Activation of Go-proteins by Membrane Depolarization
Traced by in Situ Photoaffinity Labeling of
G
o-proteins with
[
32P]GTP-azidoanilide*
Yosef
Anis
,
Bernd
Nürnberg§,
Leonid
Visochek
,
Nachum
Reiss¶,
Zvi
Naor¶, and
Malka
Cohen-Armon
From the
Department of Physiology and The Cardiac
Research Institute, Sackler School of Medicine and ¶ Department of
Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv
University, 69978 Tel-Aviv, Israel and § Institut fur
Pharmakologie, Freie Universitat Berlin, Thielallee 67-73, D-14195 Berlin, Federal Republic of Germany
 |
ABSTRACT |
Evidence for depolarization-induced activation of
G-proteins in membranes of rat brain synaptoneurosomes has been
previously reported (Cohen-Armon, M., and Sokolovsky, M. (1991)
J. Biol. Chem. 266, 2595-2605; Cohen-Armon, M., and
Sokolovsky, M. (1993) J. Biol. Chem. 268, 9824-9838).
In the present work we identify the activated G-proteins as
Go-proteins by tracing their depolarization-induced in situ photoaffinity labeling with
[
32P]GTP-azidoanilide (GTPAA). Labeled GTPAA was
introduced into transiently permeabilized rat brain-stem
synaptoneurosomes. The resealed synaptoneurosomes, while being
UV-irradiated, were depolarized. Relative to synaptoneurosomes at
resting potential, the covalent binding of [
32P]GTPAA
to G
o1- and G
o3-proteins, but not to
G
o2- isoforms, was enhanced by 5- to 7-fold in
depolarized synaptoneurosomes, thereby implying an accelerated exchange
of GDP for [
32P]GTPAA. Their depolarization-induced
photoaffinity labeling was independent of stimulation of
Go-protein-coupled receptors and could be reversed by
membrane repolarization, thus excluding induction by transmitters
release. It was, however, dependent on depolarization-induced activation of the voltage-gated sodium channels (VGSC), regardless of
Na+ current. The
subunit of VGSC was cross-linked and
co-immunoprecipitated with G
o-proteins in depolarized
brain-stem and cortical synaptoneurosomes. VGSC
subunit most
efficiently cross-linked with guanosine
5'-O-2-thiodiphosphate-bound rather than to guanosine
5'-O-(3-thiotriphosphate)-bound G
o-proteins in isolated synaptoneurosomal membranes. These findings support a
possible involvement of VGSC in depolarization-induced activation of
Go-proteins.
 |
INTRODUCTION |
GTP-binding trimeric proteins have been implicated in signal
transduction from receptors in the cell membrane to intracellular effectors and ion channels in a variety of cells (1-5). The mechanism involves signal-induced G-protein activation initiated by an exchange of GDP for GTP on the
subunit of the protein (5-7). Subsequent GTPase activity of the G
subunit converts the activated G-proteins into their inactive, GDP-bound state (7). Activation of G-proteins has been induced experimentally by stimulation of G-protein-coupled receptors in the cell membrane (2, 3, 8, 9). Evidence indicating
activation of G-proteins in response to membrane depolarization were
previously observed in brain stem synaptoneurosomes (10-12).
G
o-proteins are widely distributed in the central
nervous system (15-17). Three subtypes showing marked homology but
exhibiting functional differences have been identified (13, 14, 18). The G
o1 subtype appears to be involved in the coupling
of muscarinic receptors to Ca+2 channels, and the
G
o2 subtype mediates inhibition of Ca+2
current activated by somatostatin receptors (19). The function of the
G
o3 subtype is not clear (14). Phospholipase C
activation mediated by activation of Go-proteins has been
demonstrated in the cell-free system (20), and
Go-protein-mediated activation of protein kinase C has been
observed in Chinese hamster ovary (CHO) cells (21).
In the present study, in situ photoaffinity labeling with
[
32P]GTPAA1
(22, 23) indicated a depolarization-induced accelerated exchange of GDP
for [
32P]GTPAA in G
o1- and
G
o3-proteins, implying a depolarization-induced activation of these Go-proteins.
[
32P]-GTPAA was introduced into transiently
permeabilized synaptoneurosomes as described before (10). Unlike the
endogenously bound guanine nucleotides, [
32P]GTPAA,
covalently bound to G
-proteins by photoaffinity labeling, was not
displaced during SDS-polyacrylamide gel electrophoresis, providing a
possible tool for identification of in situ activated G-proteins.
In view of findings indicating a reciprocal influence of
depolarization-induced activation of VGSC and uncoupling of G-proteins from muscarinic receptors (12, 24, 25), we examined the possibility
that VGSC can be involved in depolarization-induced activation of
G
o-proteins. Our results indicated that
depolarization-induced activation of Go-proteins can be
prevented by preventing VGSC activation. In addition, in depolarized
brain-stem and cortical synaptoneurosomes, the
subunit of VGSC
cross-linked most efficiently with G
o-proteins. In
isolated synaptoneurosomal membranes, VGSC-
subunit cross-linked
most efficiently with GDP
S-bound rather than GTP
S-bound
G
o-proteins. These findings suggest repeated interactions between VGSC-
subunit and G
o-proteins as
long as membrane depolarization lasts.
 |
MATERIALS AND METHODS |
Reagents--
ATP (grade I), GDP
S, GTP
S, tetrodotoxin,
dithiothreitol, carbamylcholine, atropine, serotonin, spiperone,
propranolol, naloxone, and yohimbine were all purchased from Sigma.
4-Azidoaniline hydrochloride was supplied by Fluka (Switzerland).
D-2-amino-5-phosphovaleric acid was purchased from
Cambridge Research Biochemicals. DPI-205-429 (DPI R
enantiomer; see Footnote 1) was kindly supplied by Dr. E. Rissi and Dr.
D. Romer of Sandoz Ltd., Pharmacological Division, Preclinical Research
(Basel, Switzerland).
