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J. Biol. Chem., Vol. 279, Issue 28, 29628-29638, July 9, 2004

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Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors^*

Séverine Chaumont, Lin-Hua Jiang¶, Aubin Penna, R. Alan North¶, and Francois Rassendren||

From the Département de Pharmacologie, Laboratoire de Génomique Fonctionnelle, CNRS UPR2580, 141 Rue de la Cardonille, 34396 Montpellier, France and ^¶Institute of Molecular Physiology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom

Received for publication, April 8, 2004 , and in revised form, April 29, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Extracellular ATP-gated channels (P2X receptors) define the third major family of ionotropic receptors, and they are expressed widely in nerve cells, muscles, and endocrine and exocrine glands. P2X subunits have two membrane-spanning domains, and a receptor is thought to be formed by oligomerization of three subunits. We have identified a conserved motif in the cytoplasmic C termini of P2X subunits that is necessary for their surface expression; mutations in this motif result in a marked reduction of the receptors at the plasma membrane because of a rapid internalization. Transfer of the motif to a reporter protein (CD₄) enhances the surface expression of the chimera, indicating that this motif is likely involved in the stabilization of P2X receptor at the cell surface. In neurons, mutated P2X₂ subunits showed reduced membrane expression and an altered axodendritic distribution. This motif is also present in intracellular regions of other membrane proteins, such as in the third intracellular loop of some G protein-coupled receptors, suggesting that it might be involve in their cellular stabilization and polarization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
P2X receptors are extracellular ATP-gated ion channels; they define the third major class of ligand-gated channels together with the glutamate and the nicotinic superfamilies (1). P2X receptors are involved in synaptic transmission in the central and peripheral nervous systems, but in contrast to other ligand-gated channels, they are also widely expressed in peripheral tissues where they participate in physiological processes as diverse as smooth muscle contraction, secretion, and bone resorption. ATP-induced currents have been recorded from a large number of different cell types (2), but the molecular identity of P2X receptors responsible for these currents often remains elusive because of limitations of pharmacological tools to discriminate the different subtypes of P2X channels.

Seven P2X subunit genes (P2X₁-P2X₇) are found in the human genome; this number seems to be an accurate estimation of the extent of the P2X family in mammalian species, although in zebrafish two additional subunits appear to exist (2, 3). The basic structural determinants of P2X channels have been established by numerous molecular studies (4). P2X subunits have a membrane topology with two transmembrane domains linked by a large extracellular loop; N and C termini are localized intracellularly (5, 6). To form a channel, P2X subunits are thought to associate as homo- or hetero-oligomers, composed of three subunits (7, 8) organized in a head-to-tail orientation around a central pore (9). This model is consistent with studies that show that the permeation pathway is associated with transmembrane regions (10-12), and with the fact that the gating of the channel is in part due to movements of the subunits relative to the each other (12). Conserved charged residues in the extracellular loop have been implicated in ATP binding (13, 14), and desensitization is tightly linked to transmembrane domains and intracellular regions localized immediately below the plasma membrane (15, 16).

Signals that regulate intracellular trafficking of P2X receptors are not well understood. In P2X₄ subunits, an atypical endocytosis motif responsible for the rapid recycling of the receptor has been identified in the C terminus of the channel (17, 18); however, the existence of subcellular trafficking signals in other P2X subunits has not been reported. Yet evidence suggests that additional trafficking signals are present in P2X subunits. For example, transiently expressed P2X₆ subunits are not properly addressed to the cell surface presumably because of a glycosylation defect (19), and the presence of a retention signal in the rat P2X₅ subunit has been suggested to explain its poor functional expression (20). Different mutations that alter surface expression of P2X subunits have also been reported. Disrupting some of the conserved disulfide bridges of the extracellular loop of P2X₁ subunits alters their normal trafficking to the cell surface (21); similarly, deletion of the three glycosylated asparagines of the P2X₂ subunits induces an intracellular retention and a complete loss of function of the receptors (5, 22). However, these mutations are likely to alter the folding of the protein, and the trafficking defects might rather be due a failure to pass the quality check test that normally takes place in the endoplasmic reticulum than due to a trafficking defect per se. In the human P2X₇ receptor a dibasic amino acid motif (23) and a polymorphism (Ile⁵⁶⁸ to Asn) (24), both located in the C-terminal tail of the protein, have been shown to be necessary for the proper trafficking of the channel. No mechanistic explanations have been put forward to explain these trafficking defects.

In the present study, we have identified a motif in the C-terminal tail of P2X subunits that is conserved in almost all subunits known to date. By using a chemiluminescence assay (25, 26) to measure the cell surface expression of P2X subunits, we show that in HEK¹ cells and in neurons this motif is involved in the stabilization at the plasma membrane of all P2X subunits tested (P2X₂, P2X₃, P2X₄, P2X₅, and P2X₆). We also provide evidence that this motif might be important for the polarized expression of P2X₂ receptors in neurons.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Insertions of Extracellular Tags on P2X Receptors—Tags were introduced by overlapping PCR as described previously (8) in the extracellular loop of P2X subunits, all at similar positions; FLAG tags were inserted in P2X₂ and P2X₆ between Asp⁷⁸-Lys⁷⁹ and Glu⁸³-Lys⁸⁴, respectively; P2X₃ has a Myc tag inserted between residues Asn¹⁰⁴ and Arg¹⁰⁵; P2X₄ has an HA tag between Gln⁷⁸ and Leu⁷⁹; and P2X₅ has either a Myc or an HA tag inserted between Thr⁷⁸ and Thr⁷⁹. In all cases tag insertions did not alter the function of the different P2X subunits measured electrophysiologically. All constructs were confirmed by DNA sequencing.

Mutagenesis—Mutations were introduced as described previously (10) by using the QuickChange (Stratagene) protocol. A mutagenized fragment was excised by restriction digestion and subcloned into the respective backbone P2X subunit. All mutants were confirmed by sequencing.

