Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510
Submitted 29 January 2003 ; accepted in final form 22 April 2003
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ABSTRACT |
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ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals
Although [Ca2+]i measurements are sensitive enough to record the activity of native and recombinant P2XR in single nonexcitable and excitable cells (22, 32, 38, 43, 47), the majority of studies with P2XRs are done using current measurements (28, 30). Patch-clamp techniques provide powerful tools in evaluating the status of P2XR pores and thus are extremely useful in biophysical characterization of these receptors. With respect to physiology and pharmacology of P2XR, Ca2+ rather than current measurements have potential to provide valuable information. [Ca2+]i measurements were also occasionally used in evaluating the kinetics of receptor activity (2, 16, 20, 22, 24). The main advantage in using single-cell [Ca2+]i imaging in such studies is that measurements can be done simultaneously in tens of cells, enabling reliable statistical analysis, whereas current measurements are done in one cell per experiment, and slow recovery of receptors from desensitization makes repeated measurements in the same cell difficult. Single-cell [Ca2+]i imaging provides the same advantages in studies on pharmacological profiles of P2XRs and testing new compounds. Because Ca2+ signaling by P2XRs is incompletely characterized, however, [Ca2+]i measurements could be viewed as an inadequate method for studies on physiology, pharmacology, and kinetics of P2XR activity.
Here, we studied the spatiotemporal aspects of ATP-induced Ca2+ signals in P2XR-expressing cells and the dependence of the pattern of Ca2+ signaling on the kinetics of channel activity. Experiments were done with several wild-type and chimeric P2XRs in homomeric configuration. The selection of P2XRs was based on the kinetics of their desensitization during the sustained ATP stimulation, from rapidly desensitizing P2X3R to nondesensitizing P2X7R. These include the recently constructed P2X2aR + X4R and P2X2b + X4R chimeras, having the Val66-Tyr315 ectodomain sequence of P2X4R in the backbones of P2X2aR and P2X2bR and differing from parental receptors with respect to rates of signal desensitization (17). To express P2XRs, we selected two cell types: nonexcitable human embryonic kidney cells (hereafter HEK-293 cells) and excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (hereafter GT1 cells). Because the majority of previous studies with recombinant P2XR currents were done in HEK-293 cells (4, 14, 26, 27, 36, 46), we used these cells for current measurements to provide a valid comparison with the literature.
Initially, we characterized the extent to which the P2XR current properties differ in the two selected cell types. In further studies, we focused on the spatiotemporal characteristics of Ca2+ signaling by P2XRs expressed in GT1 cells, because the coactivation of endogenously expressed G protein-coupled P2YRs in HEK-293 cells (31) interferes with measurements of P2XR-mediated Ca2+ influx. We also compared the peak current and [Ca2+]i responses (commonly used for calculating the EC50 and IC50 values) and the rates of signal desensitization during prolonged ATP stimulation. In the final stage, we analyzed the usefulness of Ca2+ measurements as a method in studies on pharmacological properties of P2XRs. The results indicate that single-cell Ca2+ measurements not only provide a valuable method for characterizing the spatiotemporal aspects of Ca2+ signaling by P2XRs, but the relationship between current and Ca2+ and the sensitivity of Ca2+ signals to changes in extracellular pH and Zn2+ concentrations proves them to be useful in studies on channel activity.
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MATERIALS AND METHODS |
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GT1 cells and HEK-293 cells were used to examine the patterns of desensitization in P2XRs as previously described (19). GT1 cells were routinely maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1), containing 10% (vol/vol) fetal bovine serum and 100 µg/ml gentamicin (Invitrogen, Carlsbad, CA) in a water-saturated atmosphere of 5% CO2-95% air at 37°C. HEK-293 cells were cultured in MEM 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% wt/vol; Sigma, St. Louis, MO)- coated coverslips at a density of 0.751 x 105 cells per 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, transfection mixture was replaced with normal culture medium. Cells were subjected to experiments 2448 h after transfection.
