From the Department of Otolaryngology, Division of
Sensory Biophysics, Röntgenweg 11 and the § Department
of Physiology II, Gmelinstrasse 5,
D-72076 Tübingen, Germany
Received for publication, February 15, 2001, and in revised form, March 20, 2001
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ABSTRACT |
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The P2X3 receptor is an
ATP-gated ion channel predominantly expressed in nociceptive neurons
from the dorsal root ganglion. P2X3 receptor channels are
highly expressed in sensory neurons and probably contribute to the
sensation of pain. Kinetics of P2X3 currents are
characterized by rapid desensitization (<100 ms) and slow recovery
(>20 s). Thus, any mechanism modulating rate of desensitization and/or
recovery may have profound effect on susceptibility of nociceptive
neurons expressing P2X3 to ATP. Here we show that currents
mediated by P2X3 receptor channels and the heteromeric
channel P2X2/3 composed of P2X2 and
P2X3 subunits are potentiated by the neuropeptides
substance P and bradykinin, which are known to modulate pain
perception. The effect is mediated by the respective neuropeptide
receptors, can be mimicked by phorbol ester and blocked by inhibitors
of protein kinases. Together with data from site-directed mutagenesis
our results suggest that inflammatory mediators sensitize nociceptors
through phosphorylation of P2X3 and P2X2/3 ion
channels or associated proteins.
P2X receptors mediate fast responses of excitable cells to
application of ATP. So far, seven different P2X subunits have been cloned (P2X1-7), which form a gene family (1). They have two transmembrane domains with intracellular termini and a rather large
extracellular loop (2, 3) and form non-selective cation channels with a
high permeability to Ca2+. Various P2X receptors show
differences both in the affinity to ATP and in the kinetics of
activation and inactivation. P2X1 and P2X3 show
high agonist affinity (EC50 The desensitization properties of the rapidly activating and
inactivating P2X1 and P2X3 are likely to be of
physiological importance as synaptic transmission takes place in a few
milliseconds. Modulation of the rate of desensitization and/or recovery
of these receptors may contribute to synaptic efficacy.
P2X3 is almost exclusively expressed in sensory neurons (7,
8), mostly in capsaicin-sensitive nociceptors (9-11).
P2X3-mediated currents in sensory neurons are large (12),
and P2X3 shows the strongest expression level in dorsal
root ganglion (DRG) neurons compared with other P2X receptors (10). The
P2X3 protein is found on the sensory endings as well as on
the presynaptic membrane in inner lamina II of the spinal horn (9), and
its activation by ATP might contribute to the sensation of pain. In the
dorsal horn a presynaptic mechanism is involved, which leads to a
potentiation of the excitatory postsynaptic potential (13).
Nociceptive neurons express homomeric P2X3 as well as
heteromeric P2X2/3 receptors (5). Both types of channels
can be expressed separately or together in individual neurons (14);
recent data suggest that homomeric P2X3 is predominantly
expressed on small diameter sensory neurons and heteromeric
P2X2/3 on medium diameter sensory neurons (15-17).
Application of Here we show that the inflammatory mediators substance P (SP) and
bradykinin (Bk) can potentiate currents through P2X3 and P2X2/3 expressed in Xenopus oocytes. Both induce
an increase in peak current as well as in steady-state current with
repeated application of ATP. Phorbol ester had a similar, non-additive effect, suggesting a pathway involving protein kinase C. Mutagenesis experiments suggest that a conserved threonine at the intracellular N
terminus, which had been shown to be phosphorylated in P2X2 (19), is crucial for this effect. Together, our results suggest that
inflammatory mediators potentiate ATP-gated currents in nociceptors through a mechanism involving either direct phosphorylation of P2X3 receptors or phosphorylation of a so far unidentified protein.
cDNAs and Site-directed Mutagenesis--
cDNAs coding
for P2X2 and P2X3 are contained in the
BamHI/NotI (P2X2) or the
EcoRI/XhoI (P2X3) site of vector
pCDNA3 (Invitrogen, Groningen, Netherlands). Splice variant
P2X2-2 has been described previously (20). cDNAs for
SP and Bk receptors are contained in the HincII (SP
receptor) or the EcoRI (B2 receptor) site of vector
pBluescript. Chimeric molecules between P2X2 and P2X3 and point mutations were constructed by recombinant
polymerase chain reaction using standard protocols. All polymerase
chain reaction-derived fragments were entirely sequenced.