Construction of CD₄ Chimeras—Human cDNA genes (hCD₄-AAXX, hCD₄-KKXX, and hCD₄-GFP-KKXX) were the kind gift of B. Schwappach (Zentrum fur Molekulare Biologie, University of Heidelberg). The C termini of P2X₂, P2X_2b, and P2X₃ were amplified with a forward primer containing an in-frame NotI site. NotI-XbaI fragments were generated and subcloned in the different CD₄ plasmids (CD₄-AAXX, -KKXX, and CD₄-GFP-KKXX). All the clones were subsequently verified by sequencing.

Chemiluminescent Assay—Cells were transfected in 35-mm dishes. Twenty four hours after transfection, each plate was divided into 4 wells of a 12-well plate. Assays were carried out 48 h after transfection. Cells were fixed in 4% paraformaldehyde for 5 min, and the cells in two of four wells in each case were permeabilized using 0.1% Triton X-100. Cells were incubated in blocking solution PBS, 1% fetal calf serum for 30 min and incubated with primary antibody for 1 h at room temperature. After extensive washing, cells were incubated for 20 min at room temperature with a secondary antibody coupled to peroxidase (1/1000, anti-mouse peroxidase-conjugated, Amersham Biosciences). Luminescence was measured using Supersignal enzyme-linked immunosorbent assay femto-maximum sensitivity substrate (Pierce) and quantified in a Victor 2 luminometer (PerkinElmer Life Sciences).

For FLAG- and HA-tagged constructs, we used a primary antibody directly coupled with peroxidase (1/4000, anti-FLAG M2-peroxidase (Sigma), and 1/2000, anti-HA peroxidase, clone 12CA5 (Roche Applied Science)), thus eliminating the need for a secondary antibody. For other constructs, we used Myc antibody (1/1000, monoclonal anti-Myc antibody clone 9E10, Developmental Studies Hybridoma Bank) or CD₄ antibody (1/100, monoclonal anti-hCD₄, Immunotech). Surface expression was calculated as the ratio between the signal obtained for nonpermeabilized cells (representing the amount of proteins at the cell surface) and the signal obtained for permeabilized cells (which represents the total cellular amount of proteins).

Cell Culture and Transfections—HEK293 cells and COS cells were cultured in Dulbecco's modified Eagle's medium/Hepes containing 10% fetal calf serum, 1% GlutaMAX, and 1% penicillin plus streptomycin. Transfections were carried out in 35-mm dishes with a maximum of 1 µg of plasmid DNA, using LipofectAMINE 2000 (Invitrogen) for HEK293 cells and JetPei (Qbiogen) for COS cells, according to the manufacturers' protocols. Hippocampal cultures were prepared from E17 to E18 mice and grown in B27-supplemented neurobasal medium (Invitrogen) on poly-L-ornithine and laminin pre-coated coverslips as described previously. Ten-day-old cultures were transfected with P2XGFP fusion protein using LipofectAMINE 2000 (Invitrogen); 1.5 µg of DNA and 1.5 µl of LipofectAMINE 2000 were each mixed with 50 µl of neurobasal medium for 15 min and then mixed together and incubated for 30 min at room temperature. After addition of 150 µl of neurobasal B27, the mixture was applied to the neuronal culture for 2 h at 37 °C, and then replaced by fresh neurobasal B27. Neurons were studied 48 h later. Data were collected from at least three different dishes prepared from at least three different cultures and transfections.

Immunofluorescence on HEK293 Cells—Immunofluorescence experiments were carried out 48 h after transfection. Cells were fixed in 4% paraformaldehyde for 5 min, blocked in PBS, 0.5% BSA for 30 min, and incubated for 1-2 h at room temperature with the primary antibody (1/1000, anti-FLAG M2 biotinylated, Sigma) in blocking solution, and then for 20 min at 37 °C with streptavidin Texas Red (1/2000, Amersham Biosciences). For labeling living cells, cells were incubated in complete culture medium for 1 h with the primary antibody at 37 °C followed by incubation for 20 min at 37 °C with streptavidin Texas Red prior to fixation.

Immunofluorescence on COS Cells and Hippocampal Neurons—Forty eight hours after transfection, COS cells were fixed for 10 min at 37 °C in 4% paraformaldehyde, 4% sucrose. Cells were then washed in PBS, 0.3% BSA, and 50 mM glycine and permeabilized at 37 °C in blocking solution (PBS, 0.3% BSA, and 0.05% saponin). Cells were incubated in blocking solution containing primary antibody at room temperature for 2 h, and after an extensive wash, cells were incubated for 20 min at 37 °C with secondary antibody. For cells transfected with P2X₂-GFP fusion, anti-calreticulin (1/200, Alexis Biochemicals) and Cy3-conjugated anti-rabbit secondary antibody (1/1000, Jackson ImmunoResearch) were used. For cells transfected with CD₄-GFP-KKXX and FLAG-P2X₂ receptors, we used biotinylated anti-FLAG M2 antibody (1/1000, Sigma) and streptavidin-Texas Red (1/2000, Amersham Biosciences). Fluorescence was visualized on a Leica DMRA2 epifluorescent microscope using a x40 oil immersion objective. Images were acquired using a cool-snap HQ (photometrics) digital camera.

Internalization Experiments—HEK293 cells stably expressing P2X₂, P2X₂[K366A], or P2X₂[Y362A] were grown on polyornithine-coated coverslips for 24 h. To monitor receptor internalization, cells were incubated at 37 °C for 30 min in Dulbecco's modified Eagle's medium/Hepes containing monoclonal anti-FLAG M2 antibody (1/1000, Sigma) and 100 µg/ml leupeptin. The cells were then placed on ice and fixed in 4% paraformaldehyde for 5 min. Coverslips were transferred in a PBS solution containing 0.3% BSA and 192 mM glycine for 15 min at room temperature to quench paraformaldehyde fluorescence. After an additional 30 min of blocking in PBS, 0.3% BSA, surface staining was achieved for 20 min at 37 °C with an FITC-conjugated anti-mouse secondary antibody (1/1000, Chemicon) in PBS, 0.3% BSA blocking solution. Cells were then permeabilized for 5 min in 0.01% Triton X-100 and internalized receptors-antibodies complexes were measured following a 20-min incubation at 37 °C in the presence of a Cy3-conjugated anti-mouse secondary antibody (1/2000, The Jackson Laboratory) in PBS, 0.3% BSA. Fluorescence was visualized using a Leica TCS SP2 confocal inverted microscope with a Nikon 63 x 1.32 HCX planapochromatic objective. FITC was excited with an argon laser at 488 nm; Cy3 was excited with a helium/neon at 543 nm. Images were collected sequentially to avoid cross-contamination between fluorochromes. Series of optical sections were scanned at a 1024 x 1024 pixels resolution and collected in the Leica confocal software. 8-Bit TIFF images were imported and analyzed with ImageJ 1.62 (NIH software).