N,N'-1,4-Phenylenedimaleimide (PDM) was purchased
from Aldrich. Pertussis toxin (PTX) and the A-protomer of PTX, cholera
toxin (CTX), and the A-protomer of CTX were purchased from List
Biological Laboratories, Campbell, CA. Guanosine-5'-triphosphate
tetra(triethylammonium) salt, [
32P]GTP) (800 Ci/mmol),
and antibodies against peptide derived from the amino-terminal domain
of G
o-proteins (CG/2) were purchased from NEN Life
Science Products. [3H]Acetyl-choline
([3H]AcCh) (96 Ci/mmol, 98% pure) and
[phenyl-3H]tetraphenylphosphonium bromide
([3H]TPP+) (33.6 Ci/mmol) were purchased from
Amersham Pharmacia Biotech. Phosphorus-[32P]H3PO4 (400-800
mCi/ml) was purchased from ICN Pharmaceuticals. [3H]N-Methyl-4-piperidylbenzilate was prepared
by catalytic tritium exchange as described in Cohen-Armon et
al. (26). For detection of G
o-isoforms, three
polyclonal peptide antibodies from rabbit (AS 6, AS 201, and AS 248)
were generated as described elsewhere (14, 19). AS 6, which detects all
known G
o-isoforms, is directed against amino acids 22 to
35 of all three G
o isoforms. AS 248, which recognizes
G
o1 and the closely related isoform G
o3
(14), was directed against amino acids 310 to 323 of
G
o1. AS 201, which reacts exclusively with rodent
G
o2 (14, 18), was directed against amino acids 293 to
308 of G
o2. Antibodies directed against the
carboxyl-terminal decapeptide of G
i-proteins were kindly supplied by Professor G. Milligan, Glasgow University, UK. Antibodies detecting four splice variants of G
s-proteins in
mammalian brain AS 348 (27) have been used. Polyclonal antibodies
raised against peptide correlating to residues 1491-1508 of
subunit of rat type I VGSC (28) (SP19) were purchased by Alomone
Laboratories, Jerusalem, Israel.
Preparation of Synaptoneurosomes--
Adult male rats of the CD
strain were obtained from Levenstein's Farm, Yokneam, Israel, and
maintained as described before (26). Synaptoneurosomes were prepared
from pooled brain stems obtained from 2- to 3-month-old rats according
to Hollingsworth et al. (29). Synaptoneurosomes consist of a
presynaptic sac (synaptosome) attached to a resealed postsynaptic sac
(neurosome) and contain the original content of cytoplasm (29). The
synaptoneurosomes were resuspended in modified Krebs-Henseleit buffer
(118.5 mM NaCl, 4.7 mM KCl, 1.18 mM
MgCl2, 24.9 mM NaHCO3, 10 mM glucose, and 1.18 mM
KH2PO4, [Ca+2] < 10 µM, pH 7.4) in an atmosphere of 95% O2, 5%
CO2 at 25 °C and pelleted (1000 × g).
Under these conditions, membrane resting potential was not
significantly changed within 4 h, as indicated by
[3H]TPP+ accumulation (10).
Preparation of Membranes--
Brain stems were dissected and
homogenized in ice-cold hypotonic-buffered solution (50 mM
Tris-HCl, pH 7.4) containing the following protease inhibitors: 5 units/ml aprotinin, 5 µg/ml pepstatin A, and 0.1 mM
phenylmethylsulfonyl fluoride. The homogenates were centrifuged at
1000 × g for 20 min. Supernatants were recentrifuged at 30,000 × g for 20 min, and the pellet was
resuspended in the modified Krebs-Henseleit buffer described above.
Preparation of
[
32P]GTPAA--
[
32P]GTPAA was
synthesized by incubation of [
32P]GTP with
azidoaniline and purified by ion-pairing chromatography according to
Pfeuffer (22). The 4-azidoaniline hydrochloride was converted to the
free base. Approximately 1 mCi of [
32P]GTP was
evaporated to dryness and mixed with unlabeled GTP to obtain 200 Ci/mmol and with 4-azidoanilide (free base) in peroxide-free dioxane.
After 4 h at room temperature in the dark, the mixture was loaded
on a DEAE-cellulose column (DE52, Whatman) and eluted with 50-450
mM triethylammonium-HCO3/10% EtOH in a dark
cold room. The last large labeled peak is the
[
32P]GTPAA.
In Situ Photoaffinity Labeling of G
-proteins with
[
32P]GTPAA in Transiently Permeabilized
Synaptoneurosomes--
Synaptoneurosomes were permeabilized in the
presence of 6 mM ATP in low ionic strength buffered
solution, pH 8.3, in the absence of Ca2+ and
Mg2+ ions (10). Permeabilization is apparently achieved
under these conditions because of the chelator characteristics of
ATP
4 (30, 31). At low ionic strength (10), chelation of
Ca+2 ions bound to the cell membrane disrupts its
structure, thereby causing a nonspecific membrane permeabilization
(32). Before being lyzed (10), synaptoneurosomes were washed and
resuspended in Krebs-Henseleit (pH 7.4) containing the normal
concentrations of Ca+2 and Mg+2. This restored
membrane selective permeability and the resting potential, as indicated
by measuring the accumulation of [3H]TPP+ in
resealed synaptoneurosomes, resuspended in Krebs-Henseleit containing
various [K+] (10). Thus, a transient permeabilization was
achieved in synaptoneurosomes incubated with ATP (6 mM) in
isotonic buffer containing 169 mM glucose, 40 mM NaCl, 4.7 mM KCl, 1.18 mM
KH2PO4, 24.9 mM NaHCO3, 0.5 mM succinate, with the pH adjusted to 8.3-8.4 at
25 °C, as described before (10). [
32P]GTPAA (0.2 µM) was added to this solution. After precisely 40 min,
the permeabilized synaptoneurosomes were resealed by two successive
cycles of incubation (10 min) with Krebs-Henseleit containing 2.5 mM Ca+2 and 1.18 mM
Mg+2 followed by centrifugation (1000 × g,
5 min) (10). Membrane permeabilization and resealing were confirmed by
measuring the accumulation of [3H]TPP+ in
each step (10, 33). The resealed synaptoneurosomes were then
resuspended in Ca+2-free ([Ca+2] < 10 µM) modified Krebs-Henseleit buffer containing either 4.7 or 50 mM [K+], inducing either resting
potential or membrane depolarization, respectively. Samples of the
resealed synaptoneurosomes, containing approximately 100 µg of
protein, were UV-irradiated (300 nm, 350 W) for 5 min on ice.
GTP-binding proteins were photolabeled by [
32P]GTPAA.
For estimation of the nonspecific binding of
[
32P]GTPAA, synaptoneurosomes were permeabilized in
the presence of [
32P]GTPAA and GTP
S (200 µM). The nonspecific photoaffinity labeling of membrane
proteins with [
32P]GTPAA was negligible. For
estimation of the extracellular binding of [
32P]GTPAA
to synaptoneurosomal membranes, samples of nonpermeabilized synaptoneurosomes were subjected to photoaffinity labeling under the
above conditions. Photoaffinity labeling of membrane proteins with
[
32P]GTPAA in nonpermeabilized synaptoneurosomes was
negligible. Membrane proteins were separated by SDS-PAGE and
autoradiographed. [32P]GTPAA-labeling of proteins was
quantified by densitometry using a laser densitometer (LKB Bromma Ultrascan).
