Neurological and Urological Diseases Research, Pharmaceutical
Products Division, Abbott Laboratories, Abbott Park, Illinois
TNP-ATP has become widely recognized as a potent and selective P2X
receptor antagonist, and is currently being used to discriminate between subtypes of P2X receptors in a variety of tissues. We have
investigated the ability of TNP-ATP to inhibit 
,
-methylene ATP
(,-meATP)-evoked responses in 1321N1 human astrocytoma cells expressing recombinant rat or human P2X2/3 receptors.
Pharmacological responses were measured using electrophysiological and
calcium imaging techniques. TNP-ATP was a potent inhibitor of
P2X2/3 receptors, blocking both rat and human receptors
with IC50 values of 3 to 6 nM. In competition studies, 10 to 1000 µM ,-meATP was able to overcome TNP-ATP inhibition.
Schild analysis revealed that TNP-ATP was a competitive antagonist with
pA2 values of 
8.7 and 8.2. Inhibition of
P2X2/3 receptors by TNP-ATP was rapid in onset, reversible,
and did not display use dependence. Although the onset kinetics of
inhibition were concentration-dependent, the TNP-ATP off-kinetics were
concentration-independent and relatively slow. Full recovery from
TNP-ATP inhibition did not occur until 
5 s after removal of the
antagonist. Because of the slow off-kinetics of TNP-ATP, full
competition with ,-meATP for receptor occupancy could be seen
only after both ligands had reached a steady-state condition. It is
proposed that the slowly desensitizing P2X2/3 receptor
allowed this competitive interaction to be observed over time, whereas
the rapid desensitization of other P2X receptors (P2X3) may
mask the detection of competitive inhibition by TNP-ATP.
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Introduction |
P2X receptors comprise a family
of seven distinct gene products that encode ATP-gated ion channel
subunits (P2X1-7, Ralevic and Burnstock, 1998
).
The protein subunits combine as either homomultimers (i.e.,
P2X3) or heteromultimers (i.e.,
P2X2/3) to form functional membrane-spanning
multimeric receptors. Inclusion of multiple subunits into a functional
receptor can confer a distinct biophysical as well as pharmacological
identity to a particular receptor subtype (Lewis et al., 1995; Torres
et al., 1998). For example, the ATP analog, ,-methylene ATP
(,-meATP), displays a nanomolar EC50 value
for activation of the rapidly desensitizing rat
P2X3 homomultimeric receptor, whereas the
EC50 value for activation of the nondesensitizing
P2X2/3 receptor is in the micromolar range (Bianchi et al., 1999). Although the exact stoichiometry of P2X receptors is not known, there is some evidence that they may exist as
trimers or multiples of these (Nicke et al., 1998; Ding and Sachs,
1999; Stoop et al., 1999).
Historically, pharmacological investigation of P2X receptors has been
hampered by the lack of potent, subtype-selective ligands. Antagonists
such as suramin, pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid, reactive blue 2, and their analogs have traditionally been used as P2X receptor antagonists (Connolly, 1995; Bultmann et al.,
1996). However, these compounds are relatively nonselective and exhibit
high nanomolar-to-high micromolar affinities for P2X receptors (Bianchi
et al., 1999). The ATP analog 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) has been used as a probe for more
than two decades to label ATP-binding sites on a variety of tissues
(Hiratsuka and Uchida, 1973; Watanabe and Inesi, 1982; Mockett et al.,
1994). Mockett et al. (1994) and King et al. (1997) were among the
first to describe the antagonist effects of TNP-ATP at P2X receptors.
However, it was not until recently that Virginio et al. (1998)
described the selective antagonism of P2X1,
P2X3, and P2X2/3 receptors
by low nanomolar concentrations of TNP-ATP. This potent and relatively
selective P2X receptor antagonist has now been used to characterize a
variety of native P2X receptors (Lewis et al., 1998; Thomas et al.,
1998; Burgard et al., 1999; Grubb and Evans, 1999; Zhong et al., 2000).
TNP-ATP is a close structural analog of ATP, suggesting that it may
bind in the extracellular ATP binding pocket on P2X receptors, and may
act as a competitive antagonist. This appeared to be the case when
nondesensitizing ATP responses on cochlear hair cells were blocked by
TNP-ATP in a competitive manner (Mockett et al., 1994). However, a more
recent characterization (Virginio et al., 1998) contained strong
evidence that TNP-ATP was a noncompetitive antagonist of rapidly
desensitizing recombinant rP2X3 receptors. The
apparent discrepancy between competitive and noncompetitive inhibition
by an antagonist at receptor subtypes with different desensitization
kinetics has been previously investigated using nicotinic receptor
subtypes (Alkondon et al., 1992; Briggs and McKenna, 1996). These
studies indicated that rapid receptor desensitization kinetics can make
a competitive antagonist appear to be noncompetitive in functional assays.
