From the Department of Physiology and Pharmacology, Sackler School
of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel, the
Department of Molecular and Cellular Pharmacology,
University of Miami School of Medicine, Miami, Florida
33156, and the § Department of Integrative Biology
and Pharmacology, University of Texas-Houston Medical School, Houston,
Texas 77030
Received for publication, October 31, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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G protein-gated K+
channels (GIRK, or Kir3) are activated by the direct binding of
G GIRK1 (Kir3) channels
are crucial for the regulation of heartbeat and for inhibitory actions
of many neurotransmitters in the brain. They are activated by direct
binding of G GIRKs are usually heterotetramers composed of two pairs of homologous
subunits. GIRK1/GIRK4 is predominant in the heart; GIRK1/GIRK2 is
abundant in the brain. An aspartate, which is absent in GIRK1 but
present in the proximal C terminus of GIRK2 (Asp-226) and GIRK4
(Asp-223), is crucial for fast direct gating by Na+ (6,
8).
We have noticed an additional, slow activating effect of
Na+ on GIRK channels in excised patches of
Xenopus oocytes. The slow activation occurred both in
wild-type (WT) GIRK channels and, surprisingly, in GIRK mutants that
lack the fast direct Na+ regulation. It did not require GTP
but was blocked by a G cDNA Constructs and RNA--
RNA was synthesized in
vitro from the following DNAs: GIRK1F137S (9), GIRK1,
GIRK2, myristoylated c Xenopus Oocyte Preparation and
Electrophysiology--
Xenopus oocytes were prepared,
injected with RNA, and incubated in NDE-96 solution (96 mM
NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 2.5 mM sodium pyruvate,
50 µg/ml gentamycin, 5 mM Hepes/NaOH, pH = 7.6).
Patch-clamp measurements of GIRK activity were done at Biochemistry and Immunocytochemistry--
The DNA of
GST-G
Surface plasmon resonance (SPR) analysis of
GST-G
The level of GIRK1F137S protein in plasma membrane (Fig.
4C) was measured as described previously (10). Briefly,
large oocyte membrane patches were attached to coverslips,
fixated, stained by a specific GIRK1 antibody (Alomone Labs,
Jerusalem, Israel), and visualized using a Cy3-conjugated rabbit IgG
(Jackson Immunoresearch Laboratories) using a Zeiss LSM 410 confocal microscope.
Statistics--
Data are presented as mean ± S.E.;
pairwise comparisons were done using a two-tailed t test.
WT GIRK1/GIRK2 channels were expressed in Xenopus
oocytes, and Na+-induced activation was studied in excised
patches (Fig. 1A). GIRK
activity was first recorded in cell-attached configuration. Then the
patch was excised ("inside-out") with its intracellular membrane
surface facing the bath solution, which was Na+- and
GTP-free but contained Mg-ATP to preserve membrane phosphatidylinositol 4,5-bisphosphate (4). After excision, the basal activity declined to a
new steady level within 0.5-2 min (10). 3-4 min after excision, 40 mM NaCl was added to the bath solution, causing a 6.7 ± 2.5-fold increase (n = 8) in channel activity. In
agreement with previous studies (7), this fast
Na+ activation was not affected by coexpression of the
G or of cytosolic Na+. Na+ activation is
fast, G
-independent, and probably via a direct, low affinity
(EC50, 30-40 mM) binding of Na+ to
the channel. Here we demonstrate that an increase in intracellular Na+ concentration,
[Na+]in, within the physiological
range (5-20 mM), activates GIRK within minutes via an
additional, slow mechanism. The slow activation is observed in GIRK
mutants lacking the direct Na+ effect. It is inhibited by a
G
scavenger, hence it is G
-dependent; but it
does not require GTP. We hypothesized that Na+ elevates the
cellular concentration of free G
by promoting the dissociation of
the G
heterotrimer into free G
GDP and G
.
