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INTRODUCTION |
Purinergic receptors
(P2XRs)1 are a family of
ligand-gated receptor channels that open in response to the binding of
extracellular ATP. Like other ligand-gated receptor channels, P2XRs
also become refractory to the stimulus during the sustained agonist
occupancy. This process, called desensitization, was initially
characterized in acetylcholine receptors by Katz and Thesleff (1) and
occurs because receptors enter stable desensitized states in which ion permeation is blocked or attenuated although ligand remains bound. Significant progress has been made recently in characterizing the
ectodomain architecture of other ligand-gated receptor channels and the
relationship between ligand-binding domain occupancy and receptor
activity (2-4). However, the boundaries of the ectodomain and
ATP-binding domain of P2XRs and the molecular mechanisms of transduction of information from ligand-binding domain to the pore of
channels are largely unknown (5).
Modification at the triphosphate moiety of ATP served well in
identification of native P2XR subtypes and provided useful information about the putative ligand-binding domain. For example, the substitution of bridging oxygen between
- and
-phosphorus with a methylene group resulted in a ligand, called 
-meATP, which is a high
potency agonist for P2X1R and P2X3R, low
potency agonist for P2X2R, and partial agonist for
P2X4R (6). High sensitivity of P2X3R to 
-meATP can be transferred to P2X2aR and
P2X2bR subtypes by generating the extracellular chimeras
having the ectodomain of the P2X3 subunit in the
P2X2-based backbone (7). These chimeras also exhibited enhanced rates of desensitization (7, 8). Within this region, single
residue mutation studies have identified positively charged Lys68, Lys70, and Lys309 of
P2X1R and the corresponding Lys69 and
Lys71 of P2X2R as contributing to the
ATP-binding site and control of rate of receptor desensitization (9,
10). In general agreement with these observations, the N-terminal half
of the P2X3 ectodomain, from Val60 to
Arg180, is necessary for high 
-meATP sensitivity of
the receptor. The attempt to further narrow this region was obstructed,
indicating that the ectodomain is sensitive to modification by
site-directed mutagenesis (7).
P2XRs activate and desensitize in a receptor- and
ligand-specific manner. When stimulated with ATP, P2X1R and
P2X3R desensitize very rapidly (in an ms time scale),
P2X4R and P2X6R desensitize with a moderate
rate (within a few seconds), and P2X2R, P2X5R, and P2X7R show little or no desensitization (11, 12). Two main hypotheses emerged from previous work on desensitization of P2XRs,
one based on the structure of channels and the other based on the
actions of intracellular messengers. Heteromultimerization results in
P2XRs that desensitize with different kinetics from those seen in cells
expressing homomeric receptors (13-15). The site-directed mutagenesis
experiments indicated the relevance of C-terminal structure on
the desensitization of P2XR (15-19), as well as the relevance of a
highly conserved N-terminal site for protein kinase C in functional
desensitization of receptors (19-21). Phosphorylation of a protein
kinase A site in C-terminal of P2X2aR may also participate
in receptor desensitization (22). In parallel to other ligand-gated
receptor channels (2, 23-28), the ligand-binding domain of P2XRs may
also contribute to the control of rates of desensitization (7, 8).
Here, we extended investigations on the relevance of the ectodomain
structure on ligand selectivity and the pattern of P2XR desensitization. Experiments were done with wild-type
P2X2aR, P2X2bR, and P2X4R and
chimeric P2X2a + X4R and P2X2b + X4R containing the ectodomain sequence of P2X4R
instead of the corresponding P2X2R sequence. The reverse
P2X4 + X2R chimera was also constructed, as
well as the P2X4 + X7R chimera containing
the ectodomain sequence of P2X7R. To clarify the possible
interaction between the ectodomain and C-terminal domain in control of
agonist specificity and receptor activity, we also constructed
P2X2a-6aa + X4R mutant chimera containing the
C-terminal 6-residue sequence of P2X4R instead of the
corresponding sequence of P2X2a + X4R. Both
whole-cell patch clamp current recordings and calcium measurements were
used to estimate the activity of receptors in response to ATP, the
native agonist for P2XRs, and two agonist analogs, BzATP and

-meATP. The results of these investigations clearly indicate the
C-terminal-independent influence of the ectodomain structure on agonist
potency and rate of P2XR desensitization.
