Activation of Go-proteins by Membrane Depolarization Traced by in Situ Photoaffinity Labeling of Galpha o-proteins with [alpha 32P]GTP-azidoanilide*

Yosef AnisDagger , Bernd Nürnberg§, Leonid VisochekDagger , Nachum Reiss, Zvi Naor, and Malka Cohen-ArmonDagger parallel

From the Dagger  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
Top
Abstract
Introduction
References

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 [alpha 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 [alpha 32P]GTPAA to Galpha o1- and Galpha o3-proteins, but not to Galpha o2- isoforms, was enhanced by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an accelerated exchange of GDP for [alpha 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 alpha  subunit of VGSC was cross-linked and co-immunoprecipitated with Galpha o-proteins in depolarized brain-stem and cortical synaptoneurosomes. VGSC alpha  subunit most efficiently cross-linked with guanosine 5'-O-2-thiodiphosphate-bound rather than to guanosine 5'-O-(3-thiotriphosphate)-bound Galpha o-proteins in isolated synaptoneurosomal membranes. These findings support a possible involvement of VGSC in depolarization-induced activation of Go-proteins.

    INTRODUCTION
Top
Abstract
Introduction
References

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 alpha  subunit of the protein (5-7). Subsequent GTPase activity of the Galpha 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).

Galpha 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 Galpha o1 subtype appears to be involved in the coupling of muscarinic receptors to Ca+2 channels, and the Galpha o2 subtype mediates inhibition of Ca+2 current activated by somatostatin receptors (19). The function of the Galpha 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 [alpha 32P]GTPAA1 (22, 23) indicated a depolarization-induced accelerated exchange of GDP for [alpha 32P]GTPAA in Galpha o1- and Galpha o3-proteins, implying a depolarization-induced activation of these Go-proteins. [alpha 32P]-GTPAA was introduced into transiently permeabilized synaptoneurosomes as described before (10). Unlike the endogenously bound guanine nucleotides, [alpha 32P]GTPAA, covalently bound to Galpha -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 Galpha 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 alpha  subunit of VGSC cross-linked most efficiently with Galpha o-proteins. In isolated synaptoneurosomal membranes, VGSC-alpha subunit cross-linked most efficiently with GDPbeta S-bound rather than GTPgamma S-bound Galpha o-proteins. These findings suggest repeated interactions between VGSC-alpha subunit and Galpha o-proteins as long as membrane depolarization lasts.

    MATERIALS AND METHODS

Reagents-- ATP (grade I), GDPbeta S, GTPgamma 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, [alpha 32P]GTP) (800 Ci/mmol), and antibodies against peptide derived from the amino-terminal domain of Galpha 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 Galpha 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 Galpha o-isoforms, is directed against amino acids 22 to 35 of all three Galpha o isoforms. AS 248, which recognizes Galpha o1 and the closely related isoform Galpha o3 (14), was directed against amino acids 310 to 323 of Galpha o1. AS 201, which reacts exclusively with rodent Galpha o2 (14, 18), was directed against amino acids 293 to 308 of Galpha o2. Antibodies directed against the carboxyl-terminal decapeptide of Galpha i-proteins were kindly supplied by Professor G. Milligan, Glasgow University, UK. Antibodies detecting four splice variants of Galpha s-proteins in mammalian brain AS 348 (27) have been used. Polyclonal antibodies raised against peptide correlating to residues 1491-1508 of alpha  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 [alpha 32P]GTPAA-- [alpha 32P]GTPAA was synthesized by incubation of [alpha 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 [alpha 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 [alpha 32P]GTPAA.

