members of the Kir3.0 family of
K+ channels (16-23). It is now accepted that activation of
native and cloned Kir3.0 channels involves a direct interaction with the G In our previous studies we have developed a series of molecular tools
to study these issues including stable cell lines expressing the
channel complex along with GPCRs and both fluorescent and non-fluorescent G Molecular Biology, Cell Culture, and
Transfection--
PTx-resistant Gi/o mutant
The methods for cell culture and the generation of stable cell lines
were as previously described (26, 30). In addition to our established
cell lines (Kir3.1+3.2A channel plus either the A1
adenosine receptor (HKIR3.1/3.2/A1) or the D2S dopaminergic receptor (HKIR3.1/3.2/D2)), we made a further four dual receptor plus
channel stable lines that were designated as follows:
Transiently transfected cells suitable for patch clamping were
identified by epifluorescence from co-transfection of 100 ng of the
enhanced variant of the green fluorescent protein (pEGFP-N1, Clontech). Data were obtained from at least two
independent transfections.
Radioligand Binding--
Radioligand binding was performed on
crude membrane preparations isolated from the relevant stable lines
(HKIR3.1/3.2/A1, HKIR3.1/3.2/ Electrophysiology--
Whole-cell membrane currents were
recorded using an Axopatch 200B amplifier (Axon Instruments). Patch
pipettes were pulled from filamented borosilicate glass (Clark
Electromedical) and had a resistance of 1.5-2.5 M Rapid Drug Application and Barium Calibration--
Drugs were
applied using a "sewer pipe" system (Rapid Solution Changer
RSC-160, Bio-Logic) whereby an array of perfusion capillaries was
placed in the bath ~40 µm from the recorded cell. This system allowed rapid solution switching between capillary tubes and localized application of drugs due to the laminar flow over the studied cell from
the tubes as previously described (31). A number of parameters were
determined using this system (Fig. 1A, part ii). Upon agonist application current activated with an initial delay (lag)
followed by a rapid rise to peak amplitude (time-to-peak (ttp)).
Current subsequently became desensitized during continued agonist
application. In this study agonist was applied for 20 s. Upon
agonist removal currents deactivated back to baseline levels.
For each cell we assessed whether there were any flow artifacts
resulting from the pressure of drug application. We did this by
applying bath solution from one of the sewer pipes and recording any
flow-induced currents. If such a current was observed, then the
position of the perfusion head was moved to minimize it. Furthermore, to control for variations in positioning of the sewer pipe system relative to the cell, we calibrated this system using the kinetics of
channel block by barium. The cell was positioned in the center of the
field using crosshairs in the microscope eyepieces. Barium (1 mM) was applied to the cell in the presence of agonist when the agonist-induced current had reached a plateau phase. Block of the
current occurred with an initial delay before reaching equilibrium. It
was assumed that this lag reflected the intrinsic delivery time to the
cell. A barium calibration was performed prior to the start of
experiments to ascertain correct positioning of the sewer pipe and was
repeated on several cells during each recording session. In general the
results were highly reproducible (the lag time for barium block was
237.3 ± 11.7 ms (n = 73)).
Confocal Microscopy and Western Blotting--
Confocal
microscopy and acquisition of images were as previously described (28).
