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Originally published In Press as doi:10.1074/jbc.M307772200 on December 1, 2003

J. Biol. Chem., Vol. 279, Issue 8, 6967-6975, February 20, 2004
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Cross-talk and Co-trafficking between {rho}1/GABA Receptors and ATP-gated Channels*

Éric Boué-Grabot{ddagger}§, Michel B. Émerit¶, Estelle Toulmé{ddagger}, Philippe Séguéla||, and Maurice Garret{ddagger}

From the {ddagger}CNRS Unité Mixte de Recherche 5543, Université Victor Segalen Bordeaux 2, 33076 Bordeaux cedex, France, INSERM U288, Hopital de la Salpétrière, 75013 Paris, France, and the ||Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada

Received for publication, July 18, 2003 , and in revised form, November 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory {rho}1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of {rho}1 subunits. We also show a physical interaction between P2X2 and {rho}1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular {rho}1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal activity is regulated by a number of transmitters acting on different receptor types (1). Fast neurotransmission is achieved through different classes of transmitter-gated channels, including the P2X and nicotinic receptor superfamilies (1, 2). The family of P2X ATP-gated cation channels is composed of seven genes coding for subunits with two transmembrane domains, intracellular N and C termini, and a large extracellular loop (2). The nicotinic superfamily includes the GABA-gated1 channels along with the acetylcholine, 5-HT3, and glycine receptors that share several structural features, including a large extracellular N-terminal domain, four hydrophobic transmembrane domains (M1-M4), and a long cytoplasmic loop connecting M3 and M4 (1). GABA receptor channels have been classified into two subtypes based on their pharmacological properties. GABAA receptors are inhibited by bicuculline, whereas GABAC receptors are insensitive to this antagonist (1). Diversity of GABAA receptors is achieved by pentameric assembly of multiple subunits, including {alpha}1-6, {beta}1-3, {gamma}1-3, {delta}, {theta}, and {epsilon}. GABAC receptors are composed of {rho}1-3 subunits that can assemble into homo-oligomers (3, 4). Recent data suggest that a {rho}1 subunit could co-assemble with a GABAA subunit (5, 6). Neuronal ATP and GABAC ionotropic receptors are involved in fast excitatory and inhibitory synaptic transmission, respectively, and display overlapping distribution in many regions of the nervous system, including DRG, dorsal horn of the spinal cord (7, 8), retina (9-13), hippocampus (14-15), cerebellum (14, 16), and anterior pituitary (17, 18).

Recently, Jo and co-workers described ATP and GABA co-release from the same axon terminals into the dorsal horn of the spinal cord and into hypothalamic neurons where they activate P2X and GABAA receptors, suggesting that a single synapse can be excitatory or inhibitory (19, 20). Functional cross-inhibition between receptors activated by ATP and either acetylcholine or GABA has been reported lately in neurons or transfected cells (21-25), and we recently demonstrated that a physical interaction between P2X and 5-HT3 receptors leads to an activity-dependent cross inhibition between the two cationic channels (26). We decided to explore the possibility that ligand-gated chloride channels may also interact with P2X ATP-gated channels through similar mechanisms.

Because P2X and {rho}1/GABA receptors are co-expressed in the central nervous system (7-18), we investigated potential molecular and functional interactions between these two types of transmitter-gated channels in heterologous expression systems. We report here an intracellular inhibitory cross-talk between P2X2 and GABAC {rho}1 receptors. Moreover, we also show a link between the physical coupling of these channels and their subcellular localizations in mammalian neurons.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructions—Wild-type rat P2X2, a C-terminal truncated form of P2X2 (P2X2TR), and wild-type rat {rho}1 clones were available from previous work (18, 27). Carboxyl-terminal epitope-tagged P2X2 subunits with either hexahistidine (His6)-tagged or enhanced YFP-tagged sequences and a C-terminal His6-tagged {rho}1 subunit ({rho}1-HIS) were obtained as described previously (28). N-terminal GFP-tagged {rho}1 subunit ({rho}1-GFP) was generated by insertion of two enhanced GFPs in tandem into a unique BamHI restriction site. This artificial BamHI site was created at amino acids positions Gly49-Ala50 by site-directed mutagenesis (QuikChange, Stratagene). The intracellular C-terminal domain of P2X2 (P2X2-CT) and the main intracellular loop (IL2) of the {rho}1 subunit were amplified by PCR using 5'- and 3'-primers incorporating an initiation methionine and a stop codon, respectively. All constructs were subcloned into pcDNA3 (Invitrogen) and verified by automatic dideoxy DNA sequencing.

Cell Culture—Oocytes were prepared as described previously (26). Stage V and stage VI oocytes were manually defolliculated before the microinjection (Nanoject II, Drummond Scientific) of 0.2 ng of cRNA coding for P2X2, YFP-tagged P2X2 (P2X2-YFP), or hexahistidine-tagged P2X2 (P2X2-HIS) and 15-25 ng of RNA coding for P2X2TR, GABA {rho}1, the HIS-tagged {rho}1 subunit ({rho}1-HIS), or the GFP-tagged {rho}1 subunit ({rho}1-GFP) to reach similar levels of expression. Then the oocytes were incubated in Barth's solution containing 1.8 mM CaCl2 and gentamycin (10 µg/ml; Sigma) at 19 °C for 1-5 days prior to electrophysiological recordings. For competition experiments, RNAs coding for minigenes were injected (50-60 ng of each to reach a 2:1 minigene/receptor ratio) independently, immediately after the injection of receptor RNA.

Neuronal cultures were performed according to a modification of the procedure described by Goslin et al. (29). Briefly, hippocampi of rat embryos were dissected at day 18. Dissociation was achieved with a Pasteur pipette after trypsinization. Cells were plated on poly-D-lysine-coated coverslips (Electron Microscopy Sciences) in complete Neurobasal medium supplemented with B27 (Invitrogen) containing 1 mM L-glutamine, penicillin G (10 units/ml), and streptomycin (10 mg/ml). Four hours after plating, the coverslips were transferred to dishes containing conditioned medium obtained by incubating the complete medium described above on glial cultures (70-80% confluency) for 24 h. The medium was partially changed every 3-4 days.