In Situ Photoaffinity Labeling of Galpha -proteins with [alpha 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). [alpha 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 [alpha 32P]GTPAA. For estimation of the nonspecific binding of [alpha 32P]GTPAA, synaptoneurosomes were permeabilized in the presence of [alpha 32P]GTPAA and GTPgamma S (200 µM). The nonspecific photoaffinity labeling of membrane proteins with [alpha 32P]GTPAA was negligible. For estimation of the extracellular binding of [alpha 32P]GTPAA to synaptoneurosomal membranes, samples of nonpermeabilized synaptoneurosomes were subjected to photoaffinity labeling under the above conditions. Photoaffinity labeling of membrane proteins with [alpha 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 Galpha -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 Galpha o-proteins (residues 22-35 (AS 6) (13, 14), and residues 2-16 (GC/2) (39)) and to antibodies detecting carboxyl-terminal domains of Galpha i1-proteins, Galpha i2-proteins (AS/7) (40), and Galpha s-proteins (As 348) (27). In addition, polyclonal antibodies against carboxyl-terminal domains of Galpha o1 and Galpha 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 alpha -Subunit of VGSC by anti-Galpha 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 Galpha 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 alpha  subunits of VGSC situated in the amino terminus (28). This epitope (SP19) was used as a negative control peptide in the immunolabeling of VGSC alpha subunit. Concomitantly, the immunoprecipitated product immunoreacted with antibodies directed against the carboxyl terminus of Galpha 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 Galpha o-proteins with [alpha 32P]GTPAA-- [alpha 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.

[alpha 32P]GTPAA-labeled Galpha 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 Galpha o-proteins was not observed in synaptoneurosomes permeabilized in the presence of [alpha 32P]GTPAA and GDPbeta S (100 µM), i.e. under conditions preventing exchange of GDP for GTP in Galpha -proteins (1-3). The isoelectric pH of the photolabeled Galpha 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 [alpha 32P]GTPAA in Galpha o-proteins (5, 23) in depolarized synaptoneurosomes.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of membrane depolarization on the specific in situ photoaffinity labeling of Galpha o-proteins with [alpha 32P]GTPAA in membranes prepared from transiently permeabilized synaptoneurosomes. [alpha 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 [alpha 32P]GTPAA-labeled proteins were immunolabeled by antibodies directed against the amino-terminal domain in Galpha o-proteins (GC/2). Immunolabeled Galpha o-proteins in membranes isolated from depolarized synaptoneurosomes (B) and from synaptoneurosomes at resting potential (D) were detected. The 32P-labeling of autoradiographed Galpha o-proteins was quantified by densitometry (E). The upper and lower traces present changes in the relative optical density produced by autoradiographed labeled Galpha o-proteins in A and C, respectively. Data are from a typical experiment, one of five performed. Each sample contained 200 µg of protein.

A further identification of the photolabeled Galpha o-proteins has been enabled by immunolabeling with antibodies directed against the carboxyl-terminal domains of two Galpha o-protein subtypes: Galpha o1- and its isoform, Galpha o3 subtype, the most abundant Galpha -proteins in the brain, and Galpha o2-protein (see "Materials and Methods"). Immunolabeling indicated a depolarization-induced shift in the isoelectric pH of Galpha o1- and Galpha o3-proteins toward a more acidic pH (Fig. 2), which could result from a depolarization-induced exchange of GDP for [alpha 32P]GTPAA. The isoelectric pH of Galpha o2-proteins was not altered (Fig. 2).


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of high [K+]-induced membrane depolarization on Galpha o1-, Galpha o3-, and Galpha o2-proteins. Transiently permeabilized synaptoneurosomes were loaded with [alpha 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 Galpha o1- and Galpha o2-proteins (AS 248 and AS 201, respectively). Immunolabeled Galpha 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.

Photoaffinity-labeled Galpha -proteins were also immunolabeled with antibodies directed against a common carboxyl-terminal domain of Galpha i1- and Galpha i2-proteins (see "Materials and Methods"). Under the experimental conditions employed, the isoelectric pH of these Galpha i-protein subtypes was not significantly altered by membrane depolarization (Fig. 3).


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of membrane depolarization on the isoelectric pH of Galpha i-proteins. Proteins in transiently permeabilized synaptoneurosomes were exposed to photoaffinity labeling with [alpha 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 Galpha i1- and Galpha i2-proteins (AS/7) are presented (n = 3). Each sample contained 200 µg of protein.

