AMPA receptor activation induces association of G-beta protein with the alpha subunit of the sodium...

AMPA receptor activation induces association of G-betaprotein with the alpha subunit of the sodium channel inneurons

Philippe Marin,1 Laurent Fagni,2 Yvette Torrens,1 GiseÁ le Alcaraz,3 FrancËois Couraud,3 JoeÈ l Bockaert,2 JacquesGlowinski1 and JoeÈ l PreÂmont1¶1INSERM U114, ColleÁge de France, 11, Place Marcelin Berthelot, 75231 Paris Cedex 05, France2CNRS UPR 9023, CCIPE, 141, rue de la Cardonille, 34094 Montpellier Cedex 5, France3INSERM U464, FaculteÂ de MeÂdecine Nord, UniversiteÂ Aix-Marseille II, Boulevard Pierre Dramard, 13916 Marseille Cedex 20,

France

Keywords: AMPA receptor, Gb protein, mitochondrial calcium, mouse, sodium channel

Abstract

Glutamatergic transmission is mediated by ionotropic receptors that directly gate cationic channels and metabotropic receptors

that are coupled to second messenger generating systems and to ionic channels via heterotrimeric guanine-nucleotide binding-

(G) proteins. This distinction cannot be made for the ionotropic receptor subclass activated by a-amino-3-hydroxy-5-

methylisoxazole-4-propionic acid (AMPA), which has been shown to be physically associated with the a-subunit of Gi1 proteinand activates this G-protein. Here, we report that, in addition to a Ca2+ in¯ux, AMPA induces the mobilization of Ca2+ from the

mitochondrial pool by reversing the mitochondrial Na+/Ca2+ exchanger in mouse neurons in primary culture. Both processes

required the activation of tetrodotoxin-sensitive Na+ channels. AMPA receptor activation modi®ed the gating properties of theNa+ channel, independently of the AMPA current, suggesting a G-protein-mediated process. Indeed, co-immunoprecipitation

experiments indicated that AMPA receptor activation induced the association of Gb with the a-subunit of the Na+ channel. These

results suggest that, in addition to its ionic channel function, the AMPA receptor is coupled to Na+ channels through G-proteinsand that this novel metabotropic function is involved in the control of neuronal excitability.

Introduction

Neuronal voltage-gated Na+ channels [NaCh(s)] are heterotrimeric

complexes composed of a, b1 and b2 subunits (Catterall, 2000). Four

a-subunit genes (Nav1.1, Nav1.2, Nav1.3 and Nav1.6) are expressed

in the brain and the presence of RNA transcripts for 1.1, 1.2 and 1.3

genes have been evidenced in cultures of rat cortical neurons (Giraud

et al., 1998). Expression of the a-subunit alone is suf®cient to form

functional voltage-gated NaCh(s) (Noda et al., 1986; Goldin et al.,

1986; Smith & Goldin, 1998; Smith et al., 1998) and coexpression of

b1 and b2-subunits modulates channel expression and gating (Isom

et al., 1995a, b). It is now well established that ion channels including

NaCh can be regulated by a-subunits (Ga) and/or bg-subunits (Gbg)

of heterotrimeric guanine-nucleotide binding-(G) proteins. Activation

of brain NaCh is enhanced by G-proteins in hippocampal neurons and

CHO cells expressing type IIA a-subunit of the Na+ channel (aNaCh

IIA) (Ma et al., 1994). Moreover, in non-neuronal cells coexpressing

aNaCh IIA and different Gbg subunit combinations, a direct

association of Gbg to the C-terminal domain of the channel prolongs

inactivation of the Na+ current (Ma et al., 1997).

Fast excitatory synaptic transmission is mainly mediated by the

a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)

subtype of excitatory amino acid receptors at most synapses of the

central nervous system. One important consequence of AMPA

receptor activation is a marked increase in cytosolic free Ca2+

concentration, a necessary step in several physiological processes

including synaptic plasticity and many neuropathological disorders

such as neurotoxicity associated with cerebral ischemia, in which

AMPA receptors are implicated (Gill, 1994; Zamanillo et al., 1999).

Ca2+ entry in neurons exposed to AMPA can be mediated directly by

Ca2+-permeable AMPA receptor subtypes or indirectly by depolar-

ization-induced activation of voltage-gated Ca2+ channels (Murphy &

Miller, 1989; Weiss et al., 1990; Brorson et al., 1992).

