Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Mu-Lan He,* Hana Zemkova,* Taka-aki Koshimizu, Melanija Tomic, and Stanko S. Stojilkovic

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals


ATP-GATED PURINERGIC RECEPTOR CHANNELS (P2XRs) are expressed in many nonexcitable and excitable cells, including neurons, neuroendocrine and endocrine cells, epithelia, endothelia, bone, muscle, and hemopoietic tissues (28). These receptor channels participate in the control of numerous cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis (30). The major physiological mechanism by which activated P2XRs control cellular functions is elevation in intracellular Ca2+ concentration ([Ca2+]i). The pores of all P2XRs are permeable to small monovalent and divalent cations, including significant permeability to Ca2+ (12, 13, 35, 39, 42). In nonexcitable cells, Ca2+ influx through the pores of channels is the most important, if not exclusive, pathway for Ca2+ signaling. In excitable cells, activated receptors also promote voltage-gated Ca2+ influx due to ion influx-induced depolarization of cell membrane (24).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA constructs, cell culture, and transfection. The coding sequences of the rat P2X2a, P2X2b, P2X3, P2X4, and P2X7 subunits, isolated by RT-PCR from pituitary, were subcloned into the biscistronic enhanced fluorescent protein expression vector, pIRES2-EGFP (Clontech, Palo Alto, CA), as described previously (19). P2X2a + X4R and P2X2b + X4R chimeras were directly constructed by overlap extension PCR by using the corresponding wild-type P2XR cDNA as templates. Mutagenesis primers were pairs of chimeric sense and anti-sense that were 36-mer long, with the joint sites positioned in the center, i.e., 18 nucleotides of P2X2 or P2X4 to each side, as described in He et al. (17). The constructed chimeric subunits contain the Val66-Tyr315 extracellular domain of P2X4R instead of the native sequence Ile66-Tyr310 of P2X2R. These chimeric P2XRs were also subcloned into GFP-expression vector pIRES2-EGFP. The identity of all constructs was verified by dye terminator cycle sequencing (Perkin Elmer, Foster City, CA), performed by the Laboratory of Molecular Technology (NCI, Frederick, MD). The large-scale plasmid DNAs for transfection were prepared using a QIAGEN Plasmid Maxi kit (Qiagen, Germany).

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 0.75–1 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 24–48 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 15–50 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 3–5 M{Omega}. 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 282–287 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 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). 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 (10–90% 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 ({tau} = 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor- and cell type-specific responses. ATP-induced currents were measured using whole cell patch-recording mode in HEK-293 and GT1 cells expressing homomeric P2XRs. In both cell types, activation of all channel subtypes by ATP evoked depolarizing inward currents. The pattern of current response during the sustained ATP stimulation was specific for the expressed receptor subtype and was not affected by changing the host cells. Figure 1 illustrates typical patterns of current responses by seven receptors expressed in HEK-293 cells. Current desensitized rapidly in P2X3R- and P2X2b + X4R-expressing cells, with moderate rates in P2X2bR-, P2X2a+X4R-, and P2X4R-expressing cells, slowly in P2X2aR-expressing cells, and did not desensitize in P2X7R-expressing cells. ATP-induced currents by P2XR expressed in GT1 cells desensitized in the same order as in HEK-293 cells. The rates of their desensitization were also comparable in two cell types. Figure 2 shows the pattern of current responses in HEK-293 (B) and GT1 (A) cells expressing P2X2aR and P2X2bR, and mean values for half times of current desensitization are shown.



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Fig. 1. The receptor-specific pattern of ATP-induced current signals in HEK-293 cells expressing extracellular nucleotide-activated purinergic receptors (P2XRs). Experimental records are representative of at least 5 traces per receptor type. Means ± SE for peak current responses are shown in Table 1. In this and following figures, horizontal bars indicate the exposure time to 100 µM ATP and the current traces shown are from cells clamped at -60 mV.

 


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Fig. 2. Comparison of the pattern of current signals in HEK-293 (B) and GT1 (A) cells expressing P2X2aR (lower traces) and P2X2bR (upper traces) in response to 100 µM ATP. Peak amplitudes of current responses were normalized. Bars indicate means ± SE for half-time ({tau}) values, which were calculated as stated in MATERIALS AND METHODS.

 


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Table 1. Peak amplitudes of 100 µM ATP-induced calcium and current responses

 

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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Fig. 3. Characterization of native HEK-293 and GT1 cells for Ca2+ measurements. A: the lack of effects of high-potassium-induced depolarization of HEK-293 cells on intracellular calcium concentration ([Ca2+]i) (left), and extracellular Ca2+-dependence of high-potassium-induced rise in [Ca2+]i in GT1 cells (right). B: 100 µM ATP-induced rise in [Ca2+]i in native HEK-293 cells in Ca2+-containing (upper trace) and Ca2+-deficient medium (bottom trace)(left), and the lack of effects of 100 µM ATP on [Ca2+]i in GT1 cells (right). In this and the following experiments, cells were loaded with fura 2, and recordings were done in Krebs-Ringer buffer with 1.2 mM Ca2+. Experimental data are shown by circles and are mean values from at least 15 records in representative experiments.