Ca2+ measurements. Transfected GT1 cells were
preloaded with 1 µM fura 2 acetoxymethyl ester (fura 2-AM; Molecular Probe,
Eugene, OR) for 60 min at room temperature in modified Krebs Ringer buffer
[(in mM) 120 NaCl, 5 KCl, 1.2 CaCl2, 0.7 MgSO4, 15
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES), and 1.8 g/l glucose (pH 7.4)]. After dye loading, cells were washed
in the same medium 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). All cells were stimulated with 100 µM ATP, and
the dynamic changes of [Ca2+]i were examined under a
x40 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 1550 single cells at a rate of 1 point/s. Spatial
resolution was estimated to be
1 µm. All experiments were done at room
temperature.
Current measurements. Electrophysiological experiments were
performed on GT1 and HEK-293 cells at room temperature using whole cell
patch-clamp recording techniques
(15). ATP-induced currents
were recorded using an Axopatch 200B patch-clamp amplifier (Axon Instruments,
Union City, CA) and filtered at 2 kHz using a low-pass Bessel filter. Forty to
seventy percent 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 35 M. All current records were captured and stored using the
pCLAMP 8 software packages in conjunction with the Digidata 1322A
analog-to-digital converter (Axon Instruments). Patch electrodes were filled
with a solution containing (in mM) 140 KCl, 0.5 CaCl2, 1
MgCl2, 5 EGTA, and 10 HEPES, pH adjusted with 1 M KOH to 7.2. The
osmolarity of the internal solutions was 282287 mosM. The bath solution
contained (in mM) 142 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, 10
glucose, and 10 HEPES, pH adjusted to 7.4 with 1 M NaOH. The osmolarity of
this solution was 285295 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). The application tip was routinely positioned
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 (1090% rise time). All experiments were done at room
temperature.
Calculations. The time course of the [Ca2+]i
and current evoked by sustained ATP stimulation were fitted to a single
exponential function (ae-kt + b) using GraphPad Prism
(GraphPad Software, San Diego, CA) and pCLAMP 8, respectively, to generate the
half-time for signal desensitization ( = ln2/k). Linear
and log-linear regression analyses were used to correlate variables, and the
strength of correlation was expressed as Pearson's R coefficient
(KaleidaGraph, Reading, PA). Significant differences, with P <
0.05, were determined by one-way ANOVA with Newman-Keuls multiple-comparison
test. Each experiment was repeated five or more times to ensure the
reproducibility of the findings.
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RESULTS |
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In further studies, we compared the potential usefulness of two cell types for analyzing the P2XR activity by single-cell Ca2+ measurements. Native HEK-293 cells do not express voltage-gated Ca2+ channels, as indicated by inability of high-potassium-induced depolarization of cells to elevate [Ca2+]i (Fig. 3A, left upper trace), whereas depolarization of GT1 cells increased the [Ca2+]i (Fig. 3A, right upper trace). The response was abolished by removal of extracellular Ca2+ (Fig. 3A, right bottom trace). On the other hand, ATP induced a rapid and transient increase in [Ca2+]i in HEK-293 cells, but not in GT1 cells, bathed in Ca2+-containing (Fig. 3B, left upper trace) and Ca2+-deficient (left bottom trace) medium. These results confirm that native HEK-293 cells express Ca2+-mobilizing P2Y purinergic receptors (31) and, thus, are not a good cell model for measurements of P2XR-mediated Ca2+ influx.
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In additional experiments, we further characterized GT1 cells as a potential cell model for studies on activity of P2XRs. ATP-induced elevation in the [Ca2+]i in GT1 cells expressing P2X2aR was abolished by removal of extracellular Ca2+, indicating that Ca2+ influx exclusively accounts for the rise in [Ca2+]i (Fig. 4A). The substitution of extracellular Na+ with N-methyl-D-glucamine reduced the amplitude of Ca2+ signals but did not affect the rates of Ca2+ signal desensitization (Fig. 4B). This is probably the receptor-specific feature, because an increase in peak amplitude of Ca2+ signals was observed in P2X4R-expressing cells bathed in sodium-deficient medium (47).
