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 Previous Article | Next Article 
The Journal of Neuroscience, September 15, 1998, 18(18):7152-7159
Central P2X4 and P2X6 Channel Subunits
Coassemble into a Novel Heteromeric ATP Receptor
Khanh-Tuoc
Lê,
Kazimierz
Babinski, and
Philippe
Séguéla
Cell Biology of Excitable Tissue Group, Montreal Neurological
Institute, McGill University, Montreal, Quebec, Canada H3A
2B4
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ABSTRACT |
Ionotropic ATP receptors are widely expressed in mammalian
CNS. Despite extensive functional characterization of neuronal homomeric P2X receptors in heterologous expression systems, the subunit
composition of native central P2X ATP-gated channels remains to be
elucidated. P2X4 and P2X6 are major central
subunits with highly overlapping mRNA distribution at both regional and
cellular levels. When expressed alone in Xenopus
oocytes, P2X6 subunits do not assemble into surface
receptors responsive to ATP applications. On the other hand,
P2X4 subunits assemble into bona fide ATP-gated channels,
slowly desensitizing and weakly sensitive to the partial agonist
 , -methylene ATP and to noncompetitive antagonists suramin and
pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. We demonstrate
here that the coexpression of P2X4 and P2X6
subunits in Xenopus oocytes leads to the generation of a
novel pharmacological phenotype of ionotropic ATP receptors.
Heteromeric P2X4+6 receptors are activated by
low-micromolar ,-methylene ATP (EC50 = 12 µM) and are blocked by suramin and by Reactive Blue 2, which has the property, at low concentrations, to potentiate homomeric P2X4 receptors. The assembly of P2X4 with
P2X6 subunits results from subunit-dependent interactions,
as shown by their specific copurification from HEK-293 cells
transiently transfected with various epitope-tagged P2X channel
subunits. Our data strongly suggest that the numerous cases of neuronal
colocalizations of P2X4 and P2X6 subunits
observed in mammalian CNS reflect the native expression of heteromeric
P2X4+6 channels with unique functional properties.
Key words:
purinoceptor; nucleotide; transmitter-gated cation
channel; , methylene ATP; suramin; PPADS
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INTRODUCTION |
Fast purinergic neurotransmission is
mediated by nonselective cation channels gated by extracellular ATP.
These transduction proteins, designated P2X receptors, constitute a
distinct class of neurotransmitter-gated channels on the basis of their
primary cDNA sequences and their predicted transmembrane protein
topology. Currently, seven mammalian P2X genes have been identified
with either expression or homology cloning assays (Buell et al., 1996 ). Among the neuronal P2X receptors, only P2X4 and
P2X6 isoforms are predominantly expressed in the adult rat
brain in which they show an overlapping pattern of regional and
cellular distribution at the mRNA level (Collo et al., 1996). Homomeric
rat P2X4 receptors expressed in HEK-293A cells or
Xenopus laevis oocytes and homomeric P2X6
receptors silent in oocytes (Soto et al., 1996) but functional in
HEK-293A cells (Collo et al., 1996) are weakly responsive to ,-methylene ATP (mATP) and to P2 antagonists suramin and
pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (North
and Barnard, 1997). Yet, native ionotropic purinergic responses from
rat medial habenula, cerebellum, and hippocampus were blocked by P2
antagonists, and most native ATP receptors are activated by mATP
(Edwards et al., 1992; Mateo et al., 1998; Ross et al., 1998).
Moreover, high-affinity [3H]mATP
autoradiographic binding sites have been localized in specific but
widespread regions within the brain and spinal cord (Bo and Burnstock,
1994; Michel and Humphrey, 1994; Balcar et al., 1995).
Discrepancies between pharmacological profiles of heterologously
expressed homo-oligomeric P2X subunits and electrophysiological recordings from neuronal preparations likely reflect the existence of
native heteromeric phenotypes of P2X receptors in peripheral nervous
system as well as the CNS. Indeed, one such hybrid P2X phenotype was
recorded in sensory neurons (Khakh et al., 1995; Lewis et al., 1995)
and has been proposed to result from the association between
coexpressed P2X2 and P2X3 subunits (Chen et
al., 1995; Lewis et al., 1995; Radford et al., 1997). We describe in
this report a novel P2X heteromeric receptor containing central
P2X4 and P2X6 subunits. This phenotype of
ATP-gated channel is endowed with a unique pharmacology characterized
by increased sensitivities to mATP, 2-methylthio-ATP (2MeSATP),
suramin, PPADS, and Reactive Blue 2 (RB-2) in Xenopus
oocytes.