Direct biochemical measurements showed that Na+ causes a
moderate decrease (~2-fold) in the affinity of interaction between
G
GDP and G
. Furthermore, in accord with the
predictions of our model, slow Na+ activation was enhanced
by mild coexpression of G
i3. Our findings reveal a
previously unknown mechanism of regulation of G proteins and
demonstrate a novel G
-dependent regulation of GIRK by
Na+. We propose that Na+ may act as a
regulatory factor, or even a second messenger, that regulates effectors
via G
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
released from heterotrimeric G proteins following
activation of G protein-coupled receptors (GPCR) (1-3). GIRK activity
also crucially depends on the presence of membrane phosphatidylinositol
4,5-bisphosphate (4). Cytosolic Na+ has been shown
to activate GIRK by a direct, G protein-independent mechanism. The
direct activation by Na+ is fast and exhibits low affinity
for Na+ with an EC50 of 30-40 mM
Na+ (4-7).
scavenger, suggesting mediation by
G
. We hypothesized that Na+ promotes dissociation of
the heterotrimeric G
GDP
complex into free
G
GDP and G
; the latter activates GIRK. This
hypothesis was supported by direct biochemical measurements. Our
findings shed new light on mechanisms of regulation of G proteins and
GIRK channels by Na+ and suggest that Na+ may
act as a second messenger that regulates effectors via G
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ARK, and G
i3 (10). Amounts of
injected RNA were as follows: GIRK1, GIRK2, and GIRK2D226N, 0.1-0.2 ng/oocyte; GIRK1F137S, 1-5 ng/oocyte;
G
i3, 0.5-2 ng/oocyte; c
ARK, 5 ng/oocyte. In
GIRK1F137S experiments, an antisense oligonucleotide (50 ng) against the endogenous GIRK5 subunit was injected to prevent the
formation of GIRK1/5 channels (11).
80 mV as
described previously (10). The electrode solution contained 146 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM NaCl, 10 mM Hepes/KOH. The bath (500 µl) contained a
Na+-free solution (130 mM KCl, 2 mM
MgCl2, 10 mM Hepes/KOH, 1 mM EGTA,
2 mM Mg-ATP). The pH of all solutions was 7.4-7.6. NaCl was added in 50 µl of bath solution and mixed manually. Patches that
showed >50% rundown of activity within 6 min after addition of
Na+ (about 10% of all patches) were excluded from the
study. Currents were filtered at 2 kHz and sampled at 5 kHz using the
Axotape software (Axon Instruments). The results were analyzed as
described previously (10). In each patch, the Na+-induced
changes in activity (-fold activation) were calculated as -fold change
in total open probability, NPo, relative to
basal NPo measured during the last minute before
the addition of Na+.
i3 was constructed by inserting the coding sequence
of human G
i3 into the EcoRI-NotI
sites of pGEX-4T-1 vector (Amersham Biosciences). The protein was
amplified and purified from Escherichia coli using
glutathione-Sepharose affinity beads. For pull-down assays,
GST-G
i3 was incubated for 30 min at 4 °C in binding
buffer (50 mM Tris, 5 mM MgCl2, 1 mM EDTA, 0.05% Tween 20, pH 7.0 with 150 mM
KCl or 150 mM NaCl) with either 100 µM GDP or
GTP
S. [35S]Methionine-labeled G
was synthesized
in reticulocyte lysate, diluted 1:4, and incubated with
GST-G
i3 in 300 µl of the above buffer (with 150 mM NaCl or KCl) for 30 min at room temperature in the
presence of GDP or GTP
S. The mixture was incubated with glutathione-Sepharose beads and washed, and bound proteins were eluted
with 15 mM glutathione and analyzed on a 12%
SDS-polyacrylamide gel followed by Coomassie Blue staining and
autoradiography as described previously (12).
i3-G
interaction was carried out on Biacore
2000 (Biacore AB, Uppsala, Sweden) using anti-GST antibodies (90 ng)
covalently attached to the surface of a CM5 sensor chip as described
previously (13). Following the capture of GST-G
i3,
G
was injected across the sensor surface at a flow rate of
15 ml/min in series of concentrations in a buffer containing 50 mM Tris-Cl, 5 mM MgCl2, 1% CHAPS
buffer, and either 100 mM KCl or 100 mM KCl.