Several studies suggest that, in addition to their ionic channel

function, AMPA receptors transduce some of their effects through

interaction with heterotrimeric G-proteins. Exposing rat cortical

neurons to AMPA induces the dissociation of the a-subunit from the

Gai1bg heterotrimeric complex, its association with the GluR1

AMPA receptor subunit, as well as the inhibition of adenylyl cyclase

activity (Wang et al., 1997). Moreover, AMPA receptors activate a

G-protein that suppresses a cGMP-gated current in retinal ganglion

cells (Kawai & Sterling, 1999).

In an attempt to identify other metabotropic functions of AMPA

receptors, we found that AMPA induced the mobilization of Ca2+

from the mitochondrial pool in cultured neurons. This effect occurred

through a mechanism involving tetrodotoxin- (TTX) sensitive

voltage-gated NaChs and the reverse function of the mitochondrial

Na+/Ca2+ exchanger. This process was accompanied by a prolonged

modi®cation of the gating properties of the TTX-sensitive NaChs that

Correspondence: Dr JoeÈl PreÂmont, 1INSERM U114, as above.E-mail: [email protected]

Received 6 August 2001, accepted 29 October 2001

European Journal of Neuroscience, Vol. 14, pp. 1953±1960, 2001 ã Federation of European Neuroscience Societies

was independent of the AMPA current. Additional experiments

indicated that AMPA receptor activation increased association of Gbproteins with aNaCh in neurons.

Experimental procedures

Primary cultures

Pregnant mice were killed by prolonged exposure to a rising

concentration of CO2 and embryos (15 days old) were rapidly

removed from the uteri and placed into a phosphate-buffered saline

supplemented with glucose (33 mM). Primary cultures of mouse

cortical or cerebellar granule neurons were prepared as previously

described (Van Vliet et al., 1989; Marin et al., 1997). Cells were

maintained at 37 °C in a humidi®ed atmosphere containing 8% CO2

for 10±12 days without medium change. Under these conditions,

cultures were shown by immunocytochemistry experiments, using an

anti-Microtubule-Associated Protein 2 monoclonal antibody (Clone

HM2, Sigma, France), to be highly enriched in neurons and less than

7% of the cells exhibited immunoreactivity with a rabbit antibody

raised against glial ®brillary acid protein (Dakopatts, Glostrup,

Denmark) (data not shown).

Cytosolic Ca2+ measurement

Cytosolic free Ca2+ was monitored in cortical neurons cultured on

glass slides by quantitative ratio imaging of the ¯uorescent Ca2+

probe INDO-1 (Molecular Probes, Eugene, OR), as described

previously (Murphy et al., 1994). Cells were loaded for 60 min

with 12 mM of INDO-1 acetoxymethylester in HEPES buffer. After

loading, the glass slide was placed in a perfusion chamber where cells

were exposed to tested substances using a multichannel superfusion

device allowing the delivery of drugs to cells in less than 100 ms.

Cells were excited with a 75-W Xenon light, ®ltered at 340 nm with a

10-nm wide interferential ®lter. Excitation and emission spectra were

separated by a 380-nm dichroic long pass ®lter and the emission

spectra were divided in two halves by a 455-nm dichroic long pass

®lter (opticals were obtained from Nikon and Hamamatsu, Japan).

Two discriminant bands were selected by interferential ®lters from

the two halves at 400±410 nm and 470±480 nm and both ¯uorescent

images were digitized (eight video frames per digitized image,

permitting the recording of one image per second). The camera dark

noise was subtracted from the recorded crude image (camera and

digitizing system were from Hamamatsu). Results were expressed

as the ratio between ¯uorescence measured at 405 and 480 nm

(F405/F480).

Whole-cell patch-clamp recordings

Whole-cell, patch-clamp currents were recorded at room temperature

in cortical and cerebellar granule neurons cultured in 35-mm culture

dishes. The bathing medium contained (in mM): Na-gluconate, 140;

CaCl2, 2; KCl, 3; MgCl2, 2; HEPES, 10; D-glucose, 10; pH adjusted

to 7.4 with NaOH and to 330 mOsm with Na-gluconate. Drug

solutions were prepared in this bathing medium and the pH was

readjusted to 7.4. Patch pipettes were made from borosilicate glass,

coated with Sylgard, and the tip ®re polished. Pipettes had resistances

of 3±5 MW when ®lled with the following internal solution

(containing, in mM): Cs-acetate, 100; MgCl2, 2; HEPES, 10; glucose,

15, CsCl, 20; EGTA, 20 mM; Na2ATP, 2 mM and cAMP, 1 mM,

adjusted to pH 7.2 and to 300 mOsm with CsOH. Drugs were applied

by means of a gravity-driven, fast perfusion system as previously

described (Manzoni et al., 1992). Na+ currents were evoked by

voltage-clamp depolarizing pulses of 15 ms duration, from a holding

potential of ±60 mV. Voltage pulses were applied at a rate of 0.1 Hz.