In the current study we used a 40× oil objective, 40% laser power,
gain was set to 50%, and iris aperture was opened to 1.5 nm (optimum
aperture, 1.1 nm). Western blotting of CFP-tagged G Data Analysis--
Membrane currents were measured at
Materials and Drugs--
Solutions were as follows
(concentrations in millimolar): pipette solution, 107 KCl, 1.2 MgCl2, 1 CaCl2, 10 EGTA, 5 HEPES, 2 MgATP, 0.3 Na2GTP (KOH to pH 7.2, ~140 mM total
K+); bath solution, 140 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.4). Cell culture materials were from
Life Technologies, Inc. and Invitrogen. All chemicals were from Sigma
or Calbiochem. Drugs were made up as concentrated stock solutions and
kept at In our previous studies (26, 27), we generated an HEK293 stable
cell line expressing the Kir3.1+3.2A channel complex. On this
background of channel expression we subsequently generated dual
receptor-plus-channel stable lines in which we have investigated the
kinetic properties of receptor-mediated currents (see "Experimental Procedures"). Fig. 1A shows
the profile of current activation following stimulation of the
A1 adenosine receptor by a concentration of agonist (NECA,
1 µM) that would lead to full receptor occupancy. We
observed an initial lag followed by a rapid rise to a peak amplitude of
current. With prolonged agonist application current amplitude wanes as
the response desensitizes, and upon removal of agonist it deactivates
back to baseline levels. In this study we focus upon the initial
activation phase, which we measured as the sum of the "lag" plus
the "time-to-peak" (lag plus ttp) and investigate the effects of
the ternary complex on this response. Fig. 1B shows
representative current recordings from three stable lines
(A1: HKIR3.1/3.2/A1,
subunit to the channel complex to report, in real-time, using the patch
clamp technique the activity of the "ternary complex" of
agonist/receptor/G-protein. This analysis is further facilitated by the
use of pertussis toxin-resistant fluorescent and non-fluorescent
G
i/o subunits and a series of HEK293 cell lines
stably expressing both channel and receptors (including the adenosine
A1 receptor, the adrenergic
2A receptor, the
dopamine D2S receptor, the M4 muscarinic receptor, and the dimeric GABA-B1b/2 receptor). We systematically
analyzed the contribution of the various inputs to the observed kinetic
response of channel activation. Our studies indicate that the
combination of agonist, GPCR, and G-protein isoform uniquely specify
the behavior of these channels and thus support the importance of the
whole ternary complex at a kinetic level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dimer not the G
subunit (16, 24, 25).
i/o subunits engineered to be
resistant to the action of pertussis toxin (26-28). In this study we
combine the use of these tools with whole-cell patch clamping to record
Kir3.1+3.2A currents in response to GPCR stimulation by agonists
applied using a rapid and localized drug application system to assay
the G-protein cycle on the hundreds of millisecond time scale. We focus
on how quickly the channel activates and use this parameter to examine how the ternary complex dictates the final channel response.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits
and CFP-tagged PTx-resistant G
i/o were generated and
used as previously described (27, 28). GABA-B1b and
GABA-B2 were expressed in the dual promoter vector
pBudCE4.1 (Invitrogen). Standard molecular cloning techniques were used
to excise the relevant clones from the previous vector (GABA-B1b was excised from pcDNA3.1/neo/(+) with
PmeI/XhoI and GABA-B2 from
pcDNA3.1/neo/(+) with KpnI/XhoI), and they
were introduced into the two polylinkers in
ScaI/SalI sites for GABA-B1b and
KpnI/XhoI sites for GABA-B2.
Inducible expression of G
i3-CFP was achieved using the
TRex system (Invitrogen). G
i3-CFP was removed from the
previously described construct (28) and subcloned into pcDNA5/TO using a KpnI/NotI digest. The
A1-Gi1
(C351G) fused construct was as previously
described (29), and cDNA was excised from pcDNA3 and subcloned
in to pcDNA3.1/Zeo/(+).
2A adrenergic receptor, HKIR3.1/3.2/
2; GABA-B1b/2 receptor,
HKIR3.1/3.2/GGB; M4 muscarinic receptor, HKIR3.1/3.2/M4; and
A1-G
i1 fusion,
HKIR3.1/3.2/A1-G
i1. Monoclonal cell lines were
established by picking single colonies of cells following transfection
and growth under selective pressure. For all the dual receptor and
channel expressing lines we used a dual selection strategy with 727 µg/ml G418 and 364 µg/ml Zeocin (Invitrogen). Stable cell lines,
expressing a fluorescent G-protein
subunit (G
i3-CFP)
in an inducible system, were made after transfection of
G
i3-CFP in pcDNA5/TO and pcDNA6/TR (both
Invitrogen) into the HKIR3.1/3.2/A1 and subsequent selection with 727 µg/ml G418, 364 µg/ml Zeocin, 400 µg/ml hygromycin, and 5 µg/ml
blasticidin (Invitrogen). This stable line was designated as
HKIR3.1/3.2/A1/G
i3-T.