We have re-examined the antagonist profile of TNP-ATP and have
determined that it is a competitive antagonist of nondesensitizing rP2X2/3 receptors. Here we show that, because of
the slow off-kinetics of TNP-ATP at P2X receptors, competition between
agonist and antagonist can best be measured using nondesensitizing
P2X2/3 receptors. TNP-ATP appears to be a
noncompetitive antagonist at rP2X3 receptors, but
we propose that the rapid desensitization of
rP2X3 receptors prevents a competitive
interaction from being measured. Preliminary results from these studies
have been presented in abstract form (Niforatos et al., 1999).
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Materials and Methods |
Cell Culture.
Stably transfected 1321N1 human astrocytoma
cells expressing either rat P2X2/3
(rP2X2/3) or human P2X2a/3
(hP2X2a/3) receptors have previously been
described (Bianchi et al., 1999; Burgard et al., 1999; Lynch et al.,
1999). Briefly, these heteromultimeric cell lines were constructed by
transfecting rP2X2 or
hP2X2a cDNA into stably transfected rat or human
P2X3-expressing cells using standard
lipid-mediated transfection methods. Cell lines were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and antibiotics as follows: hP2X3, 300 µg
ml
1 G418; hP2X2a, 100 µg ml1 hygromycin; and
hP2X2a/3, 150 µg ml1
G418 and 75 µg ml1 hygromycin.
Electrophysiology.
Patch-clamp recordings were performed as
described previously (Burgard et al., 1999). Briefly, 1321N1 cells
expressing rP2X2/3 receptors were plated on
polyethylenimine-coated coverslips and grown to approximately 50%
confluence. Whole-cell patch-clamp recordings were obtained using a
modified extracellular saline consisting of 155 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 10 mM
HEPES, and 12 mM glucose. The intracellular patch pipette solution consisted of 140 mM potassium aspartate, 20 mM NaCl, 10 mM EGTA, and 5 mM HEPES. All cells were voltage-clamped at 60 mV, and series
resistance compensated 85 to 90% using an Axopatch 200B amplifier
(Axon Instruments, Foster City, CA).
Drugs were applied to the cells using a piezoelectric-driven glass
theta tube positioned near the cell. The solution interface between
barrels was swept across the cell. Solution exchange times were often
recorded by application of a 90% extracellular saline (10% water
added) across an open pipette tip. A single exponential function was
fitted to the resulting junction current transient, giving an open-tip
response time constant (
). Alternatively, whole-cell exchange s
were recorded in whole-cell patch mode by application of ,-meATP
(10 µM) to activate rP2X2/3 receptors, and
switching into and out of a high (55 mM) potassium-containing solution.
The resulting potassium-generated current relaxation through open P2X
receptors gave an indication of the whole-cell exchange time. During
experiments, agonists were usually applied every 30 to 60 s.
Agonist applications were kept short (most <3 s) to minimize pore
dilation of P2X2/3 receptors (Khakh et al., 1999). No evidence of pore dilation was observed.
Responses were acquired and digitized at 3 kHz, and analyzed using
pClamp software (Axon Instruments). Current amplitudes were always
measured at the end of the agonist application pulse. Current on- and
off-responses were fitted with a single exponential function, and the
resulting s were calculated. Agonist concentration-response curves
were fitted by least-squares regression to the logistic equation:
where Y, min, and max represent the measured, minimum, and
maximum responses, respectively; EC50 is the
ligand concentration giving half-maximal response; X is the
concentration of ligand used; and nH is
the Hill coefficient (Prism; GraphPad Software, San Diego, CA).
Antagonist concentration-response curves were fitted in the same manner
to determine IC50 values. The inhibition constant
(Ki) for the competitive antagonist TNP-ATP
was estimated from its IC50 value using the
Cheng-Prusoff relationship (Cheng and Prusoff, 1973; Craig, 1993):
Ki = IC50 / (1 + A/EC50), where A represents the concentration of
agonist, and EC50 represents the agonist
EC50.
Competitive inhibition was determined using Schild analysis
(Arunlakshana and Schild, 1959). The log concentration ratio log (A' /
A 1) was plotted against TNP-ATP concentration. A and A'
represent agonist EC50 values obtained in the
absence and presence of TNP-ATP, respectively.
pA2 values were determined from least-squares linear regression fitted to the Schild plots. Throughout the text, data
are expressed as mean ± S.E.M.
Calcium Imaging.