Electrophysiological Recordings—HEK293 cells were used to express the wild type and mutant P2X receptors with green fluorescent protein as described above. A plasmid ratio of 1/5 was adopted for co-expression of P2X₂ and P2X₃ subunits. Whole cell recordings were carried out at room temperature using an EPC9 patch clamp amplifier (HEKA Elektronik, Germany) as detailed previously and briefly described below. Membrane potential of the patched cells was held at -60 mV. Extracellular solution contained (in mM) 147 NaCl, 2 KCl, 1 MgCl₂, 2 CaCl₂, 10 Hepes, and 13 glucose. Intracellular solution contained (in mM) 147 NaF, 10 Hepes, and 10 EGTA. These solutions were maintained at pH 7.3 and 300-315 mosmol^-1. Agonists were applied using an RSC 200 fast-flow delivery system (Bio-Logic Science Instruments, France). The current responses from individual cells were normalized to cell membrane capacitance (in a range of 8-20 pF) and presented as current density (in pA/pF). All data are presented where appropriate as mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of an Intracellular Motif Implicated in the Surface Expression of the P2X₂ Receptor—The C-terminal regions of rat P2X subunits have different lengths, ranging from 27 amino acids for P2X₆ to 240 amino acids for P2X₇. As shown in Fig. 1A, their alignment in the immediate juxtamembrane region shows little homology, but two amino acids are completely conserved. The motif YXXXK is located eight residues downstream of the end of the second transmembrane domain in all except the P2X₇ subunit, where an extra 18-amino acid cysteine-rich domain is intercalated. In most subunits the residue preceding the conserved lysine is also a basic residue; therefore, we investigated if this motif YXXXK could be implicated in P2X receptor trafficking. To that goal we used P2X subunits in which epitope tags were introduced in the extracellular loop in a position that did not noticeably modify receptors properties (8).

View larger version (50K): [in this window] [in a new window]   FIG. 1. A conserved motif is involved in surface expression of P2X2 subunits. A, partial alignment of the second transmembrane and proximal cytoplasmic regions of the seven rat P2X subunits. The membrane-spanning region is indicated by the horizontal bar; conserved residues are indicated by amino acids above the alignment. YXXXK is the only conserved motif found in cytoplasmic C-terminal sequence of P2X subunits. B, mutations of the conserved C-terminal motif decrease P2X2 surface expression. Wild type P2X2, P2X2[K366A], and P2X2[Y362A] were expressed in HEK cells; all subunits carried an extracellular FLAG epitope (see "Experimental Procedures"). Immunostaining was performed on living cells using a biotinylated anti-FLAG antibody and streptavidin Texas-Red prior to cells fixation. Staining of mutant P2X2 subunits was strongly reduced compared with wild type subunits. Note the typical punctated staining that is obtained when the antibody is incubated with living cells. Insets show FLAG immunostaining on fixed and permeabilized cells. No difference could be observed between cells expressing wild type or mutant PX22 subunits. C, quantification of P2X2 surface expression. Chemiluminescence assay was performed on transfected HEK cells using peroxidase-coupled anti-FLAG antibody. Black bars represent luminescence signals from permeabilized cells, and gray bars represent luminescence signals from nonpermeabilized cells transfected with Wt and mutant P2X2 subunits. On nonpermeabilized cells, the [K366A] and [Y362A] mutations induce a strong reduction of the luminescence signal compared with wild type P2X2; this is not seen with permeabilized cells. rlu, relative luminescent units. D, alanine-scanning of the P2X2-proximal C-terminal tail. All residues from positions 360 to 369 were mutated to alanine, and their surface expression was expressed as the ratio of luminescence signals from permeabilized and nonpermeabilized cells; this gives a direct measure of the proportion of subunits present at the cell surface. Note that only [K366A] and [Y362A] mutants have a reduced surface expression. In all panels bars indicate means ± S.E. ***, p < 0.001, Student's t test.

As shown in Fig. 1C, in nonpermeabilized conditions the luminescent signal was strongly reduced for cells expressing P2X₂[Y362A] or P2X₂[K366A] subunits as compared with cells transfected with Wt P2X₂. No difference in the total amount of mutant protein was observed. The ratio of nonpermeabilized state/permeabilized state indicates that 73 ± 16% (n = 9) of Wt P2X₂ receptor were present at the plasma membrane but only 34 ± 5.5% (n = 7) and 28 ± 4.5% (n = 7) for P2X₂[Y362A] and P2X₂[K366A], respectively. The double mutant P2X₂[Y362A,K366A] did not show any further decrease in the receptor surface expression (data not shown).

We next studied whether other residues in the vicinity of the YXXXK motif could also interfere with the surface expression of the P2X₂ subunit. Residues downstream of the second transmembrane region, from Lys³⁶⁰ to Lys³⁶⁹, were individually mutated to alanine, and their membrane expression was examined. As shown in Fig. 1D, none of these mutants showed any decreased membrane expression nor did the mutation to alanine of the two leucines (Leu³⁵⁴ and Leu³⁵⁵) located at the end of the second transmembrane domain (data not shown). These results indicate that the YXXXK motif plays an important role in the trafficking of the P2X₂ subunit to the plasma membrane.