2, and HKIR3.1/3.2/D2). Cells were
harvested into binding buffer (50 mM Tris-HCl, pH 7.4) and
stored at
80 °C. Cells were hypotonically shocked (10 mM Tris-HCl and 10 mM EDTA) on ice and then
homogenized using a glass-on-glass Dounce homogenizer. The homogenate
was spun at 600 × g (4 °C) for 15 min to sediment nuclei and large cell debris. The membrane fraction was then obtained by spinning the supernatant at 100,000 × gav in an ultracentrifuge (Beckman, Optima
LE-80K). The pellet was resuspended in binding buffer and incubated
with radioligand at room temperature for 1 h. Specific binding was
assessed using saturating concentrations of radiolabeled receptor
antagonists: 8 nM [3H]DPCPX for adenosine
A1 receptors, 30 nM [3H]RX-821002
for adrenergic
2A receptors, and 4 nM
[3H]spiperone for dopamine D2S receptors.
Nonspecific binding was determined in the presence of a 1000-fold
excess of unlabeled antagonist: 8 µM DPCPX
(A1), 30 µM rauwolscine
(
2A), and 4 µM spiperone (D2).
Binding was performed in triplicate and repeated at least four times.
Data were corrected for total protein content in each sample and are
expressed as fmol/µg of protein (mean ± S.E.).
when filled with
pipette solution (see below). Prior to filling, tips of patch pipettes
were coated with a Parafilm/mineral oil suspension. Data were acquired
and analyzed using a Digidata 1200B interface (Axon Instruments) and pClamp software (version 6.0; Axon Instruments). Cell capacitance was
~15 picofarads, and series resistance (<10 M
) was at least 75%
compensated using the amplifier circuitry. Recordings of membrane current were carried out after an equilibration period of ~5 min. Immediately following patch rupture, a current-voltage relationship was
performed to establish that currents were inwardly rectifying. Thereafter cells were voltage-clamped at
60 mV, and agonist-induced currents were measured at this potential. For current-voltage relationships, records were filtered at 1 kHz and digitized at 5 kHz.
For continual data acquisition where cells were voltage-clamped at
60
mV, records were digitized at 100 Hz.
i/o
subunits was performed using a polyclonal rabbit GFP antibody as
previously described (28).
60 mV, and all data are presented as mean ± S.E., where
n indicates the number of cells recorded from which data
were recorded. Time measurements (lag plus ttp) were reciprocated prior
to statistical analysis, because the reciprocal of time is normally
distributed. Data are shown untransformed. Data were analyzed for
statistical significance using either Student's t test or
one-way repeated measures analysis of variance tests with Bonferroni
correction as appropriate (*, p
0.05; **,
p
0.01; and ***, p
0.001).
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A: HKIR3.1/3.2/
2,
D2S: HKIR3.1/3.2/D2) in response to 20-s applications of
maximal concentrations of agonist. To determine absolute levels of
receptor expression we used radioligand binding with tritiated
antagonists (see "Experimental Procedures") and found similar
levels in the HKIR3.1/3.2/A1, HKIR3.1/3.2/
2, and HKIR3.1/3.2/D2
clonal isolates used in Fig. 1B. These data are shown in
Fig. 1C. We found that, although activation kinetics were
quite similar through these three receptors, D2S-mediated currents did exhibit slower time courses of activation than
2A- or A1-mediated currents. We also
investigated the kinetics of activation in two other cell lines
(M4: HKIR3.1/3.2/M4 and GABA-B1b/2: HKIR3.1/3.2/GGB), and
the mean data are summarized in Fig. 1D. Representative
recordings from HKIR3.1/3.2/M4 and HKIR3.1/3.2/GGB are shown in
subsequent figures.
View larger version (20K):
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Fig. 1.
Receptor mediated kinetics. A, an
example of a NECA-induced current recorded in the HKIR3.1/3.2/A1 cell
line at 60 mV in response to a 20-s application of NECA (in this and
subsequent figures, agonist application is indicated by the solid
horizontal bar). The expanded current trace shows the parameters
measured. Channel activation kinetics is represented by
"lag" (time between onset of agonist application and
channel activation) and "time-to-peak (ttp)" (time
between onset of channel activation and peak current amplitude).