Depolarization-induced Activation of Go-Proteins Was Not Mediated by Stimulation of G-protein-coupled Receptors-- The enhanced photoaffinity labeling of Galpha o-proteins with [alpha 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 Galpha 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 [alpha 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 Galpha o-proteins with [alpha 32P]GTPAA in the depolarized synaptoneurosomes was preserved despite their treatment with antagonists (Fig. 4).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4.   A, photoaffinity labeling of Galpha o-proteins with [alpha 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 [alpha 32P]GTPAA were immunolabeled by antibodies directed against the amino-terminal domain of Galpha o-proteins (GC/2). Each lane contained 200 µg of protein. Data are from a typical experiment, one of five performed.

Furthermore, the possible effect of transmitters on photoaffinity labeling of Galpha o-proteins with [alpha 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-Galpha o-antibodies (AS 6) (Fig. 5, A and B). In isolated membranes the isoelectric pH of photoaffinity-labeled Galpha o-proteins shifted toward a more acidic pH as compared with that of photoaffinity-labeled Galpha o-proteins in synaptoneurosomes at resting potential (compare Figs. 1 and 2 with Fig. 5A). In comparison to the photoaffinity labeling of Galpha o-proteins in control membranes, a modest additional increase in their labeling with [alpha 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 Galpha o-proteins was similarly mildly enhanced by glutamate or melatonin (not shown). The [32P]photoaffinity labeling of Galpha o-proteins was quantified by densitometry (Fig. 5C).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   A, immunolabeling of Galpha o-proteins with antibodies directed against a common domain of Galpha o-proteins (AS 6). Proteins in isolated membranes were subjected to photoaffinity labeling with [alpha 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 Galpha o-proteins immunolabeled by AS 6 are presented. B, autoradiograms of the Western-blotted Galpha o-proteins, photoaffinity labeled with [alpha 32P]GTPAA. Isolated synaptoneurosomal membranes were incubated with 10 µM GTP/GDP and 1 µCi of [alpha 32P]GTPAA in the absence (control) or in the presence of carbamylcholine (100 µM) or serotonin (100 µM). Photoaffinity labeling of Galpha o-proteins with [alpha 32P]GTPAA in these membrane preparations was quantified by densitometry (C). Each sample contained 200 µg protein (n = 3).

The substantial increase in photoaffinity labeling of Galpha 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 Galpha o-proteins-- Because Galpha o-proteins are better substrates for PTX-catalyzed ADP-ribosylation when coupled to Gbeta gamma (43), their ADP-ribosylation is inhibited by activation of Go-proteins (1, 5). PTX-catalyzed ADP-ribosylation of Galpha o-proteins in depolarized synaptoneurosomes should therefore be inhibited, whereas Go-proteins are activated by membrane depolarization. In contrast, Galpha 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 Galpha o-proteins in situ with its effect on ADP-ribosylation of Galpha s-proteins by comparing their complementary [32P]ADP-ribosylation carried in membranes prepared from PTX- or CTX-pretreated synaptoneurosomes. [32P]ADP-ribosylated Galpha o-proteins were immunoprecipitated by antibody AS 6 (13, 14). [32P]ADP-ribosylated Galpha s-proteins were immunolabeled with antibody AS348 (27). (Galpha s-proteins are present as four splice variants appearing as two bands, each including Galpha s isoforms differing in only one amino acid (27). Membrane depolarization inhibited PTX-catalyzed ADP-ribosylation of Galpha o-proteins (Fig. 6A). This is in accordance with a depolarization-induced activation of Go-proteins (43). In contrast, CTX-catalyzed ADP-ribosylation of Galpha s-proteins was not affected by membrane depolarization (Fig. 6B).