Intermediate concentrations of Na+ were obtained by mixing
the two buffers. The kinetic data were analyzed using BIAEvaluation 3.1 software (Biacore AB) and Sigmaplot (SPSS Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
scavenger c
ARK (myristoylated C terminus of
-adrenergic
receptor kinase; data not shown). In addition, in many WT GIRK1/GIRK2
patches, a slow, late activation was observed. It started
after about 1 min and developed over the next 3-6 min (Fig.
1A).
View larger version (39K):
[in a new window]
Fig. 1.
Fast and slow activation of GIRK channels by
Na+. A, activation of the WT GIRK1/2
channels by 40 mM NaCl: full time course of a
representative experiment. Periods of patch excision and addition of
NaCl, accompanied by noise, are blanked. Channel openings are
downward. B, slow activation of
GIRK1F137S channels by 40 mM NaCl. Upper
trace, full time course of the experiment; lower traces
zoom on shorter periods of activity. C, activation of
GIRK1F137S channels by 40 mM NaCl is blocked by
coexpression of c ARK: a representative record. D, summary
of effects of 40 mM NaCl on GIRK1F137S. Numbers
of experiments are shown above bars. *, p < 0.05. c.a., cell-attached.
To explore the slow effects of Na+, we utilized the GIRK1
pore mutant GIRK1F137S, which forms functional
homotetrameric channels (9) and lacks the C-terminal aspartate crucial
for the direct Na+ effect. Fast activation of
homotetrameric GIRK1F137S channels by 40 mM
Na+ was very weak, 1.82 ± 0.36-fold
(n = 7) (Fig. 1B). It might reflect a weak
direct effect of Na+ on the GIRK1F137S channel.
In seven of eight patches, the activity continued to increase over the
next several minutes, reaching a maximum after 4-7 min. This
slow activation, measured during a 3-min period between 4 and 6 min after addition of Na+, was 3.9 ± 1-fold
(n = 7) above basal (Fig. 1D). It was fully blocked by coexpression of cARK (Fig. 1, C and
D), implying a G
-dependent mechanism. We
assumed that the additional 2-fold slow increase in GIRK activity
(compared with the 1st min after addition of Na+) reflects
an increase in cellular concentration of free G
, [G
].
Comparable slow activation (2.62 ± 0.45-fold, n = 6) was observed already at 10 mM Na+, which
causes little fast activation in WT GIRK channels (5, 14) and no fast
activation in GIRK1F137S (1.31 ± 0.16-fold, see Fig.
4).
How could Na+ activate GIRK in a
G-dependent manner? GPCRs activate G proteins by
promoting the exchange of GDP for GTP at the G
subunit followed by
dissociation of G
GTP from G
(15). Cl
also promotes GTP binding to G
in vitro (16), but
Cl
concentration in our bath solution was already
saturating for this effect before the addition of NaCl. Most
importantly the activation of GIRK1F137S by NaCl was
achieved in a GTP-free solution, ruling out a mechanism involving
GDP-GTP exchange. Another source of free G
, which does not
require GTP, is the basal equilibrium between the G
heterotrimer and free G
GDP and G
(17):
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To scrutinize this hypothesis, we examined the effect of
Na+ on binding of G1
2 to a
GST-fused G
i3 protein, GST-G
i3 (Fig. 2A), using a pull-down assay.
G
was synthesized in vitro in reticulocyte lysate.
Only GST-G
i3-GDP, but not GST or
GST-G
i3-GTP
S, bound G
in this assay (Fig.