B, representative examples from the three
channel-plus-receptor expressing stable cell lines: HKIR3.1/3.2/A1
(upper panel), HKIR3.1/3.2/
2 (middle panel),
and HKIR3.1/3.2/D2 (lower panel) in response to a 20-s
application of relevant agonist (A1, 1 µM
NECA;
2A, 3 µM noradrenaline; and
D2S, 10 µM quinpirole). C,
radioligand binding using tritiated receptor antagonists was used to
assess levels of receptor expression in the HKIR3.1/3.2/A1,
HKIR3.1/3.2/
2, and HKIR3.1/3.2/D2 stable cell lines. All three
receptor types were expressed at equivalent levels (p > 0.05), and these data are summarized in the bar chart. D,
we measured channel activation (lag+ttp) in the three cell
lines shown in A and additionally in a cell line expressing
the channel plus the GABA-B1b/2 variant (HKIR3.1/3.2/GGB)
and a cell line expressing the channel complex and the M4 muscarinic
receptor (HKIR3.1/3.2/M4). One-way analysis of variances with
Bonferroni's multiple comparisons test were used to compare data from
the HKIR3.1/3.2/A1, HKIR3.1/3.2/
2, and HKIR3.1/3.2/D2 cell lines in
which receptors were expressed to similar levels. Channel activation
via stimulation of the D2S receptor was significantly
slower than that via stimulation of either the A1
(p < 0.001) or the
2A receptors
(p < 0.001).
The experiments just described were performed using maximal
concentrations of agonist likely to result in full receptor occupancy. We next examined the effects of agonist concentration, and thus receptor occupancy, on channel activation. We used the HKIR3.1/3.2/GGB (Fig. 2A) and the
HKIR3.1/3.2/A1 cell lines (Fig. 2B) and used the agonists at
a high, saturating concentration and at a lower concentration, which
was approximately the EC50 value. We found that, with the
lower concentration of agonist, channel activation was significantly
slowed (Fig. 2, A and B).
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We have shown previously that by using engineered PTx-resistant G
subunits, it is possible to look exclusively at coupling between a
receptor and the channel via specific G
i/o isoforms (27). Furthermore, we have recently made a series of cyan fluorescent protein (CFP)-tagged PTx-resistant G
i/o isoforms and
have shown that they are both membrane-targeted and functional
(coupling to both
subunits and the adenylate cyclase pathway),
and we have established conditions where these constructs are expressed at equivalent levels (28). In our previous work we have shown that the
A1 receptor appears to couple to the channel with equal efficacy and potency via all the G
i/o isoforms tested
(27). We also now demonstrate that all CFP-tagged G
i/o
subunits are able to participate in A1-mediated channel
activation with similar magnitudes of response and similar kinetic
profiles except via G
oA-CFP, where we observed that
channel activation via this G-protein exhibited slower activation
kinetics (Fig. 3) (28). However, we know
that other receptors, for example the M4 muscarinic and the
GABAB heterodimeric receptors, show more selective patterns of coupling to G
i/o subunits (27), and so we examined
the dynamics of coupling via G
i2 and G
oA
with the M4 and GABA-B1b/2 receptors (using the
HKIR3.1/3.2/M4 and HKIR3.1/3.2/GGB lines, respectively). With the
HKIR3.1/3.2/M4 cell line we used the CFP-tagged G
subunits (G
i2-CFP and G
o-CFP, at equivalent
concentrations) and observed that, although channel activation via this
receptor was intrinsically slower than through the
A1 receptor, there was no significant difference in channel
activation via G
i2-CFP and G
o-CFP (Fig. 4). However with the HKIR3.1/3.2/GGB line
we were unable to rescue coupling between the GABA-B1b/2
receptor and the channel in PTx-treated cells using the CFP-tagged
G-proteins. This is the only receptor to date where we have observed
this, and such lack of coupling may be related to their unique
heterodimeric receptor formation. Instead we used the non-CFP-tagged
G
i2C352G and G
oAC351G to study channel
activation through the GABA-B1b/2 receptor. In the HKIR3.1/3.2/A1 cell line, expression of these constructs yielded comparable activation kinetics to the CFP-tagged variants (not shown).
