Unique Functional Properties of a Sensory Neuronal P2X ATP-Gated Channel from Zebrafish - Bou&-Grabot - 2000 - Journal of Neurochemistry - Wiley Online Library
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Abstract: We report here the structural and functional
characterization of an ionotropic P2X ATP receptor from the lower vertebrate
zebrafish (Danio rerio). The full-length cDNA encodes a 410-amino
acid-long channel subunit zP2X3, which shares only 54% identity
with closest mammalian P2X subunits. When expressed in Xenopus
oocytes in homomeric form, ATP-gated zP2X3 channels evoked a unique
nonselective cationic current with faster rise time, faster kinetics of
desensitization, and slower recovery than any other known P2X channel.
Interestingly, the order of agonist potency for this P2X receptor was found
similar to that of distantly related P2X7 receptors, with
benzoylbenzoyl ATP (EC50 = 5 &M) ≫ ATP
(EC50 = 350 &M) = ADP & ,&-methylene ATP
(EC50 = 480 &M). zP2X3 receptors are highly
sensitive to blockade by the antagonist trinitrophenyl ATP (IC50
& 5 nM) but are weakly sensitive to the noncompetitive antagonist
pyridoxal phosphate-6-azophenyl-2&,4&-disulfonic acid.
zP2X3 subunit mRNA is exclusively expressed at high levels in trigeminal neurons and Rohon-Beard cells during embryonic development, suggesting that neuronal P2X receptors mediating fast ATP responses were selected early in the vertebrate phylogeny to play an important role in sensory pathways.Extracellular ATP released from synaptic vesicles or lytic cells can induce membrane depolarizations through the direct opening of ATP-gated cation channels, or P2X receptors, expressed in a variety of neuronal and non-neuronal populations (). Despite the short half-life of ATP in the
extracellular space (), the gating of native P2X receptor channels evokes a fast
inward current carried by mono-valent and calcium ions
). Ionotropic
purinergic responses have been shown to be involved in a broad range of
calcium-dependent signaling events from the neurogenic control of smooth
muscle contraction () to the regulation of neurotransmitter release
seven genes coding for P2X receptor subunits identified in mammals share
38-48% identity at the protein level. The global architecture of P2X channels
with two transmembrane domains, intracellular termini, and a cysteine-rich
extracellular loop resembles that of recently discovered proton-gated channels
(). However, no significant homology of primary structure has
been observed between P2X and acid-sensing channels. From heterologous
expression studies, homomeric P2X channels can be classified in three groups
according to their functional and pharmacological properties (for review, see
first group of ionotropic ATP receptors with fast desensitization and high
sensitivity to the agonists ATP and ,&-methylene ATP
(&mATP) includes the P2X1 and P2X3 subtypes.