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MATERIALS AND METHODS |
Molecular biology. Wild-type full-length
P2X6 subunit cDNA was obtained by RT-PCR using adult rat
spinal cord RT-cDNA template, Expand DNA polymerase (Boehringer
Mannheim, Indianapolis, IN), and exact match primers based on published
primary sequences (Collo et al., 1996; Soto et al., 1996). Construction
of P2X1-Flag and P2X4-Flag was reported
previously (Lê et al., 1998). To generate epitope-tagged
P2X6-Flag and P2X4-(His)6 subunits,
an XhoI-XbaI cassette containing an in-frame
His6 epitope followed by an artificial stop codon was
grafted to the full-length HindIII-XhoI
P2X4 construct. The P2X4-(His)6
mutant was then subcloned directionally into the HindIII and
XbaI sites of pcDNAI vector (Invitrogen, San Diego, CA) for
cytomegalovirus-driven heterologous expression in mammalian cells and
Xenopus laevis oocytes. Epitope-tagged and RT-PCR constructs were subjected to dideoxy sequencing either manually with Sequenase (Upstate Biotechnology, Lake Placid, NY) or with an ALF DNA sequencer (Pharmacia, Piscataway, NJ).
Cell culture and protein chemistry. For cDNA transfections
of epitope-tagged and wild-type P2X subunits into mammalian cells, HEK-293A cells (CRL 1573; American Type Culture Collection, Rockville, MD) were cultured in DMEM and 10% heat-inactivated fetal bovine serum (FBS) (Wisent, St-Bruno, Quebec, Canada) containing penicillin and streptomycin. Freshly plated cells reaching 30-50% confluency were used for transient cDNA transfections with the calcium phosphate method on 90 mm cell culture dishes (Falcon) with 10 µg of
supercoiled plasmid cDNA/106 cells (Lê et al.,
1998). For Western blots, transfected HEK-293A cells were lifted in
Hank's modified calcium-free medium with 20 mM EDTA,
pelleted at low centrifugation, and homogenized in 10 volumes of 10 mM HEPES buffer, pH 7.4, containing protease inhibitors
phenylmethylsulfonyl fluoride (0.2 mM) and benzamidine (1 mM). Cell lysates were pelleted at 14,000 × g for 5 min, and membrane proteins in supernatants were
solubilized with SDS-containing loading buffer. Approximately 150 µg
of protein/lane were run on 12% SDS-PAGE and then transferred to
nitrocellulose. Immunoprobing was performed with mouse mAb M2 (1 µg/ml, IBI) followed by peroxidase-labeled anti-mouse secondary
antibodies for visualization by enhanced chemiluminescence (Amersham,
Oakville, Ontario, Canada). Copurification of associated P2X
subunits was performed as previously described for IRK channels (Tinker
et al., 1996) with minor modifications. Cell lysates were solubilized
with 5% Triton X-100 for 2 hr at 4°C. Unsolubilized materials were
pelleted at 10,000 × g, and supernatants were
incubated with 50 µl of 50% slurry of equilibrated Ni-NTA-Resin
(Qiagen, Hilden, Germany) for 2 hr at 4°C. Nickel beads were then
washed six times in TBS containing 25 mM imidazole and 1%
Triton X-100. Bound proteins were eluted from Ni-NTA resin with 500 mM imidazole, diluted 1:1 (v/v) with SDS-containing loading buffer, and warmed for 10 min at 37°C. Samples were then loaded onto
a 12% SDS-PAGE, transferred to nitrocellulose, and analyzed in Western
blot using chemiluminescence as above.