In contrast to both the A1 and M4 receptors, activation via
this receptor was much faster through Go than
Gi2 (Fig. 5). For comparison
the magnitude of current potentiation and kinetics of activation via
native G-proteins are included (see Figs. 3-5). Importantly, we see
different kinetic profiles of channel activation through different
receptor and G-protein combinations despite robust coupling apparent
from the magnitude of the response.
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G-protein isoforms are present at varying levels in different
cells, and thus it is important to know what role the G-protein concentration has in determining these kinetic responses. To do this we
established an inducible system whereby we could regulate the levels of
expression of G
i3-CFP in a cell line stably expressing the channel complex and the A1 receptor (referred to as
HKIR3.1/3.2/A1/G
i3-T). This was done using a
commercially available system (see "Experimental Procedures")
whereby gene expression is conditional on the addition of the
antibiotic tetracycline (Tet). We titrated the concentration of Tet
(0.01-100 µg/ml) to determine a high, medium, and low level of
G
i3-CFP expression. Fig.
6A shows the induction of
G
i3-CFP expression at the membrane in the
HKIR3.1/3.2/A1/G
i3-T cell line with increasing
concentration of Tet. Using Western blotting we showed the induction of
graded expression of G
i3-CFP (Fig. 6B). In
addition, in PTx-treated cells, increasing concentrations of Tet
progressively enhanced the amplitude of NECA-induced currents (Fig.
6C). However, importantly the kinetics of activation of the
channel response was not altered (Fig. 6D).
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Finally, we examined the potential role that diffusion of the receptor
to G-protein might have on activation kinetics. We took advantage of an
approach in which the receptor is physically tethered to an engineered
PTx-resistant G-protein, specifically the A1 receptor and
the Gi1C351G subunit. We both transiently and stably
expressed this construct in the HKIR3.1/3.2 cell line. In addition, we
characterized receptor expression density using radioligand binding
with [3H]DPCPX (8 nM) in a clonal isolate and
found that the fused A1 receptor was expressed at more that
2-fold higher levels (72.7 ± 13 fmol/µg of protein,
n = 9) than the non-fused A1 receptor (28.6 ± 7.2 fmol/µg of protein, n = 6) (Fig.
1C). We compared the activation kinetics of A1
via endogenous G-proteins to that of the fused
A1-G
i1 construct after both transient and
stable expression, with and without PTx treatment (Fig.
7). Under both sets of conditions
activation was significantly slower via the fused construct both before
and after PTx treatment. However, if G
i1C351G was
transfected into the HKIR3.1/3.2/A1 line, signaling was also slowed in
an analogous manner. Thus, it is the nature of the mutant G-protein
subunit rather than the tethering per se that determines the
change in activation kinetics. We also observed that the deactivation
rate was increased and this is discussed below.
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DISCUSSION |
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In this report we have taken advantage of the fact that Kir3.0
channels are gated directly by G subunits, and thus they act as
biosensors for G
concentration at the plasma membrane. One of the
major advantages of this approach is that the release of G
is
directly measured and the system output is not dependent upon
downstream events. In combination with electrophysiological recordings
(and rapid agonist application) this results in high temporal
resolution. We have analyzed the kinetic contribution of the ternary
complex to channel activation. We have a number of major findings,
namely that increased occupancy of the receptor accelerates activation
kinetics, that the activation kinetics via a Gi/o isoform
are determined by the particular receptor/G-protein combination, and
that receptor diffusion to the G-protein and the concentration of the
G-protein have little influence on the activation kinetics. The
G-protein amount simply determines the amplitude of the response. Our
data all support the hypothesis that the assembly of the ternary
complex of agonist, receptor, and G-protein is not rate-limiting. It
is, however, the unique conformation of the active ternary complex that
specifies the kinetic behavior of the channel response.