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We also tested the impact of expression of voltage-gated Ca2+ channels on the pattern of ATP-induced Ca2+ signaling. These experiments were done in P2X2aR-expressing cells. The addition of nifedipine, a blocker of L-type Ca2+ channels, also reduced the amplitude of Ca2+ responses without affecting the rates of signal desensitization (Fig. 4C, middle trace). Activation of voltage-gated Ca2+ influx by high-potassium-induced depolarization of cells before ATP stimulation was also ineffective in changing the rates of Ca2+ signal desensitization (Fig. 4C, bottom trace). To test the potential coupling of Ca2+ influx with Ca2+-induced Ca2+ release from intracellular pools, cells were treated with 100 µM ryanodine. The pattern of ATP-induced Ca2+ signals in ryanodine-treated cells was indistinguishable from that observed in control cells (not shown), confirming a previously published finding that these cells do not express operative ryanodine receptor channels (5).
Spatiotemporal characteristics of P2XR-generated Ca2+ signals in GT1 cells. As in current responses by recombinant P2XRs, ATP-induced Ca2+ response was composed of two phases: a relatively rapid rise in [Ca2+]i from basal levels to peak response (activation phase), followed by a gradual decline toward the plateau response (desensitization phase) (Fig. 4A). In further studies, we examined whether the temporal patterns of Ca2+ signaling by recombinant P2XRs reflect the receptor specificity of current signaling. We also examined the spatial characteristics of P2XR-generated Ca2+ signals during activation and desensitization phases. In these and following experiments, voltage-gated Ca2+ influx was not blocked and all receptors were stimulated with 100 µM ATP unless otherwise indicated.
Figure 5A shows that P2XR-generated Ca2+ signals were receptor specific with respect to the peak Ca2+ responses and signal desensitization rates. The peak amplitudes were in the following order: P2X3R < P2X7R < P2X4R = P2X2b + X4R < P2X2a + X4R < P2X2bR < P2X2aR (Table 1). In parallel to current responses to 100 µM ATP (Fig. 1), Ca2+ signals desensitized very rapidly in P2X3R- and P2X2b + X4R-expressing cells and with moderate rates in P2X2bR-, P2X2a + X4R-, and P2X4R-expressing cells, whereas Ca2+ signals showed little or no desensitization in P2X2aR and P2X7R-expressing cells. Figure 5B shows ratio images of cells before addition of agonist (left) and at the peak [Ca2+]i response (right). There was an increase in F340/F380 ratio in all regions of the central section of cells expressing all of the examined channels, indicating that generation of global Ca2+ signals is a common feature of these receptors.
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Except for P2X7R, activation phase was too rapid to be analyzed by the imaging system with the resolution time of about one image per second. Figure 6 illustrates the spatiotemporal aspects of Ca2+ signals in P2X7R-expressing cells before and during activation of receptors with 1 mM ATP. The temporal changes of Ca2+ signal (Fig. 6, bottom right) are shown as F340/F380 ratio of averaged intensities in the whole cell area. The [Ca2+]i rose during continuous ATP stimulation, reaching the plateau 25 s after the addition of ATP. Fig. 6, top, shows several ratio images taken at different time points (denoted by red circles at lower right). Consistent with the generation of global Ca2+ signals during activation phase, there was an increase in the ratio in all regions of the central section of the cell.
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The receptor-specific rates of desensitization did not affect the global nature of Ca2+ signaling. Figure 7 illustrates spatial characteristics of Ca2+ signaling in P2X2aR (Fig. 7A), P2X2bR (Fig. 7B), and P2X3R (Fig. 7C). After reaching the peak value, the [Ca2+]i declined slowly to the steady level in P2X2aR-expressing cells, at moderate rates in P2X2bR-expressing cells, and rapidly in P2X3R-expressing cells. In all three cases, the ratio values in all areas in the central section of the cells decreased but remained above the basal level, suggesting that the global nature of Ca2+ signals was preserved during receptor desensitization.
