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J Biol Chem, Vol. 274, Issue 22, 15415-15419, May 28, 1999
§,¶,
From the Department of Neurology and Neurosurgery, Cell Biology of Excitable Tissue Group, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4 Canada
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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The mammalian P2X receptor gene family encodes
two-transmembrane domain nonselective cation channels gated by
extracellular ATP. Anatomical localization data obtained by in
situ hybridization and immunocytochemistry have shown that
neuronal P2X subunits are expressed in specific but overlapping
distribution patterns. Therefore, the native ionotropic ATP receptors
diversity most likely arises from interactions between different P2X
subunits that generate hetero-multimers phenotypically distinct from
homomeric channels. Rat P2X1 and P2X5 mRNAs
are localized within common subsets of peripheral and central sensory
neurons as well as spinal motoneurons. The present study demonstrates a
functional association between P2X1 and P2X5
subunits giving rise to hybrid ATP-gated channels endowed with the
pharmacology of P2X1 and the kinetics of P2X5.
When expressed in Xenopus oocytes, hetero-oligomeric P2X1+5 ATP receptors were characterized by slowly
desensitizing currents highly sensitive to the agonist
Ionotropic ATP receptors constitute a unique class of
neurotransmitter-gated ion channels generated from the assembly of P2X subunits having two transmembrane-spanning domains and a protein architecture similar to the one of the amiloride-sensitive sodium channels (1, 2). Functional characterization studies of the seven
mammalian cloned P2X subunits heterologously expressed as homomeric
channels allowed to classify them in three groups according to their
properties of desensitization and to their sensitivity to the agonist
All P2X subunits have been detected in peripheral sensory ganglia,
reinforcing the view that synaptically or lytically released ATP could
play an important signaling role in sensory pathways (1, 11, 15). Rat
P2X3 subunits have been reported to be exclusively
expressed in small to medium-sized isolectin B4-positive nociceptive
neurons in nodose, trigeminal, and dorsal root ganglia (4, 5, 15). A
significant proportion of sensory neurons are thought to express
hetero-oligomeric P2X2+3 receptors based on their sustained
response to Rat P2X5 subunits mRNAs have the most restricted
distribution in the P2X family, but in situ hybridization
studies have indicated that P2X1 and P2X5
mRNAs are co-localized in primary sensory neurons as well as within
subsets of large motoneurons in the ventral horn of the spinal cord (1,
11). We report here the characterization of a novel heteromeric P2X
receptor with hybrid properties generated by co-expression and
co-assembly of P2X1 with P2X5 subunits in Xenopus laevis oocytes and transfected HEK-293A cells,
further strengthening arguments for a diversity of native ATP-gated
channels and purinergic phenotypes in mammalian neurons.
Molecular Biology--
Full-length wild-type rat
P2X1 and P2X5 cDNAs were obtained through
polymerase chain reaction amplification using A10 smooth muscle cells
(ATCC No. CRL 1476) and adult rat spinal cord reverse transcribed-cDNA templates, respectively. Reactions were performed with exact match oligonucleotide primers based upon published primary
sequences (3, 11, 12) using Pfu DNA polymerase (Stratagene)
to minimize artifactual mutations. Epitope-tagged P2X subunits with
carboxyl-terminal hexahistidine motif (His6) or Flag
peptide were constructed as reported previously (18). Briefly, an
XhoI-XbaI stuffer cassette containing in-frame
Flag or His6 epitopes followed by an artificial stop codon
was ligated to P2X1 and P2X5 cDNAs
previously mutated to replace their natural stop codon with a
XhoI restriction site. P2X1-Flag,
P2X1-His6, P2X5-Flag, and
P2X5-His6 were then subcloned directionally
into the HindIII-XbaI sites of pcDNAI vector
(Invitrogen, San Diego, CA) compatible with CMV-driven heterologous
expression in HEK-293A cells and Xenopus laevis oocytes.
RT-PCR products as well as mutant epitope-tagged subunits were
subjected to automatic dideoxy sequencing (Sheldon Biotechnology
Center, Montreal).
Cell Culture and Protein Chemistry--
cDNA transfections
of epitope-tagged P2X subunits were performed in mammalian cells.
HEK-293A cells (ATCC No. CRL 1573) were cultured in Dulbecco's
modified Eagle's medium and 10% heat-inactivated fetal bovine serum
(Wisent, St. Bruno, Canada) containing penicillin and streptomycin.