Current signals were recorded with an Axopatch 200 ampli®er,

®ltered at 1 kHz with an 8-pole Bessel ®lter and sampled at 3 kHz on

a Pentium II PC computer. Analyses were performed using the

pClamp6 program of Axon Instruments and Na+ currents were

measured at their peak amplitude. Capacitance of the cells was

measured by delivering square wave hyperpolarizing pulses of 5 mV

amplitude, 10 ms duration, at a frequency of 2 Hz. Average

capacitance, input resistance and resting potential of the cells

were of 7.2 6 0.3 pF (n = 33), 11.2 6 0.9 MW (n = 33) and

±60 6 11 mV (n = 33), respectively.

Co-immunoprecipitation experiments

Cortical neurons, grown in 100-mm culture dishes were washed twice

in HEPES buffer (containing, in mM: HEPES, 20; glucose, 5.5; NaCl,

120; KCl, 5.5; MgCl2, 1.2; CaCl2, 1.2; pH 7.4) and then incubated in

the same medium for 5 min (unless otherwise indicated) in the

presence of drugs. Cells were scraped off in 5 mL of homogenization

buffer (sucrose 0.32 M, Tris-HCl 10 mM, pH 7.4, containing a

protease inhibitor cocktail (Roche, France), centrifuged for 5 min at

100 g and homogenized in 1 mL of the same buffer. Samples were

centrifuged for 10 min at 1000 g and the postnuclear fraction was

centrifuged for 30 min at 100 000 g. Immunoprecipitations were

conducted on solubilized membrane fractions as described previously

(Alcaraz et al., 1997). The membrane fraction (pellet) was incubated

for 30 min in 0.5 mL ice-cold solubilization buffer containing

100 mM NaCl, 50 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% (wt/v)

Triton X-100 and the protease inhibitor cocktail, and centrifuged for

30 min at 100 000 g. Supernatant proteins (300 mg) were immuno-

precipitated with an antibody directed against a sequence identical to

that of the previously described SP20 antibody, which has been

largely used to detect NaChs in various tissues (Black et al., 1995a,

b). This antibody was obtained by immunization of rabbits with a

synthetic peptide derived from a conserved sequence in the second

intracellular loop of the a-subunit (amino acids 1111±1121, subtype

aII numbering: GESDFENLNTE). This serum was shown previously

to speci®cally immunoprecipitate aNaCh (Alcaraz et al., 1997).

Immunocomplexes were resolved on either 10% polyacrylamide gels,

to detect Gb, or 4% polyacrylamide gels to detect aNaCh. The

amount of immunoprecipitated Gb was estimated by Western blotting

using a monoclonal antibody directed against Gb (clone 3, 1 : 1000

dilution, Transduction Laboratories, Lexington, KY). Immuno-

precipitated aNaCh was detected using a protein A-puri®ed Ig

fraction from an antiserum directed against a glutathione S-

transferase fusion protein containing the entire sequence of the

third intracellular loop of the a subunit (1.5±2 mg/mL). In Western

blots of rat brain membranes, this antibody recognizes a polypeptide

around 260 kDa that displays the smear characteristic of the highly

glycosylated aNaCh. The total amount of Gb was also estimated by

Western blotting of an aliquot of crude neuronal membranes (10 mg

protein) following each treatment.

ADP-ribosylation of Gai/o proteins by PTX

Cortical neurons grown in 100-mm culture dishes were washed twice

in HEPES buffer and incubated in the same medium for 5 min in the

presence of drugs (unless otherwise indicated). After two washes,

cells were scraped off and centrifuged for 10 min at 100 g. The cell

pellet was resuspended and homogenized in an ice-cold lysing buffer

containing 50 mM Tris-HCl (pH 7.5), 3 mM EDTA and protease

inhibitors, and centrifuged for 30 min at 100 000 g. Particulate

fraction (50 mg protein) was incubated for 1 h at 30 °C in 40 mL

ADP-ribosylation medium containing 100 mM Tris-HCl (pH 8),

1954 P. Marin et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1953±1960

1 mM EDTA, 10 mM thymidine, 2 mM MgCl2, 1 mM ATP, 0.1 mM

GTP, 10 mM nicotinamide, 10 mg/mL Bordetella pertussis toxin

(PTX, preactivated with 1 mM dithiotreitol for 30 min at 37 °C), and

[a-32P]NAD+ (0.5 mM, 1 mCi, NEN-Dupont, France). The reaction

was stopped by the addition of 20 mL SDS (2%). Samples were

resolved on 10% polyacrylamide gels and subjected to autoradio-

graphy.