2B), confirming specificity and functional activity of
GST-G
i3. In three individual experiments like that shown
in Fig. 2C (a and b), we examined the
dose dependence of binding of G
to GST-G
GDP. The
binding was dose-dependent and reached saturation at about
20 µl of G
-containing lysate. The affinity of binding was lower
in high Na+ than in high K+ solution, but the
differences were too small for a statistically reliable estimation of
the affinity shift. Therefore, we compared the binding at a low
[G
] (2 µl of G
-containing lysate) and at saturating
[G
] (40 µl of lysate) (summarized in Fig. 2C, c). With 40 µl of lysate, the extent of binding in 150 mM NaCl was the same as in 150 mM KCl
(102.6 ± 8.2%, n = 10). However, at 2 µl of
lysate, the binding in the high Na+ solution was
reproducibly weaker than in high K+ (65.9 ± 4.2%,
n = 14, p < 0.001 compared with 40 µl of lysate). At doses of G
lower than 2 µl of lysate (where
a greater effect of Na+ was expected), the signal in the
autoradiograms was too weak for a reliable measurement. The ~34%
decrease in G
binding caused by Na+ at 2 µl of
lysate (which gave ~37% of maximum binding in high K+)
corresponds to a 1.9-fold change in KD. The effect of Na+ was dose-dependent (Fig. 2D,
a and b) with a Ki of 13.6 mM Na+ (Fig. 2D, c).
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The effect of Na+ on GGDP-G
interaction was further studied by SPR. Recombinant
G
1
2 was injected in buffers with
different concentrations of NaCl (substituted for KCl) across the
sensor surface with GST or GST-G
i3 immobilized via
anti-GST antibody (13). G
reveals a relatively high degree of
nonspecific binding to GST in this assay (13, 18). However, the extent
of G
binding to GST-G
i3-GDP was always greater
than to GST (Fig. 3, A and
B). Also the wash-out of G
from GST was fast as
expected for a low affinity process, whereas unbinding of G
from
GST-G
i3-GDP displayed a slower kinetic component,
characteristic for a high affinity interaction. The net specific signal
of G
-G
i3 binding, obtained by subtraction of the
GST signal (see Ref. 13), displayed rise and decay phases that were
reasonably well fitted by single exponential functions (Fig.
3C). As expected, GST-G
i3-GTP
S bound
G
much weaker than GST-G
i3-GDP (Fig.
3D). When the Na+ concentration was increased,
the net binding of G
to GST-G
i3-GDP was reduced in
a dose-dependent manner with a Ki of 8 mM Na+ (Fig. 3, E and F).
Although these data clearly show specific binding of G
to
G
i3, the contribution of nonspecific binding, particularly in the first few seconds postinjection and sample wash-out, impaired the precise determination of the kinetic constants in Na+ and K+. A limited kinetic analysis on
the subtracted traces showed that the dissociation constant of Reaction
1, KD, was increased by 10 mM
Na+ by ~2-fold (two separate sets of experiments). The
calculated 2-fold change is in very good agreement with the pull-down
results. The SPR experiments support the conclusion that
Na+ reduces the affinity of interaction between G
-GDP
and G
.
|
The findings so far support the hypothesis that the slow activation of
GIRK by Na+ is due to elevated [G] that dissociated
from G
GDP. This hypothesis not only explains the block
of the slow effect of Na+ by
ARK but also allows
testable predictions. Thus, mass law dictates that coexpression of G
should shift the equilibrium in Reaction 1 to the left, increasing
[G
] and reducing free [G
]. This should reduce the
basal activity of GIRK, but now addition of Na+ should
cause a larger relative increase in [G
] than in
control conditions. This prediction is independent of initial
conditions chosen to describe channel activation by
G
.2 However, it is
important not to overexpress G
to very high levels at which free
G
GDP can scavenge G
and blunt any G
-induced effects. We produced mild overexpression of G
i3,
3-4-fold over the resting cellular level (by injecting 5 ng of
G
i3 RNA or less), that does not hinder the activation of
GIRK by G
(10).
In agreement with the prediction, in Gi3-expressing
oocytes, slow activation of GIRK1F137S by 10 mM
Na+ was 10.3 ± 2.3-fold (n = 10),
significantly greater than without G
i3 (Fig.
4A and summarized in Fig.
4B). Slow activation by Na+ was still fully
blocked by coexpression of c
ARK (Fig. 4, A and B). Another GIRK channel mutant known to lack the direct
Na+ activation, GIRK1/GIRK2D226N (14), was also
slowly activated by 10 mM Na+ in
G
i3-expressing cells (11.6 ± 2.8-fold,
n = 9), and this effect was inhibited by c
ARK
("activation" by 1.04 ± 0.13-fold, n = 4).