Cells reaching 30-50% confluency were used for transient cDNA
transfections with the calcium phosphate method with 10 µg of
supercoiled plasmid cDNA per 106 cells. Transfected
HEK-293A cells used for Western blots were then lifted in Hanks'
modified calcium-free medium with 20 mM EDTA, pelleted at
low centrifugation, and homogenized in 10 volumes of 10 mM
HEPES buffer and 0.3 M sucrose, pH 7.40, containing
protease inhibitors phenylmethylsulfonyl fluoride (0.2 mM)
and benzamidine (1 mM). Membranes from cell lysates were
solubilized with 1% Triton X-100 (Sigma) for 2 h at 4 °C and
pelleted at 14000 × g for 5 min, and remaining
membrane proteins within supernatants were used for Western blots.
Solubilized proteins were incubated with 25 µl of equilibrated Ni-NTA
resin (Qiagen, Hilden, Germany) for 2 h at 4 °C under
agitation. Then Ni-NTA beads were washed six times in Tris-buffered
saline containing 25 mM imidazole and 1% Triton X-100.
Bound proteins were eluted from His6-binding resin with 500 mM imidazole, diluted 1:1 (v/v) with SDS-containing loading buffer. Samples were then loaded onto 10-12% SDS-PAGE and transferred to nitrocellulose. Immunostainings were performed with M2 murine monoclonal antibodies (10 µg/ml) (Sigma) or chicken anti-Flag polyclonal antibodies (1:200) (Aves) followed by incubations with corresponding species-specific peroxidase-labeled secondary antibodies (1:5000-1:20,000) for visualization by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Electrophysiology--
Electrophysiological recordings were
performed in Xenopus oocytes. Ovary lobes were surgically
retrieved from X. laevis frogs under deep tricaine (Sigma)
anesthesia. Oocyte-containing lobes were then treated for 3 h 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 chemically defolliculated
before nuclear micro-injections of 5-10 ng of cDNA coding for each
P2X channel subunit. Following 2-5 days of incubation at 19 °C in
Barth's solution containing 1.8 mM calcium chloride and 10 µg/ml gentamicin (Sigma), elicited P2X currents were recorded in
two-electrode voltage-clamp configuration using an OC-725B amplifier
(Warner Instruments). Responsive signals were low pass filtered at 1 kHz, acquired at 500 Hz using a Macintosh IIci computer equipped with
an NB-MIO-16XL analog-to-digital card (National Instruments). Recorded
traces were post-filtered at 100 Hz in Axograph (Axon Instruments).
Agonists, antagonists, and P2X co-factors (10 µM zinc
chloride, pH 6.40 and pH 8.40) were prepared at room temperature in
Ringer's perfusion solution containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES buffered at pH 7.40. Solutions were perfused onto
oocytes at a constant flow rate of 10-12 ml/min. Dose-response curves
were fitted to the Hill sigmoidal equation, and EC50 as
well as IC50 values were determined using the software
Prism 2.0 (Graphpad Software, San Diego, CA).
To assess the presence of P2X1+5 heteromers in
Xenopus oocytes co-injected with both subunits, we tested
the expression of inward currents during prolonged applications (5-10
s) of 50 µM 
,
-methylene ATP (EC50 = 1.1 µM) and to
the antagonist trinitrophenyl ATP (IC50 = 64 nM), observed with neither P2X1 nor
P2X5 alone. Direct physical evidence for P2X1+5
co-assembly was provided by reciprocal subunit-specific
co-purifications between epitope-tagged P2X1 and
P2X5 subunits transfected in HEK-293A cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,-methylene ATP
(m-ATP)1: (i) rapidly
desensitizing and m-ATP-sensitive receptors including P2X1 and P2X3 (3-5), (ii) moderately
desensitizing and m-ATP-insensitive receptors including
P2X4 and P2X6 (6-12), and (iii)
nondesensitizing as well as m-ATP-insensitive receptors including
P2X2, P2X5, and P2X7 (11-14).
Results from Northern blots and in situ hybridization data
(11) have indicated that the six neuronal P2X subunits genes are
transcribed in specific but overlapping populations in the central and
peripheral nervous system (1, 11). This strongly suggests that neuronal
P2X subunits belonging to different functional groups might co-assemble
into heteromultimeric channels.