Oocyte Expression--
Capped cRNA was synthesized using
mMessage mMachine (Ambion, Austin, TX) by T7 RNA polymerase from
cDNAs, which had been linearized by XhoI (P2X receptors)
or XbaI (SP and B2 receptors).
Xenopus laevis oocytes were surgically removed from adult
animals and manually dissected. For the investigation of a possible effect of inflammatory mediators on currents through P2X receptors, we
coexpressed the rat SP receptor (NK-1 receptor) or the human Bk (B2)
receptor with the P2X receptors in oocytes. We injected either 5 ng of P2X3 or 20 pg of P2X2, together with 5 ng
of SP or Bk receptor; for expression of the P2X2/3
heteromer, we co-expressed a mix of 0.5 ng of P2X2 and 5 ng
of P2X3 with the neuropeptide receptors. cRNA was injected
into stage V or VI Xenopus oocytes and oocytes kept in OR-2
medium (concentrations (in mM): 82.5 NaCl, 2.5 KCl, 1.0 Na2HPO4, 5.0 HEPES, 1.0 MgCl2, 1.0 CaCl2, and 0.5 g/liter polyvinylpyrrolidon, pH 7.3)
at 19 °C. One day after injection, oocytes were treated with
collagenase type II (0.33 mg/ml; Sigma, Deisenhofen, Germany) for 40 min, and on the 2nd day the follicular layer was removed, as
folliculated oocytes are known to have intrinsic P2 receptors (21).
Defolliculated oocytes from every donor animal, which had been injected
only with the cRNA encoding the SP receptor, were tested for endogenous ATP-activated currents.
Electrophysiology--
Experiments were done at room temperature
3-7 days after injection using 2-microelectrode voltage clamp.
Currents were recorded with a TurboTec 01C amplifier (npi, Tamm,
Germany), digitized at 30 Hz (ITC16, HEKA, Lamprecht, Germany) and
stored on hard disk. The bath solution had the following composition
(in mM): 115 NaCl, 2.5 KCl, 1.8 MgCl2, 10 Hepes, pH 7.3. Bath solution contained no Ca2+ in order to
avoid activation of endogenous Ca2+-activated
Cl
P2X receptors showed the pharmacological and kinetic characteristics
reported in previous studies using heterologous expression systems and
as known from native tissue (Fig. 1). We used 100 nM ATP
for activation of P2X3, 300 µM for activation
of P2X2 and 10 µM Data Analysis--
Data on graphs indicate the median of the
maximal increase of ATP-elicited peak current after stimulation by SP
or Bk. Values were normalized to the ATP stimulation just before
application of SP or Bk. Error bars indicate the range of the results
as these were not symmetrically distributed. Moreover, we compared the amplitude at the end of the 50-s ATP application (quasi-steady state)
and report this as the I50 s.
Desensitization time constants were fitted to a single exponential
function. They are reported as mean ± standard deviation.
Statistical analysis was done with the unpaired Student's t test.
Currents through ATP Receptor Channels Containing the
P2X3 Subunit Are Potentiated by the Inflammatory Mediators
Substance P and Bradykinin--
We used Xenopus oocytes as
an expression system to investigate differential regulation of P2X
receptor subtypes. SP or Bk application to oocytes expressing the SP or
the Bk receptor, respectively, yields an immediate transient (
Besides this transient current, SP and Bk had a regulatory effect on
the amplitude of ATP-induced currents mediated by P2X3 receptors. As shown in Fig. 2A, the application of SP (100 nM for 50 s) to oocytes expressing P2X3
together with the SP receptor caused an immediate and transient
(
The desensitization rate of the whole cell current can be well fitted
with a single exponential function. The time constant of
desensitization was not significantly different before and after
treatment with SP (3.9 ± 1.7 s compared to 4.6 ± 2.3 s; p = 0.44, n = 10).