Inhibition of
hP2X2a/3 or rP2X2/3
receptors by TNP-ATP was also studied by measuring changes in cytosolic
calcium levels. The fluorescent dye Fluo-4 was used as an indicator of
the relative levels of intracellular calcium in a 96-well format using
a fluorescence imaging plate reader (Molecular Devices, Sunnyvale, CA),
as described previously (Bianchi et al., 1999). TNP-ATP (50 µl of 4×
concentration) was added 3 min before the addition of ,-meATP (50 µl of 4× concentration, final volume = 200 µl). Fluorescence
intensities were measured at 1- to 5-s intervals throughout each
experimental run. Data shown are based on the peak increase in relative
fluorescence units compared with basal fluorescence.
Concentration-response curves are shown as a percentage of the maximum
,-meATP-mediated fluorescence signal measured in the absence of
TNP-ATP.
All reagents were obtained from Sigma Chemical Co. (St. Louis, MO.)
unless otherwise noted. TNP-ATP and Fluo-4 were purchased from
Molecular Probes (Eugene, OR), and suramin from Research Biochemicals
International (Natick, MA).
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Results |
Drug Application.
The chemical structures of ATP and TNP-ATP
are shown in Fig. 1A. For TNP-ATP, the
nucleotide has been modified to include a trinitrophenyl group on the
2',3' dihydroxy position. The structural similarity of TNP-ATP to ATP
suggested that both molecules may interact with the same binding site
on the P2X receptor.
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Fig. 1.
Rapid agonist and antagonist application. A, chemical
structures of the endogenous P2X receptor agonist ATP and the
structural analog TNP-ATP are shown. B, solution exchange times were
determined by whole-cell or pipette recording in control solution (open
bar) and rapidly switching into test solution (closed bar, under
Materials and Methods). A single exponential function
was fitted to each response and the resulting time constant () is
shown. All currents have been scaled for visual comparison and the
whole-cell current response has been inverted to maintain polarity.
,-MeATP (10 µM) was used to activate rP2X2/3
receptors.
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To determine the mechanism of action of TNP-ATP, a rapid application
system was used to apply agonists and antagonists to 1321N1 cells
expressing rP2X2/3 receptors (under
Materials and Methods). Solution exchange times were
determined under a variety of conditions (Fig. 1B). Exchange of the
solution interface across an open pipette tip was achieved in
approximately 1 to 2 ms (1.6 ± 0.1 ms, n = 10).
This provided an indication of how fast the solution interface could
travel across a pipette. In contrast, whole-cell exchange time
constants were 20.6 ± 0.3 ms (n = 5), indicating
that diffusion around an entire cell was approximately 10 to 20 times
slower than diffusion across an open pipette tip. Similar activation
(32 ± 5.5 ms, Fig. 1B) and deactivation (86 ± 13 ms) time
constants () for 10 µM ,-meATP-induced currents were also
recorded (n = 20), indicating that activation and
deactivation rates were close to being diffusion-limited. Because
,-meATP exchange rates approached the limits of diffusion, no
quantitative conclusions were drawn regarding the actual activation and
deactivation rates of P2X receptors. However, comparisons were made to
the slower rates of receptor antagonism by TNP-ATP (see below).
Competitive Inhibition by TNP-ATP.
TNP-ATP inhibited
P2X2/3 receptors in a concentration-dependent
manner as assayed either by patch-clamp (Fig.
2, A and B) or calcium imaging (Fig. 2B)
techniques. As can be seen in Fig. 2A, activation of
P2X2/3 receptors by ,-meATP resulted in a non- or very slowly desensitizing inward current. Because these receptors were relatively nondesensitizing, agonist could be applied and removed as frequently as every 15 s without a decrease in current amplitude. Concentration-dependent inhibition by TNP-ATP was
evident from patch-clamp recordings both as an increase in the amount
of inhibition (gradual decrease in current amplitude), as well as an
increase in the rate at which inhibition developed. Both agonist and
antagonist were coapplied in Fig. 2A, and the extent of inhibition was
measured at the end of the application. Calculated
IC50 values for TNP-ATP were obtained from
inhibition curves for both rP2X2/3 and
hP2X2a/3 receptors, using two different functional assays (Fig. 2B). For rP2X2/3
receptors, patch-clamp recordings revealed an
IC50 value of 5.5 ± 1.1 nM
(n = 3-11 cells/concentration), and intracellular
calcium responses revealed an IC50 value of 3.0 ± 1.1 nM (n = 5 experiments). For
hP2X2a/3 receptors, intracellular calcium
responses revealed an IC50 value of 4.6 ± 1.2 nM (n = 3 experiments). It should be noted that in
these series of experiments, the IC50 values for
TNP-ATP were almost identical, even when measured at different
receptors and under different assay conditions.
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Fig. 2.