They are blocked by nanomolar concentrations of the antagonist
2&-O-trinitrophenyl ATP (TNP-ATP)
second group is composed of slowly desensitizing,
&mATP-insensitive, and suramin-sensitive P2X2 and
P2X5 subtypes. The third group contains slowly desensitizing
P2X4 and nonneuronal P2X7 subtypes that are also
insensitive to &mATP and suramin. Recombinant P2X6
homomers have been expressed very rarely in transfected mammalian cells
() and are
silent in Xenopus oocytes (;
). Interestingly, functional heteromeric P2X receptor subtypes
resulting from the assembly of P2X2 + P2X3
P2X4 + P2X6
(), or P2X1 + P2X5
) subunits display hybrid phenotypes endowed with the
pharmacology of the subunit most sensitive to ATP. Despite intensive research
on their characterization in heterologous expression systems or in native
cells, most key properties of ATP-gated channels are not yet assigned to
structural features that could underlie their electrophysiological behavior or
their subtype-selective pharmacology. For instance, the residues of the second
transmembrane domain lining the hydrophilic pore have been identified by
cysteine scan mutagenesis (; ). Domains of P2X receptors involved in desensitization have
been identified (), and a phosphothreonine in an N-terminal protein kinase C
site is required for the expression of slowly desensitizing P2X2
(). Studies on the domains responsible for
heteropolymerization have also been published
(), but no
clear information is available yet on the ATP binding domain, the mechanism of
blockade by suramin and other noncompetitive antagonists, or the determinants
of the weak selectivity of P2X cation channels for small cations. Thus,
functional comparisons between mammalian P2X subunits and P2X subunits from
distant lower species would likely be helpful to investigate the specific
structure&activity relationships of this class of excitatory
neurotransmitter-gated channels, unrelated in sequence and transmembrane
topology to nicotinic acetylcholine or glutamate receptor channels. We report
here the primary structure and electrophysiological and pharmacological
properties of an ionotropic ATP receptor expressed in the nervous system of
the lower vertebrate zebrafish Danio rerio. This member of the P2X gene family is the most phylogenetically distant from the mammalian subunits known so far and defines by itself a novel phenotypic group of nucleotide-gated channels.EXPERIMENTAL PROCEDURESMolecular cloning and in situ hybridizationUsing the program tblastn () to search the GenBank database with a virtual probe
corresponding to a consensus P2X channel subunit, a novel partial P2X sequence
was identified in the nucleotide sequence AI588766 from the Washington
University zebrafish EST (Expressed Sequence Tag) project. The full-length
zebrafish P2X clone (zP2X3) corresponding to the EST was isolated
from a directional oligo dT-primed cDNA library constructed from various
pooled embryonic stages and adult liver, and complete automatic sequencing was
performed on both strands. In situ hybridization was carried out on
whole-mount preparations of embryos at various stages with 1.4-kb full-length
antisense riboprobes labeled with digoxygenin, as previously described by
Thisse et al. ().Injection of oocytes with recombinant channelsOriginal clone in pSport1 vector was transferred into pCDNA3 vector (Invitrogen) with cytomegalovirus promoter for expression in oocytes. Ovary lobes were surgically retrieved from Xenopus laevis frogs under deep
Tricaine (Sigma) anesthesia. Oocyte-positive lobes were then treated for 3 h
at room temperature with type I collagenase (Life Technologies) in
calcium-free Barth's solution under vigorous agitations. Stage V-VI oocytes
were then manually defolliculated before nuclear microinjections of 5 ng of
supercoiled plasmid coding for zP2X3, rat P2X3, or rat
P2X1 subunit. The cells were maintained in Barth's solution
containing 1.8 mM calcium chloride and 10 &g/ml gentamicin (Sigma) at 19&C for up to 5 days.Electrophysiology and data analysisTwo-electrode voltage-clamp recordings were made 1-3 days after microinjection using an OC-725B amplifier (Warner Instruments). Cells were voltage clamped at -60 mV. 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 interface (National Instruments). Recorded traces were postfiltered at 20-50 Hz in Axograph (Axon Instruments) for purpose of illustration only. Ringer's solution containing 115 mM NaCl, 2.5
mM KC1, 1.8 mM CaCl2, and 10 mM HEPES
buffered at pH 7.4 was perfused onto oocytes at a constant flow rate of 10-12
ml/min. The volume of the bath in the perfusion chamber was set to 200-250
&I. Agonists ATP, benzoylbenzoyl ATP (bzATP), &mATP, and ADP
and antagonists suramin, pyridoxal
phosphate-6-azophenyl-2&,4&-disulfonic acid (PPADS), and
2&,3&-O-trinitrophenyl ATP (TNP-ATP; Sigma) were prepared
in bath perfusion buffer at their final concentration. Dose&responses
curves and kinetic analysis were carried out using the Prism 2.0 software
(GraphPad, San Diego, CA, U.S.A.). Agonist concentration&response
curves, EC50 values, and cooperativity indexes were derived from
fittings to the Hill sigmoidal equation. Activation and desensitization curves
were fitted with monoexponential growth and monoexponential decay equations,
respectively. Time constants for 50% activation and 50% desensitization were
then derived to quantitate the differences of kinetic properties between
zP2X3, rat P2X1, and rat P2X3 receptors.RESULTSPrimary structure of zP2X3 receptor subunitA novel partial P2X subunit was identified by BLAST (Basic Local Alignment Search Tool) in the EST AI588766 located in the 3& region of a zebrafish mRNA. The 1,462-nucleotide-long full-length cDNA clone corresponding to this EST encodes a membrane protein with the characteristic pattern of features of a P2X ATP-gated channel subunit (). At the nucleotide level, this cDNA displays 59% identity with
rat P2X3 in the coding region. The predicted protein of 410 amino
acids has 54% identity with rat P2X3, 41% with rat P2X2,
33-39% with rat P2X1, P2X4, P2X5, or
P2X6, and 32% with the nonneuronal rat P2X7. On this
structural basis, we decided to name this subunit zP2X3, despite
significant functional differences with rat P2X3 (see below).