A similar potentiation of currents through P2X3 receptors
could be elicited by application of 100 nM Bk to oocytes
coexpressing the P2X3 receptor and the Bk B2 receptor
(median increase: 74%, range: 38-175%, n = 12; Fig.
2 (A and E); median increase of
I50 s: 89%, range: 33-140%,
n = 9), showing that multiple neurotransmitters can
modulate P2X3 receptors.
Using oocytes expressing the P2X2/3 heteromer (obtained by
coexpression of the P2X2 and P2X3 subunits)
together with the SP receptor, application of SP yielded a similar
transient and reversible increase of current (median increase of peak
amplitude: 38%, range: 21-64%, n = 6, Fig. 2
(B and E); median increase of
I50 s: 56%, range: 29-68%, n = 6). The effect was independent of the P2X receptor agonist; it could
be elicited by 300 µM ATP as well as by 10 µM
In contrast, the slowly desensitizing currents through
P2X2 receptors were not significantly increased by SP
receptor activation (median increase of peak current amplitude: 3%,
range: Potentiation by Inflammatory Mediators Is due to Activation of a
Protein Kinase--
It is known that the desensitization of nicotinic
acetylcholine receptors is directly accelerated by SP without the need
of the SP receptor (22). In oocytes expressing only P2X2/3
receptors but no SP receptor, the neuropeptide was not able to
sensitize currents through P2X2/3 (median increase of peak
amplitude:
Since SP and Bk are supposed to act via the phospholipase C/protein
kinase C pathway, we tested different pharmacological agents
interacting with kinases for their effect on sensitization of P2X
receptors. Oocytes expressing P2X3 and the SP receptor were
incubated with the serine/threonine-kinase inhibitor staurosporine (10 µM) for 10 to 30 min. After this incubation, application
of SP no longer increased ATP-activated peak or steady state currents (median increase in peak amplitude: The Intracellular C Terminus of P2X Receptors Controls Potentiation
by Inflammatory Mediators--
Intracellular signaling cascades most
likely act on intracellular parts of the P2X receptors. To identify
these regions, we made use of the differential effect of SP on
P2X2 and P2X3 designing chimeras between
P2X2 and P2X3. Exchanging the intracellular C termini yielded active channels. The P2X2 receptor with
P2X3 C terminus (P2X2-C-X3) formed
rather slowly desensitizing ion channels, which were, however, faster
than P2X2 wild type channels (mean of desensitization
rates ± S.D. 16.7 ± 0.7 s, n = 6, in
comparison to 95.6 ± 32.4 s with P2X2 wild type,
n = 8). As shown in Fig. 4A, the
P2X2-C-X3 receptor gained potentiation by SP.
It showed an increase in the steady state current that lasted longer
than the effect on P2X3 but was still transient (median
increase of peak current: 62%, range: 48-92%, n = 10, Fig. 4 (A and E); median increase of
I50 s: 71%, range: 48-101%,
n = 10). P2X3 receptors with
P2X2 C terminus (P2X3-C-X2) showed
faster desensitization than the P2X3 receptor and high
agonist affinity as the P2X3 receptor. Because of the fast
desensitization of the P2X3-C-X2 chimera, we
investigated this chimera as heteromer with P2X2. The
P2X3-C-X2/P2X2 receptor activated
either with
To further investigate the role of the P2X2 C terminus we
tested the P2X2-2 receptor, a P2X2 splice
variant in which 69 amino acids of the cytoplasmic C terminus are
missing (20). Fig. 4C shows that this splice variant was
also sensitized by SP (median increase of peak current: 36%, range:
18-49%, n = 4, Fig. 4E; median increase of
I50 s: 33%, range: 23-50%, n = 4). Thus, the P2X3 C terminus is not necessary for SP
modulation. Rather, it seems that the wild type P2X2 C
terminus inhibits the modulation by SP and that this inhibition is
relieved in the splice variant. This hypothesis is further supported by
a gain-of-regulation with a P2X2 receptor truncated at
position 401 (P2X2
N-terminal chimeras were also functional. A P2X2 receptor
with P2X3 N terminus (P2X2-N-X3)
was not regulated by SP, whereas a P2X3 receptor with
P2X2 N terminus (P2X3-N-X2) was
regulated by SP (data not shown), confirming that the C terminus
controls regulation of P2X receptors by SP.