Specificity of the YXXXK Motif and Functional Characterization of Mutant Subunits—Additional amino acid substitutions were introduced at the Tyr³⁶² and Lys³⁶⁶ positions (Tyr to Phe; Lys to Arg, Lys to Gly, and Lys to Gln). All mutants were individually transfected in HEK cells, and membrane expression was quantified by chemiluminescent assay. Fig. 2A shows that all mutants had reduced surface expression ranging from 22 ± 6.2% (n = 3) for K366Q to 43 ± 3.5% (n = 3) for Y362F; even when the conservative substitutions Y362F and K366R were introduced, surface expression of the receptor was still decreased. These values were not different from what was observed for membrane expression of Y362A and K366A.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Tyr362 and Lys366 are obligatory residues for surface and functional expression of P2X2 receptors. Different amino acids substitutions were introduced at the Tyr362 and Lys366 positions in the P2X2 subunit. A, quantification of the surface expression of P2X2 mutants by chemiluminescence assay. Note that even conservative changes such as Y362F or K366R show reduced surface expression. B, mutations of Tyr362 and Lys366 residues impair P2X2 channels function. Currents were recorded following applications of 30 µM ATP for 2 s (horizontal black bars). Currents could only be recorded from cell transfected with Y362A and Y362F P2X2 mutants. Note the difference in the current scale. The sensitivity to ATP for these mutants was unchanged (not shown). Note the marked desensitization of the currents for P2X2[Y362A]. C, current densities of P2X2[Y362A] and P2X2[Y362F] were reduced to 30% of Wt P2X2 currents. D, [K366A] mutation does not affect subunit oligomerization. Co-immunoprecipitation was performed between co-expressed P2X2 Wt and [K366A] subunits. The immunoprecipitation (IP) was performed with anti-FLAG antibody and immunoblot (IB) with anti-Myc antibody. Combinations of subunits that were transfected are indicated above the blot. In all situations, co-immunoprecipitation of subunits was obtained indicating that the K366A mutation does not impair subunit assembly. Bars indicate means ± S.E. ***, p < 0.001; **, p < 0.002 Student's t test.

The lack of function of P2X₂ Lys³⁶⁶ mutants might be due to a disruption of the oligomeric organization of the channel and subsequently its trafficking to the cell surface. To test this hypothesis we performed co-immunoprecipitation between Wt P2X₂ and P2X₂[K366A] subunits. Receptor complexes were immunoprecipitated through the FLAG epitope present on one subunit, and the presence of the second subunit was detected because of its Myc epitope. In all situations, including the homomeric P2X₂[K366A] complex, co-immunoprecipitation of subunits was successfully obtained (Fig. 2D). These results demonstrate that the decreased surface expression of the P2X₂[K366A] subunit and its lack of function cannot be attributed to an inability of the mutant to oligomerize with itself or with other subunits.

The YXXXK Motif Regulates Surface Expression of Other P2X Subunits—The YXXXK motif is conserved in almost all P2X subunits cloned to date; therefore, we asked whether the mutation of this motif in other P2X subunits would also impair their surface expression. Mutations to alanine of the Tyr and Lys residues were individually introduced in Myc-P2X₃, HAP2X₄, HA-P2X₅, and FLAG-P2X₆ subunits. Membrane expression of these different mutants and of their respective wild type subunits was measured by the chemiluminescence assay (Fig. 3A). Mutation of either tyrosine or lysine residues in all P2X subunits caused a large decrease of surface expression when compared with the respective wild type subunits; surface expression levels of mutated subunits were all in the same range, except for P2X₆: P2X₃[Y353A] 27 ± 5.3% (n = 3), P2X₃[K372A] 23 ± 0.3% (n = 3), P2X₄[Y367A] 36 ± 6.4% (n = 4), P2X₄[K370A] 27 ± 7.2% (n = 4), P2X₅[Y369A] 34 ± 6% (n = 5), P2X₅[K372A] 37 ± 4.3% (n = 5), and P2X₆[K365A] 58 ± 3.5% (n = 4). In all cases, the total cellular amount of protein was not altered (data not shown), indicating that a decrease of the stability mutant P2X subunits could not be accounted for the observed decrease in their surface expression.

View larger version (16K): [in this window] [in a new window]   FIG. 3. The YXXXK motif regulates surface expression of other P2X receptors. Mutations to alanine of the conserved tyrosine and lysine residues were introduced in P2X3, P2X4, P2X5, and P2X6 subunits carrying extracellular epitopes tags. A, surface expression of mutant P2X subunits were analyzed by chemiluminescence assays and normalized to the surface expression of their respective Wt subunits. All mutant P2X subunits have a reduced surface expression that ranges around 35% of the Wt subunits (note that for P2X6 60% of the protein is present at the cell surface but total amounts of the Wt P2X6 subunit were very low compared with the others). Mutant P2X3 and P2X4 subunits are not functional. Currents induced by ATP (30 µM) were recorded from HEK cells transfected with Wt and mutant P2X3 (B) or P2X4 (C) subunits. ATP did not elicit currents in any cell even at a concentration up to 1 mM. P2X5 and P2X6 Wt subunits did not express functionally. Bars indicate means ± S.E. ***, p < 0.001; **, p < 0.05 Student's t test.