Estimation of Membrane Potential in Terms of
[3H]TPP+ Accumulation--
Because
[3H]TPP+ permeates freely across cell
membranes (33), it follows that at equilibrium the transmembrane
concentration gradient of [3H]TPP+ is
proportional to membrane potential, according to the Nernst equation
(33). Changes in membrane potential were therefore estimated by
measuring the accumulation of [3H]TPP+ in the
synaptoneurosomes according to the method of Cheng et al.
(33). Synaptoneurosomes (approximately 4 mg of protein/ml in 40-µl
samples) were incubated with approximately 7 × 10
8
M [3H]TPP+ at 25 °C for 20 min. The samples were then passed through Whatman GF/C filters, and the
filters were counted for tritium using a scintillation mixture
(Hydroluma), as described in detail previously (10).
PTX-catalyzed ADP-ribosylation of G
-proteins in Brain-stem
Synaptoneurosomes--
Synaptoneurosomes in Ca+2-free
Krebs-Henseleit buffer, pH 7.4, were incubated with 200 ng/ml PTX for
2 h at 37 °C and 95% O2, 5% CO2 as
described before (11).
Binding of [3H]AcCh to Muscarinic Receptors in
Resealed Synaptoneurosomal--
To determine the effect of membrane
depolarization on the binding of [3H]AcCh to muscarinic
receptors, samples of synaptoneurosomes were incubated for 30 min in
Krebs-Henseleit buffer containing either 4.7 or 50 mM
[K+] (resting potential or depolarization, respectively)
in the presence of an inhibitor of acetylcholinesterase (34) and
various concentrations of [3H]AcCh. Na+ was
exchanged for K+ in the high [K+] buffer.
Under these experimental conditions the specific binding of
[3H]AcCh to high affinity muscarinic receptors can be
measured (34). Nonspecific [3H]AcCh binding was measured
in the presence of 1 µM atropine. The binding assay has
been described in detail previously (10, 25, 34). To prevent
interference by AcCh release, [3H]AcCh was added to
synaptoneurosomes preincubated for 30 min at room temperature in the
absence of acetylcholinesterase inhibitors (34). [3H]AcCh
binding to muscarinic receptors was measured in Ca+2-free
Krebs-Henseleit buffer (described above), with a
[Ca+2]:[Mg+2] ratio of 1:100 (35).
Two-dimensional Gel Electrophoresis--
Samples containing 200 µg of protein were applied to isoelectric focusing gels (1st
dimension) containing 1.8% ampholines at pH 5-8 and 0.2% ampholines
at pH 3-10. Isoelectric focusing followed by SDS-PAGE (2nd dimension)
was performed according to Ferro-Luzzi and Nikaido (36). In the 2nd
dimension, gels containing the electrofocused proteins were applied on
8 or 10% polyacrylamide slab gels (37), subjected to SDS-PAGE at room
temperature, and electroblotted.
Immunolabeling--
Proteins in the SDS-gels were electroblotted
(Western blots) onto nitrocellulose paper overnight at 10 °C and
with a constant current of 150 mA, as described by Towbin et
al (38). Nitrocellulose strips were exposed to specific antibodies
detecting amino-terminal domains of G
o-proteins
(residues 22-35 (AS 6) (13, 14), and residues 2-16 (GC/2) (39)) and
to antibodies detecting carboxyl-terminal domains of
G
i1-proteins, G
i2-proteins (AS/7) (40),
and G
s-proteins (As 348) (27). In addition, polyclonal
antibodies against carboxyl-terminal domains of G
o1 and
G
o2 subtypes were used (AS 201 and AS 248, respectively)
(14). Protein bands with bound antibodies were labeled by binding of
peroxidase-conjugated second antibody (38, 41).
Cross-linking of Membrane Proteins by PDM (4)--
The permeable
cross-linker PDM (20-30 µM) was added to
synaptoneurosomes at resting potential and during high
[K+]-induced membrane depolarization. PDM dissolved in
N,N'-dimethylformamide (Merck) was added to
synaptoneurosomes incubated in Ca2+-free Krebs-Henseleit
containing 4.7 or 50 mM [K+]. Cross-linking
was stopped by the addition of 8 mM 2-mercaptoethanol (4).
Immunoprecipitation of the
-Subunit of VGSC by
anti-G
o (AS 6)--
Membranes were solubilized with 4%
(w/v) SDS for 10 min at room temperature, and then buffer was added
containing 1% (w/v) Nonidet P-40, 1% (w/v) desoxycholate, 150 mM NaCl, 1 mM dithiotreitol, 1 mM
EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 units/ml
aprotinin,10 mM Tris-Cl (pH 7.4). Solubilized membranes
were immunoreacted with antisera against G
o-proteins (AS
6) (2 h, 4 °C) and then incubated overnight at 4 °C with protein
A-Sepharose beads (12.5% (w/v)). The Sepharose beads were then
pelleted and washed with high ionic strength solution, thereby
eliminating nonspecifically bound proteins. The immunoprecipitated
proteins bound to the A-Sepharose beads were eluted in SDS-PAGE sample
buffer, boiled for 1 min, then separated on SDS-PAGE (5% acrylamide)
and electroblotted (Western blot). The immunoprecipitated proteins
immunoreacted with antibodies against epitope common to all
subunits of VGSC situated in the amino terminus (28). This epitope
(SP19) was used as a negative control peptide in the immunolabeling of
VGSC
subunit. Concomitantly, the immunoprecipitated product
immunoreacted with antibodies directed against the carboxyl terminus of
G
o-proteins.
In Situ [32P]-labeled Phosphorylation of Membrane
Proteins in Synaptoneurosomes at Resting Potential and during Membrane
Depolarization--
Synaptoneurosomes were incubated for 30 min at
room temperature in Krebs-Henseleit buffer in the absence of
Ca+2 and in the presence of [32P]phosphorus
(10 µCi). Samples of the labeled synaptoneurosomes were resuspended
in Krebs-Henseleit buffer containing 4.7 or 50 mM KCl.
Membrane potential of unlabeled synaptoneurosomes in 4.7 and 50 mM [K+]-Krebs Henseleit was monitored by
[3H]TPP+ accumulation. After incubation for
5-10 min at room temperature, [32P]-labeled membrane
proteins in the synaptoneurosomes were denatured, separated by
SDS-PAGE, and blotted (Western blot). Protein blots were
autoradiographed and subjected to immunolabeling.
 |
RESULTS |
Depolarization-induced Photoaffinity Labeling of
G
o-proteins with
[
32P]GTPAA--
[
32P]GTPAA was
introduced into transiently permeabilized synaptoneurosomes (see
"Materials and Methods"). The "loaded" synaptoneurosomes were
UV-irradiated at resting potential or during membrane depolarization in
Ca+2-free Krebs-Henseleit buffer. Photoaffinity-labeled
proteins were separated by two-dimensional SDS-gel electrophoresis and electroblotted.