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Comparison of Ca2+ and current signaling in P2XR-expressing cells. To study the concentration-dependent effects of ATP on peak amplitude of current and Ca2+ responses, we selected P2X2aR-expressing cells. In both measurements, ATP was effective in the 0.5100 µM concentration range (Fig. 8A). The parallelism in the concentration-dependent curves for current and Ca2+ responses indicates that the peak amplitude of Ca2+ signals reflects well the size of currents.
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In cells expressing different P2XRs and stimulated with 100 µM ATP,
there were significant variations in the amplitudes of peak
current/Ca2+ responses (Table
1). Overall, the peak amplitudes of current and Ca2+
responses correlated reasonably well (R = 0.88 for linear
relationship; Fig.
8B), further indicating that the amplitude of current
response represents the major factor that determines the magnitude of
Ca2+ signals. We also correlated the receptor-specific half-times
of current desensitization () with the normalized amplitude of
[Ca2+]i responses. To do this, the peak amplitudes of
[Ca2+]i response were divided with the peak amplitudes
of the corresponding currents and these values were plotted against calculated
for current desensitization. Results, shown in
Fig. 8C, indicate a
strong (R = 0.97) log-linear relationship between decay in current
responses and peak amplitude of normalized [Ca2+]i
responses. These results imply that, in addition to the size of current, the
rate of current desensitization influences the peak amplitude of
[Ca2+]i response, especially in rapidly desensitizing
channels.
In further studies, we compared the rates in current and Ca2+ signal desensitization during the prolonged ATP stimulation. A closer evaluation of the current and Ca2+ decays in cells expressing four wild-type and two chimeric P2XRs is shown in Fig. 9A. Both Ca2+ and current measurements revealed the receptor-specific desensitization pattern in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. In chimeric P2X2a + X4R and P2X2b + X4R, substitutions of P2X2R ectodomain to that of P2X4 apparently accelerated desensitization rates, and these functional differences were detected in both Ca2+ and current measurements.
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Effects of extracellular pH and Zn2+ on ATP-induced Ca2+ signaling. To test the validity of Ca2+ measurements as a method in studies on pharmacological properties of P2XRs, we examined the sensitivity of ATP-induced Ca2+ responses in GT1 cells to extracellular pH and Zn2+. In accord with experiments with current measurements (18, 34, 44), acidification and alkalinization had opposite effects on ATP-induced Ca2+ responses in P2X2aR- and P2X4R-expressing cells. As shown in Fig. 10, acidification increased the amplitude of Ca2+ responses in P2X2aR-expressing cells stimulated with 1 µM ATP, whereas in alkalinized cells the response was diminished. In P2X4R-expressing cells, however, acidification attenuated 1 µM ATP-induced Ca2+ response and alkalinization facilitated response, compared with physiological pH 7.4.
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ATP-induced Ca2+ signals also reflected well the extracellular Zn2+ sensitivity of P2X2aR (9, 45). When added in 10 µM concentration for 35 min before ATP stimulation, Zn2+ increased the sensitivity of P2X2aR, which is illustrated in Fig. 11A by change in the threshold concentrations of agonist (left traces) and increase in the amplitude of Ca2+ response to 1 µM ATP (right traces). The full concentration dependence of ATP on peak amplitude of Ca2+ signals and the leftward shift in EC50 (from 1.81 ± 0.03 µM to 0.35 ± 0.03 µM) is shown in Fig. 11B. Increase in the sensitivity of P2X2aR for ATP was accompanied with increase in the rates of signal desensitization (Fig. 11C), confirming that efficacy of agonist for the ligand binding domain reflects the strength of desensitization (16, 17).