 

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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Fig. 4. Characterization of Ca2+ signaling in GT1 cells expressing P2X2aR. A: extracellular Ca2+ dependence of 100 µM ATP-induced Ca2+ signals. B: extracellular sodium independence of ATP-induced Ca2+ signals. In this experiment, sodium was substituted with equimollar N-methyl-D-glucamine. C: typical profiles of ATP-induced Ca2+ signals in controls (upper trace), in cells with blocked L-type Ca2+ channels by nifedipine (middle trace), and in high-potassium-depolarized cells (bottom trace, open circles). In this and following experiments, fitted curves are shown by full lines and are extrapolated for clarity.

 

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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Fig. 5. Common and receptor-specific characteristics of Ca2+ signaling by wild-type and chimeric P2XRs expressed in GT1 cells. A: the receptor-specific temporal patterns of 100 µM ATP-induced Ca2+ signals in cells expressing different P2XRs. The traces shown are mean values from at least 15 records in representative experiments. Means ± SE for peak Ca2+ responses are shown in Table 1. B: spatial distribution of Ca2+ signals at the peak Ca2+ response. For each channel, ratio images of cells were obtained before the addition of 100 µM ATP (left) and at the peak of response (right). The horizontal bars below images indicate the pseudocolor scale for each experiment.

 

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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Fig. 6. Spatiotemporal characteristics of Ca2+ signaling during the activation phase of P2X7R. Top shows ratio (F340/F380) images of a single cell at 7 different time points from a representative experiment. The pseudocolor scale is shown at bottom left. Bottom right shows temporal changes of [Ca2+]i recorded from the whole cell area. Red circles correspond to images at top.

 

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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Fig. 7. Independence of spatial characteristics of Ca2+ signals in P2XR-expressing GT1 cells of the rate of receptor desensitization. Representative images for slow desensitizing P2X2aR (A), moderate desensitizing P2X2bR (B), and rapidly desensitizing P2X3R (C). In each, bottom shows ratio (F340/F380) images of a single cell at 7 different time points. The pseudocolor scales are shown at upper left. Upper right shows temporal changes of [Ca2+]i recorded from the whole cell area, and red circles correspond to images at bottom.

 

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 0.5–100 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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Fig. 8. The relationship between current and Ca2+ responses in P2XR-expressing cells. A: concentration-dependent effects of ATP on peak amplitude of current and Ca2+ responses in P2X2aR-expressing cells. B: linear relationship between the peak amplitude of current and Ca2+ in P2XR-expressing cells in response to 100 µM ATP. C: log-linear relationship between {tau} values for decay in current response and peak amplitude of Ca2+ response to 100 µM ATP. To minimize the impact of the amplitude of current response on Ca2+ signaling, the values for peak [Ca2+]i were normalized by dividing the F340/F380 values with the peak values of the corresponding current response. a, P2X7R; b, P2X2aR; c, P2X4R; d, P2X2a + X4R; e, P2X2bR; f, P2X2b + X4R; g, P2X3R; and h, vector-expressing cells. R, coefficient of correlation.

 

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 ({tau}) 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 {tau} 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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Fig. 9. Comparison of decays for Ca2+ and current signals in P2XR-expressing cells during the sustained stimulation with 100 µM ATP. A: the profiles of agonist-induced Ca2+ (left) and current (right) signals in cells expressing P2X2aR and P2X2bR (top), P2X4R and P2X3R (middle), and P2X2a + X4Rs and P2X2b + X4Rs (bottom). Vertical dotted lines indicate the values for {tau}. Means ± SE for {tau} values are shown in Table 2. For easier comparison, the peak values for the [Ca2+]i and current were normalized and currents were purposely shown in the opposite direction. B: correlation between {tau} values for decay in Ca2+ and current signals in P2XR-expressing cells in response to 100 µM ATP. Means ± SE for {tau} values are shown in Table 2. a, P2X7R; b, P2X2aR; c, P2X4R; d, P2X2a + X4R; e, P2X2bR; f, P2X2b + X4R; g, P2X3R; and h, vector-expressing cells.