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of membrane depolarization on the ADP-ribosylation of Galpha o- and Galpha 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 Galpha 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 Galpha 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 Galpha o-proteins is presented in the lower frame. Galpha o1-proteins were immunodetected in the immunoprecipitates by antibody AS 248. The extent of [32P]ADP-labeling of Galpha 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 Galpha 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 Galpha 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). Galpha 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 Galpha s-proteins is presented in the lower frame. The extent of [32P]ADP labeling was quantified by densitometry (O.D., absorbance) (n = 3).

Blocking the activation of VGSC Prevented the Depolarization-induced Photoaffinity Labeling of Galpha o-proteins with [alpha 32P]GTPAA-- Depolarization-induced photoaffinity labeling of Galpha o-proteins with [alpha 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). [alpha 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. [alpha 32P]GTPAA-photolabeled membrane proteins were immunolabeled with common anti Galpha o-proteins antibodies (GC/2) (Figs. 7, A and B). The enhanced photoaffinity labeling of Galpha o-proteins with [alpha 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 alpha subunit of VGSC (45), did not interfere with the depolarization-induced photoaffinity labeling of Galpha 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 Galpha 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).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 7.   The effect of agent preventing VGSC activation on the depolarization-induced photoaffinity labeling of Galpha o-proteins with [32P]GTPAA. A, effect of membrane depolarization induced by 50 mM [K+] on the isoelectric pH of Galpha 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 [alpha 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 Galpha 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 Galpha o-proteins with [alpha 32P]GTPAA in transiently permeabilized synaptoneurosomes pretreated with DPI R enantiomer (10 µg). The migration of autoradiographed Galpha 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 (open circle ) or treated with DPI R enantiomer (black-triangle)). 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.

The effect of DPI R enantiomer on photoaffinity labeling of Galpha o-proteins with [alpha 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 alpha -Subunit of VGSC Cross-linked with Galpha o-proteins-- We examined the possibility that VGSC and Galpha 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 Galpha o-proteins co-immunoprecipitated with the alpha subunit of VGSC (see Figs. 8, B and C). The cross-linked product (approximately 300 kDa) was immunoprecipitated by a common anti-Galpha o-protein antibody (AS 6) and immunoreacted specifically with antibodies against a common epitope in the alpha  subunit of all VGSC subtypes (SP19) as well as with antibody directed against the carboxyl terminus of Galpha o1- and Galpha o3-proteins (AS 248) (Fig. 8C).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 8.   Co-immunoprecipitation of the alpha  subunit of VGSC cross-linked with Galpha 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-Galpha o antibodies (AS 6) and immunolabeled by antibodies directed against the alpha  subunit of VGSC (SP19) and antibody directed against Galpha o1- and Galpha o3-proteins. Each lane contained 100 µg of protein. A, immunolabeling of Galpha 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 GDPbeta S (100 µM) and GTPgamma S (100 µM), respectively (n = 10). B, upper lanes, immunolabeling of Galpha o-proteins by the common anti-Galpha 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). Galpha o-proteins were co-immunoprecipitated with Gbeta gamma subunit by antibodies directed against a common epitope in Gbeta -proteins. Lower lanes, immunolabeled Galpha o-proteins, co-immunoprecipitated with Gbeta gamma subunit by antibody directed against a common epitope in Gbeta -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 alpha  subunit of VGSC (lanes 1 and 2) co-immunoprecipitated with Galpha 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 Gbeta -proteins. Lanes 7 and 8, cross-linking in isolated mem-branes treated by GDPbeta S (100 µM) and by GTPgamma S (100 µM), respec tively. Lane 10, co-immunoprecipitation of VGSC-alpha was not preceded by cross-linking. Lanes 2, 5, and 9, immunolabeling of VGSC-alpha subunit was inhibited in the presence of the peptide (SP19) against which the antibody is directed. Lanes 11-14, immunolabeling by anti-Galpha 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 GDPbeta S (100 µM) and GTPgamma S (100 µM), respectively. Co-immunoprecipitated proteins were subjected to SDS-PAGE (5% polyacrylamide) and electroblotted (Western blot) (n = 10).