Hydrophobicity analysis and alignment with known P2X subunits predicted an
intracellular N-terminal domain of 33 amino acids, a first transmembrane
domain of 18 amino acids, an extracellular loop of 280 amino acids with the 10
conserved cysteines characteristic of the P2X receptor family, and a second
transmembrane domain of 26 amino acids followed by a C-terminal domain of 53
amino acids ().Figure&1. Primary structure of zP2X3 subunit and alignment with closest
mammalian P2X ATP-gated channel subunits, rat P2X3 and rat
P2X2. The two predicted transmembrane domains are underlined, and
the 10 extracellular cysteines conserved in all known P2X subunits are
indicated by dark squares. Nucleotide and protein sequences of
zP2X3 subunit have been deposited in the GenBank database (accession no. AF237683).Functional characterization of homomeric zP2X3 channelsWhen expressed in Xenopus oocytes, zP2X3 subunits
assembled in homomeric ATP-gated channels
(). A fast inward current
of 0.44 & 0.1 &A (n = 8) was evoked by applications of 100
&M extracellular ATP, followed by fast desensitization of the
channels during the application of the agonist
(). From its reversal
potential close to 0 mV (Erev = +5 mV) measured from
current&voltage relationship plots, we concluded that the
zP2X3-mediated inward current is carried by cations and that the
zP2X3 ATP-gated channels are nonselective cation channels
(). No inward or outward
rectification was observed between -60 and +60 mV, indicating the absence of a
voltage-dependent mechanism in the operation of these channels in this range
of membrane potentials. Likely related to their fast kinetics of
desensitization, the zP2X3 receptors never recovered their full
response to ATP from the first stimulation, even after several minutes of
agonist washout (). We
observed and reported this property of partial recovery with homomeric
P2X1 receptors
(). On the contrary, rat P2X3 receptors recovered
completely after 3-5 min in the same conditions
().Figure&2. Representative fast phenotype of ATP-induced currents mediated by homomeric zP2X3 nonselective cation channels. A: Typical currents
recorded from oocytes expressing zP2X3 in response to different
concentrations of ATP indicated in micromolar at the top of each trace.
B: Current&voltage relationship of homomeric zP2X3
ATP-gated channels. Peak currents (means & SEM from 7-11 oocytes)
recorded at different holding membrane potentials (illustrated in
inset) were normalized to the maximal responses obtained at -60 mV.
C: Partial recovery after 5-min washings between ATP applications and
comparison with rat P2X3 phenotype.The zP2X3 ATP receptors responded to extracellular nucleotides
with unusually fast kinetics (Figs.