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DISCUSSION |
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For Ca2+ measurements, we used immortalized gonadotropin-releasing hormone-secreting GT1, but not HEK-293 cells. Consistent with an earlier published study showing the expression of P2YRs in HEK-293 cells (31) and high amplitude but transient nature of Ca2+ signaling in other cell types expressing P2YRs (47), we show that ATP-induced peak Ca2+ mobilization in these cells exceeds or is comparable in magnitude and/or duration with Ca2+ influx by all P2XRs other than P2X2aR and P2X7R. The sensitivity of these receptors to ATP and several other agonists is in the concentration range typical for P2XRs (30). Thus Ca2+ measurements in these cells could not be effectively used to study P2XR-mediated Ca2+ influx. In contrast, GT1 cells do not express purinergic P1 and P2 receptors endogenously (21). Together with high expression efficiency for P2XRs (>70%), this provides the major advantage for their selection in this and related (16, 17, 23) studies.
GT1 cells have several other major advantages for studies on P2XR activity.
The endogenous ectoATPase activity in GT1 cells is low (Tomi et al.,
unpublished observations) compared with other neuroendocrine cells
(37), a feature important for
experiments with sustained ATP stimulation. The secretory pathway in GT1 cells
is operative (25) and provides
the potential for studies on the coupling of P2XR to exocytosis. GT1 cells
(but not HEK-293 cells) express voltage-gated Ca2+ channels and
spontaneously fire dihydropyridine-sensitive action potentials
(40,
41). Because P2XRs are
frequently expressed in excitable cells and participate in the control of
physiological processes by depolarizing the cells and activating voltage-gated
Ca2+ influx (28),
GT1 cells also provide a suitable cell model for studying the Ca2+
signaling function of these receptors. In general, the associated
voltage-gated Ca2+ influx amplifies Ca2+ signals but
does not influence the kinetics of signal desensitization. Here, this was
illustrated in three types of experiments: depolarization of cells with high
potassium, blockade of voltage-gated Ca2+ influx by nifedipine, and
substitution of extracellular sodium with
N-methyl-D-glucamine. Experiments with sodium-free media
also illustrate that sodium conductance through the pore of P2XR is not
critical for Ca2+ permeability of the pores.
When expressed as homomers in GT1 cells, both wild-type and chimeric channels responded to ATP with a global rise in [Ca2+]i but with variable and receptor-specific peak amplitudes of Ca2+ response and rates of desensitization. The global nature of Ca2+ signals was observed during the rising and declining phases in [Ca2+]i, indicating that the rate of receptor desensitization did not affect the spatial characteristics of Ca2+ signals. For the majority of P2XRs, the rising phase was rapid and more difficult to study with Ca2+ imaging at the rate of about one image per second. The exception was P2X7R, which responded to ATP with a rise in [Ca2+]i that lasted for tens of seconds. On the other hand, the rate of Ca2+ imaging in our experiments was sufficient to follow the temporal aspects of Ca2+ signaling during the declining phase in all receptors, including the rapidly desensitizing P2X3R.
The global Ca2+ signals are not only suitable to control the plasma membrane events but also the cytosolic and nuclear events (29). In that respect, the attenuation of the amplitude of global Ca2+ signals could provide an effective mechanism for graded actions of P2XRs on cytosolic and nuclear processes. In general, the global Ca2+ signaling could be generated by Ca2+ influx by two mechanisms: passive diffusion of ions within the cytosol and active propagation, known as Ca2+-induced Ca2+ release, through ryanodine receptor channels (29). The second mechanism is unlikely, because ryanodine did not affect the pattern of P2XR-mediated Ca2+ signals and was also ineffective in altering the pattern of action potential-dependent Ca2+ signaling in these cells (5). This provides an additional methodological advantage for the usage of GT1 cells in studies on P2XR activity.
In this study, we also progressed in understanding the mechanism for
generating the receptor-specific Ca2+ signaling patterns. Linear
regression analysis revealed the correlation between peak amplitudes of
current and Ca2+ response, suggesting that the size of inwardly
depolarizing current reflected on the amplitude of Ca2+ response.