Hippocampal neurons were transfected at 7-8 days in vitro as follows. For each coverslip, a total of 1.5 µg of plasmid DNA was mixed with 50 µl of Neurobasal medium without B27 supplement. For cotransfection experiments, the proportions of each plasmid were first adjusted to reach similar levels of expression, and the plasmids were thoroughly mixed prior to the addition of 15 µl of PlusTM packaging reagent (Invitrogen). After 15 min of incubation, 1.5 µl of LipofectAMINE 2000 (Invitrogen) in 50 µl of Neurobasal medium was added, and incubation was continued for another 30 min. After the addition of 150 µl of complete Neurobasal medium containing B27 supplement, the mix was applied on the neuronal culture for 3 h at 37 °C. Expression was then conducted for 48 h in the original medium that was added back to the neurons. Under these conditions, 85-90% of transfected neurons were cotransfected with two plasmids.

Electrophysiology and Data Analysis—Two-electrode voltage clamp recordings were performed using glass pipettes (1-2 megaohms) filled with 3 M KCl solution. Oocytes were perfused at a flow rate of 10-12 ml/min with Ringer's solution (pH 7.4) containing 115 mM NaCl, 5 mM NaOH, 2.5 mM KCl, 1.8 mM CaCl2 or BaCl2, and 10 mM HEPES. Membrane currents were recorded through an OC-725B amplifier (Warner Instruments) and digitized at 500 Hz. All drugs (purchased from Sigma) were dissolved in the perfusion solution and applied using a computer-driven valve system (BPS8, Ala Scientific). We compared the peak of actual responses to the peak of predicted additive responses obtained with Axograph software (Axon Instruments). All recordings were made at room temperature. Statistical differences between means were assessed using Student's t test. Dose-response curves were fitted to the Hill sigmoidal equation, and EC50 values were determined by a non-linear regression analysis test using Prism 2.0 (Graphpad, San Diego, CA).

Co-purification and Western Blotting—Following the measurement of {rho}1-GFP alone, P2X2-YFP alone, P2X2-YFP + {rho}1-HIS, or P2X2-HIS + {rho}1-GFP expression levels by electrophysiological recordings, batches of 15 oocytes were homogenized in 10 mM HEPES and 0.3 M sucrose and solubilized in 0.8% Triton X-100 and protease inhibitors (Sigma) at 4 °C for 2 h. Total proteins were incubated overnight with equilibrated nickel resin (Qiagen) under agitation at 4 °C. After several washes with 20 mM imidazole, bound His6-tagged proteins were eluted with 0.5 M imidazole solution and then loaded onto a 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Immunostainings of associated tagged receptors were performed with anti-GFP antibodies (1:2500; Molecular Probes) at room temperature for 2 h followed by 1 h of incubation with anti-rabbit peroxidase-labeled secondary antibodies (1:2000, Jackson ImmunoResearch, West Groove, PA) for visualization by enhanced chemiluminescence (ECL, Amersham Biosciences).

Immunocytochemistry and Confocal Microscopy—Immunofluorescence was performed 9-10 days after plating and 2 days after transfection. Cells were washed with phosphate-buffered saline containing 0.1 mM CaCl2 and 0.1 mM MgCl2 (PBS+) at 37 °C and then fixed with paraformaldehyde (4%) containing 4% sucrose at 37 °C in PBS+. Incubation with the primary antibodies was performed in 2% bovine serum albumin, 3% normal goat serum, 3% normal donkey serum, and 0.1% Triton X-100 in PBS for 2 h at room temperature. As the intensity of anti-{rho}1 labeling was dependent on Triton X-100 concentration, the detergent could not be completely omitted. However, we found that 0.002% Triton X-100 significantly enhanced anti-{rho}1 immunostaining without labeling intracellular organelles. For external labeling, therefore, immunocytochemistry was performed on non-permeabilized cultures using 0.002% Triton X-100. However, because the intensity of anti-{rho}1 immunolabeling decreased from 0.1 to 0.002% Triton X-100, the proportion of surface versus total receptors could not be quantified. Labeling was performed with affinity-purified anti-{rho}1 (1:200) directed against the extracellular N-terminal sequence (16) and revealed with Cy3-conjugated donkey anti-rabbit for 1 h at room temperature (1:1000, Jackson ImmunoResearch). Alexa Fluor® 488-phalloidin (1:3000 to 1:10,000 dilutions, Molecular Probes) staining was occasionally performed together with the secondary antibody. Immunofluorescence images were generated using a Leica laser-scanning confocal microscope. Contrast and brightness were chosen to ensure that all relevant pixels were within linear range.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Reciprocal Cross-inhibition between GABAC and P2X2 Receptors—We co-expressed {rho}1 and P2X2 RNAs in Xenopus oocytes to investigate receptor function by two-electrode voltage clamp recordings (Vh = -60 mV; Fig. 1A). Application of a saturating concentration of GABA (10 µM) induced non-desensitizing inward responses (IGABA = -1.8 ± 0.3 µA, n = 27). Similarly, application of a saturating concentration of ATP (100 µM) evoked slowly desensitizing responses (IATP = -2.7 ± 0.2 µA, n = 27). Kinetic profiles of individual responses are consistent with activation of GABAC and P2X2 receptors, suggesting that activation of one receptor is not modified by the presence of the other (Fig. 1A). If both receptors were functionally independent, we would expect that co-activation of both receptors evoked responses corresponding to the sum of individual responses. However, co-application of 10 µM GABA + 100 µM ATP evoked inward responses (denoted as Actual on Fig. 1A) significantly smaller (p < 0.0005) than the sum of individual responses (trace labeled Predicted on Fig. 1A is the arithmetic sum of IGABA and IATP traces). Amplitude of IATP+GABA represents 73.4 ± 3% of the predicted current (Fig. 1A; n = 27). When GABA was applied during application of ATP, and, conversely, when ATP was applied during GABA application, significant inhibition (p < 0.0005) of the responses was also observed (Fig. 1B). Amplitude of IATP and GABA was 73 ± 4.5% and IGABA and ATP was 77 ± 4% of the predicted current (n = 16).