). To quantitatively compare
several fast P2X phenotypes recorded in the same heterologous expression
system with the same protocol of agonist application (see inset in
), we measured the time
constant for half-activation (&a50) of rat P2X1, rat
P2X3, and zP2X3 by fitting the relation between time
before peak and percentage of peak response. The single exponential fitting
gave a &a50 = 20 & 5 ms for zP2X3 compared
with a &a50 = 225-230 & 60 ms for both rat
P2X1 and P2X3 receptors
(). Even taking into
account the experimental underestimation of the time necessary to reach the
peak current due to fast desensitization, these values emphasized the unique
speed of activation of zP2X3 channels at the whole-cell level.
Using a similar approach, we compared the kinetics of desensitization of
zP2X3 with rat P2X1 and rat P2X3. Again,
zP2X3 channels were found to be the fastest of all known P2X
subtypes, with a time constant of desensitization of 40 & 18 ms
compared with 374 & 55 and 665 & 75 ms for rat P2X1
and P2X3, respectively.Figure&3. zP2X3 channels display fastest kinetics of activation and
desensitization in P2X family. Shown is a comparison of the rates of
activation (A) and desensitization (B) of zP2X3
(filled circles) with rat P2X1 (filled squares) and rat
P2X3 (open circles). We measured at different times before and
after the peak the response evoked by application of 300 &M ATP
for zP2X3 (n = 10) and 50 &M ATP for rat
P2X1 (n = 7) and rat P2X3 (n = 7). Inset:
Superimposed traces of representative ATP-induced currents obtained in
Xenopus oocytes injected with zP2X3, rP2X1, and
rP2X3.We tested the potency of several agonists to activate zP2X3
receptors (). The
response to large concentrations (&500 &M) of ATP or
&mATP did not reach a maximum, so their EC50 values are
only conservative estimations. However, the kinetics of currents evoked by
equieffective doses of the different agonists were similar (see inset in
), as were the slopes of
their activation curves (). ATP (EC50 = 350 & 57 &M) was
found to be much less potent than bzATP (EC50 = 5 & 1
&M), equipotent to ADP (EC50 = 322 & 77
&M), but significantly more potent than &mATP
(EC50 = 479 & 22 &M) to gate the
zP2X3 channels. The index of cooperativity of the
dose&response curve for bzATP was nH = 2.2. We
tested also the sensitivity of rP2X3 to bzATP and measured an
EC50 of 8 & 1 &M (data not shown). Differences
in the profile of sensitivity to the agonists bzATP, ATP, &mATP,
and ADP between zebrafish and rat P2X3 receptors are represented in
.Figure&4. Sensitivity of zP2X3 receptors to the agonists ATP, ADP, bzATP,
and &mATP. A: Dose&response curves for the agonists
ATP (filled squares), ADP (open squares), bzATP (filled circles), and
&mATP (filled triangles). Mean peak currents were normalized to
the maximal response for bzATP, to the response obtained with 300
&M for ATP, with 500 &M for &mATP, and
with 800 &M for ADP. Each point corresponds to the average of data
obtained from 8-12 oocytes. Inset: Similar desensitization kinetics of
the response of zP2X3 channels to different agonists. B:
Comparison of agonist sensitivity between zP2X3 and rat
P2X3. EC50 values of ATP, &mATP, and ADP for
rat P2X3 were obtained from Chen et al.
() and Lewis et al.