Electrophysiology. For electrophysiological recordings in
oocytes, ovary lobes were surgically removed from Xenopus
laevis frogs anesthetized with Tricaine (Sigma, St. Louis, MO) and
treated for 3 hr at room temperature with type II collagenase (Life
Technologies, Gaithersburg, MD) in calcium-free Barth's solution under
vigorous agitations. Stage V-VI oocytes were then defolliculated
chemically before nuclear microinjections of 5-10 ng of cDNA coding
for each P2X channel subunit. After 2-5 d of incubation at 19°C in
Barth's solution containing 1.8 mM calcium chloride
(CaCl2) and 10 µg/ml gentamicin, P2X currents were
recorded in a two-electrode voltage-clamp configuration using an
OC-725B amplifier (Warner Institute). Signals were low-pass-filtered at
1 kHz, acquired at 500 Hz using a Macintosh IIci equipped with an
NB-MIO-16XL analog-to-digital card (National Instruments). Traces were
postfiltered at 100 Hz in Axograph (Axon Instruments). Agonists,
antagonists, and cofactors (zinc chloride, pH 6.5 and 8.0) were
dissolved in Ringer's solution containing (in mM):
115 NaCl, 2.5 KCl, and 1.8 CaCl2 in 10 HEPES, pH 7.4 standard at room temperature, and applied on oocytes at a constant flow
rate of 12 ml/min. Dose-response curves and EC50 values
were derived from fittings for the sigmoidal equation of Hill using Prism 2.0 software (Graphpad Software, San Diego, CA).
Statistical analysis. All comparisons involving two
variances were performed with Fisher's F values (variance
homogeneity requirements) and with Student's t tests for
two unpaired groups. Two-tailed statistical thresholds, for both
Fisher's F and Student's t critical values,
were set at p < 0.05.
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RESULTS |
Functional impact of P2X6 subunit expression on
ATP-induced currents
In response to 100 µM ATP, Xenopus
oocytes microinjected with a mix of P2X4 and
P2X6 cDNAs (1:1 molar ratio) gave rise to currents with
kinetic profiles similar to those observed with oocytes expressing
P2X4 alone (Fig.
1A). P2X6
by itself appeared to be silent in Xenopus oocytes, because
no current was detected during ATP applications (Fig.
1A), in agreement with what has been reported
previously (Soto et al., 1996). Comparison of peak current amplitudes
after 3 d of expression revealed, however, that currents from
cells coexpressing P2X4 and P2X6 subunits were reproducibly and significantly smaller than currents from cells expressing only P2X4 receptors (Fig.
1A,B), suggesting the possibility that the
P2X6 channel subunit can heteropolymerize with other members of the P2X family. We coexpressed P2X6 together
with P2X1 (Valera et al., 1994) or with P2X2
(Brake et al., 1994). In response to 100 µM ATP, there
were no differences between peak currents recorded from oocytes
coexpressing P2X1 and P2X6 and those expressing P2X1 alone (Fig. 1C) after 3 d of
expression. Similarly, we did not observe any functional impact of
P2X6 on the expression of P2X2 under the same
experimental conditions (Fig. 1D), eliminating the
possibility of a general inhibitory effect of P2X6 on
protein synthesis or on translocation. Thus these data indicate either that the subunit-specific interaction between P2X4 and
P2X6 isoforms generates a heteromeric P2X4+6
receptor, or that P2X6 subunits exert a specific inhibitory
function on P2X4 receptor expression. If P2X4+6
heteromers are expressed, smaller peak currents could result from a
lower affinity for ATP or a smaller single conductance in comparison
with homomeric P2X4 channels. Alternatively, smaller ATP
responses at day 3 could simply reflect a slower kinetics of
receptor expression.
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Figure 1.
Representative heteromeric P2X4+6
channel current phenotype at day 3. A, ATP-induced
currents after heterologous expression of P2X4,
P2X6, and P2X4 + P2X6 (1:1
molar ratio) subunits recorded 3 d after corresponding cDNA
nuclear microinjections in Xenopus oocytes.
Arrows indicate beginnings of ATP applications (10 sec).
B, P2X4-dependent functional impact of
P2X6 on ATP-induced response (P2X4 expressed
alone; 1x, 5 ng of cDNA; 2x, 10 ng).
C, P2X1 receptor (P2X1;
1x, 5 ng; 2x, 10 ng) functional
expression is unaffected by coexpressed P2X6 subunits.