Cross-linking of Galpha o-proteins with the alpha  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, Galpha o-proteins were, apparently, cross-linked to inactivated VGSC in depolarized synaptoneurosomes. Galpha o-proteins were also most efficiently cross-linked with the alpha  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 alpha  subunit of VGSC cross-linked most efficiently with Galpha o-proteins bound to GDPbeta S rather than to GTPgamma S (Fig. 8C). In accordance, the addition of muscarinic agonists did not induce cross-linking of Galpha o-proteins with the VGSC alpha  subunit (not shown). This may exclude the possibility that VGSC act as effectors of Go-proteins (5-7).

Without cross-linking, Galpha o-proteins and the alpha  subunit of VGSC did not co-immunoprecipitate (Fig. 8C). The VGSC alpha  subunit was not cross-linked with the beta gamma 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 alpha  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 Galpha o-proteins and the alpha  subunit of VGSC is possible once VGSC have been activated. Regarding the GTPase activity of Galpha o-proteins, these findings also suggest that Galpha o-proteins may repeatedly interact with VGSC alpha  subunits as long as membrane depolarization lasts.

Effect of Membrane Depolarization on Phosphorylation of Galpha o-proteins-- The isoelectric pH of Galpha o-proteins photolabeled with [32alpha 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 Galpha 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 Galpha o-proteins was 32P-phosphorylated (Fig. 9). Although high [K+]-induced membrane depolarization increased their photoaffinity labeling with [alpha 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.


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 9.   In situ [32P] phosphorylation of Galpha 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 Galpha 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 Galpha 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 [alpha 32P]GTPAA in membranes of depolarized synaptoneurosomes with that in synaptoneurosomes at resting potential (Figs. 1, 2, and 4). The increased photoaffinity labeling of Galpha o1 and Galpha 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 Galpha o-proteins (Fig. 6). The depolarization-induced exchange of GDP for [alpha 32P]GTPAA in Galpha 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 Galpha o1- and Galpha o3-proteins in depolarized membranes (Fig. 2) could also be associated with Galpha 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 Galpha o-proteins to phospholipids (53-59). Recent reports have indicated that modifications in the binding of Galpha o-proteins to myristate and palmitate in the phospholipid matrix affect their interaction with the Gbeta gamma subunit, (53, 57, 60) as well as with Go-protein-coupled receptors (61). Changes in palmitylation and myristoylation of Galpha 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 Galpha 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 alpha  subunit of the VGSC in the brain is covalently bound to a glycoprotein with a single transmembrane spanning segment, the beta 2 subunit of VGSC (69, 70). Evidence for a possible role of beta 2 in the anchorage of the alpha  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 alpha  subunit of VGSC may influence other membrane proteins, including Galpha o-proteins, should be considered and further examined.

DPI-enantiomers that interact with a common site in the alpha  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 [alpha 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-alpha subunit with Galpha o-proteins during depolarization (Figs. 8, A and C). In addition, GDP-bound Galpha o-proteins most efficiently cross-linked with the alpha  subunit of VGSC in isolated synaptoneurosomal membranes (Fig. 8C). Regarding the GTPase activity of Galpha o-proteins (7), these findings may suggest repeated interactions between alpha  subunit of VGSC and Galpha 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.

parallel 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; GDPbeta S, guanosine 5'-O-(2-thiodiphosphate); GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); Galpha , the alpha  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); Gbeta gamma , beta gamma subunit of G protein; Gpp(NH)p, guanosine 5'-(beta ,gamma -imido)triphosphate; NMDA, N-methyl-D-aspartic acid.