().The antagonist TNP-ATP blocks P2X1- and P2X3-
containing receptors with high affinity
). We show here that TNP-ATP also inhibits zP2X3
channels at nanomolar concentrations, when co-applied with ATP after
preincubation: 5 nM TNP-ATP suppressed 80% of the maximal response to
300 &M ATP (). The antagonist suramin spared 25% of the maximal response to
ATP when applied at 40 &M. However, contrasting with the mammalian
P2X3 receptors, &20% of maximal ATP-gated currents remained
after application of 30 &M PPADS in the same conditions, and a
significant current was still recorded at 100 &M
(). By comparison, rat
P2X3 receptors are completely blocked by 30 &M PPADS,
with an IC50 = 1.5 &M
().Figure&5. Differential blockade of zP2X3 activation by the antagonists
TNP-ATP, PPADS, and suramin. Amplitude of responses (means & SEM, n =
6-11) to co-application of 300 &M ATP is shown with different concentrations of antagonist as a percentage of the response to ATP alone (control response). In all cases, oocytes were preincubated during 30 s with the antagonist alone.Cellular distribution of zP2X3 mRNA during developmentAnatomical localization of zP2X3 transcripts using in situ
hybridization revealed that these ATP-gated channel subunits are exclusively
expressed in the nervous system during development
(). Only two subsets of
central neurons transcribe zP2X3 gene in the embryo during the
stage corresponding to 24-48 h post fertilization (hpf): the neurons from the
trigeminal ganglia in the rhombencephalon
() and the
Rohon&Beard cells in the spinal cord
(). These two
populations of neurons are primary sensory neurons. From their common set of
genetic (;
immunocytochemical (; ) markers, trigeminal and Rohon&Beard neurons seem to
share the same development program despite their different localization.
Furthermore, both types of neurons are among the earliest neurons to send
axons () and
are mechanosensory neurons that participate in the motor responses to touch,
the first behavioral response of the embryo
(). We noticed that the expression of zP2X3 is
regulated during development: Neuronal zP2X3 expression peaks at
24-48 hpf and then disappears completely from spinal cord at 72 hpf.
Rohon&Beard cells die before the adult stage when they are functionally
replaced by peripheral dorsal root ganglia
(), so the expression of zP2X3 seems to follow the
fate of these transient spinal sensory neurons. A small number of positive
neurons remained detectable in trigeminal nuclei at 96 hpf. Using RT-PCR, we
detected zP2X3 mRNA in the adult animal (data not shown), where
zP2X3 channels might participate in the excitability of peripheral
trigeminal and dorsal root ganglion neurons
().Figure&6. Neuronal localization of zP2X3 gene expression in zebrafish
embryo. A: Cells in the trigeminal ganglia (TG) and Rohon&Beard
neurons (RB) in the spinal cord (SC) express high levels of zP2X3
transcripts at 24 hpf. B: Dorsal view of a 24 hpf embryo with the yolk
sac dissected out. Characteristic bilateral rows of
zP2X3-expressing neurons are labeled in the trigeminal ganglia and
Rohon&Beard cells. C: Transverse section through the trunk of an
embryo of the same stage as in A, showing zP2X3-positive Rohon&Beard neurons located in the dorsal horn of spinal cord. Bar = 130 &m (A and B) and 30 &m (C). e, n, m, y, yolk sac.DISCUSSIONThe predicted structure of zP2X3, a novel member of the P2X ATP
receptor family, displays all the key elements that characterize these
channels activated by extracellular nucleotides. The N-terminal domain of
zP2X3 contains the highly conserved protein kinase C
phosphorylation site with the acceptor Thr17 that has recently been
shown to control the kinetic properties of P2X2 channel subtype
(). Seven Asnglycosylation consensus sites have been
counted in the extracellular domain of zP2X3, but the potential
acceptor Asn180 is the only one at the same relative position in
rat P2X3 and in other subunits. A protein kinase C site is also
present in the C-terminal domain of zP2X3. This potential site of
phosphorylation could have an impact on the activity of zP2X3
channels because a protein kinase A site located in the C-terminal domain of
P2X2 has been reported to be involved in the modulation of
desensitization (). Interestingly, the motif EKXSXDSGX(Y/F)SIG is found in the
distal part of the C-terminal domain of human
zebrafish P2X3 subunits. The presence of this conserved stretch of
amino acids in vertebrate P2X3 subunits suggests that it could play
a role in intrasubunit interactions, in a P2X3-specific mechanism
of heteromeric assembly, or in the association of P2X3 subunits with a conserved heterologous intracellular partner.Despite significant homology with rat P2X3 at the protein level,
we noticed some unique aspects of the function of homomeric zP2X3
channels during their electrophysiological characterization in
Xenopus oocytes. The small difference of EC50 for bzATP
between rat and zebrafish P2X3 was found not significant
(), and ADP has also a
similar potency for zebrafish and rat P2X3
). However,
we observed that ATP and &mATP, two high-affinity agonists for the
mammalian P2X1 and P2X3 receptors
much lower potency (&200 times) for zP2X3. These major
differences with the pharmacological profile of rat P2X3
demonstrated that the primary structure of a novel P2X channel subunit is not
currently a reliable predictor of its pharmacology. Indeed, the rank order of
agonist potency for zP2X3 was closest to the one of more distantly
related nonneuronal P2X7 receptor that is more sensitive to bzATP
than to ATP in recombinant () or native
() forms in macrophages.The homomeric zP2X3 channels thus define a novel phenotype for
ATP-gated channels with fast kinetics of desensitization, with bzATP being a
more potent agonist than ATP and with a weak sensitivity to PPADS.