A Conserved N-terminal Consensus Site Is the Most Likely Target for
Phosphorylation--
Regulation by SP might be due to direct
phosphorylation of the channel protein. We, therefore, constructed a
series of point mutations, either single or in combination, of
cytoplasmic serine or threonine residues in the P2X3
receptor. These mutations include all intracellular serines or
threonines that are in a consensus sequence for phosphorylation by
protein kinase A or C
(R/K(X0-3)S/ T(X0-3)R/K).
The effect of these mutations is summarized in Fig.
5. Combined mutation of all serines or
threonines that are in a consensus sequence for phosphorylation at the
cytoplasmic C terminus
(P2X3T364A/T365A/T369A/S371A/T382A/S387A/T388A)
leads to a functional, SP-regulated channel, rendering it rather
unlikely that direct phosphorylation of the C terminus mediates the
regulation. Moreover, there is no C-terminal consensus site conserved
between P2X3 and P2X2-2.
Single or combined amino acid substitutions of the serines or
threonines at the N terminus lead in all cases to functional, regulated
channels with the exception of P2X3T12A, which was not measurable. This threonine residue is conserved in all known P2X receptors, contained in a conserved consensus sequence for
phosphorylation (TXK/R) and has recently been shown to be
phosphorylated in the P2X2 receptor (19). Mutation of
lysine 14 in P2X3 contained in the consensus sequence
(P2X3K14Q) similarly leads to not measurable channels,
whereas mutation of threonine 13 leads to functional, regulated
channels (Fig. 5), identifying the consensus motif TXK as
crucial for normal channel function. A T12E mutant mimicking constitutive phosphorylation was also not measurable, demonstrating that it is not just the presence of a negative charge at this position,
which is important. Mutation of the corresponding threonine in either
the P2X2-C-X3 or the
P2X3-N-X2 chimera, or the P2X2-2 splice variant leads always to not measurable channels, rendering it
impossible to directly assess functional consequences of this mutation.
P2X2-2 possesses, apart from this completely conserved threonine, only one other serine/threonine at its cytoplasmic N
terminus. Mutation of this second serine (serine 11) leads to functional, regulated channels.
Together, these results suggest that the completely conserved threonine
at the cytoplasmic N terminus of P2X receptors is the best candidate
for an amino acid directly phosphorylated after stimulation by SP.
Alternatively, protein kinases might phosphorylate an unrelated
protein, which controls activity of P2X receptors.
Both P2X3 and P2X2/3 receptors are
expressed in sensory neurons, and there is accumulating evidence that
they have a specific role in nociception (17, 23-26). Moreover,
activation of P2X3 leads to a much stronger nociceptive
effect in inflamed compared with normal tissue (27, 28) and the
P2X2/3 heteromer might mediate mechanical allodynia (11).
Thus, it seems that sensitization of nociceptors leads to a bigger
response to ATP. Our study addressed the underlying mechanism by
reconstituting the relevant signaling cascades in Xenopus oocytes.