D, P2X2-mediated (P2X2;
1x, 5 ng; 2x, 10 ng) ATP-induced peak
current amplitudes are unchanged in the presence of P2X6
subunits. (Averages ± SEM from 3 to 15 oocytes in 2-4
independent experiments; double asterisks denote significant
difference; p < 0.01).
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To further characterize a time-dependent effect, we studied the time
course of expression, daily recording peak currents in response to 100 µM ATP from oocytes expressing either P2X4
and P2X6 cDNAs or P2X4 cDNA alone. Figure
2 demonstrates that ATP receptors in
oocytes coexpressing P2X4 and P2X6 subunits,
compared with P2X4 alone, needed a longer time to reach the
same levels of ATP-induced currents. However, between days 2 and 5 after injection, there was a dramatic sevenfold increase in peak
current amplitudes in oocytes coexpressing P2X4 and
P2X6 subunits (Fig. 2A). This profile is
in striking contrast with the time course of P2X4
expression that slowly decayed over the same period (Fig.
2B).
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Figure 2.
A, Potentiation of ATP response
represented by P2X4+6 channel current phenotype at day 5. Arrows indicate beginnings of ATP applications (10 sec).
B, Time course of heteromeric P2X4+6
expression. Kinetics of appearance of functional ATP receptors on
plasma membranes is strikingly different in oocytes coinjected with
P2X4 and P2X6 subunits compared with those
injected with P2X4 subunits (averages ± SEM from 3 to
15 oocytes in 2-8 independent experiments).
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Agonist sensitivity profile of P2X4+6
heteromeric receptors
No significant difference was detected between the
EC50 values derived from ATP dose-response profiles of
P2X4+6 (6.3 ± 0.9 µM) channel phenotype
and homomeric P2X4 (4.2 ± 1.1 µM) receptors (Fig. 3A) expressed
in oocytes. However, the partial agonist 2MeSATP had EC50
values of 7.67 ± 1.01 and 26 ± 1.8 µM for
P2X4+6 and P2X4 receptors, respectively, a
statistically significant difference (Fig. 3B). Even more
striking, in response to 100 µM mATP on day 3 after
injection, oocytes expressing P2X4+6 heteromeric channels
gave rise to peak current amplitudes of 0.7 ± 0.13 µA compared
with 0.12 ± 0.02 µA only from oocytes expressing
P2X4 homomeric receptors, in marked contrast with the situation observed in response to ATP (compare Figs.
4A, 1B). The mATP
EC50 values were found to be 12 ± 2 µM
for P2X4+6 and 55 ± 2 µM for
P2X4 channel phenotypes (Fig. 4B).
Therefore, mATP shows more potency and has a higher affinity on
P2X4+6 receptors than on P2X4 receptors. These
different sensitivities to 2MeSATP and mATP constitute more
experimental evidence for a functional association between
P2X4 and P2X6 subunits coexpressed in
Xenopus oocytes.
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Figure 3.
Sensitivity of P2X4+6 receptors to the
agonists ATP and 2MeSATP. A, Similar ATP dose-response
profile between heteromeric P2X4+6 channels and homomeric
P2X4 receptors in Xenopus oocytes.
B, Heteromeric P2X4+6 receptors showed
increased sensitivity to 2MeSATP compared with P2X4
receptors. Values are normalized to the response to 300 µM ATP (averages ± SEM from 3 to 7 oocytes per
point in 2 independent experiments).
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Figure 4.
Sensitivity of P2X4+6 receptors to the
agonist mATP. A, Differential mATP
responsiveness measured in peak current amplitudes between
Xenopus oocytes expressing either P2X4+6
channels or P2X4 at day 3 after injection; see Figure
1B for comparison of ATP-induced peak currents.
B, Normalized dose-response curves of
P2X4+6 and P2X4 receptor species for
mATP. Values are normalized to the response to 100 µM ATP; double asterisks denote
significant difference; p < 0.01 (averages ± SEM from 5 to 8 oocytes per point in 2 independent experiments).