    REFERENCES
Top
Abstract
Introduction
References
  1. Gilman, A. G. (1995) Biosci. Rep. 15, 65-97[Medline] [Order article via Infotrieve]
  2. Pfaffinger, P. J., Martin, J. M., Hunter, D. D., Nathanson, M. M., and Hille, B. (1985) Nature 317, 536-538[Medline] [Order article via Infotrieve]
  3. Ross, E. M. (1989) Neuron 3, 141-152[Medline] [Order article via Infotrieve]
  4. Coulter, S., and Rodbell, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5842-5846[Abstract]
  5. Rens-Domiano, S., and Hamm, H. E. (1995) FASEB J. 9, 1059-1065[Abstract/Free Full Text]
  6. Cassel, D., and Selinger, Z. (1978) Proc. Natl. Acad. Sci. U. S. A. 79, 4155-4159
  7. Kleuss, C., Raw, A. S., Lee, E., Sprang, S. R., and Gilman, A. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9828-9831[Abstract/Free Full Text]
  8. Cerione, R. A., Regan, J. W., Nakata, H., Codina, J., Benovic, J. L., Gierschik, P., Somers, R. L., Spiegel, A. M., Birnbaumer, L., Lefkowitz, R. J., and Caron, M. G. (1986) J. Biol. Chem. 261, 3901-3909[Abstract/Free Full Text]
  9. Florio, V. A., and Sternweis, P. C. (1989) J. Biol. Chem. 264, 3909-3915[Abstract/Free Full Text]
  10. Cohen-Armon, M., and Sokolovsky, M. (1991) J. Biol. Chem. 266, 2595-2605[Abstract/Free Full Text]
  11. Cohen-Armon, M., and Sokolovsky, M. (1991) Neurosci. Lett. 126, 87-90[Medline] [Order article via Infotrieve]
  12. Cohen-Armon, M., and Sokolovsky, M. (1993) J. Biol. Chem. 268, 9824-9838[Abstract/Free Full Text]
  13. Wilcox, M. D., Dingus, J., Balcueva, E. A., McIntire, W. E., Mehta, N. D., Schey, K. L., Robishwa, J. D., and Hildebrandt, J. D. (1995) J. Biol. Chem. 270, 4189-4192[Abstract/Free Full Text]
  14. Nurnberg, B., Spicher, K., Harhammer, R., Bosserhoff, A., Frank, R., Hilz, H., and Schultz, G. (1994) Biochem. J. 300, 387-394[Medline] [Order article via Infotrieve]
  15. Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H., Suzuki, K., and Kaziro, Y. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3776-3780[Abstract]
  16. Avigan, J., Murtagh, J. J., Stevens, L. A., Angus, C. W., Moss, J., and Vaughan, M. (1992) Biochemistry 31, 7736-7740[Medline] [Order article via Infotrieve]
  17. Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J., and Fishman, M. C. (1990) Nature 344, 836-841[CrossRef][Medline] [Order article via Infotrieve]
  18. Spicher, K., Klinz, F.-J., Rudolph, U., Codina, J., Birnbaumer, L., Schultz, G., and Rosenthal, W. (1991) Biochem. Biophys. Res. Commun. 175, 473-479[Medline] [Order article via Infotrieve]
  19. Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schoultz, G., and Witting, B. (1991) Nature 353, 43-48[CrossRef][Medline] [Order article via Infotrieve]
  20. Banno, Y., Nagao, S., Katada, T., Nagata, K., Ui, M., and Nozawa, Y. (1987) Biochem. Biophys. Res. Commun. 146, 861-869[Medline] [Order article via Infotrieve]
  21. van Biesen, T., Hawes, B. E., Raymond, J. R., Luttrell, L. M., Koch, W. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 1266-1269[Abstract/Free Full Text]
  22. Pfeuffer, T. (1977) J. Biol. Chem. 252, 7224-7234[Medline] [Order article via Infotrieve]
  23. Offermans, S., Schafer, R., Hoffmann, B., Bombien, E., Spicher, K., Hinsch, K.-D., Schultz, G., and Rosenthal, W. (1990) FEBS Lett. 260, 14-19[CrossRef][Medline] [Order article via Infotrieve]