Channel activation kinetics through a number of different
receptors with saturating agonist concentration occurs rapidly via the
mixed pool of G-proteins endogenously expressed in HEK293 cells.
However, there are significant differences with activation, with
that through M4 being the slowest and 2A being the most rapid. Indeed, the nature and numerical details of channel kinetics are
very similar to those occurring with the channel expressed in neurons
(15, 32). Receptor-mediated currents elicited using high agonist
concentration have a typical profile comprising an initial lag followed
by a subsequent sharp rise to a peak amplitude after drug application.
This pattern likely reflects the occurrence of a number of sequential
steps. In a classic "collision" coupling view these steps might
consist of agonist binding to receptor followed by diffusion of the
agonist/receptor complex to the G-protein, activation and dissociation
of the G-protein heterotrimer, diffusion of G
to the channel, and
finally, activation and opening of the channel. Binding of G
and
channel activation are assumed to be fast and not rate-limiting. Indeed
there is also evidence that channel activation is intrinsically
cooperative, because the Hill coefficient for G
-mediated channel
stimulation is between 1.5 and 3 and more than one G
subunit
needs to occupy one of the four equivalent binding sites to initiate
channel opening (33, 34). Such considerations would account for why
there is a small discrepancy between the point at which induced
G-protein is detected and the point at which significant coupling
begins to occur in Fig. 6.
As might be expected from the principles of mass action, agonist
concentration clearly influences the onset kinetics. Agonist binding to
receptors is diffusion-limited, agonists bind preferentially to active
receptor conformations, and thus within the timescale of signaling
events the establishment of this equilibrium between active and
inactive receptor conformations will not be rate-limiting. It is the
proportion of active species that will subsequently determine the
kinetics of the downstream response. But, what about other steps in the
G-protein signaling pathway? To address the role of receptor diffusion
to the G-protein we fused the A1 receptor directly to
Gi1. It is possible that a receptor might activate sequentially multiple G-proteins resulting in signal amplification. In
this case, a slower response would ensue following stimulation of a
fused receptor G-protein construct. Alternatively, diffusion might be a
rate-limiting step in which case fusion should accelerate signaling.
Experimentally, we observed that channel activation kinetics was slower
(despite more than 2-fold higher levels of receptor expression in
stable lines expressing the A1-G
i1 fused complex) compared with non-fused receptor signaling via endogenous G-proteins. However if channel activation via the
A1-G
i1 construct is compared with activation
when the A1 receptor is simply constrained to (but not
tethered to) G
i1C351G, then there is no significant difference between these two conditions. Thus it is the
G
i1 itself rather than the lack of amplification that
influences kinetics. It has been previously noted that the nature of
the cysteine mutation does result in lowered affinity for the receptor
(35, 36), and our finding appears to be a kinetic reflection of this
observation. Although this may be an issue with this approach, it is
essentially unavoidable. A second caveat is that the levels of receptor
expression are higher in our system than might reasonably be expected
to occur with natively expressed receptors: under conditions of lower expression amplification may be important. Thus, it seems that diffusion of the receptor to the G-protein is not kinetically important; it is the subsequent intrinsic activation process
that is rate-limiting. Our results with the
A1-G
i1 fusion are consistent with
biochemical studies using this construct (36), but a study on a
receptor-Gz fusion found diffusion to be key, although this may be related to the unusual properties of that particular G-protein (37). Furthermore, we show that deactivation rates are increased and
this too is consistent with data of Waldhoer et al. (36). They find that the A1-G
i1C351G fusion
releases bound radioligand agonist more readily than the A1
receptor and argue that the ternary complex is less stable (36). It is
difficult to be quite so categorical with our approach, but our data
are potentially consistent with this hypothesis.