Sensitization of nociceptors is due to the release of inflammatory
mediators such as the neuropeptides SP (29) and Bk (30). SP is
expressed in small diameter afferent neurons (31, 32) and is released
upon peripheral nociceptive stimulation in the periphery as well as in
the superficial dorsal horn (33). It binds with high affinity to the
tachykinin receptor (NK-1), which is expressed in neurons of the spinal
cord (34, 35). There are conflicting data, however, on expression of
the SP (NK-1) receptor in primary afferent neurons from the DRG (34,
36-38). Although the receptor has not been identified by
immunocytochemistry (34) SP can elicit an inward current in DRG
neurons, which is mediated by a non-selective cation channel (37).
Moreover, it had been shown that application of SP to sensory neurons
can potentiate currents, which are gated by ATP (39). This suggests a
modulation of P2X receptors by SP also in sensory cells. So far,
neither the P2X subunit involved nor the intracellular signaling
cascade was known.
The main receptor for Bk in primary afferent neurons is the B2
receptor, which couples similar to the SP receptor to a signaling pathway using Gq and phospholipase C and leading to a rise
in intracellular Ca2+.
Our study demonstrates that the inflammatory mediators SP and Bk can
potentiate currents through P2X3 and P2X2/3
receptors in Xenopus oocytes.
Heteromeric P2X2/3 receptor channels showed a decrease of
the desensitization rate after treatment with inflammatory mediators, which could explain the potentiation. As the potentiation was only
transient, we were not able to measure the time constant for recovery.
Rate of desensitization of P2X3 was not significantly changed, but the fast desensitization rate of this channel may not be
reliably determined in whole oocytes. We, therefore, propose that an
effect on desensitization rate is the underlying mechanism of current potentiation.
Evidence that protein kinase C mediates potentiation of P2X receptors
in oocytes is severalfold. (i) Both the SP and the Bk receptor are
coupled to phospholipase C activation; (ii) phorbol ester can mimic the
effect; (iii) stimulation by inflammatory mediators and phorbol ester
is not additive; and, finally, (iv) the serine/threonine kinase
inhibitor staurosporine blocks potentiation of P2X receptors.
We could not unequivocally identify a phosphorylation site on
P2X3 receptors responsible for the modulation. Our chimeras between P2X2 and P2X3 implicated the
cytoplasmic C terminus as crucial. The results from
site-directed-mutagenesis suggest, however, that there is no
phosphorylation site on the cytoplasmic C terminus of P2X3
implicated in the effect. (i) Mutation of all the serine/threonine residues in any consensus site at the same time (7 out of a total of 12 serine/threonine) yielded functional, regulated channels; (ii) there is
no serine or threonine in any consensus site conserved between
P2X2-2 and P2X3; and (iii) there is only one
serine/threonine that is contained in both P2X2-2 and
P2X2 Mutants at the N terminus were either functional and regulated or not
measurable. The non-measurable mutants all concerned a highly conserved
consensus site for phosphorylation by PKC (TXR/K). Interestingly, Séguéla and colleagues (19) recently
demonstrated phosphorylation of P2X2 on this threonine.
Their results suggested that the receptor is constitutively
phosphorylated and that this phosphorylation leads to the slow
desensitization rate of P2X2. Accelerated desensitization
of the already rapidly desensitizing P2X3 receptor would
explain that we were not able to measure the corresponding
P2X3T12A mutant. These findings strongly support phosphorylation of this site also in P2X3 and speak in
favor of a decreased desensitization rate as the underlying mechanism
of current potentiation. This would imply that the C terminus interacts with the N terminus to control phosphorylation. In this model the C
terminus of P2X2 would stabilize phosphorylation at the N-terminal TXK site leading to constitutive phosphorylation,
whereas the C terminus of P2X3 and the splice variant
P2X2-2 would destabilize phosphorylation, allowing
phosphorylation to be regulated. This would also imply that splicing at
the C terminus of P2X2 would be a means to control
modulation of receptor activity by phosphorylation at the N terminus.
Interaction of cytoplasmic termini has already been shown for other
structurally related channels (40). As we cannot directly prove the
implication of the N-terminal phosphorylation site in the regulation of
P2X receptor activity, we still must consider, however, the possibility
that a different, so far unidentified protein may be phosphorylated and
control activity of P2X receptors.