Surface Expression of Mutant P2X Subunit Is Rescued by Wild Type Subunits—P2X₂ and P2X₃ subunits form well characterized hetero-oligomeric receptors with pharmacological and biophysical properties that are clearly distinct from homo-oligomeric P2X₂ and P2X₃ channels (28, 29). We studied the trafficking of hetero-oligomeric P2X receptors formed by the association of wild type and trafficking deficient subunits. As show in Fig. 4A, when FLAG-P2X₂[K366A] was co-expressed with nontagged P2X₃ subunits, a strong surface immunostaining of the mutant P2X₂ was recovered. Quantification of the recovery by chemiluminescent assay confirmed that when P2X₂[K366A] was co-expressed with P2X₃,80 ± 6.7% (n = 4) of the total cellular amount of the subunit was expressed at the cell surface, compared with only 32 ± 1.0% (n = 4) when P2X₂[K366A] was expressed alone. Similar results were found when P2X₂[Y362A] was co-expressed with P2X₃ (data not shown). In the converse experiment, when the Myc-tagged P2X₃[K357A] subunit was co-expressed with the nontagged P2X₂ subunit, its surface expression rose from 23 ± 0.3% (n = 3) (P2X₃[K357A]) to 79 ± 4.9% (n = 3) (P2X₃[K357A]/P2X₂) (Fig. 4C).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Surface expression of trafficking deficient P2X subunits are rescued by co-expression with wild type subunits in heteromeric channels. Wt and mutant P2X2 and P2X3 subunits were co-expressed in HEK cells; mutant subunits carried an extracellular epitope tag. A, surface immunostaining of P2X2[K366A] is rescued by co-expression with Wt P2X3. Live cell immunostaining was performed as described in Fig. 1. Almost no P2X2[K366A] staining is observed when the subunit is expressed alone (left panel), whereas strong immunoreactivity appears when P2X2[K366A] and P2X3 are co-expressed (right panel). B, differential functional rescue of P2X2[K366A] and P2X3[K357A] mutants in heteromeric P2X2/3 channels. Wt or mutant P2X2 and P2X3 subunits were co-expressed in HEK cells. Currents induced by -me-ATP (30 µM; horizontal bars) were recorded 2 days later. From left to right, -me-ATP induced stable currents at Wt P2X2/3 and P2X2[K366A]/3 heteromeric channels. No current could be induced at P2X2/3[K357A] receptors even when concentrations of -me-ATP up to 1 mM were used. -me-ATP currents at homo-oligomeric P2X2 and P2X3 channels are shown; note that P2X2[K366A]/3 currents present faster desensitization compared with Wt P2X2/3. C, surface expression of P2X2[K366A] and P2X3[K357A] subunits is rescued in the hetero-oligomeric channel. Surface expression of mutated P2X2 and P2X3 was quantified as indicated in Fig. 1. When subunits were co-expressed, only the mutated one carried an extracellular FLAG (P2X2) or Myc (P2X3) tag. Both P2X3 and P2X2 mutant surface expressions are rescued in hetero-oligomeric channels. Results are expressed relative to the surface expression of Wt subunits. Bars indicates means ± S.E., ***, p < 0.001 Student's t test.

Mutation of the YXXXK Motif Does Not Induce Retention of P2X₂ Subunits in the Endoplasmic Reticulum—Immunofluorescence and luminescence data clearly indicated that P2X₂[Y362A] and P2X₂[K366A] subunits as well as the other P2X mutant subunits were not expressed at the cell surface but were present intracellularly. One possibility is that the mutation of the YXXXK motifs impaired the transport of the receptor to the plasma membrane and induced its retention in an intracellular compartment. We therefore investigated if Wt and mutant P2X₂ subunits showed different intracellular localization. COS-7 cells were transiently transfected with P2X₂-GFP fusion constructs containing or not containing the [Y362A] or the [K366A] mutations. Cells were fixed 48 h after transfection, and endoplasmic reticulum was labeled with an anti-calreticulin antibody for co-localization experiments. As shown in Fig. 5A, in COS-7 cells expressing P2X₂-GFP, the protein is localized at the plasma membrane as well as in intracellular compartments that are labeled by the anti-calreticulin antibody. In cells transfected with mutant P2X₂-GFP subunits, no GFP signal could be resolved at the plasma membrane, whereas it clearly co-localizes with calreticulin. No difference was observed in the intracellular localization of Wt and mutant subunits. This was further observed when a CD₄-GFP fusion protein that carried a C-terminal KKXX ER retention motif (25) was co-expressed with FLAG-P2X2 Wt or mutant subunits (data not shown). In addition, co-labeling experiments with markers of the trans-Golgi network did not reveal any differences between Wt and mutant P2X₂ subcellular localization. Because in transient transfection the synthesis of the recombinant protein is constant, it is difficult to discriminate using classical immunofluorescent approaches between proteins that are retained in intracellular compartments such as the endoplasmic reticulum from proteins being normally shipped to their terminal location. To circumvent this problem, we performed glycosylation analyses to monitor a possible ER-to-Golgi transport defect in mutant P2X₂ subunits. Fig. 5B illustrates pulse-chase assays that demonstrate that both Wt and mutant P2X₂ subunits were able to acquire endoglycosidase H-resistant carbohydrates after a 1- or 3-h chase period. No differences in the endoglycosidase H sensitivity could be observed between Wt and mutant subunits at these different time points, ruling out a difference in the rate of transport between compartments of mutant and Wt subunits. These results indicate that mutant P2X subunits are not retained in the ER and that their transport is not likely to be affected.

View larger version (42K): [in this window] [in a new window]   FIG. 5. Mutant P2X2 subunits are not retained in the endoplasmic reticulum. A, co-localization of P2X2-GFP C-terminal fusion proteins with the ER marker calreticulin. Co-localization experiments were performed 48 h after transfection of COS cells with either wild type P2X2-GFP, [K366A], and [Y362A] mutants. Wt P2X2-GFP fluorescence is observed at the cell surface as well as in intracellular compartments where it overlaps with calreticulin staining. P2X2-GFP mutant subunits are not present at the plasma membrane. No difference could be observed in the intracellular localization of Wt and mutant P2X2 subunits that clearly co-localized with calreticulin in the Golgi apparatus (perinuclear staining). Cells that have no GFP fluorescence have not been transfected. B, mutant P2X2 subunits acquire mature glycosylation. Pulse-chase analysis was performed in transfected HEK cells using S35-labeled methionine. After different times, chase protein extracts were treated or not with endoglycosidase H. At time 0, glycosylation of all P2X2 subunits can be removed by endoglycosidase H as indicated by the mobility shift on the autoradiograph. Endoglycosidase H-resistant sugars are added after an hour chase. Note that Wt and mutant subunits show a similar pattern of glycosylation.