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MATERIALS AND METHODS |
DNA Constructs, Cell Culture, and Transfection--
The
coding sequences of the rat P2X2a,
P2X2b, P2X4 subunits were isolated by reverse
transcription-PCR (15) and subcloned into the biscistronic enhanced
fluorescent protein expression vector, pIRES2-EGFP
(Clontech), at the restriction enzyme sites of XhoI/PstI for P2X2aR and
P2X2bR and XhoI/EcoRI for
P2X4R. Chimera P2X2a + X4R,
P2X2b + X4R, P2X4 + X2R, and P2X4 + X7R were directly constructed by overlap extension PCR using the corresponding wild-type P2XRs cDNA as templates. Mutagenesis primers were pairs of chimeric sense and antisense that were 36-mer long with the joint sites positioned at the center. The constructed P2X2a + X4R, P2X2b + X4R chimeric subunits
replace Ile66-Tyr310 with
Val66-Tyr315 extracellular domain of
P2X4R, whereas P2X4 + X2R and
P2X4 + X7R contain the
Ile66-Tyr310 and
Val61-Phe313 sequence of P2X2R and
P2X7R, respectively, instead of the native Val66-Tyr315 of P2X4R. We also
constructed a mutant of chimera P2X2a + X4R, termed P2X2a-6aa + X4R, by overlap extension
PCR, as described previously (15). This mutant contains the
Glu376-Gly381 sequence of P2X4R
instead of the corresponding Arg371-Pro376
sequence of P2X2a + X4R. These chimeric P2XRs
were subcloned into GFP expression vector pIRES2-EGFP. The identity of
all constructs was verified by dye terminator cycle sequencing
(PerkinElmer Life Sciences), performed by the Laboratory of Molecular
Technology (NCI, National Institutes of Health, Frederick, MD). The
large scale plasmid DNAs for transfection were prepared using a Qiagen Plasmid Maxi kit (Qiagen).
Mouse immortalized gonadotropin-releasing hormone-secreting cells
(hereafter GT1 cells) and human embryonic kidney cells (hereafter HEK293 cells) were used in functional studies of wild-type and mutant
P2XRs, as described previously (15). GT1 cells were routinely maintained in Dulbecco's modified Eagle's medium/Ham's F12 medium (1:1) containing 10% (v/v) fetal bovine serum and 100 µg/ml
gentamicin (Invitrogen) in a water-saturated atmosphere of 5%
CO2 and 95% air at 37 °C. HEK293 cells were
cultured in minimum Eagle's medium supplemented with 10% horse serum
and 100 µg/ml gentamicin. Before the day of transfection, cells were
plated on 25-mm poly-L-lysine (0.01% w/v; Sigma)-coated
coverslips at a density of 0.75-1 × 105 cells/35-mm
dish. For each dish of cells, transient transfection of expression
constructs was conducted using 1 µg of DNA and 7 µl of
LipofectAMINE 2000 reagent (Invitrogen) in 3 ml of serum-free Opti-MEM.
After 6 h of incubation, the transfection mixture was replaced
with normal culture medium. Cells were subjected to experiments 24-48
h after transfection.
Calcium Measurements--
Transfected GT1 cells were preloaded
with 1 µM Fura-2 acetoxymethyl ester (Fura-2/AM;
Molecular Probes, Eugene, OR) for 60 min at room temperature
in modified Krebs-Ringer buffer: 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, and 15 mM HEPES plus 1.8 g/liter glucose (pH 7.4). After dye loading, cells were incubated in
Modified Krebs-Ringer buffer and kept in the dark for at least 30 min
before single-cell [Ca2+]i measurements.
Coverslips with cells were mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor
digital fluorescence microscopy system (Atto Instruments, Rockville,
MD). Cells were stimulated with various doses of agonists, the dynamic
changes of [Ca2+]i were examined under a ×40 oil
immersion objective during exposure to alternating 340- and 380-nm
light beams, and the intensity of light emission at 520 nm was
measured. The ratio of light intensities,
F340/F380, which reflects changes in
[Ca2+]i, was simultaneously followed in several
single cells. GFP was used as a marker for cells with P2XR expression
as described previously (15). Cells expressing GFP were optically
detected by an emission signal at 520 nm when excited by a 488-nm
ultraviolet light. Experiments were done in cells with comparable GFP
fluorescence signals (about 60 arbitrary units), and no repetitive
stimulation was done to avoid the possible impact of desensitization on
the amplitude and pattern of [Ca2+]i signals.