Results

Role of TTX-sensitive Na+ channels in AMPA-induced Ca2+

increase in neurons

As observed previously in several neuronal types (Murphy & Miller,

1989; Brorson et al., 1992), exposing mouse cortical neurons to a

maximally effective concentration of AMPA (30 mM) (Williams et al.,

1995) induced a prolonged increase in cytosolic Ca2+ in the presence

of extracellular Ca2+ (Fig. 1A). This effect was blocked by 6,7-

dinitroquinoxaline-2,3-dione (DNQX, 100 mM), an antagonist of

AMPA receptors (not shown). The AMPA-induced elevation of

cytosolic Ca2+ was also entirely suppressed by the coapplication of

TTX (1 mM, Fig. 1B).

Interestingly, AMPA also induced a transient (less than 1 min)

elevation of cytosolic Ca2+ in the absence of extracellular Ca2+

(Fig. 1C) and this response could be reliably observed following

successive applications of the agonist (Fig. 1D). This mobilization of

intracellular Ca2+ was totally suppressed by DNQX (not shown). It is

worth noting that washout of AMPA in a Ca2+-containing medium

was accompanied by a delayed Ca2+ increase (Fig. 1D). This Ca2+

response was probably mediated by activated voltage-gated Ca2+

channels, as it was suppressed by nifedipine, whereas, the transient

Ca2+ elevation detected in the absence of extracellular Ca2+ was not

altered (not illustrated).

Several pieces of evidence suggest that the mobilization of

intracellular Ca2+ induced by AMPA results from the reverse

function of the Na+/Ca2+ mitochondrial exchanger following the

presumed Na+ in¯ux through TTX-sensitive NaCh. The Ca2+

response was suppressed by TTX (Fig. 1E) and strongly reduced

when extracellular NaCl was replaced by choline chloride (120 mM)

or N-methyl-D-glucamine (240 mM) (84 6 11% and 81 6 13% of

decrease of the Ca2+ pike, compared with that measured in the

presence of extracellular Na+, n = 35, respectively); it was unaltered

by pretreating the neurons with thapsigargin (10 mM), which specif-

ically depletes endoplasmic reticulum Ca2+ stores (Fig. 1F); it was

suppressed in the presence of CGP 37 157 (25 mM), a speci®c

inhibitor of the mitochondrial Na+/Ca2+ exchanger (Fig. 1G) (Cox

et al., 1993; White & Reynolds, 1997). Finally, AMPA-induced

mitochondrial Ca2+ mobilization was mimicked by direct activation

of voltage-gated NaCh. Indeed, exposure of neurons to veratridine

(10 mM, in the absence of extracellular Ca2+), induced a transient

elevation of cytosolic Ca2+ (Fig. 1H), which was also suppressed by

CGP 37 157 (not shown).

Altogether, these results indicate that both Ca2+ in¯ux and

mobilization of mitochondrial Ca2+ induced by AMPA are strictly

dependent on the activation of TTX-sensitive NaCh.

Effects of AMPA on the gating properties of the Na+ channel

Additional experiments were performed to examine the effect of

AMPA receptor activation on the properties of the voltage-gated

NaChs. Neurons exhibited stable Na+ currents (±145 6 10 pA/pF at

±15 mV, n = 11) that activated rapidly in response to depolarization

and then inactivated rapidly and virtually completely in less than

5 ms (Fig. 2A). The Na+ current was entirely suppressed by TTX

(1 mM, not shown). Exposing the neurons to AMPA (30 mM) induced

an inward current (2.4 6 0.6 pA/pF at ±60 mV membrane potential,

n = 11) in about 80% of the cells tested (n = 14), which rapidly

decayed after drug washout (Fig. 2B). Na+ currents were not recorded

during the application of AMPA because of the shunting effect of

AMPA-operated channels. The amplitude of the Na+ current was thus

compared in the same cell before and after a 1-min application of

AMPA followed by washout of the agonist (Fig. 2A and C).