View larger version (28K): [in this window] [in a new window]   FIG. 6. P2X2 mutant subunits are not stabilized at the cell surface. A, subunit internalization was studied in HEK stable cell lines expressing P2X2 Wt or mutant subunits. All subunits carried an extracellular FLAG epitope. Cells were incubated with anti-FLAG antibody for 30 min at 37 °C. Surface-expressed receptor was detected using a FITC-labeled secondary antibody prior to cell fixation. Internalized receptors were visualized following fixation with a Cy3-labeled secondary antibody. Images are confocal optical sections of the different fluorophores. Both P2X2[Y362A] and [K366A] subunits show intense intracellular staining compared with Wt P2X2-expressing cells. Line scan fluorescence analysis (right panel) indicates that surface expression of mutant subunits is drastically reduced, whereas the intracellular signal is increased compare with Wt subunits. A.U., arbitrary units. B, the P2X YXXXK motif enhances CD4 surface expression. Fusion between CD4 and the C-terminal tails of P2X subunits was expressed in HEK cells. Surface expression of CD4 and CD4-P2X C-terminal fusions were analyzed by chemiluminescence assay. Top panel, schematic describing the different CD4 constructions used and their orientation in the plasma membrane. CD4 carries an AAXX sequence at its C-terminal extremity and is normally exported to the cell surface. Wt and mutated C-terminal tail of P2X2, splice variant P2X2b, and P2X3 were fused to the C terminus of CD4. Bottom panel shows surface expression of CD4 C-terminal fusions expressed as % of CD4-AAXX membrane expression. All CD4 fused to the Wt C-terminal P2X tails showed an enhanced surface expression. When the YXXXK motif was mutated surface expressions of CD4 fusions were not different from CD4-AAXX (CD4-P2X3 and CD4-P2X2b) or decreased in the case of CD4-P2X2. In this latter case the use of the splice variant P2X2b indicates that a retention signal in the splice exon is likely to exist. Bars indicates means ± S.E. **, p < 0.005 Student's t test. Tmd, transmembrane domain.

Trafficking Deficient P2X₂ Subunits Have Altered Surface Expression and Axodendritic Localization in Neurons—P2X receptors are expressed in different types of neurons in the central and peripheral nervous systems. We have therefore determined whether the surface expressions of mutant P2X₂ subunits were similarly affected in neurons. Cultures (9-10 days) of embryonic hippocampal neurons were transfected with Wt or mutant FLAG P2X₂-GFP cDNAs and studied 48 h later. Surface expression of receptors was studied using an anti-FLAG antibody on nonpermeabilized neurons, whereas total receptor expression was visualized using GFP fluorescence. As shown in Fig. 7A, in neurons transfected with Wt P2X₂-GFP, FLAG staining (corresponding to cell surface-expressed receptors) and GFP fluorescence (corresponding to all receptors) were found to be nearly identical in appearance. Fluorescence was observed in varicosities even in distal regions of neuronal processes. However, when neurons were transfected with either P2X₂[Y362A]-GFP or P2X₂[K366A]-GFP subunits, a strong reduction of extracellular FLAG staining was observed in the varicosities; the GFP signal remained not different from the control. The number of varicosities that were double positive for FLAG and GFP signals were quantified in neurons transfected with Wt and mutants P2X₂ receptors. Only 14 ± 2.9% (n = 13) and 11 ± 2.4% (n = 24) of varicosities were double positive for P2X₂[Y362A] or P2X₂[K366A] mutants, respectively, whereas the corresponding figure for Wt P2X₂ was 43 ± 4.2% (n = 25). This clearly indicates that in neurons mutant P2X₂ subunits have a reduced membrane expression, although their transport to varicosities did not seem to be affected.

View larger version (50K): [in this window] [in a new window]   FIG. 7. Mutant P2X2 subunits have a reduced surface expression and an altered polarization in transfected neurons. 10-Day cultures of embryonic hippocampal neurons were transfected with Wt and mutant P2X2-GFP subunits carrying an extracellular FLAG epitope. Neurons were fixed 48 h after transfection. A, reduced surface expression of P2X2 mutant subunits in neurons. Left panel, total and cell surface distribution of P2X2 subunits were visualized through GFP fluorescence and FLAG immunostaining on nonpermeabilized cells, respectively. Both Wt and mutant P2X2 subunits localized in varicosities even in distal regions of processes. Wt but not mutant subunits are expressed at the cell surface of varicosities. Arrows indicate processes that are only GFP positives. Right panel, the number of double fluorescent varicosities were quantified using the Image J software as indicated under "Experimental Procedures." Bars indicate means ± S.E. **, p < 0.005 Student's t test. B, altered polarization of mutant P2X2 subunits in neurons. Co-localization of Wt and mutant P2X2 subunits and the dendritic marker MAP2 was analyzed in transfected neurons. Wt P2X2 subunit P2X2 is expressed only in MAP2-positive processes; however, both mutated P2X2 subunits were present in MAP2-negative processes as indicated by arrows. Images are representative of results obtained from at least three independent cultures.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A Conserved Intracellular Motif Regulates the Surface Expression and the Function of P2X Subunits—Sequences of the cytoplasmic C-terminal regions of P2X subunits are quite variable in length and are minimally related to each other. However, alignment of the subunits in the region close to the end of the second transmembrane domain shows that a tyrosine and a lysine separated by three amino acids are conserved. This defines a YXXXK motif present in 39 of the 41 full-length P2X sequences found in data bases. This motif is not found only in two zebrafish subunits (zP2X₂ and zP2X₅₁₄) (3), but it is found in the three Dictyostelium P2X-related sequences.

Our present results demonstrate that the YXXXK motif is responsible for appropriate surface expression of P2X₂, P2X₃, P2X₄, P2X₅, and P2X₆ receptors. Mutation to alanine of either conserved residue also leads to a loss of function of most the mutant channels. Although we cannot exclude that the loss of function and the reduced surface expression of mutant P2X subunits are directly related, several lines of evidence suggest that this is not the case. Indeed, three mutants, P2X₂[Y362A], P2X₂[Y362F], and P2X₁[Y363F] (31), retained residual activity (see Fig. 2) even though their surface expression was reduced. In addition, hetero-oligomeric channels formed by the association of Wt P2X₂ and mutant P2X₃ subunits are still not functional, although both subunits have a normal surface expression (see below). This excludes a direct causality between the lack of functional expression of mutated subunits and their trafficking deficit. We cannot interpret the lack of function of the mutant subunits. One possibility is that these residues are involved in the regulation of the desensitization of the channels. Indeed, in the case of the P2X₂[Y362A] mutant, we observed that a single application of 30 µM ATP completely desensitizes the channel. Similar findings were made previously on the P2X₁[Y363F] mutant (31). In addition, several studies (15, 27) have demonstrated that the rate of desensitization of P2X₂ currents is modulated when mutations are introduced in the region downstream of the second transmembrane domain. It is possible that mutations introduced in the YXXXK motif in P2X subunits induce conformational changes to the receptor that corresponds to a desensitized state. Alternatively, the YXXXK motif might be necessary for interactions with cytoskeletal proteins that normally regulate the desensitization of these channels. Such interactions have been shown to regulate the desensitization of P2X₁ receptors (32).