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  		FIG. 1.
Reciprocal cross-talk between {rho}1 GABAC and P2X2 channels expressed in Xenopus oocytes. A, co-application of ATP + GABA induced currents (Actual) significantly smaller than the arithmetic sum (Predicted) of the individual ATP and GABA responses. ***, p < 0.0005, n = 27. B, non-additivity occurs whether ATP application begins before or after the start of GABA application, n = 16. Amplitudes of responses are normalized to the predicted response from each cell. Holding membrane potential (Vh = -60 mV) was monitored during recordings as illustrated in panel A.

 
We showed previously, by recordings of oocytes expressing {rho}1 GABAC receptors and 5-HT3A receptors, that co-activation by simultaneous application of GABA (10 µM) and 5-HT (100 µM) evoked responses (IGABA + 5-HT = -2.7 ± 0.6 µA) corresponding to the sum of IGABA = -1.2 ± 0.4 µA and I5 - HT = -1.5 ± 0.2 µA, demonstrating that responses mediated by these two independent receptors are additive under similar experimental conditions (26). These results showed that {rho}1 and P2X2 channels do not function independently during co-activation and that the subunit-specific cross-talk induces rapid and reciprocal current occlusion.

Current Occlusion Was Observed at Low Receptor Densities and Was Not Due to Cross-activation—To check if the cross-inhibition was dependent on the density of receptors, the expression level of both receptors was decreased while the ratio of P2X2/{rho}1 subunits was kept constant. Co-application of ATP (100 µM) and GABA (10 µM) evoked currents with an amplitude (IATP + GABA = -0.4 ± 0.1 µA) that was significantly lower than the expected sum of currents evoked separately by ATP (IATP = -0.3 ± 0.1 µA) and by GABA (IGABA = -0.3 ± 0.1 µA; Fig. 2A). The amplitude of actual ATP + GABA responses represented 61.5 ± 2% of the predicted current (n = 8), indicating that current occlusion during co-activation of GABAC and P2X2 receptors was not related to artificially high receptor densities.



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  		FIG. 2.
Non-additivity at low channel densities and no cross-modulation or shift in agonist sensitivity in oocytes co-expressing P2X2 and GABAC channels. A, co-application of ATP + GABA induced currents (Actual) significantly smaller than the arithmetic sum (Predicted) of the individual ATP (100 µM) and GABA (10 µM) responses in oocytes expressing low levels of each receptor. B and C, superimposed current traces obtained with 10 µM of GABA, 100 µM of ATP, or a mixture of ATP and GABA from oocytes expressing P2X2 receptors alone (B) and homomeric {rho}1 receptors (C). D, ATP dose-response curves from oocytes expressing P2X2 alone (filled squares) or with {rho}1 (filled circles). Normalized ATP responses obtained from oocytes expressing P2X2 only and P2X2 + {rho}1 are presented in the inset. Duration of application was 5 s. E, GABA dose-response curves from oocytes expressing {rho}1 alone (filled squares) and with P2X2 (filled circles). Normalized GABA (10 µM) responses obtained from oocytes expressing {rho}1 only and P2X2 + {rho}1 are presented in the inset. Duration of application was 5 s. In panels D and E, mean peak currents were normalized to the maximal response (mean ± S.E.) from 5-7 oocytes. Vh = -60 mV.

 
Oocytes injected with P2X2 RNA only did not respond to 10-100 µM application of GABA, and responses to 100 µM ATP (IATP = -4.6 ± 0.1 µA, n = 6) were not modulated by co-application of GABA (IATP + GABA = -4.65 ± 0.1µA, n = 6) (Fig. 2B). Similarly, in oocytes expressing {rho}1 GABAC receptors only, the application of saturating concentration of ATP did not activate GABAC channels, and co-application of ATP + GABA-induced currents (IATP + GABA = -1.9 ± 0.4 µA, n = 5) were identical in kinetics and amplitude to GABA responses (IGABA = -1.9 ± 0.4 µA, n = 5; Fig. 2C). The ATP concentration-response curve of the P2X2 channel was not modified by co-expression with {rho}1 receptors (EC50 for ATP was 2.6 ± 1.2 and 4.3 ± 1.3 µM, n = 7, respectively, for oocytes expressing P2X2 alone and the P2X2 + {rho}1 subunit; Fig. 2D). Normalized ATP (100 µM) responses obtained from oocytes expressing P2X2 alone or {rho}1 + P2X2 indicated that P2X2 channel kinetics were not affected by the presence of {rho}1 receptors (Fig. 2D). Conversely, GABA concentration-response curves determined from oocytes expressing either {rho}1 receptor alone or {rho}1 + P2X2 (Fig. 2E) had similar EC50 (2.5 ± 1.9 and 1.7 ± 1.5 µM, respectively, n = 5). Kinetics of GABAC receptors were identical in the absence or presence of P2X2 receptors (Fig. 2E). These results showed that the cross-inhibition between GABAC and P2X2 receptor is not mediated by agonist cross-modulation or by a shift in agonist sensitivity and further indicated that functional cross-talk occurs only when both channels are co-activated.

Voltage, Ionic Flux Direction, and Ca2+ Independence—To investigate a potential voltage-dependence of the cross-talk, we recorded responses obtained from the same oocyte expressing {rho}1 and P2X2 channels after successive applications of ATP (100 µM), GABA (10 µM), and a mixture of both agonists at different holding potentials. Currents induced by co-application of ATP + GABA (actual) were significantly lower than the expected sum of individual ATP and GABA responses (prediction) at holding potential ranging from -80 to 20 mV (Fig. 3, A and B). Amplitude of IATP + GABA was 65 ± 6% at -80 mV, 70 ± 5% at -60 mV, 75 ± 4% at -40 and -20 mV, 60.3 ± 9% at 0 mV, and 52 ± 10% at 20 mV of the corresponding predicted current (n = 7).



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  		FIG. 3.
Current occlusion is not dependent on membrane potential or ion permeation. A, representative currents recorded from one oocyte co-expressing P2X2 and {rho}1 channels elicited by successive applications of ATP (100 µM), GABA (10 µM), and ATP + GABA at different holding potentials ranging from -80 to 20 mV in 20-mV steps. Prediction (Predicted) represents the arithmetic sum of individual ATP and GABA responses at each potential. B, co-application of ATP + GABA induced currents smaller than the sum of IATP and IGABA. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; n = 7. C, current-voltage relationship of P2X2 receptors (open squares), {rho}1 channels (filled squares), and from co-activation of both channels (gray filled circle). The predicted current-voltage relationship of {rho}1 + P2X2 channels is represented in black filled circles. Each point corresponds to the current amplitude mean ± S.E. obtained from 7 oocytes. Reversal potentials are indicated by arrows. D and E, superimposed currents induced by application of ATP (100 µM), GABA (10 µM), or a mixture of both agonists recorded from oocytes co-expressing P2X2 and GABAC receptors (n = 6). Non-additivity of ATP and GABA responses were observed at 30 mV holding membrane potential (D) and in calcium-free extracellular solution (E); ***, p < 0.0005.