Current Measurements--
Electrophysiological experiments were
performed on HEK293 cells at room temperature using whole-cell patch
clamp recording techniques (29). ATP-induced currents were recorded
using an Axopatch 200B patch clamp amplifier (Axon Instruments, Union
City, CA) and were filtered at 2 kHz using a low pass Bessel filter. 40-70% series resistance compensation was used. Patch electrodes, fabricated from borosilicate glass (type 1B150F-3; World Precision Instruments, Sarasota, FL) using a Flaming Brown horizontal puller (P-87; Sutter Instruments, Novato, CA), were heat-polished to a
final tip resistance of 3-5 megaohms. All current records were captured and stored using the pClamp 8 software packages in conjunction with the Digidata 1322A A/D converter (Axon Instruments). Patch electrodes were filled with a solution containing 140 mM
KCl, 0.5 mM CaCl2, 1 mM
MgCl2, 5 mM EGTA, and 10 mM HEPES;
the pH was adjusted with 1 M KOH to 7.2. The osmolarity of
the internal solutions was 282-287 mosM. The bath solution
contained 142 mM NaCl, 3 mM KCl, 1 mM MgCl2, 2 mM CaCl2,
10 mM glucose, and 10 mM HEPES; the pH was
adjusted to 7.3 with 1 M NaOH. The osmolarity of this
solution was 285-295 mosM. A 3 M KCl agar
bridge was placed between the bathing solution and the reference
electrode. ATP was applied for 60 s using a fast
gravity-driven microperfusion system (BPS-8, ALA Scientific
Instruments, Westbury, NY). The application tip was routinely
positioned about 500 µm above the recorded cell. Less than 600 ms was
required for complete exchange of solutions around the patched cells,
as estimated from altered potassium current (10-90% rise time). The
time between each ATP application was about 10 min to allow recovery
from receptor desensitization.
Calculations--
The time course of the
[Ca2+]i was fitted to a single exponential
function (ae
ktb+b)
using GraphPad Prism (GraphPad Software, San Diego, CA) to generate the
rates of signaling desensitization (k) and half-times of
decay (
= ln2/k). The time courses of
currents evoked by sustained ATP stimulation were fitted to a single
exponential function using the pClamp 8 program (Axon
Instruments). Significant differences, with p < 0.05, were determined by one-way analysis of variance with Newman-Keuls
multiple comparison test. Concentration-response relationships were
fitted to a four-parameter logistic equation using a non-linear
curve-fitting program, which derives 50% efficient concentrations
(EC50) and 50% desensitizing concentrations
(DC50) (Kaleidagraph, Synergy Software, Reading, PA).
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RESULTS |
Characterization of Wild-type and Chimeric P2XRs--
When
expressed in GT1 cells under identical experimental conditions,
parental receptors P2X2aR, P2X2bR, and
P2X4R responded to ATP, BzATP, and 
-meATP stimulation
with a rapid rise in [Ca2+]i followed by a
gradual decline toward steady plateau levels. In accordance with
previously published data (8, 30), in all agonist concentrations
studied, the peak [Ca2+]i responses were
comparable in P2X2aR- and P2X2bR-expressing cells. Fig. 1A illustrates the
concentration dependence of three agonists on the peak amplitude of
[Ca2+]i responses for both receptor subtypes
combined. The calculated EC50 values (Fig. 1, dotted
vertical lines) were in general agreement with the data in the
literature (6): ATP was the most potent agonist for P2X2Rs,
followed by BzATP, whereas 
-meATP acted as a low potency
agonist.

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Fig. 1.
Concentration-dependent
effects of agonists on peak calcium response in single cells expressing
homomeric P2XRs. A-C, comparison of the effects of ATP
and its two analogs, BzATP and  -meATP, on peak calcium response
in cells expressing P2X2aR and P2X2bR
(A), P2X4R (B), and P2X2a + X4R and P2X2b + X4R
(C). Data shown are means ± S.E. derived from 3 to 11 experiments per dose, each done in at least 15 single cells. The
results for P2X2aR and P2X2bR (A)
and P2X2a + X4R and P2X2b + X4R (C) are combined because no differences in
peak [Ca2 + ]i responses were observed between
them. Dotted vertical lines indicate the calculated
EC50 values for three agonists. P2X2 + X4R chimeras were constructed as described under
"Materials and Methods."