[
32P]GTPAA-labeled G
o-proteins were
immunoidentified in membranes of synaptoneurosomes UV-irradiated at
either resting potential or during membrane depolarization (5-10
min). Their photoaffinity labeling in depolarized
synaptoneurosomes was 5- to 7-fold higher than that measured in
unstimulated synaptoneurosomes (Fig. 1). The intensity of 32P-labeling was quantified by
densitometry (Fig. 1E). Depolarization-induced enhancement
in photoaffinity labeling of G
o-proteins was not observed in synaptoneurosomes permeabilized in the presence of [
32P]GTPAA and GDP
S (100 µM),
i.e. under conditions preventing exchange of GDP for GTP in
G
-proteins (1-3). The isoelectric pH of the photolabeled
G
o-proteins in depolarized synaptoneurosomes was shifted
by approximately 0.5 pH unit toward a more acidic pH relative to that
in synaptoneurosomes at resting potential (Fig. 1). These results are
consistent with an accelerated exchange of GDP for [
32P]GTPAA in G
o-proteins (5, 23) in
depolarized synaptoneurosomes.

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Fig. 1.
Effect of membrane depolarization on the
specific in situ photoaffinity labeling of
G o-proteins with
[ 32P]GTPAA in membranes prepared
from transiently permeabilized synaptoneurosomes.
[ 32P]GTPAA was introduced into permeabilized
synaptoneurosomes. Membrane proteins were separated by two-dimensional
gel electrophoresis (pH 4-8 in the 1st dimension, 10% polyacrylamide
in the second dimension). Autoradiograms of photoaffinity-labeled
proteins in synaptoneurosomes exposed to 50 mM
[K+]-Krebs-Henseleit buffer (depolarization)
(A) or 4.7 mM [K+] (resting
potential) (C) are presented. Western-blotted
[ 32P]GTPAA-labeled proteins were immunolabeled by
antibodies directed against the amino-terminal domain in
G o-proteins (GC/2). Immunolabeled
G o-proteins in membranes isolated from depolarized
synaptoneurosomes (B) and from synaptoneurosomes at resting
potential (D) were detected. The 32P-labeling of
autoradiographed G o-proteins was quantified by
densitometry (E). The upper and lower
traces present changes in the relative optical density produced by
autoradiographed labeled G o-proteins in A and
C, respectively. Data are from a typical experiment, one of
five performed. Each sample contained 200 µg of protein.
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A further identification of the photolabeled G
o-proteins
has been enabled by immunolabeling with antibodies directed against the
carboxyl-terminal domains of two G
o-protein subtypes:
G
o1- and its isoform, G
o3 subtype, the
most abundant G
-proteins in the brain, and
G
o2-protein (see "Materials and Methods").
Immunolabeling indicated a depolarization-induced shift in the
isoelectric pH of G
o1- and G
o3-proteins
toward a more acidic pH (Fig. 2), which could result from a depolarization-induced exchange of GDP for [
32P]GTPAA. The isoelectric pH of
G
o2-proteins was not altered (Fig. 2).

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Fig. 2.
Effects of high
[K+]-induced membrane depolarization on
G o1-,
G o3-, and
G o2-proteins. Transiently
permeabilized synaptoneurosomes were loaded with
[ 32P]GTPAA and UV-irradiated. Membrane proteins were
separated by two-dimensional gel electrophoresis and electroblotted
(Western blots). The blotted proteins were labeled by Ponceau and
exposed to antibodies directed against the carboxyl-terminal domains of
G o1- and G o2-proteins (AS 248 and AS 201, respectively). Immunolabeled G o-protein subtypes in
membranes prepared from synaptoneurosomes at resting potential
(A and C) and from depolarized synaptoneurosomes
(B and D) are presented. Each sample contained
200 µg of protein.
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Photoaffinity-labeled G
-proteins were also immunolabeled with
antibodies directed against a common carboxyl-terminal domain of
G
i1- and G
i2-proteins (see "Materials
and Methods"). Under the experimental conditions employed, the
isoelectric pH of these G
i-protein subtypes was not
significantly altered by membrane depolarization (Fig.
3).

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Fig. 3.
Effect of membrane depolarization on the
isoelectric pH of
G i-proteins. Proteins in
transiently permeabilized synaptoneurosomes were exposed to
photoaffinity labeling with [ 32P]GTPAA. Proteins in
membranes prepared from resting potential and depolarized
synaptoneurosomes were separated by two-dimensional gel electrophoresis
(see Fig. 1) and electroblotted (Western blot). Blotted proteins
(40-41 kDa) that were immunolabeled by antibodies directed against a
common carboxyl-terminal domain in G i1- and
G i2-proteins (AS/7) are presented (n = 3). Each sample contained 200 µg of protein.
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|
Depolarization-induced Activation of Go-Proteins Was
Not Mediated by Stimulation of G-protein-coupled Receptors--
The
enhanced photoaffinity labeling of G
o-proteins with
[
32P]GTPAA in depolarized synaptoneurosomes may
suggest that, apart from being activated by stimulation of
Go-protein-coupled receptors, Go-proteins were
activated by membrane depolarization. However, this effect could
conceivably be attributable to a depolarization-induced release of
transmitters (42) evoking receptor stimulation as well.
To exclude a possible effect of transmitters released by membrane
depolarization on the exchange of GDP for GTPAA in
G
o-proteins, all experiments were conducted under
conditions preventing transmitter release (see "Materials and
Methods"). Moreover, depolarization-induced activation of
Go-proteins was examined in synaptoneurosomes treated with
antagonists of Go-proteins-coupled and abundantly spread receptors in the central nervous system. Resealed synaptoneurosomes preloaded with [
32P]GTPAA were depolarized and
photolabeled in the presence of antagonists of muscarinic,
NMDA-glutamate, dopaminergic, serotonergic, or adrenergic receptors in
concentrations that inhibit 90% of agonist binding to these receptors.
However, the increase in the photoaffinity labeling of
G
o-proteins with [
32P]GTPAA in the
depolarized synaptoneurosomes was preserved despite their treatment
with antagonists (Fig. 4).

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Fig. 4.
A, photoaffinity labeling of
G o-proteins with [ 32P]GTPAA in
transiently permeabilized synaptoneurosomes at resting potential
(lane 1) and during membrane depolarization (lane
2), in the presence of antagonists of muscarinic and adrenergic
receptors (atropine (1 µM), propranolol (1 µM), and yohimbine (1 µM)), at resting
potential (lane 3) and during membrane depolarization
(lane 4), and in the presence of antagonists of the
dopaminergic, serotonergic, and NMDA-glutamate receptors (naloxone (10 µM), spiperone (1 µM) and
D-2-amino-5-phosphovaleric acid(100 µM)) at
resting potential (lane 5) and during depolarization
(lane 6). Membrane proteins were separated by SDS-PAGE (10%
acrylamide), blotted (Western blot), and autoradiographed.