 
P2X2 channels are non-selective cationic channels and display a current-voltage relationship with marked inward rectification and a reversal potential close to 0 mV (30), whereas {rho}1 channels are mainly permeable to Cl- and display a linear I-V relationship with a reversal potential close to -20 mV from oocytes recorded in normal ringer solution (31). In oocytes co-expressing GABAC and P2X2 channels, the experimental current-voltage relationship for P2X2 was inwardly rectifying, whereas that for {rho}1 channels was linear with reversal potential of +3.4 ± 1.3 mV for ATP and -19 ± 2 mV for GABA respectively (Fig. 3, A and C). Current-voltage relationship for actual and predicted ATP + GABA currents were linear with a slight inward rectification and displayed no difference in reversal potential (-6.7 ± 6 mV for actual and -7.6 ± 4 mV for predicted). These data indicated that both channels participated to inhibited ATP + GABA currents induced by co-application of ATP and GABA.

It has been proposed that cross-inhibition between P2X and GABAA receptors expressed in DRG neurons was mainly due to action of Cl- efflux (generated by GABA inward currents) on P2X channels and by Ca2+ entry on GABA receptors (25). We expressed P2X2 and {rho}1 in order to obtain outward currents with similar amplitudes. In these conditions, at 30-mV holding potential, we obtained similar responses to each agonist (IATP = 0.3 ± 0.1 µA and IGABA = 0.4 ± 0.1 µA, n = 6). ATP + GABA induced responses with amplitudes (IATP + GABA = 0.4 ± 0.1, n = 6) significantly smaller (p < 0.0005) than the arithmetic sum of IATP and IGABA (Fig. 3D). The ATP + GABA current represents only 51 ± 4% of the prediction, showing that current occlusion occurred during Cl- entry. P2X2 and {rho}1 GABA receptors also elicited non-additive responses when calcium ions (Ca2+) were removed (or substituted with Ba2+, not shown) from extracellular Ringer solution (Fig. 3E). ATP + GABA responses represent 56.7 ± 7% of the prediction (n = 6). Taken together, these results indicated that cross-inhibition is voltage-independent and did not arise from Cl- efflux via {rho}1 channels or Ca2+ entry via P2X2 channels.

Intracellular Domains of Both Receptors Are Involved in Non-additivity—To determine whether intracellular domains of receptors were involved in cross-inhibition between P2X2 and GABAC receptors, we first co-expressed {rho}1 subunits with P2X2TR, a C-terminal truncated form of P2X2 (Fig. 4A). Activation of P2X2TR with 100 µM ATP evoked inward responses with slow to fast desensitizing kinetics, depending on oocytes as shown previously (27). As for wild-type P2X2, P2X2TR receptors expressed alone in oocytes were not gated or modulated by the application of GABA (Fig. 4B). Co-activation of P2X2TR and {rho}1 channels evoked currents (IATP + GABA = -0.8 ± 0.2 µA), not significantly different (p > 0.5, n = 11) from the sum of the individual responses (IATP = -0.3 ± 0.1 µA and IGABA = -0.4 ± 0.1 µA). IATP + GABA represented 107 ± 3.5% of the prediction (Fig. 4C). Similarly, additive responses were recorded when GABA was applied during application of ATP and when ATP was applied during application of GABA (Fig. 4D), accounting for 97.2 ± 1 and 98.5 ± 0.6% of the predicted currents respectively (n = 5). These results showed that P2X2TR and {rho}1 receptors were independent, indicating a prominent role of the cytoplasmic domain of these channels in the cross-talk.



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  		FIG. 4.
Loss of cross-inhibition between truncated P2X2 and {rho}1 GABAC responses. A, schematic diagrams showing full-length P2X2 and C-terminal truncated P2X2 (P2X2TR) subunits. Boxes represent the transmembrane domains. B, representative superimposed currents obtained with individual or combined application of GABA (10 µM) and ATP (100 µM) from an oocyte expressing P2X2TR alone. C and D, current traces showing co-expression of P2X2TR and {rho}1 channels. C, simultaneous application of ATP + GABA (Actual) elicited responses that were not significantly different (ns; p > 0. 5, n = 11) than the arithmetic sum of individual ATP and GABA responses (Predicted). D, additives responses were also recorded when GABA was applied during ATP application (ATP and GABA) and when ATP was applied during GABA responses (GABA and ATP). Amplitudes of responses normalized to the predicted response from each oocyte. Vh = -60 mV.

 
To determine whether both intracellular domains were involved in cross-inhibition between P2X2 and {rho}1 channels, we examined the ability of minigenes coding for P2X2-CT, the C-terminal domain of P2X2 receptors (amino acids 374-472; Fig. 5A), and minigenes encoding the intracellular loop between the M3 and M4 transmembrane domains of {rho}1 subunits ({rho}1-IL2, amino acids 366-457; Fig. 5A) to disrupt the functional cross-talk by competition. As illustrated in Fig. 5, B and C, over-expression of P2X2-CT (Fig. 5B) or {rho}1-IL2 (Fig. 5C) significantly blocked the cross-inhibition observed between P2X2 and GABAC receptors. Co-application of ATP + GABA elicited currents that were not significantly different (p > 0.5) from the arithmetic sum of individual ATP and GABA applications. IATP + GABA, recorded in the presence of either P2X2-CT or {rho}1-IL2, represented 101.4 ± 6% (n = 10) and 100.4 ± 3% (n = 7), respectively, of the predicted sum. These data confirm that interactions between cytoplasmic domains are critical for the reciprocal current occlusion between P2X2 and GABAC receptors.