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Sensitivity of P2X4+6 receptors to suramin, PPADS,
and RB-2
It is widely recognized that neither P2X4 nor
P2X6 homomeric receptors (in HEK-293 cells) are completely
blocked by suramin or PPADS up to 100 µM without
preincubation (Buell et al., 1996; Collo et al., 1996). In response to
100 µM ATP and 10 µM suramin coapplications
without preincubation, oocytes expressing P2X4+6 gave rise
to residual currents of 61 ± 3% (Fig.
5) of the response to 100 µM ATP (100%). Under the same experimental conditions, oocytes expressing P2X4 receptors alone were almost
unaffected (93 ± 3%; Fig. 5). We have also found that 10 µM PPADS coapplied with 100 µM ATP gave
rise to peak current amplitudes of 83 ± 7 and 103 ± 6% for
P2X4+6 and P2X4 receptor phenotypes,
respectively (Fig. 5B), but we did not find any significant
difference between this 17% inhibition on P2X4+6 and no
effect on P2X4. We have also investigated the effects of
RB-2 by coapplying 10 µM of the antagonist with 100 µM ATP: oocytes expressing P2X4+6 receptor phenotypes were characterized by residual peak currents of 60 ± 9% compared with potentiated peak currents of 123 ± 18% from oocytes expressing P2X4 receptors alone (Fig.
5A). Preincubation of the cell with antagonist during 1 min
before coapplication with ATP resulted in even more dramatic
phenotypical differences between P2X4 and
P2X4+6 for suramin (23% blockade vs 41%) and PPADS (19%
blockade vs 38%) (Fig. 5B). Furthermore, in conditions of
preincubation, 10 µM RB-2 blocked P2X4+6
heteromeric channels by up to 26% but increased P2X4
response by >45% (Fig. 5B). A potentiating effect of RB-2
on P2X4 homomeric receptors has been reported in oocytes,
albeit to a smaller extent (Bo et al., 1995).
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Figure 5.
Sensitivity of P2X4+6 to P2X
antagonists. Suramin, PPADS, and RB-2 were tested for their blocking
properties on heteromeric P2X4+6 channels and on homomeric
P2X4 receptors. Antagonists were coapplied with ATP
(A) or preincubated before coapplication
(B). Note that P2X4+6 receptors are
inhibited, whereas P2X4 receptors are potentiated by 10 µM RB-2. Values are normalized to the response to ATP
only (averages ± SEM from 5 oocytes per experiment;
single and double asterisks denote
significant difference; p < 0.05 and
p < 0.01, respectively).
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Sensitivity of P2X4+6 receptors to coagonists zinc ions
and protons
We have reported previously that 10 µM extracellular
zinc ions coapplied with 10 µM ATP potentiated
P2X4 peak currents by almost twofold (Séguéla
et al., 1996). In addition, it has also been shown that the sensitivity
to ATP of homomeric P2X4 channels is modulated by external
pH: pH <7 inhibits ATP responses, whereas pH >8 has no significant
effects (Stoop et al., 1997). Therefore, we checked whether these
coagonists applied with ATP could discriminate between
P2X4+6 and P2X4 receptor phenotypes. In
response to 10 µM zinc ions and 10 µM ATP
coapplications, there were no significant differences between
potentiating factors of 1.8 ± 0.19 and 1.8 ± 0.21 for
P2X4+6 heteromeric channels and P2X4 homomeric
receptors, respectively (Fig.
6A). There was also no significant difference between these two receptor phenotypes with respect to ATP (20 µM) applied at pH 6.5. In both cases,
residual peak current amplitudes were 46 ± 4% of control values
measured at pH 7.4 (Fig. 6B). When 20 µM ATP was applied at pH 8.0, it elicited peak currents
of 121 ± 4 and 106 ± 4% for P2X4+6 heteromers
and P2X4 homomers, respectively (Fig. 6C). Thus,
contrary to mATP, 2MeSATP, and antagonists suramin, PPADS, and
RB-2, cofactors zinc and protons did not discriminate between
P2X4+6 and P2X4 receptors on a pharmacological
basis.
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Figure 6.
Sensitivity of P2X4+6 to the
extracellular cofactors zinc ions and pH. Extracellular
Zn2+, pH 6.5 and 8.0, coapplied with ATP, did not
allow differentiation between P2X4+6 and P2X4
receptors. Values are normalized to the response to ATP only
(averages ± SEM from 4 oocytes per experiment).