B, blotted proteins photoaffinity-labeled with
[ 32P]GTPAA were immunolabeled by antibodies directed
against the amino-terminal domain of G o-proteins (GC/2).
Each lane contained 200 µg of protein. Data are from a
typical experiment, one of five performed.
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Furthermore, the possible effect of transmitters on photoaffinity
labeling of G
o-proteins with [
32P]GTPAA
was examined in isolated synaptoneurosomal membranes rather than in
synaptoneurosomes, thereby elliminating possible effects induced by
changes in membrane potential. Photoaffinity-labeled proteins in these
preparations were specifically immunolabeled by
anti-G
o-antibodies (AS 6) (Fig.
5, A and B). In
isolated membranes the isoelectric pH of photoaffinity-labeled
G
o-proteins shifted toward a more acidic pH as compared
with that of photoaffinity-labeled G
o-proteins in
synaptoneurosomes at resting potential (compare Figs. 1 and 2 with Fig.
5A). In comparison to the photoaffinity labeling of
G
o-proteins in control membranes, a modest additional increase in their labeling with [
32P]GTPAA and a
slight additional shift in their isoelectric pH were observed in
membranes treated with agonists of muscarinic or serotonergic
receptors (Fig. 5B). Photoaffinity labeling of G
o-proteins was similarly mildly enhanced by glutamate
or melatonin (not shown). The [32P]photoaffinity labeling
of G
o-proteins was quantified by densitometry (Fig.
5C).

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Fig. 5.
A, immunolabeling of
G o-proteins with antibodies directed against a common
domain of G o-proteins (AS 6). Proteins in isolated
membranes were subjected to photoaffinity labeling with
[ 32P]GTPAA and were separated by two-dimensional
SDS-PAGE (pH 4-8 in the 1st dimension, 10% acrylamide in the second
dimension) and electroblotted (Western blots). Blotted proteins labeled
with Ponceau and G o-proteins immunolabeled by AS 6 are
presented. B, autoradiograms of the Western-blotted
G o-proteins, photoaffinity labeled with
[ 32P]GTPAA. Isolated synaptoneurosomal membranes were
incubated with 10 µM GTP/GDP and 1 µCi of
[ 32P]GTPAA in the absence (control) or in the presence
of carbamylcholine (100 µM) or serotonin (100 µM). Photoaffinity labeling of G o-proteins
with [ 32P]GTPAA in these membrane preparations was
quantified by densitometry (C). Each sample contained 200 µg protein (n = 3).
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The substantial increase in photoaffinity labeling of
G
o-proteins during membrane depolarization, as compared
with the slight effect of receptors stimulation on their photoaffinity
labeling (compare Figs. 1E and 5C), further
supports the assumption that depolarization-induced activation of
Go-proteins is not mediated by receptors stimulation.
Effect of Depolarization on PTX-catalyzed ADP-ribosylation of
G
o-proteins--
Because G
o-proteins are
better substrates for PTX-catalyzed ADP-ribosylation when coupled to
G
(43), their ADP-ribosylation is inhibited by activation of
Go-proteins (1, 5). PTX-catalyzed ADP-ribosylation of
G
o-proteins in depolarized synaptoneurosomes should
therefore be inhibited, whereas Go-proteins are activated by membrane depolarization. In contrast, G
s-proteins are
better substrates for CTX-catalyzed ADP-ribosylation when GDP is
replaced by GTP, i.e. under conditions activating
Gs-proteins (44). A possible activation of
Gs-proteins by membrane depolarization would therefore
accelerate their ADP-ribosylation.
We compared the effect of membrane depolarization in
[Ca+2]-free Krebs-Henseleit buffer on ADP-ribosylation of
G
o-proteins in situ with its effect on
ADP-ribosylation of G
s-proteins by comparing their
complementary [32P]ADP-ribosylation carried in membranes
prepared from PTX- or CTX-pretreated synaptoneurosomes.
[32P]ADP-ribosylated G
o-proteins were
immunoprecipitated by antibody AS 6 (13, 14).
[32P]ADP-ribosylated G
s-proteins were
immunolabeled with antibody AS348 (27). (G
s-proteins are
present as four splice variants appearing as two bands, each including
G
s isoforms differing in only one amino acid (27).
Membrane depolarization inhibited PTX-catalyzed ADP-ribosylation of
G
o-proteins (Fig.
6A). This is in accordance
with a depolarization-induced activation of Go-proteins (43). In contrast, CTX-catalyzed ADP-ribosylation of
G
s-proteins was not affected by membrane depolarization
(Fig. 6B).

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Fig. 6.
Effect of membrane depolarization on the
ADP-ribosylation of G o- and
G s-proteins. A,
synaptoneurosomes were pretreated in the presence of atropine (1 µM) with PTX (200 ng/ml, 37 °C, 2 h, 95%
O2, 5%CO2) (lanes 1 and
2) during depolarization (lane 1) and at resting
potential (lane 2). PTX-catalyzed
[32P]ADP-ribosylation of G o-proteins was
subsequently performed in membranes isolated from the PTX-pretreated
synaptoneurosomes (lanes 1 and 2) and from
control synaptoneurosomes preincubated without PTX during
depolarization (lane 3) and at resting potential (lane
4). [32P]ADP-ribosylated G o-proteins
were immunoprecipitated by antibodies AS 6, subjected to SDS-PAGE, and
blotted (Western blot). Their autoradiograms are presented
in the upper frame. Immunolabeling of the immunoprecipitated
G o-proteins is presented in the lower frame.
G o1-proteins were immunodetected in the
immunoprecipitates by antibody AS 248. The extent of
[32P]ADP-labeling of G o-proteins was
quantified by densitometry (n > 5). B,
synaptoneurosomes were pretreated in the presence of propranolol (1 µM) with CTX (10 µg/ml, 37 °C, 2 h,
95%O2/5%CO2) during depolarization
(lane 1) or at resting potential (lane 2).
[32P]ADP-ribosylation of G s-proteins was
subsequently performed in CTX-pretreated (lanes 1 and 2) and
in untreated synaptoneurosomes (lane 3, depolarized during
preincubation; lanes 4 and 5, preincubated at
resting potential. In lane 4, synaptoneurosomes were
pretreated with CTX in the presence of isoproterenol (10 µM) in the absence of propranolol).
[32P]ADP-ribosylation of G s-proteins was
catalyzed by CTX-A protomer in the presence of Gpp(NH)p (100 µM). Membrane proteins were separated by SDS-PAGE and
electroblotted (Western blot). G s-proteins were
immunolabeled by antibodies AS 348. Autoradiograms of the
[32P]ADP-ribosylated proteins are presented in the
upper frame. Immunolabeling of
[32P]ADP-labeled G s-proteins is presented
in the lower frame. The extent of [32P]ADP
labeling was quantified by densitometry (O.D., absorbance)
(n = 3).