Inhibition of P2X2/3 responses by
TNP-ATP. A, individual rP2X2/3 currents recorded in
response to 10 µM ,-meATP (denoted by the bar). Increasing
concentrations of TNP-ATP were coapplied (0-100 nM) with ,-meATP
and traces were superimposed. B, TNP-ATP concentration-response curves
from two different P2X2/3 receptors. Inhibition of 10 µM
,-meATP responses by TNP-ATP at rP2X2/3 receptors was
measured using either patch-clamp recording ( ) or calcium imaging
( ). TNP-ATP inhibition at hP2X2a/3 receptors was
measured using calcium imaging techniques ( ). Data are expressed as
percentage of a corresponding 10 µM (rP2X2/3) or 3 µM
(hP2X2a/3) ,-meATP response.
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To evaluate the competitive nature of TNP-ATP antagonism, competition
curves were constructed for TNP-ATP inhibition of
rP2X2/3 (electrophysiology, Fig.
3A) and
hP2X2a/3 (calcium imaging, Fig. 3B) receptors. As
can be seen from both sets of graphs, increasing concentrations of
,-meATP were able to fully overcome inhibition produced by
TNP-ATP over a range of concentrations. A similar rightward shift of
the ,-meATP concentration-response curve was observed when
calcium imaging experiments were performed using rP2X2/3 receptors (data not shown). Schild
analysis of both rat and human receptors (Fig. 3, A and B, insets)
revealed plots best fitted with a regression line of slope 1.0 (hP2X2a/3) or 1.1 (rP2X2/3). pA2 values
obtained from Schild analysis were 8.2 (6 nM) for hP2X2a/3 and 8.7 (2 nM) for
rP2X2/3. Parallel right-shifted competition curves with a Schild plot of slope = 1 were consistent with
competitive antagonism of ,-meATP with TNP-ATP at both human and
rat P2X2/3 receptors
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Fig. 3.
Competitive inhibition by TNP-ATP at different
P2X2/3 receptors. A, concentration-response curves for
,-meATP in the presence of increasing concentrations of TNP-ATP
were measured using electrophysiological techniques on cells expressing
the rP2X2/3 receptor. Data (n = 3-14
cells/data point) are plotted as percentage of 1 mM
,-meATP-induced current amplitude. Boxed inset, Schild plot of
the data shown in A. B, concentration-response curves for ,-meATP
in the presence of increasing concentrations of TNP-ATP were measured
using calcium imaging techniques on cells expressing the
hP2X2a/3 receptor. Data (n = 3 experiments/data point) are plotted as percentage of maximal
,-meATP-induced Fluo-4 fluorescence. A maximal concentration of
100 µM ,-meATP was used for these experiments. Boxed inset,
Schild plot of the data shown in B. For A and B, upper and lower limits
of curves were constrained to 100 and 0% of response, respectively.
For Schild plots, concentration ratios were calculated and plotted as
detailed under Materials and Methods. pA2 values were
determined from the x-intercept of a regression line
fitted to the data. C, concentration-response curves for ,-meATP
in the presence of increasing concentrations of TNP-ATP were measured
using calcium imaging techniques on cells expressing the
hP2X3 receptor. Data (n = 2 experiments/data point) are plotted as ,-meATP-induced Fluo-4
fluorescence units as recorded in the assay. For A to C, TNP-ATP
concentrations and their corresponding symbols are displayed in the
upper left corner of each graph.
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Assuming competitive binding at the P2X2/3
receptor, Ki values were calculated from
TNP-ATP IC50 values using the Cheng-Prusoff relationship (under Materials and Methods). Agonist
concentrations of 3 µM (hP2X2a/3) and 10 µM
(rP2X2/3) ,-meATP were used in these
experiments. The EC50 values for ,-meATP
were 3.4 µM (rP2X2/3, Bianchi et al., 1999) and
1.3 µM (hP2X2a/3; H. McDonald and E. C. Burgard, unpublished observations), as determined from agonist concentration-response curves. Calculated
Ki values for TNP-ATP were 1.4 nM for both
rP2X2/3 and hP2X2a/3. This
was very similar to IC50 as well as calculated
pA2 values, in that all were between 1 and 10 nM.
The high affinity of TNP-ATP for P2X2/3 receptors was independent of species or assay conditions.
In contrast, calcium imaging experiments performed using the
hP2X3 receptor (Fig. 3C) revealed TNP-ATP
competition curves characterized by a decrease in the maximal
agonist-induced current, with a rightward shift in the agonist
EC50 value. The apparent inability of
,-meATP to overcome TNP-ATP block is in agreement with a
published report (Virginio et al., 1998) that describes TNP-ATP as a
noncompetitive antagonist at desensitizing P2X3
receptors. However, this apparent discrepancy could be explained by
differences in desensitization rates between the two receptor subtypes
(see below).
Kinetics of TNP-ATP Inhibition.