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Subunit-specific association of P2X4 with
P2X6 subunits
Before testing their biochemical interaction, the expression of
Flag-tagged P2X1, P2X4, and
P2X6 subunit proteins in transiently transfected HEK-293A
cells was confirmed by immunoblot of total membrane proteins (Fig.
7A, lanes
1-6). Homogenates from HEK-293A cells transiently
cotransfected with cDNA templates encoding
P2X4-(His)6 and either P2X1-Flag,
P2X4-Flag, or P2X6-Flag constructs were analyzed for copurification. After solid-phase binding of
P2X4-(His)6 proteins on poly His-binding resin,
we detected the coprecipitation of P2X4-Flag subunits,
confirming that P2X4 subunits interacted between themselves
to generate a homomultimeric complex (positive controls, Fig.
7B, lane 1). Coexpression of
P2X4-(His)6 with P2X6-Flag subunits
gave a positive band corresponding to the expected size of
P2X6 (51 kDa; Fig. 7B, lane 3),
demonstrating directly for the first time that P2X4 and
P2X6 subunits do physically interact in a multimeric
complex. Coexpression of P2X4-(His)6 with
P2X1-Flag subunits did not give any signal when probed with
anti-Flag M2 antibodies after purification, confirming that
P2X4 and P2X1 subunits do not heteropolymerize
(Fig. 7B, lane 5). All control coexpressions including wild-type P2X4 (lacking the poly-His motif)
cotransfected with Flag-tagged P2X4,
P2X6, or P2X1 subunits were negative
after purification on poly His-binding resin (Fig. 7B,
lanes 2, 4, 6).
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Figure 7.
Subunit specificity of P2X4 and
P2X6 heteropolymerization. A, Immunoblot of
Flag-tagged P2X1, P2X4, and
P2X6 subunits probed with anti-Flag M2 monoclonal
antibodies in total membrane proteins from transiently transfected
HEK-293A cells. B, From the same samples, immunoblot of
Flag-tagged P2X1, P2X4, and
P2X6 subunits probed with M2 antibodies after
copurification through P2X4-(His)6 subunits.
Molecular weight markers (in A): 104, 82, and 48 kDa.
Cotransfections: lane 1,
P2X4-(His)6 + P2X4-Flag;
lane 2, P2X4-wt + P2X4-Flag;
lane 3, P2X4-(His)6 + P2X6-Flag; lane 4, P2X4-wt + P2X6-Flag; lane 5,
P2X4-(His)6 + P2X1-Flag;
lane 6, P2X4-wt + P2X1-Flag.
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DISCUSSION |
Functional identification of P2X4+6
heteromeric receptors
In the present study, we first observed an apparent inhibition of
P2X6 subunits on ATP-induced currents in oocytes expressing P2X4 subunits (Fig. 1B). However, neither
ATP-induced currents mediated by P2X1 subunits (Fig.
1C) nor currents mediated by P2X2 subunits (Fig.
1D) were affected, strongly suggesting that
P2X4 and P2X6 isoforms associate together, in a
subunit-specific manner, into a novel heteromeric P2X channel.
Functional P2X4+6 protein assembly and/or plasma membrane
channel targeting appeared to be on a different time scale compared
with P2X4 receptors. Indeed, in response to 100 µM ATP, P2X4+6 heteromultimers gave rise to
increasing peak currents even after 5 d of expression (Fig. 2A), whereas P2X4 homopolymers yielded
decreasing peak current amplitudes under identical conditions (Fig.
2B). We did not find any difference between the
EC50 values of ATP for both homomeric and heteromeric
receptor isoforms (Fig. 4A), so we concluded that the
apparent inhibitory effect of P2X6 on P2X4
recorded 3 d after injection was mainly attributed to a slower
expression of P2X4+6 receptors on the cell surface,
assuming similar channel conductance. These findings constituted our
first set of experimental evidence demonstrating a heteropolymerization
between P2X4 and P2X6 subunits. It has been
noticed previously that P2X6 subunits and channels express
poorly in HEK-293A cells (Collo et al., 1996) and are silent to ATP in
the Xenopus oocyte expression system (Soto et al., 1996), as
observed here. However, maximal ATP-induced peak currents were
significantly larger at day 5 in the case of P2X4+6 channels than in the case of P2X4 alone (Fig.