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  		FIG. 5.
Disruption of the functional cross-talk between P2X2 and {rho}1 GABAC channels by over-expression of cytoplasmic domains. A, schematic diagrams showing the full-length P2X2 and {rho}1 subunits and minigenes encoding the P2X2 C-terminal domain, P2X2-CT, or the large cytoplasmic loop of the {rho}1 subunit, {rho}1-IL2. B and C, inward currents evoked by ATP (100 µM), GABA (10 µM), and by both agonists (Actual) in oocytes co-expressing wild-type P2X2 and {rho}1 receptors in the presence of a minigene encoding either P2X2-CT (B) or {rho}1-IL2 (C). ATP + GABA (Actual) responses were not significantly different from the arithmetic sum of individual ATP and GABA responses (Predicted ns; p > 0.5) in the presence of minigenes. Numbers in parentheses indicate numbers of recorded cells.

 
Physical Coupling and Co-trafficking of {rho}1 and P2X2 Receptors—To assess the existence of complexes containing P2X2 and GABAC receptors by using affinity purification and Western blot, we first verified cross-inhibition in oocytes expressing P2X2-HIS and {rho}1-GFP channels (Fig. 6A1) or, conversely, expressing {rho}1-HIS and P2X2-YFP channels (Fig. 6B1). Currents recorded by co-application of ATP + GABA represented 69.7 ± 6.7% (Fig. 6, A1) and 77.6 ± 4% (Fig. 6, B1) of the prediction (p < 0.05, n = 5), showing that the addition of tags did not affect the functional cross-talk between P2X2 and GABAC receptors. After affinity purification of P2X2-HIS in total protein extracts from oocytes on nickel resin and labeling using polyclonal anti-GFP antibodies, we detected a band corresponding to the expected size of {rho}1-GFP (Fig. 6A2). Reciprocally, after purification of {rho}1-HIS, a band corresponding to P2X2-YFP was revealed by anti-GFP antibodies (Fig. 6B2), suggesting that GABAC receptors form a stable complex with P2X2 receptors.



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  		FIG. 6.
Physical association between P2X2 and {rho}1 GABAC receptor channels. A1 and B1, nonadditive currents recorded in oocytes co-expressing P2X2-HIS and {rho}1-GFP (A1) and P2X2-YFP and {rho}1-HIS during the application of ATP (100 µM), GABA (10 µM), and both agonists (Actual). Bars show means ± S.E. of response amplitudes normalized to the predicted response from each cell (*, p < 0.05, n = 5 for each). A2 and B2, Western blot with anti-GFP antibodies. B1, before and after purification (IP) of P2X2-HIS on nickel resin in total protein extracts from oocytes injected (+) with {rho}1-GFP alone, with {rho}1-GFP + P2X2-HIS, or non-injected (-). B2, before and after purification (IP) of {rho}1-HIS on nickel resin in total protein extracts from oocytes injected (+) with P2X2-YFP alone, with {rho}1-HIS + P2X2-YFP, or non-injected (-). Numbers on the right indicate molecular masses in kilodaltons.

 
To challenge the hypothesis that the cross-inhibition implicated receptor-receptor interactions and, consequently, subunit colocalization, we visualized GABAC and P2X2 receptors in transfected hippocampal neurons. Wild-type and tagged {rho}1 and P2X2 receptors were first expressed in hippocampal neurons at 7 days in vitro to compare their subcellular distribution. No significant differences in addressing were noticed between wild-type and tagged receptors, indicating that the GFP/YFP tags did not affect receptor trafficking (not shown). The homomeric GABAC receptors exhibited a punctate distribution restricted to the close vicinity around cell bodies and proximal dendrites in agreement with an earlier report (32). Confocal microscopy, associated with Alexa Fluor 488-phalloidin staining to label the F-actin cytoskeleton and reveal the outline of the cell, showed that most puncta were intracellular (Fig. 7A). This observation was confirmed by surface labeling of the extracellular {rho}1 epitope. At lower concentrations of Triton X-100 (0.002%), the extracellular component of the {rho}1 immunolabeling could be visualized (Fig. 7B). In this condition, very low levels of uniform immunoreactivity were detected on cell bodies and proximal dendrites, suggesting that only a minor fraction of {rho}1 subunits were located at the cell surface. Thus, GABAC receptors expressed in hippocampal neurons exhibit a distribution compatible with a predominant intracellular localization.



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  		FIG. 7.
Colocalization of GABAC {rho}1 receptors with P2X2 in transfected neurons and modification of {rho}1 receptors targeting in the presence of P2X2. Hippocampal neurons were transfected with {rho}1 subunits alone (A and B) or cotransfected with P2X2-YFP subunits (C-E). GABAC receptors were labeled with anti-{rho}1/Cy3 antibodies in permeabilized (A, C, and D) or non-permeabilized (B and E) neurons. In the absence of P2X2-YFP subunits, neurons were counterstained with Alexa Fluor 488-phalloidin to label the F-actin cytoskeleton and visualize neuronal shape. Note the co-clustering of {rho}1 and P2X2-YFP (indicated by arrowheads) and the redistribution of {rho}1 subunits to the plasma membrane in neurons expressing P2X2-YFP subunits by comparison of panels A and B or D and E. Scale bars, 10 µm.

 
In contrast to GABAC receptors, we found P2X2-YFP receptors uniformly distributed throughout the neurons, with a predominant surface localization consistent with previous reports (33). YFP fluorescence was not restricted to proximal dendrites but extended to the very extremities of neuronal processes (Fig. 7C, middle). This localization was divided into diffuse and clustered distribution, with a typical topology suggestive of P2X receptors at the plasma membrane, occasionally associated in dendritic clusters (Fig. 7D, middle).