Constructions of functional chimeras containing domains of mammalian and
zebrafish P2X3 subunits should facilitate the identification of
regions involved in sensitivity to agonists, in high-affinity blockade by
noncompetitive antagonists, or in recovery from desensitization. We believe
that the partial but significant homology between the extracellular domains of
rat P2X3 and zP2X3 subunits (56%) will increase the
chance of getting functional chimeric receptors with informative pharmacology.
A chimera with the transmembrane domains and intracellular termini of
zP2X3 and a part of or the whole extra-cellular domain of rat
P2X3 would be helpful to pinpoint the structural elements conferring the high sensitivity to ATP and &mATP as well as the determinants of high sensitivity to blockade by PPADS.All known neuronal P2X receptor subunits are expressed in peripheral sensory ganglia in mammals (). From our in situ hybridization results, the expression of
zP2X3 restricted to primary sensory neurons appears to mirror the
sensory-specific anatomical localization of rodent P2X3 subunits
(). Such a
conserved pattern of cellular expression suggests strongly that the
P2X3 subunit/subtype of ATP receptors has been selected early
during vertebrate phylogeny, and likely before the vertebrates arose, for the
detection of sensory inputs. The fast kinetics of activation and
desensitization of zebrafish and mammalian P2X3 channels could have
been one of the main physiological properties maintained in vertebrates by
selective pressure. The high sensitivity to ATP of mammalian P2X3,
not found in zebrafish receptors, would then correspond to a more recent
functional acquisition. The low sensitivity of zP2X3 receptors to
the endogenous agonist candidate ATP could indicate also that other ligands
activate them more efficiently or that co-agonists are required. Several
neuronal P2X subtypes are sensitive to extracellular protons and zinc ions
tested the effects of co-application of alkaline (pH 8.5) and acidic (pH 6.3)
solutions and 50 &M zinc chloride ions with 100 &M ATP
on zP2X3, but we did not record any significant difference with ATP
alone (data not shown). In a subset of rodent small-diameter sensory neurons,
P2X3 subunits associate with P2X2 to generate functional
P2X2+3 receptors wi thus, it is probable that
zP2X3 subunits assemble natively with other P2X partners during
development and in the adult. Other P2X genes expressed in trigeminal and
Rohon&Beard sensory neurons of zebrafish remain to be identified and
cloned to test subunit combinations and investigate how zP2X3
contributes to the phenotype of heteromeric channels. Thanks to a short life
cycle in autonomous stages, the zebrafish has been chosen as a particularly
good experimental model to dissect the role of specific genes in vertebrate
development. With use of dominant negative transgenes or antisense technology,
it will be interesting to study the impact of a knockout of the functional
expression of zP2X3 subunits during development to increase our understanding of the role of ATP-gated channels in sensory transmission in lower vertebrates and in mammals.
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