The dramatic effect of G
i3 on slow
Na+-induced activation strongly supports the G
protein-dependent character of this phenomenon. The extent
of slow activation by 10 mM Na+, in the
presence of G
, is comparable to the direct effect of 40 mM Na+, although it is still severalfold
smaller than the 20-800-fold activation by saturating doses of G
(10). A simple kinetic model based on our hypothesis and on known or
estimated parameters of G
GDP-G
and G
-GIRK
interactions correctly described the slow GIRK activation by
Na+. The calculations showed that a ~1.8-fold increase in
KD of Reaction 1 by 10 mM
Na+ fully accounted for the 2-fold slow activation of
GIRK1F137S under control conditions and for an ~8-fold
activation after coexpression of G
i3.2
|
It appeared (although a systematic study has not been conducted) that
coexpression of Gi3 did not cause the expected reduction in basal activity of GIRK1F137S (e.g. compare
the cell-attached levels of channel activity in Fig. 4A,
a and b). The unexpectedly high basal activity
could reflect an increase in membrane levels of GIRK1F137S
caused by G
i3 as described for WT GIRK1/GIRK2 (10). Indeed immunostaining of the expressed channels in large plasma membrane patches (Fig. 4C) showed a net 6.4-fold increase of
GIRK1F137S protein by G
i3 (after subtraction
of background observed in uninjected oocytes) compared with channels
expressed alone.
The dose dependence of the slow Na+ effect was studied in
oocytes coexpressing GIRK1F137S and Gi3.
Maximal activation was observed at 10 mM Na+
(Fig. 4D). At higher [Na+] the effect was
smaller, consistent with the previously reported inhibition, at high
[Na+], of GIRK channel constructs lacking the fast
Na+ activation (6). The mechanism of this inhibition is
unknown. The half-maximal activation dose (EC50) appeared
to be slightly above 5 mM Na+, but considering
the interference of the inhibitory effect at high [Na+],
the actual EC50 of slow Na+ activation is
probably higher.
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DISCUSSION |
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Na+ Regulates Basal Equilibrium between Free and
GGDP-bound G
--
Our results strongly suggest
that Na+ reduces the affinity of interaction between
G
GDP and G
. This hypothesis initially arose
following the observation of slow, G
-dependent
activation by Na+ of mutant GIRK channels lacking the
direct Na+ modulation. We then demonstrated weakening of
G
GDP-G
binding using direct in vitro
binding assays. Although the detected change in affinity was small, the
results obtained by two independent methods (a pull-down assay and
surface plasmon resonance) were in good agreement. Both methods, as
well as estimates obtained from patch-clamp data, indicate that
Na+ causes a ~2-fold decrease in the affinity of
interaction between G
i3-GDP and G
. The
EC50 or Ki of the Na+ effect
estimated by electrophysiological and the two biochemical methods are
also in good agreement (~6-14 mM), falling within the
physiological range of [Na+]in. The proposed
hypothesis made it possible to explain the observed inhibition
of the slow Na+-induced activation by the G
scavenger, c
ARK, and to predict a novel physiological effect: a
dramatic enhancement of slow Na+ activation of GIRK by
coexpressed G
. Interestingly Xenopus oocytes possess an
unusually high basal level of free G
(19, 20), and coexpression
of G
may actually mimic the "usual" cellular condition of low
[G
].
Na+ has long been known to modulate the binding of agonists
to many GPCRs, uncoupling these receptors from G proteins. Initially Na+ had been suspected to regulate G proteins
directly, but later studies demonstrated a pivotal role of
Na+ interaction with a conserved aspartate residue located
in the transmembrane region of many GPCRs (for review, see Ref. 21). Finally, although NaCl has been shown to promote GDP-GTP exchange at
the G protein, the active agent was Cl rather than
Na+ (16). Thus, no direct effects of Na+ on G
proteins are known, and this report is the first demonstration of such regulation.