Hu et al. (39) reported that application of 100 nM SP leads to a transient potentiation by 127.2 ± 6.7% of ATP-gated currents in sensory neurons. This potentiation was
blocked by the protein kinase inhibitor H7 and the SP (NK1) receptor
antagonist spantide, resembling our own results and suggesting that the
underlying mechanism is active in sensory neurons.
For Bk it is well established that it acts directly on primary sensory
neurons, and several studies have suggested that PKC mediates this
effect (41-43). Moreover, Bk sensitizes heat-activated currents in
isolated nociceptors, and this sensitization can be mimicked by phorbol
ester and blocked by staurosporine (44). Our results now suggest that
another target for PKC in nociceptors may be P2X3 and that
Bk may sensitize different pain-related ion channels using similar
mechanisms. At the same time, we show that different inflammatory
mediators may converge on the same ion channel.
Together, we show that modulation of P2X3 and
P2X2/3 activity by the inflammatory mediators SP and Bk may
account for sensitization of nociceptors to the action of ATP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 µM) and
rapid activation and desensitization with full activation in less than
10 ms and almost complete desensitization in less than 1 s (4, 5). Moreover, repeated application of the agonist leads to the
disappearance of the ATP-gated currents (4, 5). In contrast,
P2X2 and P2X4 through P2X7 have a
low agonist affinity (EC50
10 µM), are
slowly activating with time constants in the order of 100 ms, and show
only partial desensitization (1, 6). Heterooligomeric P2X2/3 channels show a mixed phenotype with high affinity
to the agonist
,
-methylene ATP
(
,
-meATP)1 like
P2X3 receptors and incomplete desensitization like
P2X2 receptors (5).
,
-meATP leads to nociceptive behavior in
vivo (18), confirming a role for P2X3 and
P2X2/3 receptors in nociception.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-channels. ATP,
,
-methylene ATP (
,
-meATP),
SP, Bk, phorbol 12-myristate 13-acetate (PMA), and staurosporine were
all purchased from Sigma-Aldrich (Deisenhofen, Germany).
,
-meATP or 300 µM ATP for activation of P2X2/3, in order to
get sufficient current amplitude at low desensitization rate for all
subunit combinations injected. To investigate intracellular regulation
of ATP-gated currents, we repeatedly applied the agonist for 50 s
with 50-s intervals. During the first four to eight ATP pulses, current
peak amplitudes decreased; thereafter, they remained constant (Fig.
1). We took three equal current peak
amplitudes as the base line for judging regulatory effects on current
amplitude.
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Fig. 1.
Kinetic properties of P2X receptors expressed
in Xenopus oocytes. Agonist application is
indicated by horizontal bars. A,
oocytes expressing P2X2 channel receptors showed slowly and
incompletely desensitizing currents after application of 300 µM ATP. B, oocytes expressing P2X3
channel receptors showed fast and almost completely desensitizing
currents after application of 100 nM ATP. C,
oocytes expressing P2X2/3 channel receptors showed slowly
and incompletely desensitizing currents after application of 10 µM ,
-meATP. Membrane potential was
70 mV. The SP
receptor was always co-expressed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 s)
inward current (arrows in Figs.
2-4)
resulting from activation of endogenous Ca2+-activated
chloride channels. This represents a typical response of the oocyte
membrane in response to Ca2+ release from internal stores,
providing evidence for the functional expression of the receptors and
their potency to rise intracellular Ca2+ levels.
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Fig. 2.
Behavior of ATP-gated currents through P2X
channels after activation of SP receptor or Bk receptor. P2X
receptors were activated by a pulse protocol as shown in Fig. 1. Once
ATP-gated currents showed reproducible peak currents, inflammatory
mediators were applied. Arrowheads indicate Cl
currents due to the activation of endogenous Ca2+-activated
Cl
channels. A, transient potentiation of
currents through P2X3 homomeric channels following
application of SP (upper panel, 100 nM agonist) or Bk (lower panel; 100 nM agonist). B, P2X2/3 heteromeric
channels showed a similar potentiation by SP (upper
panel) and Bk (lower panel).