If TNP-ATP and ,-meATP
compete for the same binding site at P2X2/3
receptors, then analysis of a competitive interaction should be
performed when both ligands have achieved steady-state binding. This
assumes that both ligands have had a sufficient amount of time to bind
to (or unbind from) the receptor. The agonist-induced activation and
deactivation kinetics of P2X2/3 receptors
approached the experimental limits of solution diffusion (Fig. 1B),
suggesting that 10 µM ,-meATP probably reached steady-state
binding in tens of milliseconds. However, it appeared that TNP-ATP on-
and off-kinetics were much slower. To evaluate the on- and off-kinetics of TNP-ATP, three different drug application protocols were used in
patch-clamp experiments to apply TNP-ATP to cells expressing rP2X2/3 receptors (Fig.
4). TNP-ATP was either 1) preapplied, to
measure the steady-state effects of TNP-ATP on subsequent activation by
agonist; 2) postapplied, to measure both the on- and off-kinetics of
TNP-ATP in the presence of agonist; or 3) coapplied, to measure TNP-ATP
kinetics during simultaneous application with agonist.
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Fig. 4.
Inhibition and kinetics of TNP-ATP are similar
regardless of application protocol. TNP-ATP was applied with
,-meATP using three different protocols in a single cell
expressing rP2X2/3 receptors. The control response to 10 µM ,-meATP is shown along with the preapplication protocol
trace. For preapplication, TNP-ATP (open bar) was applied at least
30 s before, during, and after ,-meATP (solid bar)
application. For postapplication, ,-meATP was applied for 5 to
10 s before, during, and after TNP-ATP application. The asterisk
denotes a baseline shift due to slow agonist-induced desensitization of
the response. For coapplication, TNP-ATP and ,-meATP were applied
and removed at the same time. The on of single
exponential functions fitted to the TNP-ATP traces for post- and
coapplication are shown.
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The preapplication protocol is the conventional application method used
to study antagonist potency and efficacy. Using this method,
preapplication of TNP-ATP (30 s) allowed the antagonist to reach a
steady-state interaction with the receptor before agonist was applied.
As shown in Fig. 4, preapplication of TNP-ATP (100 nM) produced a
decrease in both amplitude and apparent activation rate of the
,-meATP (10 µM) response. This response resembled that of a low
,-meATP concentration, and is consistent with the profile of a
competitive interaction at the receptor. Because the ,-meATP
on-kinetics are fast, the slow activation seen in the presence of
TNP-ATP is an indirect reflection of the slow off-kinetics of TNP-ATP
(Benveniste et al., 1990; see below) because the competitive antagonist
must unbind for agonist to activate the receptor. Here 100 nM TNP-ATP
also inhibited the ,-meATP-induced response to <10% of control,
consistent with the concentration-response curve in Fig. 2B. In
addition, the first agonist response was always inhibited after TNP-ATP
application, so there was no evidence for a use-dependent mechanism of
action of TNP-ATP. Although the preapplication method reveals the
extent of steady-state inhibition by an antagonist, it provides only
indirect information about the antagonist off-kinetics, and no
information regarding the on-kinetics of antagonist interactions with
the receptor.
To investigate the on- and off-kinetics of antagonists, the
postapplication protocol was used because it allowed the antagonist kinetics to be measured entirely during steady-state receptor activation by agonist. As shown in Fig. 4, on- and off-response kinetics were recorded when rP2X2/3 receptors
were first activated by ,-meATP (10 µM), and then TNP-ATP (100 nM) was applied and removed during steady-state receptor activation
(postapplication). All on- and off-responses were adequately described
by a single exponential function. Using this protocol, the 100 nM
TNP-ATP on-response time constant on was
320 ± 74 ms (n = 9), and the off-response time
constant (off) was 2390 ± 315 ms
(n = 7). Even at this high IC90
concentration of TNP-ATP, it is evident that the on- and off-kinetics
are 10-fold slower than 10 µM ,-meATP kinetics. Antagonist
steady state was reached after a sufficiently long TNP-ATP application,
and the extent of inhibition was measured under these conditions. One
minor drawback to this approach was the presence in some cells of very
slow ,-meATP-induced desensitization during "steady-state"
agonist application. Agonist was often applied for >10 s before
applying TNP-ATP, and the steady-state current amplitude decreased
slightly in a proportion of cells. Although this did not affect either
the TNP-ATP kinetics or extent of inhibition, it did shift the baseline
amplitude of the agonist response during TNP-ATP application. This
effect can be seen in Fig. 4 (postapplication trace, asterisk).