Treatment with AMPA increased the amplitude of the Na+ current

(mean increase at ±15 mV, 31 6 6%, n = 9) in 80% of the neurons

examined (n = 11, Fig. 2A). This effect was rapid (less than 1 min)

and lasted for several minutes (Fig. 2C). Current±voltage relation-

ships of the Na+ currents obtained after the AMPA treatment

(Fig. 2D) shifted toward positive membrane potentials, showing that

the increase in current amplitude occurred only at potentials above ±

25 mV. Neither the time course of inactivation (Fig. 2A), nor the

steady-state inactivation curve (Fig. 2E) of the Na+ current were

signi®cantly affected by the AMPA treatment.

AMPA increases the association of Gb with aNaCh

A previous study demonstrated that heterotrimeric G-protein acti-

vation induced an increase in Na+ currents recorded in acutely

dissociated rat hippocampal neurons (Ma et al., 1994). Subsequent

experiments performed in non-neuronal cells coexpressing rat brain

aNaCh IIA and different Gbg subunit combinations indicated that the

binding of Gbg subunits to aNaCh IIA is responsible for the increase

of a component of the Na+ current displaying normal voltage-

dependence of activation but with a strongly slowed and incomplete

inactivation (Ma et al., 1997). Thus, we have examined whether

AMPA receptor activation stimulates the association of Gbg with

aNaCh in cortical neurons.

Co-immunoprecipitation experiments demonstrated that an anti-Gbantibody was able to detect a variable amount of Gb in

immunocomplexes obtained with an antibody directed against the

aNaCh in membranes from cortical neurons (Fig. 3A). Treating the

neurons with AMPA (30 mM) for 5 min increased the amount of Gbpresent in the immunoprecipitate, compared to that detected in both

untreated neurons and neurons exposed to DNQX (100 mM, Fig. 3A

and C). As expected, the effect of AMPA was totally suppressed by

DNQX (data not illustrated). In most experiments, exposing the

neurons to DNQX decreased the amount of Gb immunoprecipitated

with the anti-aNaCh antibody relative to untreated neurons (Fig. 3A).

This suggests that the association of Gb with aNaCh in untreated

neurons resulted from activation of AMPA receptors by ambient

glutamate. Thus, in subsequent experiments, the basal level of

immunoprecipitated Gb was determined in the presence of DNQX.

The observed changes in the amount of Gb associated to aNaCh were

not related to an altered association of Gb to neuronal membranes

after the treatments, as an equal amount of protein was found in crude

neuronal membranes (Fig. 3A, bottom).

In the presence of TTX (1 mM), the AMPA receptor-mediated

coprecipitation of Gb with aNaCh was signi®cantly increased

(Fig. 3C). This increase may be the consequence of a conformational

change of aNaCh induced by TTX (Tejedor et al., 1988). This result

also indicates that the association of Gb with aNaCh is not the result

of Na+ ¯ux through activated NaCh following AMPA receptor-

mediated depolarization. Further supporting this hypothesis, the

depolarization of neurons with KCl (50 mM) did not induce

association of Gb with aNaCh (Fig. 3C). Moreover, this treatment

did not further increase the level of Gb bound to aNaCh in AMPA-

exposed neurons (Fig. 3C). The association of Gb with aNaCh was

Modulation of sodium channels by AMPA through Gb protein 1955


also independent of AMPA receptor ionic channel activity. Indeed,

the effect of AMPA persisted when neurons were incubated in a

Ca2+- and Na+-free buffer (Fig. 3C).

Part of Gbg that associates with aNaCh upon AMPA receptor

stimulation might arise from PTX-sensitive G-proteins (Gi/Go).

Indeed, treating mouse cortical neurons with AMPA reduced the

FIG. 1. Role of TTX-sensitive Na+ channels in the AMPA-induced increase in cytosolic Ca2+ Variations in cytosolic Ca2+ concentration (ratio between the¯uorescence measured at 405 and 480 nm (F405/F480) were measured in INDO-1-loaded neurons. Horizontal black bars represent the periods of superfusionof drugs, performed in the presence (+ Ca2+) or absence (± Ca2+) of Ca2+. Between these periods, extracellular Ca2+ was present throughout the recordings.The following concentrations were used: AMPA, 30 mM; TTX, 1 mM; thapsigargin, 10 mM; CGP 37 157 (generously provided by Novartis, France), 25 mM;veratridine, 10 mM. The coapplication of CGP 37 157 with AMPA did not yield a signi®cant decrease of the AMPA-evoked response, a 30- s pre-exposure ofthe cells to the drug being required for full inhibition. The AMPA-evoked response was fully restored only 1 min after the removal of CGP 37 157. Tracesrepresent the mean of those obtained in at least 35 neurons originating from two sets of cultured neurons.