C, P2X2 homomeric channels showed only very weak
potentiation. D, application of SP to oocytes that had only
been injected with cRNA coding for P2X2 and
P2X3 receptors but not for SP receptors induced no current
potentiation. E, columns indicate median increase
of peak current amplitudes after application of SP (filled
columns) or Bk (open columns)
normalized to the last ATP stimulation before SP or Bk application. The
error bars represent the range between which the
results were distributed. The number, n, of independent
measurements is indicated above each
column.
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Fig. 3.
Pharmacology of current potentiation through
P2X receptors. Protocols were as described in legend to Fig. 2.
Arrowheads indicate Cl currents due to the
activation of endogenous Ca2+-activated Cl
channels. A, staurosporine blocked current increase through
P2X3 receptors. Oocytes had been incubated in extracellular
solution containing 10 µM staurosporine for 10-30 min
before the measurements were done. SP induced no longer any current
increase. B, phorbol ester mimicked the effect of
inflammatory mediators. Application of 4
-PMA (10 nM) for
50 s yielded current potentiation through P2X3
receptors very similar to that evoked by SP or Bk. C, if SP
was applied during 4
-PMA application, SP elicited no further
stimulation. The strong rundown of ATP-gated currents was only observed
with long application of PMA. D, columns indicate median
increase of peak current amplitudes after application of effector
substances normalized to the last ATP stimulation before effector
application. The error bars represent the range
between which the results were distributed. The number, n,
of independent measurements is indicated above each
column.
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Fig. 4.
Effect of the cytoplasmic C terminus of P2X
receptors on potentiation of currents. Protocols were as described
in legend to Fig. 2. Arrowheads indicate Cl
currents due to the activation of endogenous Ca2+-activated
Cl
channels. A, a P2X2 receptor
whose C terminus had been replaced by the P2X3 C terminus
gained potentiation by SP. B, a P2X3 receptor
whose C terminus had been replaced by the P2X2 C terminus
lost potentiation by SP. This mutant has been expressed as a heteromer
together with P2X2. C, the P2X2-2
splice variant that lacks 69 amino acids within the C terminus gained
potentiation by SP. D, the C-terminally truncated
P2X2
401 receptor gained potentiation by SP. The
respective P2X receptors are schematically shown at the top
of each trace. E, columns indicate median
increase of peak current amplitudes after application of SP (100 nM) normalized to the last ATP stimulation before SP
application. The error bars represent the range
between which the results were distributed. The number, n,
of independent measurements is indicated above each
column.
150-s duration) increase in peak amplitude (median increase: 61%,
range: 33-165%, n = 10; Fig. 2E) and
steady state current (I50 s, current amplitude at the end of the 50-s ATP pulse, median increase: 50%, range: 4-129%, n = 10) of ATP induced currents through
P2X3 receptors. After the potentiation phase, current
amplitudes occasionally became smaller than before SP stimulation (not
quantified). Short (2-5 s) applications of SP, repeated after about 10 min, induced each time current increase, suggesting that the underlying
alteration of P2X receptors by SP was reversible (data not shown). The
current increase following SP application could also be elicited using saturating ATP concentrations (300 µM), demonstrating
that SP does not potentiate ATP currents by changing P2X receptors'
affinity to ATP.
-meATP. A similar effect was again seen with
application of Bk when the Bk receptor was coexpressed (median increase
of peak amplitude: 112%, range: 22-140%, n = 9; Fig.
2 (B and E); median increase of
I50 s: 136%, range: 17-199%,
n = 9). The desensitization rate could not be well
fitted but showed an apparent decrease after application of
inflammatory mediators (see Fig. 2B).