  24. Cohen-Armon, M., and Sokolovsky, M. (1986) J. Biol. Chem. 261, 12498-12505[Abstract/Free Full Text]
  25. Cohen-Armon, M., Garty, H., and Sokolovsky, M. (1988) Biochemistry 27, 368-374[Medline] [Order article via Infotrieve]
  26. Cohen-Armon, M., Henis, Y. I., Kloog, Y., and Sokolovsky, M. (1985) Biochem. Biophys. Res. Commun. 127, 326-332[Medline] [Order article via Infotrieve]
  27. Spicher, K., Kalkbrenner, F., Zobel, A., Harhammer, R., Nurnberg, B., Soling, A., and Schultz, G. (1994) Biochem. Biophys. Res. Commun. 198, 906-914[CrossRef][Medline] [Order article via Infotrieve]
  28. Noda, M., Ikeda, T., Suzuki, H., Takeshima, H., Takahashi, T., Kuno, M., and Numa, S. (1986) Nature 322, 826-828[Medline] [Order article via Infotrieve]
  29. Hollingsworth, E. B., McNeal, E. T., Burton, J. L., Williams, R. J., Daly, J. W., and Creveling, C. R. (1985) J. Neurosci. 5, 2240-2253[Abstract]
  30. Fabiato, A., and Fabiato, F. (1979) J. Physiol. Paris 75, 463-505[Medline] [Order article via Infotrieve]
  31. Fabiato, A., and Fabiato, F. (1981) J. Gen. Physiol. 78, 457-497[Abstract]
  32. Tasaki, I., Watanable, A., and Lerman, S. (1967) Am. J. Physiol. 213, 1465-1474[Free Full Text]
  33. Cheng, K., Haspel, H. C., Vallano, M. L., Osotimehin, B., and Sonenberg, M. (1980) J. Membr. Biol. 56, 191-201[Medline] [Order article via Infotrieve]
  34. Gurwitz, D., Kloog, Y., and Sokolovsky, M. (1985) Mol. Pharmacol. 28, 297-305[Abstract]
  35. Drapeau, P., and Blaustein, M. P. (1983) J. Neurosci. 3, 703-713[Medline] [Order article via Infotrieve]
  36. Ferro-Luzzi, A. G., and Nikaido, K. (1976) Biochemistry 15, 617-623
  37. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  38. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4353[Abstract]
  39. Spiegel, A. (1990) in ADP-ribosylating Toxins and G-proteins: Insights into Signal Transduction (Moss, J., and Vaughan, M., eds), pp. 207-217, American Society of Microbiology, Washington, D. C.
  40. Goldsmith, P., Rossiter, K., Carter, A., Simonds, W., Unson, C. G., Vinitsky, R., and Spiegel, A. M. (1988) J. Biol. Chem. 263, 6476-6479[Abstract/Free Full Text]
  41. Walker, G. R., Feather, K. D., and Hines, K. K. (1995) J. NIH Res. 7, 76 (abstr.)
  42. Betz, H. (1990) Biochemistry 29, 3591-3599[Medline] [Order article via Infotrieve]
  43. Thomas, T. C., Schmidt, C. J., and Neer, E. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10295-10298[Abstract]
  44. Bobak, D. A., Tsai, S.-C., Moss, J., Vaughan, M., and Spiegel, A. (1990) in ADP-ribosylating Toxins and G-proteins: Insights into Signal Transduction (Moss, J., and Vaughan, M., eds), pp. 439-452, American Society of Microbiology, Washington, D. C.
  45. Romey, G., Quast, U., Pauron, D., Frelin, C., Renaud, J. F., and Lazdunski, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 896-900[Abstract]
  46. Daly, J. W. (1982) J. Toxicol. Toxin Rev. 1, 33-86
  47. Khodorov, B. I. (1985) Prog. Biophys. Mol. Biol. 45, 57-148[Medline] [Order article via Infotrieve]
  48. Moore, J. W., Blaustein, M. P., Anderson, N. C., and Narahashi, T. (1967) J. Gen. Physiol. 50, 1401-1411[Abstract/Free Full Text]