PTX-induced ADP-ribosylation of both Gai and Gao (respective

molecular weights of 41 and 39 kDa) in a subsequent in vitro assay

(Fig. 3B), as compared to that measured in membranes from DNQX-

treated neurons. As the heterotrimeric structure of the Gi/o-proteins is

essential for the PTX-induced ADP-ribosylation of their a-subunit

(Huff & Neer, 1986), this result suggests that AMPA does induce the

dissociation of both ai and ao subunits from the ai/obg heterotrimeric

complexes in these neurons. However, pretreatment of cortical

neurons with PTX (1 mg/mL, 24 h), which leads to ADP-ribosylation

of all Gai,o subunits (assessed by back ADP-ribosylation experi-

ments, Fig. 3B), did not prevent the AMPA-induced association of

Gb with aNaCh (Fig. 3C). Rather, PTX pretreatment signi®cantly

increased the stimulated level of Gb coprecipitated with aNaCh. In

agreement with these results, PTX did not inhibit the increase in

cytosolic Ca2+ evoked by AMPA (mean values of F405/F480 evoked

by AMPA in control and PTX-pretreated neurons in the presence of

extracellular Ca2+: 0.28 6 0.05 and 0.27 6 0.03, respectively,

n = 200 cells tested in each experimental condition).

Discussion

In this study, we demonstrate that AMPA receptor activation in

mouse neurons in culture induces the association Gb with aNaCh,

independently of its ionic channel activity. Indeed, the effect of

AMPA persisted in neurons incubated in a Ca2+- and Na+-free buffer.

This metabotropic function of AMPA receptors is in agreement with

previous observations indicating that AMPA receptors exert some of

their effects by a process involving heterotrimeric G-proteins. In

retinal ganglion cells, AMPA receptor activation leads to the

FIG. 2. Effect of AMPA receptor activation on the gating properties of Na+ current. (A)Na+ currents evoked in the same cell by a depolarizing pulse to ±15 mV, from a holding potential of ±60 mV, after a 1-min sham perfusion [Control (1)], and 1 min after a 1-min application of 30 mM AMPA [Post-AMPA(2)]. (B)AMPA-evoked current recorded in the same neuron. (C)Time course of the effect of AMPA on voltage-gated Na+ current in the same cell. Thehorizontal bar indicates the period of AMPA application. (D)Na+ current-voltage relationships obtained in another cell before and after AMPA exposure.(E)Normalized, steady-state inactivation curves obtained in another cell. Peak current generated by test pulses to ±15 mV was plotted as a function ofprepulse (2 s) potential.



inhibition of cationic channels gated by cGMP through PTX-sensitive

G-proteins (Kawai & Sterling, 1999). In these neurons, AMPA

receptor activation opposes its cationic channel activity by inhibiting

the opening of another cationic channel through heterotrimeric G-

proteins. In rat cortical neurons, AMPA induces the dissociation of

the ai1 subunit of Gi-protein from the abg heterotrimeric complex

and increases its association with the AMPA receptor subunit GluR1.

This process is probably involved in the inhibition of adenylyl

cyclase activity (Wang et al., 1997). Another subclass of ionotropic

glutamate receptors, preferentially activated by kainate, appears to be

similarly coupled to heterotrimeric G-proteins and, thus, to display a

metabotropic function. Pharmacological manipulations that uncouple

G-proteins from their receptors have been shown to decrease the

binding of kainate receptor-selective agonists to hippocampal mem-

branes, as observed for agonists of classical metabotropic receptors

(Cunha et al., 1999). Moreover, activation of kainate receptors

decreases the release of g-aminobutyric acid in the hippocampus by a

mechanism sensitive to PTX and inhibitors of protein kinase C

(Rodriguez-Moreno & Lerma, 1998)´

The decreased ADP-ribosylation of Gai/o proteins by PTX,

following AMPA treatment, indicates that AMPA receptors are

capable of activating these G-protein subclasses in mouse cortical

neurons. In contrast, the association of Gb with aNaCh was totally

insensitive to the toxin. One possible explanation may be that, unlike

classical metabotropic receptors, the interaction of AMPA receptors

with the Ga subunit is not impaired by its ADP-ribosylation.