9% to 15%, n = 13, p > 0.01, Fig. 2 (C and E); median increase of
I50 s: 2%, range:
7% to 8%,
n = 13, p > 0.05) nor by Bk receptor
activation (median increase of peak current amplitude: 6%, range:
11% to 17%, n = 12, p > 0.05; Fig.
2 (C and E); median increase of
I50 s: 1%, range:
25% to 17%,
n = 12, p > 0.05). Thus, it seems that modulation by the neuropeptides is subunit-specific and that the P2X3 subunit is responsible for modulation of the
P2X2/3 heteromer.
2%, range:
5% to 10%, n = 6, Fig. 2
(D and E); median increase of I50 s: 6%, range:
3% to 17%,
n = 6). This shows that the effect is mediated by the
SP receptor and suggests pathways downstream of the receptor to be
responsible for sensitization of P2X receptors.
6%, range:
16% to 9%,
n = 10, Fig. 3 (A and D); median
increase in I50 s: 3%, range:
12% to 16%,
n = 7), suggesting the involvement of protein kinase in
the potentiating process. In addition, the effect of SP could be
mimicked using the phorbol ester 4
-PMA (10 nM for
50 s; median increase of peak current: 66%, range: 7-337%,
n = 22; Fig. 3 (B and D); median
increase of I50 s: 94%, range: 17-265%, n = 22) but not using the biologically inactive
stereoisomer 4
-PMA (data not shown). Moreover, as shown in Fig.
3C, during PMA application SP was unable to further
sensitize the current (median increase of peak current: 4%, range:
6% to 10%, n = 4; Fig. 3 (C and
D); median increase of I50 s: 8%,
range:
9% to 42%, n = 4), suggesting that the same
mechanism underlies both effects. Together, these experiments suggest
that protein kinase C mediates the stimulation of ATP-gated currents
through P2X3 and P2X2/3.
-meATP or with ATP lost SP regulation (median
increase of peak current:
7%, range:
21% to
3%,
n = 4; Fig. 4 (B and E)),
implicating the cytoplasmic C terminus as an important region for
regulation by SP.
401) deleting 72 of the about 116 cytoplasmic amino acids at the C terminus. This mutant showed kinetics
similar to that of the P2X2 wild type but gained SP
regulation (median increase of peak current: 21%, range: 16-23%,
n = 3, Fig. 4 (D and E); median
increase of I50 s: 21%, range: 16-24%,
n = 3).
View larger version (27K):
[in a new window]
Fig. 5.
Site-directed mutagenesis of putative
PKC-phosphorylation sites within the intracellular N and C termini of
the P2X3 receptor. Left, scheme of the
intracellular N and C termini of the P2X3 receptor. All
serine/threonine residues are enlarged. The residues that were mutated
are highlighted in bold. Residues that had been mutated in
combination are marked by lines. Positively charged amino
acids (Arg, Lys) are indicated by a + sign. Right, table
summarizing the phenotype of the mutants. Left
column, list of mutants that were generated.
Right column, results of tests for regulation by
SP using a measurement protocol as described in Fig. 2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
401. Still, it might be that phosphorylation sites
are not contained in a classic consensus site and are not conserved
between P2X2 and P2X3.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. J. Krause and W. Müller-Esterl for the gift of the SP and Bk receptors, respectively.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the "Graduiertenkolleg Zellbiologie in der Medizin" (to M. P.) and Grant FG 1-0-0 of the Attempto Research Group Program of the Universitätsklinikum Tübingen (to S. G.).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.
¶ Present address: Center for Hearing Sciences, Dept. of Otolaryngology/Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
To whom correspondence should be addressed. Tel.:
49-7071-29-84827; Fax: 49-7071-22917; E-mail:
stefan.gruender@uni-tuebingen.de.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M101465200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
,
-meATP,
,
-methylene ATP;
Bk, bradykinin;
SP, substance P;
PMA, phorbol
12-myristate 13-acetate;
DRG, dorsal root ganglion.
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REFERENCES |
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