Experiments with P2X2aR stimulated with increasing concentrations
of ATP are also in line with this hypothesis. From a physiological point of
view, variations in single-channel conductivity of P2XRs
(10,
11,
46) and the receptor-specific
conductivity for Ca2+
(12,
13,
35,
39,
42) should be the major
sources contributing to the variations in the peak amplitude of current
response among receptors, if they are expressed at comparable levels. To
account for impact of the size of current on Ca2+ signaling, the
peak amplitudes of Ca2+ responses were normalized and plotted
against values for decay in current response. The results of these
investigations indicate a linear-log relationship between the two parameters.
Therefore, the rate of current desensitization is also likely to influence the
magnitude of Ca2+ response.
Furthermore, we show that the patterns of current and Ca2+
signal decays are highly comparable. Linear regression analysis revealed a
strong correlation between the values for current and Ca2+
signals, indicating that rates of current desensitization also determine the
rates of Ca2+ signal desensitization. This in turn suggests that
both measurements could be used to compare the rates of receptor
desensitization. For example, the same conclusion would be reached in analysis
of the dependence of receptor desensitization on ectodomain structure by
comparing wild-type P2X2aR and P2X2bR and chimeric
P2X2a + X4R and P2X2b + X4R pairs
in current and Ca2+ measurements.
However, the times needed to reach the half- and steady desensitized states
for P2XRs were significantly longer in Ca2+ than in current
measurements. Slower Ca2+ signal desensitization reflects the slow
kinetics of cation elimination from the cytosol. The present data confirmed
that Ca2+ handling is not a channel-but rather a cell-dependent
process. In general, Ca2+ is removed from the cytosol by several
mechanisms, including Na+/Ca2+ exchanger, plasma
membrane and endoplasmic reticulum membrane Ca2+-ATPases, and
uptake of Ca2+ by mitochondria
(29). In this respect, it is
reasonable to speculate that the ratio between values for current and
Ca2+ could vary among variable cells, depending on the cell
type-specific Ca2+ handling mechanisms.
Finally, our results show that Ca2+ signals reflect well the receptor-specific pharmacology. We have previously reported that agonists other than ATP stimulate Ca2+ influx in GT1 cells in a receptor-specific manner and with EC50 values comparable to those in current measurements (16, 17, 23). Here, we show that EC50 values for ATP estimated in current and Ca2+ responses are comparable. Our results further indicate that Ca2+ measurements report accurately about the receptor-specific sensitivity to extracellular pH and Zn2+, found previously by others in current (1, 79, 34, 44, 45) and Ca2+ (47) measurements. Experiments with Zn2+ are also in accord with our recent findings that the ectodomain structures of P2XRs, i.e., the efficacy of agonist for receptors, influence the rates of receptor desensitization (16, 17). Thus a leftward shift in the EC50 for ATP in P2X2aR-expressing cells bathed in Zn2+-containing medium increased the rates of receptor desensitization.
In conclusion, here we show that recombinant P2XRs can generate global Ca2+ signals when expressed as homomers in GT1 cells. Ca2+ influx through the pores of P2XRs and voltage-gated Ca2+ channels, and the subsequent diffusion of this cation within the cell, accounts for the generation of global signals. This is a common feature of all P2XRs, whereas the peak amplitude of Ca2+ response and the temporal aspects of Ca2+ signaling are receptor specific. Parallelism in current and Ca2+ signaling patterns observed under different experimental conditions also indicate the potential application of single-cell Ca2+ measurements in studies of activity of P2XRs. There are several advantages of single-cell Ca2+ measurements in such studies, including the number of cells that can be examined simultaneously, the preserved interior of the cells compared with the whole cell patch-clamp recording, and the possibility of studying the spatial aspects of signaling and physiological role of P2XRs in intact cells. This method also has its limitations, including the temporal dissociation between Ca2+ signaling and P2XR activity and the selection of a cell model with respect to the endogenous expression of P1 and P2YRs, the coupling of Ca2+ influx to Ca2+-induced Ca2+ release from intracellular stores, and the cell type specificity in pathways controlling Ca2+ efflux.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* M.-L. He and H. Zemkova contributed equally to this work.
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