Another approach used to study TNP-ATP kinetics was to apply both
agonist and antagonist simultaneously, and then measure the effect of
the antagonist under these conditions. This coapplication protocol
eliminated any potential effects of slow agonist-induced desensitization on TNP-ATP kinetic measurements. When TNP-ATP was
coapplied with ,-meATP (Figs. 2 and 4, coapplication), currents activated rapidly, but a time-dependent inhibition of current developed
(relatively slowly). Both the extent of block and the apparent rate at
which inhibition occurred increased with increasing concentrations of
TNP-ATP. Because agonist steady state was reached within the first 50 ms, it was assumed that antagonist on-kinetics could be reliably
estimated if they were slower than 50 to 100 ms. Indeed, the
,-meATP activation on was the same
whether applied alone (26 ± 10 ms, n = 5) or
coapplied with 100 nM TNP-ATP (27 ± 12 ms, n = 5), indicating that TNP-ATP inhibition developed more slowly than
,-meATP activation. As shown in Fig. 4, the 100 nM TNP-ATP
on for both coapplication (387 ± 91 ms,
n = 5) and postapplication (320 ± 74 ms,
n = 9) protocols was similar. Although the overall
on was slower at lower TNP-ATP concentrations (10 and 30 nM), these TNP-ATP on were also not
significantly different between co- and postapplication protocols.
Because the TNP-ATP on-kinetics were slow, and a relatively long (2
s) application was used, a reasonable estimate of both
on and extent of inhibition was obtained using
the coapplication protocol.
All three protocols resulted in similar on
and/or off values for TNP-ATP. It is important
to note that for concentrations of TNP-ATP 
IC90, on and
off kinetics were 10 times slower than
,-meATP kinetics. At these concentrations, TNP-ATP inhibition developed slowly, and recovery from inhibition was correspondingly slow.
Of the three protocols, measurements using the postapplication method
best represented the true on- and off-kinetics of TNP-ATP inhibition.
Using this protocol, the concentration dependence of
on and off was
further investigated. As shown in Fig.
5A, the on
decreased with increasing concentrations of TNP-ATP. This concentration-dependent increase in the apparent rate of inhibition was
consistent with a mass action increase in binding probability. However,
the off did not change with increasing TNP-ATP
concentrations, indicating that the off-response kinetics were
concentration-independent. The on- and off-kinetics of the P2X receptor
antagonist suramin are shown in Fig. 5B. In comparison, suramin
exhibits an IC50 value of approximately 800 nM at
rat P2X2/3 receptors (Bianchi et al., 1999). In
this cell at equimolar concentrations (10 µM), suramin displayed much
faster off-kinetics (off = 775 ms) than TNP-ATP (off = 5951 ms), consistent with its
lower affinity for the P2X2/3 receptor. A summary
of the effects of TNP-ATP concentration on on
and off from 15 cells is shown in Fig.
6. At all concentrations of TNP-ATP, the
mean off values were very slow (>2000 ms). It is reasonable to assume that the high affinity of TNP-ATP for rP2X2/3 receptors is predominantly determined by
its slow off.
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Fig. 5.
Concentration-dependent kinetics of TNP-ATP. A,
postapplication responses of TNP-ATP. ,-meATP (10 µM) was
applied before, during, and after TNP-ATP applications of 10 and 100 nM. Note the concentration dependence of TNP-ATP on-response only.
Individual traces recorded from the same cell. B, equimolar
concentrations (10 µM) of TNP-ATP or suramin show difference in
kinetics. Both antagonists were applied to the same cell using the
postapplication protocol. Note the faster on- and slower off-kinetics
of TNP-ATP. For A and B, traces have been scaled to equal amplitudes
for visual comparison.
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Fig. 6.
Summary of TNP-ATP on- and off-kinetics. A,
on of TNP-ATP inhibition is concentration-dependent
(n = 4-11 cells/data point). B, off
of TNP-ATP inhibition is not concentration-dependent
(n = 3-10 cells/data point). For A and B, s
were determined from single exponential curves fitted to the TNP-ATP
on- or off-response as in Fig. 5.
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In contrast to rP2X2/3 receptors,
rP2X3 receptors exhibit rapid desensitization in
the presence of agonist (Lewis et al., 1995). The relationship between
receptor desensitization and TNP-ATP time course can be seen in the
traces in Fig. 7. Here ,-meATP induced a nondesensitizing inward current at
rP2X2/3 receptors (,-meATP control trace).
Preapplication of TNP-ATP (,-meATP + TNP-ATP trace) produced a
decrease in the ,-meATP activation rate due to competition of
,-meATP with TNP-ATP. The slow off of
TNP-ATP is evident here because it takes >2 s to approach a steady-state interaction between agonist and antagonist. Only after
this time can a competitive interaction be observed where agonist can
replace the slowly dissociating antagonist at the receptor. The
relatively nondesensitizing rP2X2/3 receptor
remains open long enough to detect the competition. In contrast, the
rapid desensitization of rP2X3 (light
P2X3 trace) receptors occurs before TNP-ATP can
dissociate from the rP2X2/3 receptor, precluding
a steady-state competition to be recorded. Apparent noncompetitive inhibition would be observed at rapidly desensitizing
P2X3 receptors, due to the inability of TNP-ATP
to dissociate before the current desensitized.