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Blocking the activation of VGSC Prevented the
Depolarization-induced Photoaffinity Labeling of
G
o-proteins with
[
32P]GTPAA--
Depolarization-induced photoaffinity
labeling of G
o-proteins with [
32P]GTPAA
was prevented by the R enantiomer of the cardiotonic and antiarrhythmic drug DPI 201-106 (10 µM), which prevents
activation of VGSC (45) (Fig. 7).
[
32P]GTPAA was introduced into transiently
permeabilized control synaptoneurosomes and synaptoneurosomes treated
with the R enantiomer of DPI. After resealing,
synaptoneurosomes were UV-irradiated at either resting potential or
depolarization in Ca+2-free buffered Krebs-Henseleit.
Photolabeled membrane proteins were separated by two-dimensional
SDS-PAGE and electroblotted. [
32P]GTPAA-photolabeled
membrane proteins were immunolabeled with common anti
G
o-proteins antibodies (GC/2) (Figs. 7, A and
B). The enhanced photoaffinity labeling of
G
o-proteins with [
32P]GTPAA in
depolarized synaptoneurosomes, as well as the shift in their
isoelectric pH (see Figs. 1 and 2), were eliminated following pretreatment of the synaptoneurosomes with the R-enantiomer
of DPI, i.e. when activation of VGSC had been prevented
(Figs. 7, A and B). In contrast, the neurotoxin
batrachotoxin (46) and the S-enantiomer of DPI (45), both of
which prolong the activation of VGSC (45-47) although they share a
common binding site with DPI R-enantiomer on the
subunit
of VGSC (45), did not interfere with the depolarization-induced
photoaffinity labeling of G
o-proteins or with the
coupling of G-proteins to muscarinic receptors (12). These findings may
suggest that voltage-induced activation of VGSC is essential for
depolarization-induced activation of Go-proteins. Blocking
of the voltage-dependent Na+ current by
tetrodotoxin (48) did not affect the depolarization-induced photoaffinity labeling of G
o-proteins, ruling out a
possible involvement of Na+-current in the activation of
Go-proteins. This is in consistence with previous findings
(10, 12).

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Fig. 7.
The effect of agent preventing VGSC
activation on the depolarization-induced photoaffinity labeling of
G o-proteins with
[32P]GTPAA. A, effect of membrane
depolarization induced by 50 mM [K+] on the
isoelectric pH of G o-proteins in control
synaptoneurosomes (lower frame) and in synaptoneurosomes
treated with DPI R enantiomer (10 µM)
(upper frame). Membrane proteins in transiently
permeabilized and depolarized synaptoneurosomes were subjected to
photoaffinity labeling with [ 32P]GTPAA, separated by
two-dimensional gel electrophoresis, and electroblotted (Western
blots). The migration of proteins immunolabeled by antibodies directed
against the amino-terminal domain of G o-proteins (GC/2)
is presented. Each sample contained 200 µg of protein
(n = 3). B, effect of membrane
depolarization induced by high [K+] (50 mM)
on the photoaffinity labeling of G o-proteins with
[ 32P]GTPAA in transiently permeabilized
synaptoneurosomes pretreated with DPI R enantiomer (10 µg). The migration of autoradiographed G o-proteins in
two-dimensional gel electrophoresis is presented. Data are from a
typical experiment, one of three performed. Each sample contained 200 µg of protein (n = 3). C, effect of DPI
R enantiomer (10 µM) on the specific binding
of [3H]AcCh to muscarinic receptors in their high
affinity state at resting potential (filled
symbols) and during membrane depolarization (open
symbols) in transiently permeabilized synaptoneurosomes.
Nonspecific binding of [3H]AcCh was measured in the
presence of atropine (1 µM) (dashed line).
Data are the mean of values measured in five experiments. D,
specific binding of the muscarinic antagonist
[3H]N-methyl-4-piperidylbenzilate
([lsqb]3H]NMPB) to muscarinic
receptors in transiently permeabilized synaptoneurosomal membranes
(control ( ) or treated with DPI R enantiomer ( )).
Nonspecific binding of [lsqb]3H]NMPB
was measured in the presence of 1 µM atropine
(dashed line). Data are the mean of values measured in five
experiments.
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|
The effect of DPI R enantiomer on photoaffinity labeling of
G
o-proteins with [
32P]GTPAA in
depolarized synaptoneurosomes (Figs. 7, A and B)
was accompanied by prevention of the previously described high to low
affinity conversion in muscarinic receptors of depolarized synaptoneurosomes (10) (Fig. 7, C and D). Both
effects of DPI R enantiomer may be attributable to the
prevention of depolarization-induced activation of
Go-proteins (10, 12).
Co-immunoprecipitation of the
-Subunit of VGSC Cross-linked with
G
o-proteins--
We examined the possibility that VGSC
and G
o-proteins interact with each other. Membrane
proteins of unstimulated and depolarized brain-stem and cortical
synaptoneurosomes were cross-linked by the permeable cross-linker, PDM
(4), which reacts with SH sulfhydryls, producing uncleavable products
(4) (Fig. 8A). As a result, a
fraction of G
o-proteins co-immunoprecipitated with the
subunit of VGSC (see Figs. 8, B and C). The
cross-linked product (approximately 300 kDa) was immunoprecipitated by
a common anti-G
o-protein antibody (AS 6) and
immunoreacted specifically with antibodies against a common epitope in
the
subunit of all VGSC subtypes (SP19) as well as with antibody
directed against the carboxyl terminus of G
o1- and
G
o3-proteins (AS 248) (Fig. 8C).

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Fig. 8.
Co-immunoprecipitation of the
subunit of VGSC cross-linked with
G o-proteins in the membrane of
brain synaptoneurosomes. Brain-stem and cortical synaptoneurosomes
were treated with the permeable cross-linker PDM (25 µM).
The cross-linked product was immunoprecipitated by
anti-G o antibodies (AS 6) and immunolabeled by
antibodies directed against the subunit of VGSC (SP19) and antibody
directed against G o1- and G o3-proteins.
Each lane contained 100 µg of protein. A,
immunolabeling of G o-proteins in 39-kDa protein band
(10% polyacrylamide SDS-PAGE) following cross-linking during 30-s and
1-, 2-, and 3-min incubation with PDM in synaptoneurosomes,
unstimulated (a), depolarized (60 mM
[K+] (b), pretreated with 1 µM
batrachotoxin (c), or with batrachotoxin (1 µM) and tetrodotoxin (1 µM) (d).