2A). This situation is reminiscent of epithelial
sodium-selective channels, belonging to another family of
two-transmembrane-domain cation channels, whereby a fully functional
channel requires the heteropolymerization of subunits with and
 subunits, both inactive when expressed alone (Canessa et al.,
1994).
Unique pharmacological profile of P2X4+6
heteromeric receptors
We made the assumption that the association between
P2X4 and P2X6 subunits should be reflected in
some unique aspects of the pharmacological profile of the resulting
heteromeric receptor. Although P2X4 seemed the dominant
subunit for the sensitivity to ATP in the heteromers, we observed a
statistical difference between EC50 values of 2MeSATP for
P2X4+6 heteromeric channels and P2X4 receptors
(Fig. 4B). Furthermore, in response to 100 µM mATP applications, oocytes coexpressing
P2X4 and P2X6 subunits gave rise to larger
maximal peak currents than oocytes expressing P2X4 isoforms
alone (Fig. 3A), despite slower kinetics of expression. Indeed, we measured a lower EC50 of mATP for
P2X4+6 than for P2X4 channel species (Fig.
3B). Therefore, in addition to opposite protein expression
profiles between P2X4+6 and P2X4 channels,
these observations strongly indicate that P2X4 and
P2X6 subunits generate a novel receptor phenotype
characterized by a unique agonist profile, namely increased 2MeSATP and
mATP sensitivity. Moreover, these data provide for the first time
experimental evidence for moderately desensitizing mATP-activated
ionotropic responses.
Furthermore, we probed the sensitivity of P2X4+6 heteromers
to P2 antagonists suramin, PPADS, and RB-2 coapplied with ATP. We found
that suramin significantly blocked P2X4+6 activity without
inhibiting significantly P2X4 homomeric receptors (Fig. 5A); 10 µM suramin coapplied with 100 µM ATP decreased P2X4+6 heteromeric receptor
peak current amplitudes by up to 40% compared with 7% for
P2X4 homomeric channels. Coapplied PPADS inhibited P2X4+6 weakly, although it had no measurable effects on
oocytes expressing P2X4 subunits alone (Fig.
5A). After preincubation, low concentrations of RB-2
provided the most dramatic differential effect by inhibiting
P2X4+6 heteromers while potentiating P2X4
channel activity (Fig. 5B). Suramin and RB-2 would thus be useful pharmacological tools to investigate the expression of native
P2X4+6 heteromers in mATP-sensitive neuronal
preparations.
Biochemical evidence of P2X4+6 heteropolymers
We demonstrated direct interactions between the two predominant
brain P2X4 and P2X6 isoforms through the use of
an established copurification assay (Tinker et al., 1996). Based on our
coprecipitation results with epitope-tagged subunits in nondenaturing
conditions, P2X4 associates with P2X6 subunits
(Fig. 7B). In Xenopus oocytes, this
heteropolymerization underlies the specific pharmacological and
electrophysiological phenotype of a novel heteromeric channel distinct
from either P2X4 or P2X6 homomeric receptors.
On the other hand, P2X4 and P2X1 subunits did
not seem to interact significantly with each other (Fig.
7B). Furthermore, the absence of obvious phenotypical
differences between oocytes coexpressing P2X6 + P2X1 and P2X1 subunits alone (Fig.
1C), or between P2X6 + P2X2 and P2X2 homomers (Fig. 1D), indicate that
structural determinants of association between P2X4 and
P2X6 isoforms are subunit-dependent. A similar biochemical
approach using copurification of P2X4 with chimeric
subunits based on P2X6 and P2X1 structures
could lead to the identification of the domain(s) involved in specific
heteropolymerization.