In 40-50% of neurons that were cotransfected with {rho}1 and P2X2-YFP, significant differences in GABAC receptor distribution were noticed (Fig. 7C) when compared with neurons transfected with {rho}1 alone (Fig. 7A). In conditions of co-expression, whereas P2X2-YFP receptor topology seemed unaffected by the presence of the {rho}1-subunit, GABAC receptors were translocated to the cell surface and colocalized with P2X2-YFP clusters (Fig. 7, C and D). Co-clustering of {rho}1 and P2X2-YFP subunits was particularly obvious on proximal dendrites (Fig. 7D). The external immunolabeling of {rho}1 subunits appeared to overlap with P2X2-YFP fluorescence, indicating that the addressing of GABAC receptors was changed by the presence of P2X2-YFP receptors and that the two receptors were largely colocalized in surface clusters (Fig. 7E). Furthermore, {rho}1 immunofluorescence was found more distally to the cell body, indicating that the targeting of GABAC receptors was also following the one exhibited by P2X2-YFP subunits. The specificity of this effect was verified by transfecting neurons with {rho}1 GABAC and 5-HT3A receptors; no changes in subcellular labeling were observed following co-transfection (not shown). It can be concluded from these results that, in neurons expressing both receptors, P2X2 receptors regulate the addressing of GABAC receptors in a dominant way by inducing their translocation from an internal vesicular compartment to surface clusters common to both receptors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
No functional or biochemical interaction was previously described between identified subtypes of excitatory and inhibitory ligand-gated channels. We demonstrate here a functional cross-talk and a physical coupling between P2X2 and {rho}1 GABAC receptors. A reciprocal current inhibition (25-50%) was recorded from oocytes co-expressing homomeric P2X2 and {rho}1 channels when they were simultaneously activated, showing that these two unrelated subtypes of receptor channels are not functionally independent. The percentage of current occlusion is identical whatever the sequence of agonist application (co-application, GABA during application of ATP, or vice versa), indicating that each type of receptor has an equal propensity to inhibit the other one. Similarities in kinetics (i.e. activation and desensitization) between recorded and predicted currents (see Fig. 1A) induced by the co-application ATP + GABA suggested that the cross-talk closed an important proportion of both receptors. Application of ATP did not gate or modulate GABAC receptors, and GABA did not activate or modulate P2X2 receptors, showing that nonspecific cross-activation or cross-desensitization between one type of transmitter and the other type of receptor could not explain the cross-inhibition observed during co-application. A similar reciprocity of cross-inhibition was observed between P2X2 and nicotinic or 5-HT3 receptors (24, 26). Interestingly, an inhibitory cross-talk between P2X and GABAA channels reported in dorsal root ganglion neurons indicates a more pronounced inhibitory effect of GABA receptors on P2X channels than the reverse (25). It was proposed that the chloride efflux generated by GABA-gated channels could inhibit P2X receptors and, inversely, that the Ca2+ influx mediated by P2X channels could inhibit the GABA-gated channels (25). It is unlikely that these mechanisms play a major role in the functional interaction between the P2X2 and GABAC receptors studied here, because we observed a reciprocal current occlusion when the oocytes co-expressing both channels are clamped between -80 to 30 mV (above the reversal potential of chloride ions) and in the absence of extracellular calcium ions. The cross-talk was also recorded at low channel densities, giving rise to small current amplitudes at saturating concentrations of agonists and suggesting a significant affinity of coupling between P2X2 and GABAC receptors.

Co-expression of {rho}1 subunits with a functional truncated version of the P2X2 subunit lacking most of its C-terminal domain resulted in receptor independence, as indicated by current additivity. Moreover, an excess of minigene coding for the C-terminal domain of P2X2 or for the main intracellular loop (IL2) of {rho}1, co-expressed with P2X2 and GABAC receptors, resulted also in the loss of functional cross-talk despite normal individual current phenotypes. The results of these mutagenesis and competition experiments underlined the importance of the intracellular domains of both receptor subunits in this cross-inhibition. The crucial role of cytoplasmic domains was also demonstrated in the functional coupling between P2X and 5-HT3 receptor channels. However, in competition experiments these cytoplasmic domains were not able to disrupt physical association between 5-HT3 and P2X receptors (26). We also observed co-purification of GABAC and P2X2 receptors expressed in oocytes. No clear sequence homology exists between the sequences of the large intracellular loops of members of the nicotinic receptor superfamily and between the C-terminal domains of the different P2X family members. Taken together, this led us to formulate the hypothesis of the existence of two distinct mechanisms involved in the physical and functional coupling between these two families of ligand-gated channels. To identify putative sequences required in both mechanisms, the propensity of other members of P2X family to interact with GABAC or different subunit combinations of GABAA receptors should now be investigated.

Our immunolocalization experiments in the primary culture of neurons revealed that expression of the {rho}1 subunits was mainly intracellular and compatible with a prominent vesicular localization. Whether these vesicular compartments are part of the routing mechanism involved in {rho}1 subunit trafficking, an early endosomal compartment involved in subunit recycling, or a lysosomal network remains to be determined. Co-expression with P2X2 resulted in a dramatic change in the distribution of GABAC receptors that were translocated to the cell surface and colocalized with P2X2 clusters. When co-expressed with P2X2, the {rho}1 receptor was also found more distally to the cell body, indicating that the radial topology of GABAC was driven by P2X2 receptors. The co-targeting in transfected neurons and the co-purification of P2X2 and {rho}1 receptor channels expressed in oocytes demonstrate the existence of physical complexes formed by the constitutive association of P2X2 and GABAC receptor channels in a different expression system.

Our findings suggest also that, besides the regulation of neuronal activity by modulation of current amplitude during co-activation, physical association between receptors could regulate the location and the number of surface receptors, which is an important means for synaptic neuronal regulation. Recent studies showed that P2X1 and P2X4 receptors undergo rapid agonist-dependent internalization and recycling, contrary to P2X2, which appeared to be more stable at the cell surface (33-36). The molecular mechanism of P2X receptor trafficking has yet to be elucidated despite identification of some associated intracellular proteins (37, 38). Dynamic regulation of GABAA and GABAC receptor numbers at the membrane is regulated by interactions with several proteins. For example, gephyrin and dystrophin have been shown to play a role in the anchoring of GABAA receptors, although no physical interaction with gephyrin has been demonstrated in vitro (39, 40). GABARAP, a microtubule-associated protein, and Plic-1, an ubiquitin-related protein, interact with GABAA receptor subunits and are involved in receptor trafficking (41, 42). MAP1B is a large protein that binds actin and tubulin and is the only GABAC receptor binding partner identified so far. Whereas expression of the {rho}1 subunit in COS cells produced diffuse surface and intracellular labeling, coexpression with MAP1B resulted into a punctuate intracellular localization of {rho}1 receptor (43). Proteins associated with P2X or GABA receptors bind directly to the same cytoplasmic domains critical for their cross-inhibition, suggesting that these proteins and/or cytoskeleton could also participate in the functional coupling.