Trafficking Rescue of Mutant P2X Subunits—P2X receptors are oligomers probably composed of three subunits (7) that assemble either as homo-oligomers or hetero-oligomers. P2X_2/3 hetero-oligomeric channels have been extensively studied (28, 29). Based on biochemical and functional evidence, it has been proposed recently that the P2X_2/3 receptor is formed by the head-to-tail association of one P2X₂ and two P2X₃ subunits (9). Our results show that the surface expression of P2X₃[K357A] can be efficiently rescued by co-expression with the Wt P2X₂ or P2X₃ subunits. By taking the proposed arrangement of the P2X_2/3 subunits as valid, this suggests that one P2X₂ subunit is sufficient to stabilize the whole channel complex at the cell surface. Similar rescue of the surface expression of trafficking deficient ion channels by Wt subunits has been reported for the homo-oligomeric Kir2.1 and hetero-oligomeric Kir3 inward rectifier potassium channel (33). Kir channels have a tetrameric subunit organization; however, the number of Wt Kir subunits that are sufficient to drive the surface expression of the channel complex has not been documented. Our results suggest that one could be sufficient if the trafficking of P2X receptors and Kir channels follows the same general rules.

Functional Rescue of Trafficking Deficient P2X Receptors Is Subunit-dependent—Hetero-oligomeric P2X_2/3 channels have unique biophysical and pharmacological properties (sensitivity to the agonist -me-ATP and slow desensitization kinetics) that make them easily distinguishable from the homo-oligomeric P2X₂ and P2X₃ receptors. Our results show that co-expression of P2X₃ with P2X₂[K366A] rescues the lack of function of the mutated P2X₂ subunits in a heteromeric channel. A similar rescue is observed with zebrafish P2X₂ and P2X₃ subunits (3). zP2X₂ has no function when expressed as a homo-oligomeric channel, presumably because it lacks the trafficking motif in its C-terminal tail and/or a critical lysine implicated in ATP binding (13); on the other hand, ATP induces typical P2X₃-like fast desensitizing currents at zP2X₃. When both zP2X₂ and zP2X₃ subunits are co-expressed, ATP induces non-desensitizing currents typical of P2X_2/3 receptors. These results as well as our presents findings provide strong evidence that in the hetero-oligomeric P2X_2/3 channel the P2X₂ subunit is not directly implicated in ATP-induced gating but rather that binding to the P2X₃ subunit alone is responsible for the channel activation. This is further supported by the observation that hetero-oligomeric P2X_2/3[K357A] channels could not be activated by -me-ATP. The most probable interpretation for the lack of functional rescue of the P2X₃[K357A] subunit by Wt P2X₂ is that P2X₂ and P2X₃ subunits do not participate equally in the receptor complex and that at least two functional subunits are necessary for a channel to gate normally. An alternative explanation is that the P2X₃[K357A] channel is not able to confer -me-ATP sensitivity to the hetero-oligomeric channel but can still be activated by ATP. This is unlikely because homo-oligomeric P2X₃[K357A] channels are not activated by 30 µM or 1 mM ATP (see Fig. 3, and data not shown). However, this question will be difficult to address experimentally because in co-expression experiments ATP does not discriminate between homo-oligomeric P2X₂ and hetero-oligomeric P2X_2/3 channels.

Mutation of the YXXXK Motif Destabilized Surface Expression of P2X₂ Subunits—Mutations of the YXXXK motif reduce the surface expression of all homomeric P2Xs tested. Such a reduction may have different causes such as a misfolding of the protein leading to its degradation, a reduction in the forward trafficking of the protein during its biosynthesis, or a lack of stabilization of the receptors at the plasma membrane. The two first possibilities seem unlikely. Thus, we never noticed any decrease in the total cellular content of mutated subunits when compared with wild type subunits as assayed either by Western blotting or by luminescence assay (see Fig. 1C); this rules out a misfolding and/or degradation of mutated P2X subunits. By comparison, a P2X₂ subunit that lacks glycosylation sites presents a trafficking defect (5) that can be attributed, at least in part, to a misfolding of the protein, because a reduction in the total cellular content of the nonglycosylated channel was observed.² In addition, it is likely that a misfolded subunit would not have formed an oligomeric receptor nor retained any channel activity. Our results show that mutant P2X₂ subunits are able to co-immunoprecipitate and to some extent can be activated by ATP (see Fig. 2). The forward trafficking of mutant P2X receptors is not impaired. Indeed, our pulse-chase experiments clearly demonstrate that mutant P2X receptors acquire complex sugars resistant to endoglycosidase H. This clearly indicates that these proteins are able to travel from the ER to the Golgi apparatus where endoglycosidase H-resistant sugars are added. The rate of forward trafficking of mutant P2X subunits is not likely to be affected because we did not observe any differences in the rate at which mature glycosyl residues are added to WT or trafficking deficient subunits. A reduced forward trafficking rate can therefore not explain the residual membrane expression of mutant P2X subunits that we consistently observed. We cannot exclude that mutant P2X subunits accumulate in the Golgi network; however, such subcellular location would have been noticed in our immunostaining experiments as a marked difference compared with the Wt P2X subunit cellular distribution.