In e and f, cross-linking was performed in
isolated synaptoneurosomal membranes treated with GDP S (100 µM) and GTP S (100 µM), respectively
(n = 10). B, upper lanes,
immunolabeling of G o-proteins by the common
anti-G o antibody (Ab.) AS 6 following a 2-min
cross-linking of membrane proteins by PDM in depolarized (60 mM [K+] (lane 1) and unstimulated
synaptoneurosomes (lane 2). G o-proteins were
co-immunoprecipitated with G subunit by antibodies directed
against a common epitope in G -proteins. Lower lanes,
immunolabeled G o-proteins, co-immunoprecipitated with
G subunit by antibody directed against a common epitope in
G -proteins, from membranes of depolarized and unstimulated
synaptoneurosomes (lanes 1 and 2, respectively).
Co-immunoprecipitation was not preceded by cross-linking.
Immunoprecipitated proteins were subjected to SDS-PAGE (10%
polyacrylamide) and electroblotted (Western blot) (n = 3). C, immunolabeling of the subunit of VGSC
(lanes 1 and 2) co-immunoprecipitated with
G o-proteins by AS 6 antibody (lanes 3-5 and
7-9) after a 2-min cross-linking by PDM (25 µM) under the following treatments. Lanes 3,
5, and 6, cross-linking conducted during
depolarization. Lane 4, cross-linking conducted at resting
potential. Lane 6, immunoprecipitation of the cross-linked
product by a common antibody directed against G -proteins.
Lanes 7 and 8, cross-linking in isolated
mem-branes treated by GDP S (100 µM) and by GTP S
(100 µM), respec tively. Lane 10, co-immunoprecipitation of VGSC-
was not preceded by cross-linking. Lanes 2, 5,
and 9, immunolabeling of VGSC- subunit was inhibited in
the presence of the peptide (SP19) against which the antibody is
directed. Lanes 11-14, immunolabeling by
anti-G o antibody. Lanes 11 and 12,
cross-linking performed for 2 min during depolarization and at resting
potential, respectively. Lanes 13 and 14,
cross-linking in isolated membranes pretreated with GDP S (100 µM) and GTP S (100 µM), respectively.
Co-immunoprecipitated proteins were subjected to SDS-PAGE (5%
polyacrylamide) and electroblotted (Western blot) (n = 10).
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Cross-linking of G
o-proteins with the
subunit of
VGSC was much more efficient when conducted during depolarization than at resting potential (Fig. 8C). Cross-linking occurred
within seconds (Fig. 8A), whereas VGSC inactivation follows
their short activation, lasting for ms (49). Thus,
G
o-proteins were, apparently, cross-linked to
inactivated VGSC in depolarized synaptoneurosomes. G
o-proteins were also most efficiently cross-linked with
the
subunit of VGSC in synaptoneurosomes pretreated with
batrachotoxin in the presence or absence of the Na+ current
blocker tetrodotoxin, i.e. when activation of VGSC had been
prolonged (46, 47) (not shown). When cross-linking was conducted in
isolated synaptoneurosomal membranes, the
subunit of VGSC
cross-linked most efficiently with G
o-proteins bound to
GDP
S rather than to GTP
S (Fig. 8C). In accordance, the
addition of muscarinic agonists did not induce cross-linking of
G
o-proteins with the VGSC
subunit (not shown). This
may exclude the possibility that VGSC act as effectors of
Go-proteins (5-7).
Without cross-linking, G
o-proteins and the
subunit
of VGSC did not co-immunoprecipitate (Fig. 8C). The VGSC
subunit was not cross-linked with the 
subunit of G-proteins in
depolarized synaptoneurosomes (Figs. 8, B and
C).
Because reportedly VGSC inactivation does not interfere with their
depolarization-induced activation (49, 50) (different parts in the VGSC
subunit undergo conformational changes that are responsible for the
depolarization-induced activation and their successive fast
inactivation (50-52)), our findings may conclude that an interaction
between G
o-proteins and the
subunit of VGSC is
possible once VGSC have been activated. Regarding the GTPase activity
of G
o-proteins, these findings also suggest that G
o-proteins may repeatedly interact with VGSC
subunits as long as membrane depolarization lasts.
Effect of Membrane Depolarization on Phosphorylation of
G
o-proteins--
The isoelectric pH of
G
o-proteins photolabeled with
[32
P]GTPAA was lower by approximately 0.5 pH units in
depolarized synaptoneurosomes than in synaptoneurosomes at resting
potential (Figs. 1 and 2). This depolarization-induced shift in
their pI may be attributable, as mentioned above, to a
depolarization-induced acceleration in the exchange of GDP for
covalently bound GTPAA. Alternatively or in addition it might indicate
a depolarization-induced phosphorylation of G
o-proteins.
This possibility was examined by in situ labeled phosphorylation of membrane proteins in depolarized synaptoneurosomes.
Synaptoneurosomes were incubated for 30 min with
[32P]phosphorus (10 µCi) and then resuspended in either
4.7 or 50 mM [K+] in Ca+2-free
Krebs-Henseleit buffer. Membrane proteins were separated by SDS-PAGE,
electroblotted (Western blot), and examined by autoradiography and
immunolabeling (GC/2). Only a small fraction of the immunolabeled G
o-proteins was 32P-phosphorylated (Fig.
9). Although high
[K+]-induced membrane depolarization increased their
photoaffinity labeling with [
32P]GTPAA by
approximately 5-7-fold (Figs. 1 and 2), it did not significantly alter
their 32P-phosphorylation (Fig. 9), suggesting that the
effect of membrane depolarization is not attributable to
phosphorylation of these proteins.

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Fig. 9.
In situ [32P]
phosphorylation of G o-proteins in
synaptoneurosomes at resting potential and during membrane
depolarization. Brain-stem synaptoneurosomes preincubated for 30 min with [32P]phosphorus (10 µCi/sample, 400-800
µCi/ml) in Krebs-Henseleit buffer were incubated for 10 min in
Krebs-Henseleit buffer containing 4.7 or 50 mM
[K+]. Membrane proteins were separated by SDS-PAGE (10%
acrylamide), electroblotted (Western blot), and immunolabeled with
antibodies directed against the amino-terminal domain of
G o-proteins (GC/2). Lanes 1 and 2,
autoradiogram of 32P-phosphorylated proteins in the
membranes of synaptoneurosomes at resting potential (lane 1)
and during depolarization (lane 2). Lanes 3 and
4, immunolabeled G o-proteins at resting
potential and during depolarization, respectively. Each lane
contained 200 µg of protein (n = 3).
|
|
 |
DISCUSSION |
The experiments described in this study provide evidence for a
depolarization-induced activation of Go1-protein and its
isoform Go3-protein in membranes of rat brain-stem
synaptoneurosomes. The voltage-induced effect was clearly demonstrated
by comparing their in situ photoaffinity labeling with
[
32P]GTPAA in membranes of depolarized
synaptoneurosomes with that in synaptoneurosomes at resting potential
(Figs. 1, 2, and 4). The increased photoaffinity labeling of
G
o1 and G
o3 subtypes in depolarized
synaptoneurosomes and the shift in their isoelectric pH toward a more
acidic pH (Figs. 1 and 2) are attributable to an accelerated exchange
of GDP for GTPAA. This is also consistent with the
depolarization-induced inhibition of PTX-catalyzed ADP-ribosylation of
G
o-proteins (Fig. 6). The depolarization-induced
exchange of GDP for [
32P]GTPAA in
G
o-proteins was reversed by repolarization and was independent of transmitters release and stimulation of muscarinic, NMDA-glutamate receptors, dopaminergic, or serotonergic receptors (2,
3, 8, 9) (Figs. 4 and 5). Thus, it is apparently not a consequence of
depolarization-induced release of these transmitters into the synaptic
cleft (42).