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On the other hand, the estimated EC50 values for ATP,
BzATP, and 
-meATP in GT1 cells expressing rat P2X4R
(Fig. 1B) differed significantly from previously published
data (reviewed in Ref. 6). In our experiments, ATP was a highly potent
agonist for these receptors, with an EC50 of about 1 µM, and the calculated EC50 for BzATP was 2.5 µM as compared with >500 µM reported in other expression systems. At supramaximal concentrations, the peak
amplitudes of ATP- and BzATP-induced [Ca2+]i
responses in P2X4R-expressing cells were about 40% of
those observed in P2X2R-expressing cells, whereas the peak amplitudes of current responses were comparable (Fig. 1).

-meATP-induced peak current/[Ca2+]i
responses were 44% as compared with those in ATP- and BzATP-stimulated
cells, consistent with the partial agonist action of this ATP analog
observed in other expression systems (31), but the calculated
EC50 was 4 µM (Fig. 1B).
The P2X2a + X4R and P2X2b + X4R chimeras having the ectodomain of P2X4R in
the backbone of P2X2aR and P2X2bR,
respectively, were functional and responded to three agonists in a
concentration-dependent manner with highly comparable peak
amplitudes. Fig. 1C illustrates the combined results for
both receptors. Chimeric receptors differed from parental receptors in
two respects. First, although the structure of the pore was not
altered, the peak amplitudes of [Ca2+]i responses
in chimeric receptors were 80% of those observed in
P2X2aR- and P2X2bR-expressing cells
(p < 0.01 in the 10-1000 µM
concentration range of ATP). Second, chimeric receptors exhibited
higher sensitivity to ATP as compared with both parental receptors, and

-meATP exhibited the full agonistic action as compared with
partial agonistic action in P2X4R- expressing cells.
The reverse P2X4 + X2R chimera, having the
ectodomain of P2X2R inserted into the backbone of
P2X4R, was expressed at the levels comparable with those
observed in experiments with P2X2a + X4R and
P2X2b + X4R, as estimated by GFP fluorescence
intensity. However, the receptor did not respond to ATP in the 1-1000
µM concentration range. The lack of effects of ATP was
not due to the endogenous desensitization of receptors because ATP was
also ineffective in cells cultured in the presence of apyrase, an
ectoATPase. Also, the P2X4 + X7R chimera having
the ectodomain of P2X7R instead of P2X4R was
expressed but was not functional in the presence or absence of apyrase.
All together, experiments with chimeras suggested that although the
ectodomain sequences we selected contained several residues critical
for ligand binding, they were not sufficient to preserve intact
ligand-binding domains. The P2X4R-specific ligand potency
was enhanced, whereas the P2X2R- and
P2X7R-specific ligand potency was abolished in chimeric
channels, indicating the interaction between the ectodomain and nearby residues.
Receptor-specific Desensitization Pattern--
To characterize the
pattern of receptor desensitization and its impact on calcium
signaling, both current and calcium measurements were used. Fig.
2A illustrates typical
profiles of ATP (100 µM)-induced current responses in
cells expressing wild-type P2X2aR, P2X2bR, and
P2X4R. The peak amplitudes of current responses were in
high pA (P2X4R) to low nA (P2X2aR and
P2X2bR) range. Consistent with previously published data
(16), P2X2aR desensitized slowly and incompletely, reaching
the steady levels with a
of 22 s, whereas P2X2bR
desensitized rapidly with a calculated
of about 4 s. P2X4R desensitized to the steady levels comparable with
P2X2bR but with a
of about 9 s.

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Fig. 2.
Receptor-specific desensitization pattern of
P2XRs. A and B, comparison of the effects of
ATP on the pattern of current (A) and calcium (B)
responses in cells expressing wild-type P2X2aR (left
traces), P2X4R (central traces), and
P2X2bR (right traces). In this and following
figures, experimental records for current responses are shown by
solid lines and are representative from at least 20 traces
per receptor, whereas experimental records for calcium response are
shown by open circles (mean values from at least 15 traces
in representative experiments). Horizontal bars indicate the
time of exposure to 100 µM ATP. The current traces shown
are from cells clamped at 50 mV.
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The significance of receptor-specific desensitization pattern on
[Ca2+]i response is illustrated in Fig.