The P2X2 receptor subunit is widely expressed in the nervous system, whereas the {rho}1 subunit shows a restricted expression profile. Both of these subunits have been detected in the same regions, suggesting that they are co-localized in the same neurons (7-18). Lately, the {rho}1 subunit was immunolocalized in DRG neurons and in lamina I and II of the spinal superficial dorsal horn, and {rho}1-containing receptors have been shown to modulate nociceptive transmission (8). However, it is still unclear if {rho}1 forms homooligomeric receptors or if it associates with GABAA subunits to form a hybrid receptor/channel (8). P2X2 receptors are also expressed in these neuronal populations and have also been shown to be involved in modulation of pain/sensory pathways in spinal cord (44). Physical association between {rho}1 and P2X2 receptors could ensure a co-clustering on the soma of DRG neurons, on primary nociceptive afferent fibers, or in interneurons constituting lamina II. The resulting cross-inhibition could modify the balance between ATP and GABA signaling and, consequently, could contribute to normal or pathological pain/sensory transduction.

Accumulating data suggest that, besides modulation through intracellular second messenger cascades, direct interactions may regulate receptor signaling. This has been illustrated by interactions between different G-protein-coupled receptors (45), between GPCRs and ligand-gated channels (46, 47), and between different ligand-gated channels (Refs. 26 and 48 and this work). Moreover, our findings show also that, in addition to a functional role in modulation of receptor activity, receptor-receptor interaction may regulate receptor trafficking and targeting. Thus, interactions between distinct neuronal ligand-gated channels, excitatory or inhibitory, appear to be a widespread mechanism for the fine regulation of fast transmission in peripheral and central synapses.


   FOOTNOTES
 
* This work was supported by grants from CNRS and Région Aquitaine (to E. B.-G. and M. G.), INSERM (to M. B. E.), and INSERM-Fonds de Recherche en Sante du Quebec (FRSQ) (to E. B.-G. and P. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Laboratoire de Neurophysiologie, CNRS UMR 5543, Université Victor Segalen Bordeaux 2, 33076 Bordeaux cedex, France. Tel.: 33-5-57-57-16-86; Fax: 33-5-56-90-14-21; E-mail: eric.boue-grabot{at}umr5543.u-bordeaux2.fr.