m-ATP applications (5). However, recent
immunocytochemistry results have demonstrated that P2X2 and
P2X3 subunits in rat dorsal root ganglia are rarely co-localized at the level of central primary afferents in the dorsal
horn of the spinal cord, despite their high degree of co-localization in somata, indicating different subunit-specific subcellular targetings (16). Altogether, these data suggest that physiologically relevant associations of neuronal P2X subunits, giving rise to phenotypes that
are not mediated by the previously described P2X2+3 (5, 17)
or P2X4+6 (18) receptors, remain to be discovered.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
m-ATP, exploiting the fact that
homomeric P2X5 ATP-gated channels are almost insensitive to
this agonist when applied at concentrations below 100 µM
(Fig. 1) (11, 12). Whereas homomeric P2X1 receptors desensitize strongly in the first seconds of
agonist application, a slowly desensitizing response induced by 50 µM m-ATP was observed in oocytes co-injected with
P2X1 and P2X5 subunits at a 1:1 cDNA molar
ratio (Fig. 1). This hybrid phenotype was the unambiguous trademark of
the expression of heteromeric P2X1+5 receptors. Oocytes
expressing P2X1+5 receptors showed robust 50 µM m-ATP-induced whole-cell currents with
amplitudes in the range of 3-15 µA at Vh = 
50 mV after 2-5 days of post-injection time, similar to currents
recorded from oocytes expressing P2X1 alone (Fig. 1).
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Fig. 1.
Co-expression of P2X1
with P2X5 (P2X1+5)
yields a slowly desensitizing current that is activated by
m-ATP. Whole-cell currents were
recorded from oocytes after nuclear injections with P2X1
cDNA alone, P2X5 cDNA alone, and P2X1 + P2X5 cDNAs (P2X1+5) on prolonged
applications of 50 µM m-ATP. Fast desensitization
of the m-ATP-induced current occurs in oocytes expressing
P2X1 alone but not in oocytes expressing P2X1
and P2X5 together. P2X5-expressing oocytes
showed weak currents to 50 µM ATP and no detectable
response to 50 µM m-ATP. Oocytes were
voltage-clamped at Vh = 100 mV.
Bars represent the durations of agonist applications.
P2X1+5 receptors slowly desensitized during agonist
application but showed complete recovery in 2 min (Fig.
2), a noticeable difference with
homomeric P2X5 receptors that do not desensitize in
heterologous systems (Fig. 1) (11, 12). However, P2X1+5 receptors (Fig. 2, B and D) recovered
significantly faster than P2X1 receptors, the latter
recovering less than 50% of their initial response after 5 min of
washout (Fig. 2, A and C). We noticed slight
differences in the rate of desensitization of P2X1+5 receptors between oocytes (Fig. 2). These variations of phenotype could
be because of the expression of populations of heteromeric channels
with different stoichiometries, a cell-dependant variable that is not
controlled in these experiments of co-injection. The kinetic properties
of P2X2 receptors have been shown to be modulated by
protein kinase A activity (19). Thus it is possible that inter-individual differences in the levels of endogenous kinase activity present in oocytes could have some impact on the properties of
desensitization of P2X1+5 receptors. Furthermore, the correlation between the number of P2X5 subunits and the
kinetic properties of the oligomeric complex, which has been reported to be a trimer for homomeric P2X1 channels (20), is not yet known.
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P2X1+5 receptors were challenged with ATP, m-ATP, and
ADP at various concentrations for comparison with the pharmacology of
homomeric P2X1 and P2X5 receptors. We measured
EC50 values for P2X1+5 heteromers of 0.4 ± 0.2 µM for ATP, 1.1 ± 0.6 µM for
m-ATP and 13 ± 4 µM for ADP (Fig.
3). These EC50 values were
not significantly different from those obtained with homomeric P2X1 receptors in the same experimental conditions:
0.7 ± 0.1 µM for ATP, 2.4 ± 1 µM for m-ATP, and 47 ± 9 µM for
ADP (Fig. 3), in good agreement with previously published data (3).
Differences in the apparent Hill coefficient nH
(cooperativity index) of ADP activation between P2X1
(nH = 4.9 ± 2.3) and P2X1+5
(nH = 1.6 ± 0.8) (Fig. 3C)
could be because of the fact that we record from a heterogeneous
population of P2X1-containing receptors with varying
stoichiometries. The amplitudes of peak currents from P2X5-expressing oocytes were too small to carry out
complete dose-response curve experiments with these agonists (Fig. 1).
No significant differences were observed between P2X1+5 and
P2X1 receptors during co-applications of extracellular zinc
ions (10 µM), protons (pH 6.4), or alkaline solutions (pH
8.4) with sub-saturating concentrations of ATP (0.1 µM)
(data not shown). Our results suggest that P2X1 subunits
confer their high m-ATP sensitivity to the P2X1+5 heteromers. Another specific pharmacological property of
P2X1 subunits, the potent inhibitory effect of
trinitrophenyl-ATP (TNP-ATP) (20), is observed in the heteromeric
receptors (Fig. 4A). In conditions of co-application of TNP-application of TNP-ATP and m-ATP without pre-incubation, we measured an IC50 of
64 ± 14 nM on P2X1+5 and 200 ± 120 nM on homomeric P2X1receptors (Fig.