2B. Three receptors generated calcium signals, which
differed in profiles. The rates of signal desensitization (expressed as
) were: 96 s in P2X2aR-expressing cells, 18 s
in P2X2bR-expressing cells, and 45 s in
P2X4R-expressing cells. Differences in the
calculated
values in current and [Ca2+]i
measurements illustrate the impact of calcium-handling mechanisms of
the cells used in experiments on the rate of receptor desensitization.
On the other hand, the relative ratios in the rates of
P2X2aR, P2X2bR, and P2X4R
desensitization estimated from two measurements were highly comparable.
This clearly indicates that [Ca2+]i measurements
not only provide information about the physiological relevance of
receptor activation and desensitization but also could be used as
valuable parameters in characterizing the nature of P2XR
desensitization, at least for slower desensitizing receptors.
A comparison between the patterns of current responses in cells
expressing wild-type P2X2aR and P2X2bR and
chimeric receptors is shown in Fig. 3.
The substitution of the native P2X2aR ectodomain with
P2X4 ectodomain dramatically enhanced the rate of
receptor desensitization (Fig. 3B). Significant differences
were also observed between P2X2bR and
P2X2b + X4R (Fig. 3A). Both
chimeric receptors also desensitized more rapidly than
P2X4R (Fig. 3 versus Fig. 2). The same
conclusion was reached in experiments with single cell calcium
measurements. As shown in Fig.
4A, calcium signals desensitized more rapidly in cells expressing chimeric receptors when
stimulated with 10 µM ATP (upper panels) and
100 µM ATP (lower panels). In parallel with
changes in the EC50 values for chimeric receptors (Fig. 1),
calcium signals also desensitized with about one log unit leftward
shift in the DC50 values as compared with parental
P2X2Rs (Fig. 4B). The ratios between
values
for current and calcium measurements in cells expressing two chimeric
receptors were comparable with those observed in parental receptors.
These results clearly indicate the relevance of the ectodomain
structure on the kinetics of receptor desensitization and suggest that
the ligand potency reflects on rate of receptor desensitization.

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Fig. 3.
Acceleration of P2X2aR and
P2X2bR desensitization by substituting their common
ectodomain with the P2X4R ectodomain (current
recordings). The traces shown are representative for wild-type
P2X2bR and chimeric P2X2b + X4R (A)
and wild-type P2X2aR and chimeric P2X2a + X4R (B). The mean ± S.E. values are shown
above traces, and the numbers in
parentheses indicate the number of records for each
receptor. Asterisks indicate significant differences
(p < 0.05) between the pairs.
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Fig. 4.
Influence of ectodomain and C-terminal domain
on acceleration of P2X2a + X4R and
P2X2b + X4R desensitization (calcium
recordings). A, representative traces of calcium
responses in cells stimulated with 10 µM ATP (upper
traces) and 100 µM ATP (bottom traces).
In this and following figures, experimental records are shown by
open circles (mean values from at least 15 traces in
representative experiments), and fitted curves are shown by full
lines. A single exponential function was sufficient to describe
the desensitization rates. The fitted function is extrapolated for
clarity. B, concentration dependence of ATP on the rate of
P2XR desensitization. Asterisks indicate significant
differences (p < 0.01) between the pairs.
Vertical dotted lines indicate the calculated
DC50 values, and horizontal arrows indicate a
leftward shift in DC50 values for chimeric channels.
C, comparison of 100 µM ATP-induced
[Ca2+]i signals in GT1 cells expressing
P2X2a + X4R, P2X2a-6aa + X4R, and P2X2b + X4R.
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Current and calcium measurements showed that
P2X2b+X4R desensitized more rapidly than
P2X2a+X4R, consistent with the hypothesis that
the C-terminal-specific desensitization pattern was preserved in
chimeric channels. To test this hypothesis further, we generated P2X2a-6aa+X4R mutant chimera, having the
Glu376-Gly381 C-terminal sequence of
P2X4R instead of the Arg371-Pro376
sequence of P2X2a+X4R. Our earlier studies have
shown the relevance of Arg371-Pro376 in
slowing the rate of receptor desensitization (16). Substitution of this
sequence with the Glu376-Gly381 sequence of
P2X4R increased the rate of P2X2aR
desensitization (15). In accordance with these observations,
P2X2a-6aa + X4R mutant chimera showed an
increased rate of desensitization as compared with P2X2a + X4R chimera (Fig. 4C), and the ratio between the
rates of P2X2a + X4R, P2X2a-6aa + X4R, and P2X2b + X4R was comparable
with that observed in cells expressing P2X2aR,
P2X2a-6aaR, and P2X2bR (15). Thus, the
increase in the potency of receptors for ATP increases the rates of
receptor desensitization independently of the
C-terminal-controlled desensitization.