1 The abbreviations used are: GABA, {gamma}-aminobutyric acid; GABAA, GABA subtype A; GABAC, GABA subtype C; 5-HT, serotonin (5-hydroxytryptamine); DRG, dorsal root ganglia; GFP, green fluorescent protein; YFP, yellow fluorescent protein; CT, carboxyl terminal; IL2, main intracellular loop (of {rho}1); HIS, hexahistidine (His6); P2X2TR, C-terminal truncated form of P2X2; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Marie-Françoise Odessa for her technical assistance and Drs. Yassar Chakfe and Jim Deuchars for helpful comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barnard, E. A. (1996) Trends Pharmacol. Sci. 17, 305-309[CrossRef][Medline] [Order article via Infotrieve]
  2. North, R. A. (2002) Physiol. Rev. 82, 1013-1067[Abstract/Free Full Text]
  3. Bormann, J. (2000) Trends Pharmacol. Sci. 21, 16-19[CrossRef][Medline] [Order article via Infotrieve]
  4. Bonnert, T. P., McKernan, R. M., Farrar, S., LeBourdelles, B., Heavens, R. P., Smith, D. W., Hewson, L., Rigby, M. R., Sirinathsinghji, J. S., Brown, N., Wafford, K. A., and Whiting, P. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9891-9896[Abstract/Free Full Text]
  5. Pan, Z. H., Zhang, D., Zhang, X., and Lipton, S. A. (2000) Eur. J. Neurosci. 12, 3137-3145[CrossRef][Medline] [Order article via Infotrieve]
  6. Qian, H., and Ripps, H. (1999) Proc. R. Soc. Lond. Ser. B Biol. Sci. 266, 2419-2425[Medline] [Order article via Infotrieve]
  7. Vulchanova, L., Riedl, M. S., Shuster, J., Buell, G., Surprenant, A., North, R. A., and Elde, R. (1997) Neuropharmacology 36, 1229-1242[CrossRef][Medline] [Order article via Infotrieve]
  8. Zheng, W., Xie, W., Zhang, J., Strong, J. A., Wang, L., Yu, L., Xu, M., and Lu, L. (2003) J. Biol. Chem. 278, 48321-48329[Abstract/Free Full Text]
  9. Wheeler-Schilling, T. H., Marquordt, K., Kohler, K., Jabs, R., and Guenther, E. (2002) Brain Res. Mol. Brain Res. 76, 415-418
  10. Brandle, U., Guenther, E., Irrle, C., and Wheeler-Schilling, T. H. (1998) Brain Res. Mol. Brain Res. 59, 269-272[Medline] [Order article via Infotrieve]
  11. Koulen, P., Brandstatter, J. H., Kroger, S., Enz, R., Bormann, J., and Wassle, H. (1997) J. Comp. Neurol. 380, 520-532[CrossRef][Medline] [Order article via Infotrieve]
  12. Enz, R., Brandstatter, J. H., Wassle, H., and Bormann, J. (1996) J. Neurosci. 16, 4479-4490[Abstract/Free Full Text]
  13. Koulen, P., Brandstatter, J. H., Enz, R., Bormann, J., and Wassle, H. (1998) Eur J. Neurosci. 10, 115-127[Medline] [Order article via Infotrieve]
  14. Kanjhan, R., Housley, G. D., Burton, L. D., Christie, D. L., Kippenberger, A., Thorne, P. R., Luo, L., and Ryan, A. F. (1999) J. Comp. Neurol. 407, 11-32[CrossRef][Medline] [Order article via Infotrieve]
  15. Wegelius, K., Pasternack, M., Hiltunen, J. O., Rivera, C., Kaila, K., Saarma, M., and Reeben, M. (1998) Eur. J. Neurosci. 10, 350-357[CrossRef][Medline] [Order article via Infotrieve]
  16. Boue-Grabot, E., Roudbaraki, M., Bascles, L., Tramu, G., Bloch, B., and Garret, M. (1998) J. Neurochem. 70, 899-907[Medline] [Order article via Infotrieve]
  17. Stojilkovic, S. S., Tomic, M., Van Goor, F., and Koshimizu, T. (2000) Biochem. Cell Biol. 78, 393-404[CrossRef][Medline] [Order article via Infotrieve]
  18. Boue-Grabot, E., Taupignon, A., Tramu, G., and Garret, M. (2000) Endocrinology 141, 1627-1632[Abstract/Free Full Text]
  19. Jo, Y. H., and Sclichter, R. (1999) Nat. Neurosci. 2, 241-245[CrossRef][Medline] [Order article via Infotrieve]
  20. Jo, Y. H., and Role, L. W. (2002) J. Neurosci. 22, 4794-4804[Abstract/Free Full Text]
  21. Barajas-Lopez, C., Espinosa-Luna, R., and Zhu, Y. (1998) J. Physiol. 513, 671-683[Abstract/Free Full Text]
  22. Zhou, X., and Galligan, J. J. (1998) J. Physiol. 513, 685-697[Abstract/Free Full Text]
  23. Searl, T. J., Redman, R. S., and Silinsky, E. M. (1998) J. Physiol. 510, 783-791[Abstract/Free Full Text]
  24. Khahk, B. S., Zhou, X., Sydes, J., Galligan, J. J., and Lester, H. A. (2000) Nature 406, 405-410[CrossRef][Medline] [Order article via Infotrieve]
  25. Sokolova, E., Nistri, A., and Giniatullin, R. (2001) J. Neurosci. 21, 4958-4968[Abstract/Free Full Text]
  26. Boué-Grabot, E., Barajas-Lopez, C., Chakfe, Y., Blais, D., Bélanger, D., Emerit, M. B., and Séguéla, P. (2003) J. Neurosci. 23, 1246-1253[Abstract/Free Full Text]
  27. Boué-Grabot, E., Archambault, V., and Séguéla, P. (2000) J. Biol. Chem., 275, 10190-10195[Abstract/Free Full Text]
  28. Lê, K. T., Boué-Grabot, E., Archambault, V., and Séguéla, P. (1999) J. Biol. Chem., 274, 15415-15419[Abstract/Free Full Text]
  29. Goslin, K., Asmussen, H., and Banker, G. (1998) in Culturing Nerve Cells (Banker, G., and Goslin, K., eds) pp. 339-370, MIT Press, Cambridge, MA
  30. Brake, A. J., Wagenbach, M. J., and Julius, D. (1994) Nature 371, 519-523[CrossRef][Medline] [Order article via Infotrieve]
  31. Wotring, V. E., Chang, Y., and Weiss, D. S. (1999) J. Physiol. 521, 327-336[Abstract/Free Full Text]
  32. Cheng, Q., Burkat, P. M., Kulli, J. C., and Yang, J. (2001) J. Neurophysiol. 86, 2605-2615[Abstract/Free Full Text]
  33. Bobanovic, L. K., Royle, S. J., and Murrell-Lagnado, R. D. (2002) J. Neurosci. 22, 4814-4824[Abstract/Free Full Text]
  34. Li, G. H., Lee, E. L., Blair, D., Holding, C., Poronnik, P., Cook, D. I., Barden, J. A., and Bennett, M. R. (2000) J. Biol. Chem. 275, 29107-29112[Abstract/Free Full Text]
  35. Dutton, J. L., Poronnik, P., Li, G. H., Holding, C. A., Worthington, R. A., Vandenberg, R. J., Cook, D. I., Barden, J. A., and Bennett, M. R. (2000) Neuropharmacology 39, 2054-2066[CrossRef][Medline] [Order article via Infotrieve]
  36. Royle, S. J., Bobanovic, L. K., and Murrell-Lagnado, R. D. (2002) J. Biol. Chem. 277, 35378-35385[Abstract/Free Full Text]
  37. Kim, M., Jiang, L. H., Wilson, H. L., North, R. A., and Surprenant, A. (2001) EMBO J. 20, 6347-6358[CrossRef][Medline] [Order article via Infotrieve]
  38. Gendreau, S., Schirmer, J., and Schmalzing, G. (2003) J. Chromatogr. 786, 311-318[Medline] [Order article via Infotrieve]
  39. Giustetto, M., Kirsch, J., Fritschy, J. M., Cantino, D., and Sassoe-Pognetto, M. (1998) J. Comp Neurol. 395, 231-244[CrossRef][Medline] [Order article via Infotrieve]
  40. Knuesel, I., Mastrocola, M., Zuellig, R. A., Bornhauser, B., Schaub, M. C., and Fritschy, J. M. (1999) Eur. J. Neurosci. 12, 4457-4462
  41. Bedford, F., Kittler, J., Muller, E., Thomas, P., Uren, J., Merlo, D., Wisden, W., Triller, A., Smart, T., and Moss, S. (2001) Nat. Neurosci. 4, 908-916[CrossRef][Medline] [Order article via Infotrieve]
  42. Wang, H., Bedford, F. K., Brandon, N. J., Moss, S. J., and Olsen, R. W. (1999) Nature 397, 69-72[CrossRef][Medline] [Order article via Infotrieve]
  43. Hanley, J. G., Koulen, P., Bedford, F., Gordon-Weeks, P. R., and Moss, S. J. (1999) Nature 397, 66-68[CrossRef][Medline] [Order article via Infotrieve]
  44. Dunn, P. M., Zhong, Y, and Burnstock, G (2001) Prog. Neurobiol. 65, 107-134[CrossRef][Medline] [Order article via Infotrieve]
  45. Bouvier, M. (2001) Nat. Rev. Neurosci. 2, 274-286[CrossRef][Medline] [Order article via Infotrieve]
  46. Liu, F., Wan, Q., Pristupa, Z. B., Yu, X. M., Wang, Y. T., and Niznik, H. B. (2000) Nature 403, 274-280[CrossRef][Medline] [Order article via Infotrieve]
  47. Lee, F. J., Xue, S., Pei, L., Vukusic, B., Chery, N., Wang, Y., Wang, Y. T., Niznik, H. B., Yu, X. M., and Liu, F. (2002) Cell 111, 219-230[CrossRef][Medline] [Order article via Infotrieve]
  48. Li, Y., Wu, L. J., Legendre, P., and Xu, T. L. (2003) J. Biol. Chem. 278, 38637-38645[Abstract/Free Full Text]

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