4B). This subunit association is therefore reminiscent of the association between P2X2 and P2X3 in which
P2X3 is the pharmacologically dominant component both for
m-ATP sensitivity (5, 17) and blockade by TNP-ATP (21, 22).
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To demonstrate direct associations between P2X1 and
P2X5 subunits that underlie their assembly in hybrid
heteromers, we assayed their physical interaction by co-purification of
epitope-tagged subunits in transfected HEK-293A cells. Purification of
P2X5-His6 on nickel-binding resin in
nondenaturing conditions (see "Experimental Procedures" for
details) allowed the detection of co-transfected P2X1-Flag
in Western blots (Fig. 5, lane
C). Reciprocally, P2X1-His6 was shown to
co-assemble with P2X5-Flag (Fig. 5, lane D).
Positive controls included pseudo-homomeric receptors composed of
P2X1-His6 + P2X1-Flag or
P2X5-His6 + P2X5-Flag (Fig. 5,
lanes A and B). Technical controls of
transfections with one P2X subunit only or with sham-transfected
HEK-293A cells were negative (data not shown).
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Peripheral sensory neurons have been reported to express ATP-gated
channels with a slow rate of desensitization and a high sensitivity to
m-ATP characterized by EC50 in the low micromolar range (Ref. 5, and references therein). This sensory phenotype was
thought to be exclusively accounted for by the co-assembly of
P2X2 and P2X3 subunits into heteromeric
P2X2+3 receptors (5, 17). Alternatively, we propose from
our results that slowly desensitizing and m-ATP-elicited
responses could be mediated by hybrid P2X1+5 heteromeric
receptors endowed with the pharmacology of P2X1 and the
kinetics of P2X5. Our data suggest to use TNP-ATP as a
specific antagonist of P2X1-containing ATP-gated channels. In spinal motoneurons where P2X3 is absent, complete
blockade of slowly desensitizing P2X responses by 1 µM
TNP-ATP would indicate the expression of P2X1+5 heteromeric channels.
Using subunit-specific polyclonal antibodies, Vulchanova et al. (23) described a strong P2X1 immunoreactivity in the laminae I-II of spinal cord, corresponding to presynaptic labeling of central axon terminals from dorsal root ganglia sensory neurons. As P2X2 and P2X3 subunits do not appear to co-assemble in heteromeric channels in these primary afferents (16), a presynaptic localization of P2X1+5 receptors would provide sensory axon terminals with high sensitivity to ATP and slowly desensitizing voltage-independent calcium entry that could play a modulatory role in the release of central neurotransmitters glutamate or substance P (24). The effects of presynaptic P2X1+5 receptors on the release of sensory transmitters can now be experimentally challenged with application of the blocker TNP-ATP at low concentrations.
In the central nervous system, an important role for purines in
motor systems is deduced both from the distribution of several P2X
subunits mRNA within cranial and spinal motor nuclei (11) and from
the powerful cellular effects of extracellular ATP on motor outflow
(25). More specifically, a subset of large projection motoneurons in
lamina IX of rat spinal cord has been characterized by the
co-expression of P2X1 and P2X5 subunits (11).
We propose from their functional properties that highly
agonist-sensitive P2X1+5 receptors might provide a specific
excitatory function to the motor control by allowing a sustained entry
of extracellular calcium within motoneurons in response to minute
amounts of released ATP.
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ACKNOWLEDGEMENT |
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We gratefully acknowledge Kazimierz Babinski for the cloning of the rat P2X5 receptor subunit.
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FOOTNOTES |
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* This work was supported by the Medical Research Council of Canada and the Fondation des Maladies du Coeur du Québec.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ Present address: Dept. of Cellular and Molecular Physiology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536.
¶ Recipient of a Postdoctoral Fellowship from the Fondation pour la Recherche Médicale (France).
Junior Scholar from the Fonds de la Recherche en Santé
du Québec. To whom correspondence should be addressed: Montreal
Neurological Institute, 3801 University, Montreal, Quebec H3A 2B4,
Canada. Tel.: 514-398-5029; Fax: 514-398-8106; E-mail:
mips{at}musica.mcgill.ca.
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ABBREVIATIONS |
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The abbreviations used are:
m-ATP, ,-methylene ATP;
His6, hexahistidine;
TNP-ATP, trinitrophenyl-ATP;
NTA, nitrilotriacetic acid.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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