Two Mechanisms of Regulation of GIRK by
Na+--
Na+ regulation of GIRK channels is
considered of high physiological importance since it is believed to
determine part of their basal activity in intact cells and to underlie
the negative chronotropic effect of cardiac glycosides in the heart (2,
5). Our data imply that, in GIRK channels, Na+ acts both
directly and indirectly (via G) to modulate basal activity. The
EC50 of the well characterized, direct, G
-independent activation by Na+ is 30-40 mM. The slow,
G
-dependent activation of GIRK by Na+ has
a definitely lower EC50: maximal effect is attained at 10 mM Na+. Despite the small magnitude of
G
GDP-G
affinity change by Na+, 10 mM Na+ caused an impressive 4-10-fold
activation of GIRK. Therefore, the slow mechanism may contribute
substantially to the basal activity of the channel in intact cells
where the resting [Na+]in is 5-10
mM.
The observed features of slow Na+ modulation of GIRK
conform to the model in which [G], elevated because of the
direct effect of Na+ on G
dissociation, activates
the channel. Yet at present we cannot exclude contribution of
additional mechanisms, for instance activation of a nucleotide
diphosphate kinase (NDPK), which catalyzes the transfer of phosphate
from ATP to GTP. This might, in principle, promote G
dissociation and activate GIRK (see the discussion in Ref. 2). However,
Na+ has been reported to inhibit NDPK (22) and should have
inhibited GIRK if NDPK were involved.
Na+ as a Second Messenger or a Servo-type Intracellular Regulator-- Sodium ions are crucial for neuronal activity as carriers of depolarization and also regulate many physiological processes (fluid balance and secretion, cardiac contraction, glutamate-induced neuronal excitation, etc.) by a variety of molecular mechanisms from a direct binding of Na+ (GIRK and Na+-dependent transporters and exchangers (23)) to activation of Src by Na+ via an unknown intermediate (N-methyl-D-aspartate receptors (24)). Basal [Na+]in is tightly regulated by pumps and exchangers, being maintained in resting cells below 10 mM. Yet [Na+]in often widely fluctuates. In neurons, even relatively short periods of synaptic activity produce very large increases in [Na+]in, reaching ~30 mM in apical dendrites and >100 mM in dendritic spines (25). These considerations raise the possibility that Na+ may, in some cases, act as a second messenger that regulates intracellular targets when a substantial change in concentration occurs.
Our findings suggest a new mechanism of
Na+-dependent regulation of cellular processes:
via G. The effective range of Na+ concentrations that
regulate the G
GG
GDP + G
equilibrium is close to the resting physiological range of
[Na+]in. Therefore, regulation of G proteins
by Na+ may be a servo-type mechanism that sensitively
responds to bidirectional changes in [Na+]in
rather than to increases alone. We propose that Na+
regulation of the dynamic equilibrium between bound and free G
GDP and G
can have a substantial biological
effect by regulating a host of effectors of G
(1), some of which
are second messenger-generating enzymes that may further amplify
Na+ effects. This establishes a possible novel way of
communication between electrical activity and other cellular processes.
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ACKNOWLEDGEMENTS |
---|
We are grateful to A. G. Gilman for
encouragement and to Bernard Attali, Sagit Peleg, and Ilana Lotan for
critical reading of the manuscript. We thank the colleagues who kindly
provided the original cDNA constructs: D. Logothetis
(GIRK1F137S), E. Reuveny (cARK), M. I. Simon
(G protein subunits), E. Liman (pGEM-HE), and R. Murrell-Lagnado
(GIRK1D226N).
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM56260 (to N. D.), GM60419 (to C. D.), and GM60019 (to V. S.) and grants from the United States-Israel Binational Science Foundation (to N. D., V. S., and C. D.).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.
¶ To whom correspondence should be addressed. Tel.: 972-3-6405743; Fax: 972-3-6409113; E-mail: dascaln@post.tau.ac.il.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.C200605200
2 D. Yakubovich, I. Rishal, and N. Dascal, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
GIRK, G
protein-gated K+ channel;
cARK, myristoylated C-terminal
part of
-adrenergic receptor kinase;
[G
], free cellular
concentration of G
;
GPCR, G protein-coupled receptor;
WT, wild-type;
GST, glutathione S-transferase;
GTP
S, guanosine 5'-3-O-(thio)triphosphate;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
NDPK, nucleotide diphosphate kinase.
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