Ligand-specific Desensitization Pattern--
In further
experiments, we compared the patterns of calcium signals and rates of
desensitization in response to ATP, BzATP, and 
-meATP. Fig.
5 illustrates typical profiles of calcium
signals in response to these three agonists in cells expressing
parental receptors. In accordance with our previous study (8), the
C-terminal-specific desensitization pattern of wild-type
P2X2Rs observed in response to ATP (Fig. 5A,
left traces) was partially mimicked by BzATP (central
traces) but was lost in cells stimulated with 
-meATP (right traces). The ligand specificity of receptor
desensitization was also observed in cells expressing wild-type
P2X4R (Fig. 5A, upper traces). The
mean values of the rate of receptor desensitization in response to
three agonists are shown in Fig. 5B. When stimulated with
ATP, three receptors desensitized with significantly different rates
(left panel). No differences in the rates of
P2X2aR and P2X4R desensitization were observed
in response to BzATP stimulation (central panel), and all
three receptors desensitized with highly comparable kinetics when
stimulated with 
-meATP (right
panel). The plateau [Ca2+]i
levels in response to three agonists also differed (illustrated by
dotted lines in Fig. 5A and quantified in Table I).

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Fig. 5.
Ligand-specific receptor desensitization
pattern of wild-type P2XRs. A, representative traces of
[Ca2+]i response in cells expressing
P2X4R (upper traces), P2X2aR
(central traces), and P2X2bR (bottom
traces). Traces shown are representative from 3-5 independent
experiments. Horizontal dotted lines indicate differences in
the plateau [Ca2+]i. B, mean values of
rates of receptor desensitization. Asterisks indicate
significant differences (p < 0.01) between the
pairs.
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In further studies, we examined the ligand-specific desensitization
pattern of chimeric receptors. Fig. 6
compares typical calcium signal profiles in cells expressing wild-type
P2X2aR and chimeric P2X2a + X4R
(Fig. 6A) and wild-type P2X2bR and chimeric P2X2b + X4R (Fig. 6B) during the
prolonged stimulation with increasing BzATP concentrations. The
C-terminal-specific desensitization patterns of P2X2aR and
P2X2bR that are present in ATP-stimulated cells (Fig. 5),
but are lost in BzATP-stimulated cells (Figs. 5 and 6), were
reestablished in BzATP-stimulated cells expressing chimeric receptors.
Fig. 6A (left panels) shows a typical pattern of
receptor desensitization in wild-type and chimeric P2X2Rs, and bars (right panels) illustrate significant differences
in the mean values for the rate of receptor desensitization, whereas Fig. 6B (left and right panels)
illustrates more dramatic differences between wild-type and chimeric
P2X2bR.

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Fig. 6.
Comparison of BzATP-induced calcium signals
in cells expressing wild-type and chimeric receptors.
A, concentration-dependent effects of BzATP on
the pattern of calcium response in P2X2aR and
P2X2a + X4R-expressing cells (A) and
P2X2bR and P2X2b + X4R-expressing
cells (B). In A and B, left
panels illustrate representative traces, and right
panels (bars) illustrate differences in the rates of
calcium signal desensitization.
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The establishment of C-terminal-specific desensitization pattern was
also observed in chimeric receptors stimulated with 
-meATP. Fig.
7A shows that wild-type
P2X2Rs do not respond to 10 µM 
-meATP, whereas chimeric receptors do. Fig. 7B illustrates the lack
of receptor-specific desensitization pattern in wild-type
P2X2Rs and a significant difference in the rates of
chimeric receptor desensitization in response to 100 µM

-meATP. The same conclusions were also derived from cells
stimulated with 500 µM 
-meATP (Fig. 7C).
Table I summarizes the level of calcium signal desensitization in
wild-type and chimeric P2X2Rs. Thus, the introduction of
the P2X4R ectodomain in P2X2aR and
P2X2bR backbones had three obvious effects: increase of the
rate of receptor desensitization for ATP without affecting the
C-terminal-dependent desensitization pattern,
reestablishment of the C-terminal-dependent receptor desensitization in response to BzATP and 
-meATP stimulation, and
comparable plateau levels in response to three agonists.