The shift in the isoelectric pH of photoaffinity-labeled
G
o1- and G
o3-proteins in depolarized
membranes (Fig. 2) could also be associated with
G
o-protein phosphorylation. This seems unlikely, however, in view of the negligible effect of membrane depolarization on
their labeled in situ phosphorylation under the experimental conditions employed (Fig. 9).
Membrane depolarization or changes in the electric field of the
membrane would induce charge redistribution in the phospholipid matrix,
which may modify the anchorage of G
o-proteins to
phospholipids (53-59). Recent reports have indicated that
modifications in the binding of G
o-proteins to myristate
and palmitate in the phospholipid matrix affect their interaction with
the G
subunit, (53, 57, 60) as well as with
Go-protein-coupled receptors (61). Changes in palmitylation
and myristoylation of G
o-proteins apparently affect the
stability of GDP binding (55, 57, 61, 62) and thereby may affect
Go-protein activation (62). Depolarization-induced modifications of the anchorage of Go-protein subunits in
the phospholipid matrix might occur via modification of labile
thioester bonds between cysteine residues in G
o-proteins
or GAP-43 (63) and palmitate (56, 57, 64-68). Voltage-induced
modifications in the phospholipid matrix might be also mediated by
voltage-induced modifications in voltage-gated ion channels. In view of
the reciprocal effects of VGSC activation and the activation of
G-coupled muscarinic receptors (12, 24, 25), we examined the
possibility that depolarization-induced activation of VGSC is involved
in depolarization-induced activation of Go-proteins. Unlike
in other excitable tissues, the
subunit of the VGSC in the brain is
covalently bound to a glycoprotein with a single transmembrane spanning
segment, the
2 subunit of VGSC (69, 70). Evidence for a possible
role of
2 in the anchorage of the
subunit of the VGSC to
phospholipids may suggest its involvement in the interaction of VGSC
with other proteins anchored to the phospholipid matrix (70). In view
of this, the possibility that voltage-induced modifications in the
subunit of VGSC may influence other membrane proteins, including G
o-proteins, should be considered and further examined.
DPI-enantiomers that interact with a common site in the
subunit of
VGSC (45) modulate differently its voltage-dependent activation (45). Only the DPI R enantiomer, which prevents
the activation of VGSC (45), prevented both the depolarization-induced exchange of GDP for [
32P]GTPAA in
Go-proteins (Figs. 7, A and B) and
the depolarization-induced uncoupling of G-proteins from muscarinic
receptors (Fig. 7C). This supports the assumption that
depolarization-induced activation of VGSC may be involved in
depolarization-induced activation of Go-proteins. This
assumption was further supported by the efficient cross-linking of
VGSC-
subunit with G
o-proteins during depolarization (Figs. 8, A and C). In addition, GDP-bound
G
o-proteins most efficiently cross-linked with the
subunit of VGSC in isolated synaptoneurosomal membranes (Fig.
8C). Regarding the GTPase activity of
G
o-proteins (7), these findings may suggest repeated
interactions between
subunit of VGSC and
G
o-proteins, as long as depolarization lasts (Fig. 8).
Taken together, these findings support a possible depolarization-induced interaction between these proteins, which might
be involved in depolarization-induced activation of
Go-proteins.
Voltage-dependent activation of Go-proteins
would imply a voltage-induced modulation of signal transduction
cascades triggered by stimulation of Go-coupled-receptors.
An example of reciprocal influence of membrane potential and receptor
activity has been observed in muscarinic receptors (10, 12). Recent
findings indicated a voltage-dependent modulation of the
affinity of presynaptic muscarinic receptors (M2) providing an
autoregulation of transmitter release (71). Also, modulation of the
oscillatory properties of entorhinal cortex layer II neurons has been
attributed to a reciprocal effect of post-synaptic membrane potential
and the activation of muscarinic (M1) receptors (72). Thus, in addition to activation of ion channels by stimulation of G-protein-coupled receptors (1, 73-75), receptor stimulation may be modulated by
activation of voltage-gated ion channels. This in turn may result
feedback mechanisms, producing long term changes in the membrane potential.
Because receptor stimulation induces activation of several
receptor-coupled G-proteins (4), depolarization-induced activation of
even a fraction of Go-proteins may affect the activity of
other G-proteins, as well (10, 24), thereby producing additional versatility in synaptic transmissions in the central nervous system.
 |
ACKNOWLEDGEMENT |
We thank Dr. John Daly (NIH) for his generous
supply of batrachotoxin throughout many years of collaboration. We
thank Dr. E. Rissi and Dr. Romer (Sandoz Ltd.) for the gift of
DPI-201-106.
 |
FOOTNOTES |
*
This work was supported by grants from the Israel Academy of
Sciences, Slezak Fund, Israeli Ministry of Science, and the Deutsche Forschungsgemeinschaft.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 and reprint requests should be
addressed. Tel.: 972 3 5354865; Fax: 972 3 5351139; E-mail:
marmon{at}post.tau.ac.il.
 |
ABBREVIATIONS |
The abbreviations used are:
GTPAA, GTP-azidoanilide;
AcCh, acetylcholine;
CTX, cholera toxin;
DPI, 4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxyl]-1H-indole-2-carbonitrile;
GDP
S, guanosine 5'-O-(2-thiodiphosphate);
GTP
S, guanosine 5'-O-(3-thiotriphosphate);
G
, the
subunit
of G-protein;
PDM, N,N'-1,4-phenylenedimaleimide;
PTX, pertussis toxin;
PAGE, polyacrylamide gel electrophoresis;
TPP+, tetraphenyl phosphonium (bromide salt);
VGSC, voltage-gated sodium channel(s);
G
, 
subunit of G
protein;
Gpp(NH)p, guanosine 5'-(
,
-imido)triphosphate;
NMDA, N-methyl-D-aspartic acid.
 |
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