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Fig. 7.
Slow TNP-ATP kinetics can obscure competitive
inhibition at P2X3 receptors. Three superimposed traces
recorded under different conditions. The light P2X3 trace
was recorded in response to 100 µM ATP. The dark P2X2/3
responses were both recorded from a separate cell. ,-meATP (100 µM, control) was either applied alone, or was applied in the presence
of TNP-ATP (10 nM, TNP-ATP preapplication). Note the slow activation of
P2X2/3 receptors in the presence of TNP-ATP. Agonist
application is denoted by the horizontal bar.
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Discussion |
TNP-ATP is a trinitrophenyl analog of ATP that binds to a variety
of ATP-binding sites. When used as a fluorescent probe, relatively high
concentrations have been used to label ATP-binding sites and to probe
enzyme activities (Hiratsuka and Uchida, 1973; Watanabe and Inesi,
1982; Mockett et al., 1994). However, the nanomolar affinity of TNP-ATP
for P2X1, P2X3, and
P2X2/3 receptors (Virginio et al., 1998) suggests
that TNP-ATP may be a selective antagonist for these P2X receptors over
other ATP-binding sites. Using these criteria, TNP-ATP is currently the
most potent and selective P2X receptor antagonist available.
In the present study, we have confirmed the high affinity of TNP-ATP
for P2X2/3 receptors. Potent antagonism
(IC50 < 10 nM) of both human and rat
P2X2/3 receptors by TNP-ATP was demonstrated using calcium imaging as well as electrophysiological techniques. TNP-ATP was determined to be a competitive antagonist based on a number
of experimental results. First, the inhibition could be overcome by
increasing concentrations of agonist. Second, parallel right-shifted
agonist concentration-response curves were constructed in the presence
of increasing concentrations of TNP-ATP, with no change in the maximal
agonist response. Third, Schild analysis revealed a competitive
inhibition by TNP-ATP at P2X2/3 receptors. Fourth, calculated pA2 values were also <10 nM,
and correlated well with the IC50 value and
calculated Ki values. A similar competitive antagonism of nondesensitizing P2X responses in cochlear hair cells was
reported by Mockett et al. (1994), although the TNP-ATP concentrations
used were orders of magnitude higher than those used in the present
study. This was presumably due to lower affinity interactions of
TNP-ATP with nondesensitizing cochlear P2X2
receptors (Housley et al., 1999). From these two studies, it appears
that TNP-ATP is a competitive antagonist of nondesensitizing P2X receptors.
In addition to confirming the high affinity of TNP-ATP for the
P2X2/3 receptor, we have demonstrated that the
off-kinetics of TNP-ATP are very slow (>2000 ms), and is
concentration-independent. It is proposed that the slow off-kinetics
contribute to the high affinity of the antagonist, as well as
contribute to its apparent noncompetitive action at
P2X3 receptors. However, to draw conclusions based on apparent antagonist kinetics, a number of experimental assumptions must be met. In relation to the kinetics of a competitive antagonist, the solution exchange rate, the binding and unbinding kinetics of the agonist, and the channel gating kinetics must all be
fast (Benveniste et al., 1990; Benveniste and Mayer, 1991). In
addition, the channel must be relatively nondesensitizing. Under these
assumptions, agonist will reach equilibrium quickly, and the slow
kinetics of the antagonist will be measurable. In the present studies,
the on- and off-kinetics of 10 µM ,-meATP approached the limits
of whole-cell diffusion for our drug delivery system ( = 20-30
ms). In comparison, the on-kinetics for TNP-ATP at an
IC90 concentration was 10-fold slower, and the
corresponding off-kinetics were >25-fold slower than the agonist.
Although channel-gating kinetics have not been described adequately for
either P2X3 or P2X2/3
receptors, channel opening and closing rates estimated from either
recombinant or native P2X receptors indicate that these parameters are
also significantly faster than TNP-ATP kinetics (Krishtal et al., 1988;
Bean et al., 1990; Cloues, 1995; Wright and Li, 1995; Evans, 1996; Ding
and Sachs, 1999). Having met these assumptions, the TNP-ATP kinetics
measured in the present studies are a reasonable measure of apparent
antagonist binding and unbinding rates.
Preapplication of a competitive antagonist for a sufficient period of
time allows equilibrium to be reached between antagonist and receptor.
Subsequent application of a rapidly equilibrating agonist will result
in a slow increase in agonist-activated current, reflecting the
dissociation kinetics of the antagonist (Benveniste et al., 1990).