Functional correlates of native P2X4+6 heteromers
Purinergic responses from CA3 neurons in rat hippocampal slices
have been shown recently to be activated by mATP and inhibited by
suramin but not by PPADS (Ross et al., 1998). Based on in
situ hybridization results (Collo et al., 1996), P2X4
and P2X6 are the only P2X subunits expressed at significant
levels in adult rat hippocampus, namely in CA1-CA4 hippocampal
subfields and in the dentate gyrus. Thus, our functional data obtained
from recombinant receptors are in close agreement with this native
phenotype and suggest that the sensitivities to mATP and to
suramin of rat CA3 neurons might be mediated through native
P2X4+6 heteromeric channels.
Neonatal rat cerebellar Purkinje cells have been characterized as
having purinergic receptors with a P2X2-like
pharmacological profile in eliciting extracellular calcium influxes
(Mateo et al., 1998). This conclusion rested on mATP
insensitivity, the potency ratio of ATP to 2MeSATP, as well as suramin
and PPADS blockade after preincubation. However, on recombinant
P2X4+6 receptors, the concentration of mATP used by
Mateo et al. (1998) (50 µM) was ~10% as efficacious as
50 µM ATP in eliciting ionotropic responses, so
mATP-mediated intracellular calcium increases could have remained
undetected and consequently interpreted as mATP unresponsiveness.
The developmental regulation of expression levels of neuronal P2X genes
in cerebellum is not established so far. Adult rat Purkinje neurons are
known to transcribe P2X4 and P2X6 mRNA (Collo
et al., 1996) and have been shown to translate high levels of
P2X4 subunits (Lê et al., 1998), whereby
P2X2 mRNAs (Collo et al., 1996) or subunits (Vulchanova et
al., 1996) were reported previously to be absent (Kanjhan et al.,
1996). It is also possible that native P2X receptors in neonatal
Purkinje cells are composed of three subunits, namely
P2X2, P2X4, and
P2X6, assembled in a heteromeric complex in which
P2X2 is pharmacologically dominant. We have recorded in
oocytes purinergic currents mediated by P2X4+6 heteromeric
channels that were significantly more sensitive to the agonists
mATP and 2MeSATP, as well as to the antagonist suramin compared
with P2X4 homomeric receptors. So it is likely that the
moderately desensitizing mATP-activated and suramin-sensitive
postsynaptic purinergic responses recorded from medial habenula
(Edwards et al., 1992) could be accounted for by the expression of
postsynaptic P2X4+6 receptors, because in situ
hybridization results demonstrate the exclusive presence of
P2X4 and P2X6 transcripts in this region (Collo
et al., 1996). The widespread distribution of high-affinity
[3H]mATP binding sites within the rat CNS
(Bo and Burnstock, 1994; Michel and Humphrey, 1994; Balcar et al.,
1995) appears to correlate with in situ hybridization data
on P2X4 and P2X6 mRNA distributions (Collo et
al., 1996; Séguéla et al., 1996) as well as with the immunocytochemical localization of P2X4 protein (Lê
et al., 1998). This neuroanatomical evidence strongly suggests that the
P2X4+6 channel phenotype might be present in most rat brain
and spinal cord regions. Moreover, we have shown that the
P2X4 subunit is a major presynaptic purinoceptor component
in laminae I and II of spinal cord and in olfactory glomeruli (Lê
et al., 1998), two regions in which P2X6 is also expressed
(Collo et al., 1996). Therefore, the heteromeric P2X4+6
ATP-gated cation channel could play a significant role in the
regulation of excitatory transmitter release in central sensory
synapses.
| |
FOOTNOTES |
Received April 6, 1998; revised June 22, 1998; accepted July 6, 1998.
K-T.L. holds a PhD studentship from the Savoy Foundation for Epilepsy;
K.B. is a Medical Research Council-PMAC-Astra postdoctoral Fellow; and P.S. is a junior Scholar from the Fonds de la Recherche en
Santé du Québec. We thank the Medical Research Council of Canada, the Fondation des Maladies du Coeur du Québec, and the Astra Research Center in Montreal for their operating support, as well
as Michel Paquet for expert technical assistance.
Correspondence should be addressed to Dr. Philippe Séguéla,
Cell Biology of Excitable Tissue Group, Montreal Neurological Institute, 3801 University Avenue, Room 778, Montreal, Quebec, Canada
H3A 2B4.
| |
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Top
Abstract
Introduction