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Fig. 7.
Comparison of
 -meATP-induced calcium
responses in cells expressing wild-type (left panels)
and chimeric (right panels) P2X2Rs.
A, difference in the sensitivity of wild-type and chimeric
channels to 10 µM  -meATP. B and
C, comparison of the pattern of calcium signal
desensitization in cells expressing wild-type and chimeric receptors
during continuous stimulation with 100 µM  -meATP
(B) and 500 µM  -meATP (C).
Upper panels, representative traces from 3-8 independent
experiments, each done in at least 15 cells. Bottom panels,
mean values of rates of receptor desensitization.
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DISCUSSION |
In this study, we used the wild-type P2X2aR and
P2X2bR because of their identical ectodomains and rapid
activation properties, but distinct and well defined desensitization
patterns in response to sustained stimulation with ATP (30, 32, 33).
The P2X4R shares about 39% similarity with
P2X2R and desensitizes with rates comparable with those
observed in cells expressing P2X2bR, whereas P2X7R shares about 26% similarity with P2X2R
and desensitizes in a manner more comparable with the
P2X2aR subtype (6, 34). These four receptors also exhibit
highly specific ligand potency profiles, including ATP, BzATP, and

-meATP. ATP is a highly potent agonist for P2X4R, a
high to middle potency agonist for P2X2R, and a low potency
agonist for P2X7R. BzATP is considered as a high potency
agonist for P2X7R, a middle potency agonist for
P2X2R, and a low potency agonist for P2X4R.

-meATP acts as a low potency agonist for P2X2R and a
partial agonist for P2X4R, whereas P2X7R is
insensitive to this agonist (6).
The sequences Ile66-Tyr310 of
P2X2R, Val66-Tyr315 of
P2X4R, and Val61-Phe313 of
P2X7R used for construction of our chimeric channels
contain the majority of residues relevant for ATP binding identified so far. They enclose the 10 conserved cysteine residues among all known P2XRs and three N-linked glycosylated sites
(Asn182, Asn239, and Asn298 in rat
P2X2R), which might be responsible for functionality of the
channels (35-39). The ectodomain sequences we used also contain several conserved residues, which might contribute to the ATP-binding site, including Lys69 and Lys71 in rat
P2X2 sequence and corresponding to Lys68 and
Lys70 in human P2X1 (9, 10), Trp256
in rat P2X2 (40), and Lys309 in human
P2X1 (9). Because those are common residues for all seven
channels, it is obvious that other residues account for ligand
specificity among receptors. By exchanging the ectodomain sequences, we
hoped that the ligand selectivity profiles would be preserved and thus
enabled us to examine the dependence of channel activity on the
ligand-binding-specific domains. In accordance with this, our
previously published data with P2X2 + X3R
chimeras having the Val60-Phe301 ectodomain
sequence of P2X3R instead of the native
Ile66-Tyr310 sequence showed highly comparable
ligand potency with parental P2X3R (7, 8).
However, the present data clearly indicate that the transfer of these
ectodomains alters the native agonist selectivity and potency. First,
the ATP potency for P2X2a + X4R and
P2X2b + X4R chimeras was higher than that
observed in both parental receptors. In parallel to that, two chimeras
desensitized more rapidly than parental receptors. Second, 
-meATP
is a partial agonist for P2X4R (31) and a full and highly
potent agonist for P2X2a + X4R and
P2X2b + X4R chimeras. Third, P2X4 + X2R and P2X4 + X7R were expressed
but were not functioning receptors. These results suggest the effects
of transmembrane domain flanking sequences on ligand specificity and
agonistic potency. We may speculate that these sequences act as
"dominant-positive" and "dominant-negative" domains, depending
on the structure of the main ectodomain sequence, to change the
sensitivity of receptors for agonists. In accordance with this view, it
has been reported recently that point mutations in the first
transmembrane domain affect the ligand selectivity of rat
P2X2R (41).