Our results show that mutant P2X receptors have a reduced surface expression because of a reduced residency time at the cell surface. Indeed, we show that mutant P2X₂ subunits are significantly internalized over a period as short as 30 min, whereas Wt P2X₂ shows very little endocytosis as already described (17). This internalization of P2X₂ mutant subunits is not likely mediated by clathrin-mediated endocytosis because these mechanisms are based on the recognition by the endocytic machinery of tyrosine-based motifs. This is clearly not the case for mutant P2X subunits in which the deletion of a tyrosine residue promotes internalization. The most likely interpretation for these results is that mutant P2X receptors are internalized through clathrin-independent or fluid-phase endocytosis. We believe that after their endocytosis mutant subunits are recycled to the plasma membrane; this could explain the residual surface expression that we observed for all mutant P2X subunits that were analyzed. Because we did not observe any difference in the total cellular content between mutant and Wt subunits, it is not likely that internalized P2X mutant receptors are directed to the lysosomal pathway. Clathrin-independent internalization and recycling of the D₂ dopamine receptor have been demonstrated; however, the mechanism underlying this type of endocytosis remains unexplained (34).

We propose that the YXXXK motif stabilizes P2X receptors at the plasma membrane. This interpretation is supported by our experiments using CD₄ protein fused with the C terminus of either P2X₂ or P2X₃. These CD₄ fusion proteins display higher surface expression than Wt CD₄, which returns to normal levels when the YXXXK motif is mutated. Because the YXXXK is not involved in the forward trafficking of P2X receptors, it is most likely that it enhances CD₄ residency time at the plasma membrane by promoting its stabilization (presumably by interactions with cytoskeletal proteins).

There are several examples of stabilization of G protein-coupled receptors or ion channel proteins by interaction with cytoskeletal or scaffolding proteins (for review see Ref. 35). For instance, dopamine D₂ receptor or the inward rectifier Kir2.1 channels have been shown to interact with filamin A, an actin-binding protein. This interaction promotes their surface expression by increasing their half-life at the plasma membrane (35). Similarly Kv1.4 potassium channels undergo constitutive internalization unless it is stabilized by co-expression with the scaffolding protein PSD-95 (36, 37). Membrane stability of ligand-gated channels might also be promoted by interactions with intracellular proteins that regulate membrane turnover and degradation of the receptor. Indeed, and subunits of the -aminobutyric acid, type A receptor are stabilized at the plasma membrane through a direct interaction with Plic-1, a ubiquitin-like protein (38). Although we cannot exclude that P2X subunits interact through the YXXXK motif with a stabilizing protein such as Plic-1, P2X₂ subunits do not interact with Plic-1, and protease inhibitors had no effect on the number of wild type or mutant P2X₂ subunits at the cell surface (not shown).

The YXXXK Motif Is Involved in the Polarized Expression of P2X₂ in Neurons—Interactions of membrane protein with cytoskeletal or scaffolding proteins not only promote their stabilization but also determine their subcellular expression in polarized cells. We found that the YXXXK motif of the P2X₂ receptor regulates their surface expression and polarization in neurons. Because this motif is present in all P2X receptors, it is unlikely that it represents a true dendritic trafficking motif. Rather, we think that P2X receptors are stabilized at the plasma membrane through interactions with intracellular proteins that might have a polarized expression depending on the cell type. Such an hypothesis is supported by the results obtained on the polarized expression of _2B-adrenergic receptors. These receptors are stabilized at the basolateral membrane of Madin-Darby canine kidney cells through interaction with the scaffolding protein spinophilin. Mutant _2B-adrenergic receptors that do not interact with spinophilin display an enhanced rate of internalization. However, the artificial targeting of spinophilin to the apical membrane also promotes the redistribution of Wt _2B-adrenergic receptors to that surface (39). Interactions of the YXXXK motif with specific cytoskeletal proteins might promote the stabilization and the polarization of P2X receptors to specialized membrane domains.

The YXXXK motif is too degenerate to be used in data mining. We searched data bases with a more stringent pattern (LI)(LV)X₈YX₃K in which the double hydrophobic residues help to anchor the motif near the membrane (see Fig. 1). This pattern is still not very stringent, but we identified the motif in several membrane proteins, usually at the interface between a transmembrane domain and a cytoplasmic region. Most interesting, in the three ₂-adrenergic receptors, this pattern is found in the proximal region of their third intracellular loop where basolateral trafficking and stabilization signals have been located. The pattern is also present in intracellular regions of other G protein-coupled receptors such as the serotonin 5-hydroxytryptamine, type 4 receptor, the cannabinoid CB₁, or the chemokine CCR7 receptors. The pattern is also found in other membrane proteins such as the sodium channel subunit SCN8, chloride channels CLC3-5, single transmembrane domain proteins such as neuronal cell adhesion molecule or atrial natriuretic peptide clearance receptor I_B, as well as different kinds of transporters. The presence of the pattern in the third loop of ₂-adrenergic receptors suggests that it might represent a protein-protein interaction domain involved in stabilization of the subset of membrane proteins.

In summary, we have identified a conserved motif in P2X receptors that is involved in their surface expression. This motif is necessary for the stabilization of these receptors at the cell surface likely through direct interactions with intracellular proteins. In addition we provide evidence that P2X₂-polarized expression in neurons is also related to the presence of this trafficking motif.

	FOOTNOTES

 
^* This work was supported in part by Fondation pour la Recherche Medicale (to F. R.) and The Wellcome Trust (to R. A. N.). 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.

Supported by a postgraduate fellowship from Ministère de l'Education Nationale et de la Recherche.

^|| To whom correspondence should be addressed. Tel.: 33-499-61-99-78; Fax: 33-499-61-99-01; E-mail: far{at}igh.cnrs.fr.

¹ The abbreviations used are: HEK, human embryonic kidney; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; COS cells, African green monkey kidney cells; GFP, green fluorescent protein; BSA, bovine serum albumin; Cy3, cyanine 3; Wt, wild type; ,-me-ATP, ,-methylene ATP; ER, endoplasmic reticulum; HA, hemagglutinin; pF, picofarad; zP2X₂, zebrafish P2X₂.

² F. Rassendren and S. Chaumont, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank G. Roussignol for neuronal culture and transfections and Dr. B. Schwappach for the different CD₄ plasmids and for advice on the chemiluminescence assays.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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