However, several observations demonstrate that PTX prevents the

activation of different biochemical cascades mediated by AMPA or

kainate (Wang & Durkin, 1995; Wang et al., 1997; Rodriguez-

Moreno & Lerma, 1998; Kawai & Sterling, 1999). This suggests that

Gbg subunits that bind aNaCh upon AMPA receptor activation in

cortical neurons mainly arise from PTX-insensitive G-proteins.

AMPA receptors may thus be coupled to different G-protein

subclasses, sensitive and insensitive to PTX.

Evidence for membrane depolarization-induced activation of Go-

protein has been reported previously in rat brain synaptoneurosomes

(Anis et al., 1999). In this model, voltage-dependent activation of

NaCh appears to be essential for the activation of Go, regardless of

Na+ current (Anis et al., 1999). In our experiments performed on

living cortical neurons, KCl-induced cell depolarization was, alone,

unable to stimulate the binding of Gb to NaCh and did not further

increase the level of Gb bound to aNaCh in AMPA-treated neurons.

This indicates that the association of Gb with aNaCh is not the

consequence of the activation of a G-protein following Na+ in¯ux

initiated by AMPA receptor activation and prolonged by the opening

of voltage-gated NaCh.

Parallel to this metabotropic effect of AMPA, we provide

electrophysiological experiments showing that AMPA induces a

shift in the voltage-dependence of activation of TTX-sensitive NaChs

and an increase in the Na+ current amplitude, which persisted after

agonist removal. A previous study has shown that brain NaCh activity

can be increased by a G-protein-dependent mechanism (Ma et al.,

1994). Further experiments performed in non-neuronal cells trans-

fected with aNaCh IIA and different Gbg subunit combinations,

indicated that direct association of Gbg with aNaCh IIA enhanced a

late component of the Na+ current with a normal voltage-dependence

of activation but slower and incomplete inactivation (Ma et al., 1997).

We propose that the modi®cations of electrophysiological properties

of NaCh following AMPA exposure in cortical neurons may be the

consequence of Gb association with aNaCh. Supporting the role of

the binding of Gb to aNaCh in the facilitation of Na+ current by

AMPA, both phenomena persisted in the absence of AMPA. Indeed,

the binding of Gb to aNaCh was observed in neuronal membranes

that were prepared in the absence of AMPA and the facilitation of

Na+ current also lasted several minutes after the AMPA withdrawal.

Contrasting with the results obtained in transfected non-neuronal

cells, we did not observe any persistent Na+ current nor any shift in

steady-state inactivation curve of Na+ current in neurons exposed to

AMPA. These discrepancies may result from the expression of an

excess of Gbg-subunits over free Ga-subunits or from the absence of

regulatory proteins associated with NaCh in the non-neuronal cells

used for tranfection experiments (Ma et al., 1997).

Our study highlights the importance of TTX-sensitive NaChs in the

regulation of the intracellular Ca2+ concentration in central neurons.

FIG. 3. AMPA receptor activation induces the association of Gb withaNaCh. (A) Neurons were exposed to sham treatment (None), DNQX100 mM or AMPA (30 mM) for 5 min and membrane proteins, solubilizedwith 1% Triton X-100, were immunoprecipitated with a polyclonal antibodyrecognizing aNaCh. Immunoprecipitated Gb and aNaCh were detected byWestern blotting. The total amount of Gb was determined in an aliquot ofthe crude membrane fraction in each experimental condition (bottom). Theillustrated experiment is representative of three independent experimentsperformed on different sets of cultured neurons. (B) Membrane proteins ofneurons challenged for 24 h with PTX (1 mg/mL) or for 5 min with eitherDNQX or AMPA were subjected to in vitro PTX-induced ADP-ribosylation.Data are representative of three experiments performed independently. (C)Neurons were incubated for 5 min in the absence or presence of eitherDNQX, AMPA, TTX (1 mM) or KCl (50 mM). In the experiments usingPTX, neurons were pretreated for 24 h with the toxin. For Na+- and Ca2+-free condition, cells were washed and incubated for 5 min in HEPES bufferwithout Ca2+ and containing choline chloride instead of NaCl. The amountof Gb co-immunoprecipitated with the anti-aNaCh antibody was determinedby densitometric analysis. Data are the mean 6 SEM of values obtained inthree experiments performed on different sets of cultured neurons and havebeen calculated as a percentage of immunoprecipitated Gb in neuronsexposed to AMPA in each experiment (hatched line). *P < 0.01 vs. DNQXin the corresponding condition. §P < 0.05 vs. AMPA alone (ANOVA

followed by Student's Newman±Keuls test). The above treatments did notchange the amount of Gb in crude membranes (data not illustrated).