In this study, we also progressed in understanding the mechanism by
which the putative ligand-binding domain may influence the rate of
receptor desensitization and the relationship between the ectodomain
and C-terminal domain in control of desensitization. The main
conclusion that emerged from this work is that the P2XR desensitization
pattern is receptor- and ligand-specific. The P2X2aR-,
P2X2bR-, and P2X4-specific desensitization
patterns were observed in response to ATP, the native agonist for these
channels, but were less obvious when stimulated with BzATP and were
lost when receptors were stimulated with 
-meATP. The increase in EC50 values for all three agonists induced by substituting
the ectodomains indicated that the potency of agonists reflects the ligand specificity of receptor desensitization, i.e. highly
potent agonists trigger the subtype-specific desensitization pattern, whereas agonists with lower potency are less effective or are ineffective. Our effort to further establish this hypothesis by generating receptors with decreasing sensitivity for ATP was
unsuccessful because P2X4 + X2R and
P2X4 + X7R chimeras were nonfunctional.
On the other hand, we were more successful in establishing that the
ectodomain influences desensitization independently of the C-terminal
domain. Earlier studies have indicated that the 6-residue
receptor-specific sequences in C-terminal influence the rates of
P2X2aR, P2X2bR, P2X3R, and
P2X4R (15). In this study, current and calcium measurements
indicated that chimeric P2X2a + X4R and
P2X2b + X4R desensitized with different rates, i.e. a leftward shift in EC50 and
DC50 for chimeric receptors proportionally affected both
receptors. This suggests that the C-terminal-dependent
desensitization pattern was not affected by substituting the
ectodomains. To test this hypothesis further, we generated a mutant of
P2X2a + X4R chimera in which we substituted the
C-terminal Arg371-Pro376 sequence of
P2X2a + X4R with
Glu376-Gly381 sequence of P2X4R.
In parallel to the desensitization patterns of P2X2aR and
P2X2a-6aaR (15), the present data show that chimeric P2X2a-6aa + X4R desensitized more rapidly than
P2X2a + X4R when stimulated with ATP.
At the present time, we cannot speculate about the possible molecular
mechanism by which the ligand-binding domain influences receptor
desensitization. The main limitation comes from the fact that the
ligand-binding domain structure and the crystal structure of P2XRs have
not been identified. The structural similarities of P2XRs with class II
aminoacyl-tRNA synthases (42) provided a model for initial studies on
the ATP-binding domain (9, 10, 40, 41), which could not give a
rationale for the receptor behavior shown here. Based on studies with
crystallization of glutamate channels, in a recently published study,
Sun et al. (3) showed that desensitization of these
receptors occurs through rearrangement of the dimmer interface, which
disengages the agonist-induced conformation change in the binding core
from the ion channel gate. To which extent this model provides a
rationale for the observed effects with P2XRs is also difficult to
discuss, especially with respect to data on the pattern of
polymerization of these receptors (43).
The influence of the ectodomain structure on P2XR desensitization is a
novel finding for this family of receptors but is reminiscent of those
seen among subtypes of other ligand-gated receptor channels. For
example, AMPA and glutamate maximally activate AMPA receptors, whereas
kainite and domoate act as partial agonists and produce much
less desensitization than glutamate (2, 23, 24, 44). A single
amino acid mutation in the third loop of the binding pocket of the
nicotinic acetylcholine receptor produces a right shift in the
concentration-response curve but also significantly slows the rate of
channel opening (25). The desensitization of this receptor is affected
by amino acids near or within the agonist-binding domain, as well as by
residues within M2 that line the pore (26, 27). A single residue
substitution in GluR1 also gives a mutant channel that exhibits
6-20-fold lower EC50 values for kainate (28). Introduction
of the
4 residues in the agonist-binding site of nicotinic nicotinic
acetylcholine receptors produces a 100-fold increase in the apparent
agonist potency and desensitization of receptors and a 3-7-fold shift in the apparent affinity for activation (27).
In conclusion, our results show that wild-type P2X2aR,
P2X2bR, and P2X4R desensitized in a receptor-
and ligand-specific manner. The P2X4R-specific ligand
potency and desensitization patterns were not only transferred but were
enhanced in P2X2a + X4R and P2X2b + X4R, whereas the P2X2R and P2X7R
ectodomains and the backbone of P2X4R generated
nonfunctional channels. This suggests that flanking sequences around
the transmembrane domains may act as modulatory regions. A parallelism
in the leftward shift of EC50 and DC50 for ATP
further suggests that the potency of agonists underlines the
ligand-specific patterns of receptor desensitization.