Preapplication of TNP-ATP significantly slowed the subsequent agonist
response and decreased its amplitude. This protocol gave a reliable
measure of magnitude of inhibition by TNP-ATP, and allowed an indirect
estimate of the off-kinetics of TNP-ATP. A better measure of TNP-ATP
on- and off-kinetics was obtained by using the postapplication
protocol, where agonist equilibrium was achieved first, and TNP-ATP was
subsequently applied. The antagonist on-kinetics reflected the
apparent on-rate because these kinetics depend on antagonist
concentration, the intrinsic antagonist association and dissociation
rates, and the concentration of agonist (Benveniste and Mayer, 1991).
On the other hand, the measured TNP-ATP off-kinetics revealed a closer
estimate to the true dissociation rate because these were
concentration-independent and were sufficiently slow to not be
influenced by rapid association and dissociation of agonist.
Interestingly, we also found that if TNP-ATP was coapplied with
agonist, the response reached a rapid peak, but a progressive decrease
in agonist-activated current occurred with a kinetic profile that was
similar to the on-kinetics measured using the postapplication protocol.
It appeared that coapplication was a rapid way to obtain an estimate of
the on-kinetics of TNP-ATP.
TNP-ATP was originally characterized as a potent, noncompetitive
antagonist at rapidly desensitizing rat P2X3
receptors (Virginio et al., 1998). Likewise, we have observed an
apparent noncompetitive block by TNP-ATP at rP2X3
receptors (Fig. 3C). This apparent competitive (rP2X2/3) versus noncompetitive
(rP2X3) discrepancy between two receptor subtypes
could be explained by intrinsic differences in the receptor
desensitization rates. For example, a competitive antagonist of
nondesensitizing nicotinic acetylcholine receptors appeared to be
noncompetitive at rapidly desensitizing receptor subtypes (Alkondon et
al., 1992; Briggs and McKenna, 1996). This effect was also due to
rapid receptor desensitization occurring before slow dissociation of
the high-affinity antagonist.
We propose a model (Fig. 7) to explain why TNP-ATP appears to be a
noncompetitive antagonist at rapidly desensitizing
P2X3 receptors. When TNP-ATP is preapplied and
allowed to reach equilibrium binding before an agonist is applied,
agonist activation produces a P2X3 response that
desensitizes faster than TNP-ATP can dissociate from the receptor.
Because rP2X3 receptors desensitize with an initial rapid time course ( = 39 ms; Burgard et al., 1999),
desensitization will occur before a significant amount of TNP-ATP
dissociates. In addition, the desensitization rate of
P2X3 receptors increases with increasing agonist
concentration (Chen et al., 1995). Attempts to compete with TNP-ATP for
receptor binding by increasing the agonist concentration can only make
the agonist response faster, allowing even less time for antagonist
dissociation. Under these conditions, competition with TNP-ATP will not
be measured. However, competition can be measured at a relatively
nondesensitizing receptor such as P2X2/3.
The possibility that TNP-ATP can exhibit different antagonist actions
(competitive versus noncompetitive) at different P2X receptors remains
unlikely. P2X2/3 receptors are formed by
heteromultimeric combination of P2X3 and
P2X2 subunits. Although the desensitization kinetics of P2X2/3 receptors closely matches the
P2X2 phenotype, the pharmacology of the
antagonist suramin, as well as that of agonists ATP and ,-meATP,
resembles that of P2X3 receptors (Bianchi et al.,
1999). The antagonist potency of TNP-ATP at
P2X2/3 receptors also matches that of
P2X3 receptors. Assuming that ATP, ,-meATP, and TNP-ATP all bind at the same extracellular binding site on P2X2/3 receptors, we would propose that this
binding relationship is similar for P2X3
receptors. TNP-ATP would therefore show competitive antagonism at both
P2X3 and P2X2/3 receptors.
Because of the rapid desensitization of P2X3
receptors, this cannot be measured using standard electrophysiological
techniques on wild-type receptors. The development of a sensitive and
specific P2X3 radioligand-binding assay could
measure steady-state interactions between TNP-ATP and agonist, and
could determine the competitive nature of TNP-ATP binding at
P2X3 receptors. The success of this approach
would also depend on the ability of agonist to remain bound to the
receptor after desensitization. Alternatively, this issue could be
investigated electrophysiologically using a mutant
P2X3 receptor that retains P2X3 binding properties, but has altered
desensitization/gating kinetics. These approaches will require
development of new tools for studying antagonist actions at P2X receptors.
We thank Karen Alexander, Heath McDonald, and Bruce Bianchi for
assistance with experiments, and Lance Lee and Clark Briggs for
valuable discussions.
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Abstract
Introduction
![<UP>Y</UP>=<UP>min</UP>+[(<UP>max</UP>−<UP>min</UP>)/(1+10<SUP>(<UP>LogEC</UP><SUB>50</SUB>−<UP>X</UP>)n<SUB>H</SUB></SUP>)]](/content/vol58/issue6/fulltext/1502/img001.gif)
a nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels.
Br J Pharmacol
124:
1463-1466[Medline].