Indeed, we show that AMPA receptor stimulation induces a Ca2+

in¯ux and the mobilization of mitochondrial Ca2+ stores through the

reverse function of mitochondrial Na+/Ca2+ exchanger, both being

governed by a Na+ in¯ux through voltage-gated NaChs. The role of

Gb association with aNaCh in AMPA-induced Ca2+ elevation in

neurons remains to be elucidated. As the mobilization of mitochon-

drial Ca2+ is entirely dependent on the activity of NaCh, binding of

Gb to aNaCh may contribute, at least in part, to this AMPA-evoked

Ca2+ response. Experiments performed with TTX suggest that Na+

in¯ux through NaCh but not AMPA receptors is suf®cient to alter the

Na+/Ca2+ exchanger. This result is consistent with our electrophy-

siological recordings showing that the amplitude of steady-state

AMPA currents measured at ±60 mV was 50±60-fold smaller than

that of Na+ currents evoked by a depolarizing pulse. The AMPA

current was, however, suf®cient to activate voltage-gated NaChs

through membrane depolarization. In fact, Na+ concentration may

only reach a level capable of altering Na+/Ca2+ exchanger mode at

the immediate submembrane space following activation of NaCh.

Because of the limited intracellular volume of neuronal cells, in

particular in processes, one can suppose that peri-mitochondrial Na+

concentration also reaches a level that reverses the Na+/Ca2+

exchanger function. This hypothesis is supported by a recent study

showing that the increase in cytosolic Ca2+ in hippocampal neurons,

during in vitro ischemia, is mediated by a sequence of events

involving Na+ entry through voltage-gated NaCh and activation of

mitochondrial Na+/Ca2+ exchanger (Zhang & Lipton, 1999).

The physiological consequences of the mobilization of mitochon-

drial Ca2+ remain to be fully elucidated. It has been shown that the

uptake of Ca2+ into mitochondria plays a predominant role in the

buffering of intracellular Ca2+ during glutamate challenge (White &

Reynolds, 1997). This process is critically regulated by the rate of

Ca2+ release from mitochondria via the Na+/Ca2+ exchanger, which is

dictated by intracellular Na+ concentration. Indeed, the increase in

recovery time-course of cytosolic Ca2+ with the intensity and duration

of glutamate stimulation is due to an enhanced ef¯ux of mitochon-

drial Ca2+ (White & Reynolds, 1997). AMPA receptor stimulation

and Na+ entry-mediated activation of mitochondrial Na+/Ca2+

exchanger also plays a major role in the sustained increase in

neuronal cytosolic Ca2+ during in vitro ischemia in the hippocampus

(Zhang & Lipton, 1999). This observation, together with the

protective effects of both AMPA receptor antagonists and Na+

channel blockers, including TTX, in cerebral ischemia (Weber &

Taylor, 1994; Yatsugi et al., 1996; Turski et al., 1998) suggests that

the enhanced ef¯ux of Ca2+ from mitochondria following a robust

Na+ entry may be a key determinant in ischemic neuronal damage.

Physical coupling of AMPA receptors with PTX-sensitive G-

proteins as well as a direct interaction of bg subunits of G-

proteins with aNaCh had already been described (Ma et al., 1994;

Wang et al., 1997). These ®ndings are extended by the present

study, which suggests a functional coupling between AMPA

receptors and voltage-gated NaCh through the binding of Gb to

aNaCh, a process that may play an important role in the control

of neuronal excitability and contribute to neuronal death following

cerebral ischemia.

Acknowledgements

This work was supported by grants of Institut National de la SanteÂ et de laRecherche MeÂdicale (INSERM), Centre National de la Recherche Scienti®que(CNRS) and ColleÁge de France.

Abbreviations

AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; DNQX, 6,7-dinitroquinoxaline-2,3-dione; G-protein, guanine-nucleotide binding- protein;NaCh, Na+ channel; PTX, Bordetella pertussis toxin; TTX, tetrodotoxin.

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