  49. Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. (Lond.) 117, 500-544[Medline] [Order article via Infotrieve]
  50. Catterall, W. A. (1992) Physiol. Rev. 72 (Suppl. 15), 15-47
  51. Urenjak, J., and Obrenovitch, T. P. (1996) Pharmacol. Rev. 48, 21-67[Medline] [Order article via Infotrieve]
  52. Trainer, V. L., Brown, G. B., and Catteerall, W. A. (1996) j. BIOL. chem. 271, 11261-11267[Abstract/Free Full Text]
  53. Casey, P. J. (1994) Curr. Opin. Cell Biol. 6, 219-225[Medline] [Order article via Infotrieve]
  54. Hamm, H. E., and Gilchrist, A. (1996) Curr. Opin. Cell Biol. 8, 189-196[CrossRef][Medline] [Order article via Infotrieve]
  55. Neubig, R. R. (1994) FASEB J. 8, 939-946[Abstract/Free Full Text]
  56. Casey, P. J. (1995) Science 268, 221-225[Medline] [Order article via Infotrieve]
  57. Wedegaertner, P. B., Wilson, P. T., and Bourne, H. R. (1995) J. Biol. Chem. 270, 503-506[Free Full Text]
  58. Hepler, J. R., and Gilman, A. G. (1992) Trends Biochem. Sci. 17, 383-387[CrossRef][Medline] [Order article via Infotrieve]
  59. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127[CrossRef][Medline] [Order article via Infotrieve]
  60. Bigay, J., Faurobert, E., Franco, M., and Chabre, M. (1994) Biochemistry 33, 14081-14090[Medline] [Order article via Infotrieve]
  61. Iniguez-Lluhi, J., Kleuss, C., and Gilman, A. G. (1993) Trends Cell Biol. 3, 230-236[CrossRef]
  62. Mumby, S. M., Kleuss, C., and Gilman, A. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2800-2804[Abstract]
  63. Sudo, Y., Valenzuela, D., Beck-Sickinger, A. G., and Strittmatter, S. M. (1992) EMBO J. 11, 2095-2102[Abstract]
  64. Milligan, G., Parenti, M., and Magee, A. I. (1995) Trends Biochem. Sci. 20, 181-186[CrossRef][Medline] [Order article via Infotrieve]
  65. Wedegaertner, P. B., and Bourne, H. R. (1994) Cell 77, 1063-1070[Medline] [Order article via Infotrieve]
  66. Neer, E. J. (1994) Protein Sci. 3, 3-14[Abstract/Free Full Text]
  67. Dratz, E. D., Fursteneau, J. E., Lambert, C. G., Thireault, D. L., Rarick, H., Schepers, T., Pakhlevaniants, S., and Hamm, H. E. (1993) Nature 363, 276-280[CrossRef][Medline] [Order article via Infotrieve]
  68. Denker, B. M., Boutin, P. M., and Neer, E. J. (1995) Biochemistry 34, 5544-5553[Medline] [Order article via Infotrieve]
  69. Isom, L. L., Ragsdale, D. S., De Jongh, K. S., Westenbroek, R. E., Reber, B. F. X., Scheuer, T., and Catterall, W. A. (1995) Cell 83, 433-442[Medline] [Order article via Infotrieve]
  70. Sheng, M., and Kim, E. (1996) Cur. Opin. Neurobiol. 6, 602-608[CrossRef][Medline] [Order article via Infotrieve]
  71. Slutsky, I., Parnas, H., and Parnas, I. (1998) Eur. J. Neurosci. 10 (suppl.), 174 (abstr.)
  72. Klink, R., and Alonso, A. (1997) J. Neurophysiol. 77, 1813-1828[Abstract/Free Full Text]
  73. Ma, J. Y., Li, M., Catterall, W. A., and Scheuer, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12351-12355[Abstract/Free Full Text]
  74. Krapivinsky, G, Krapivinsky, L., Wickman, K., and Clapham, E. D. (1995) J. Biol. Chem. 270, 29059-29062[Abstract/Free Full Text]
  75. Stehno-Bittel, L., Krapivinsky, G., Krapivinsky, L., Perez-Terzic, C., and Clapham, D. E. (1995) J. Biol. Chem. 270, 30068-30074[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.