Receptor occupancy is not the only factor that has important
consequences for channel kinetics. We demonstrate that activation occurs faster via Gi1, Gi2, and Gi3
than GoA for the A1 receptor. This pattern was
reversed when channels were activated via the GABA-B1b/2
receptor, and a similar non-significant trend was observed with the M4
receptor, thus illustrating the particular receptor/G-protein combination dictates the response. These observations can be accounted for by greater "kinetic efficacy," i.e. some
agonist-receptor-G-protein ternary complexes promote the faster release
of GDP from the G-protein subunit. This proposal is not
unreasonable given the accumulating data supporting the idea that
different GPCRs have differing affinities for the various G
isoforms
and that even different agonists at the same receptor may couple with
varying degrees to different G-protein isoforms (2, 6, 27, 38, 39). It
has been argued on theoretical grounds that there may be significant
differences in the predicted behavior of signaling cascades when
considered in kinetic models compared with that in equilibrium models
(3, 7). Our data argue that the ternary complex uniquely determines the
kinetic as well as the steady-state properties.
Intriguingly, the G-protein amount simply modifies the amplitude of the
response and does not influence the activation kinetics. This is
consistent with the potential existence of a complex between the
G-protein subunit and channel (40) or a high degree of precoupling
between receptor and G-protein being important for channel activation
(26, 41), and it is consistent with theoretical studies (3). Our
studies here have mostly focused on the A1 receptor, and it
is possible that variations in precoupling, for example promoted by RGS
proteins, might influence activation kinetics via other receptors. It
is intriguing that a recent report has shown dopamine receptors and
Kir3.0 channels potentially existing in complexes suggesting that all
three components may be associated in a macromolecular complex (42). It
is also interesting that the kinetics of K+ and
Ca2+ channel modulation in G
o knockout mice
are slowed (43): our results suggest that this may be related to
differences in the efficacy of GPCR coupling to the remaining
G-proteins rather than changes in amount of the total G-protein pool.
Our results and conclusions depend on the use of a number of tools, in
particular that of PTx-resistant G-proteins with a point mutation at
the cysteine four amino acids from the end of the protein. Despite the
advantages of this approach, there are some essentially unavoidable
caveats that should be appreciated. It is apparent from biochemical
studies that the mutation of the cysteine and the particular amino acid
substituted affects the efficacy of the response (35, 44). Of these
mutations the C-I replacement seems to best preserve coupling and is
the one we have used in our fluorescent protein G-protein alpha subunit chimaeras (28, 35, 44). However we have previously shown little
difference in efficacy of coupling between C-I and C-G mutations of
Gi1 (27). However, using these reagents (and the G
i2C352G and G
oAC351G mutants) in our
system, it is clear that the magnitude and the time to peak activation
varies little between coupling via native G-proteins in the absence of
PTx and coupling via a selected G-protein mutant in PTx-treated cells.
For example, if one compares coupling to endogenous G
via
G
o-CFP with the M4 receptor (Fig. 4, B and
C) and via G
oAC351G with GABA-B (Fig. 5,
B and C), it can be seen that there is little
difference in magnitude and activation kinetics. The only place where
such issues may be pertinent is with the studies shown in Fig. 7, but
it is difficult to see an alternative strategy that would ensure strict coupling between the receptor and fused G-protein. A second
consideration is that different G
combinations in the
heterotrimer may also play a role (45), however, in our hands all four
G-protein
isoforms can robustly couple with endogenous G
dimers in HEK293 cells (27, 28).
In summary our data support the concept that the formation
of the ternary complex is not rate-limiting for signaling. It is the
release of GDP from the G-protein heterotrimer that is important, and
the rate at which that happens is determined by the conformation of the
ternary complex.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. F. Marshall for providing the GABA-B receptor clones.
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FOOTNOTES |
---|
* This work was supported in part by the Wellcome Trust, the Royal Society, and the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ A Royal Society Dorothy Hodgkin Fellow.
** To whom correspondence should be addressed. Tel.: 44-20-7679-6391; Fax: 44-20-7691-2838; E-mail: a.tinker@ucl.ac.uk.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M212299200
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G-protein-coupled receptor;
PTx, pertussis toxin;
CFP, cyan fluorescent
protein;
GABA, -aminobutyric acid;
GFP, green fluorescence protein;
Tet, tetracycline;
ttp, time to peak;
DPCPX, 8-cyclopentyl-1,3-dipropylxanthine;
NECA, 5'-(N-Ethylcarboxamido)adenosine.
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