 


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Table 2. Receptor-specific half-times ({tau}) for desensitization of calcium and current responses during the prolonged stimulation with 100 µM ATP

 
The calculated {tau} values were consistently smaller in current measurements than in [Ca2+]i measurements (Fig. 9B and Table 2), indicating that intracellular Ca2+ handling mechanisms influence the rates of signal decrease. Correlation analysis of {tau} values for current and Ca2+ decays showed a strong linear relationship, with a fourfold higher {tau} for [Ca2+]i signals (Fig. 9B). Two chimeric receptors fit well into the curve (Fig. 9B, filled circles), further indicating that modification of the receptor structure affects the rates of their desensitization but does not change the impact of Ca2+ handling on signaling. Thus the slower kinetics of Ca2+ elimination from the cytosol is not dependent on the channel type.

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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Fig. 10. Receptor-specific sensitivity of 1 µM ATP-induced Ca2+ signals in GT1 cells to pH. A: P2X2aR; B: P2X4R. The traces shown are mean values from at least 15 records in representative experiments.

 

ATP-induced Ca2+ signals also reflected well the extracellular Zn2+ sensitivity of P2X2aR (9, 45). When added in 10 µM concentration for 3–5 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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Fig. 11. Zn2+ modulates ATP-induced activity of P2X2aR. A: representative traces of ATP-induced Ca2+ signals in the presence and absence of 10 µM Zn2+. B: leftward shift in concentration dependence of ATP on peak amplitude of Ca2+ signals. C: the extracellular Zn2+-dependent increases in the rates of signal desensitization. Traces shown are means for at least 20 cells in representative experiments, and numbers are means ± SE for {tau} values from at least 3 experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Here, we studied the spatiotemporal aspects of Ca2+ signaling by P2XRs, the dependence of Ca2+ signaling pattern on the kinetics of current activation and desensitization, and the validation of single-cell Ca2+ measurements as a method for investigating the P2XR activity. For current measurements, we used HEK-293 and GT1 cells. A majority of previously published electrophysiological recordings with recombinant P2XRs was done in HEK-293 cells (4, 14, 26, 27, 36, 46), and we used these cells in current recordings to provide a valuable control for comparison with previous work. In full accordance with literature data (reviewed in Refs. 28 and 30), we show here that wild-type P2XRs desensitized in a receptor-specific manner. P2X3R desensitized rapidly, P2X2bR desensitized with moderate rates, P2X2aR desensitized slowly, and P2X7R did not desensitize. Furthermore, two chimeric channels, P2X2aR + X4R and P2X2b + X4R, desensitized in a manner that differed from that observed in parental receptors. Finally, the patterns of ATP-induced current signaling in mammalian HEK-293 and GT1 cells were highly comparable. The receptor-specific current responses in these two cell types were also comparable with records from receptors expressed in Xenopus oocytes (3, 6, 33, 46), further suggesting that the influence of host cells is not critical in evaluating the receptor-specific activity by current measurements.

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 (Tomic 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 {tau} 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 {tau} 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 {tau} 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.


    ACKNOWLEDGMENTS
 
Present addresses: H. Zemkova, Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague4, Czech Republic; T.-a. Koshimuzu, Dept. of Molecular Cellular Pharmacology, NCMRC, Tokyo 154, Japan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Stojilkovic, Section on Cellular Signaling, ERRB/NICHD/NIH, Bldg. 49, Rm. 6A-36, 49 Convent Drive, Bethesda, MD 20892-4510 (E-mail: stankos{at}helix.nih.gov).

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. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Acuna-Castillo C, Morales B, and Huidobro-Toro JP. Zinc and copper modulate differentially the P2X4 receptor. J Neurochem 74: 1529–1537, 2000.[ISI][Medline]

2. Bianchi BR, Lynch KJ, Touma E, Niforatos W, Burgard EC, Alexander KM, Park HS, Yu H, Metzger R, Kowaluk E, Jarvis MF, and van Biesen T. Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 376: 127–138, 1999.[ISI][Medline]

3. Brake AJ, Wagenbach MJ, and Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371: 519–523, 1994.[ISI][Medline]

4. Buell G, Lewis C, Collo G, North RA, and Surpenant A. An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J 15: 55–62, 1996.[Abstract]

5. Charles AC and Hales TG. Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT1–7) neurons. J Neurophysiol 73: 56–64, 1994.[ISI][Medline]

6. Chen CC, Akoplan AN, Sivllott L, Colquhoun D, Burnstock G, and Wood JN. A P2X purinoreceptor expressed by a subset of sensory neurons. Nature 377: 428–431, 1995.[ISI][Medline]

7. Clarke CE, Benham CD, Bridges A, George AR, and Meadows HJ. Mutation of histidine 286 of the human P2X4 purino-receptor removes extracellular pH sensitivity. J Physiol 523: 697–703, 2000.[Abstract/Free Full Text]

8. Clyne JD, Brown TC, and Hume RI. Expression level dependent changes in the properties of P2X2 receptors. Neuropharmacology 44: 403–412, 2003.[ISI][Medline]

9. Clyne JD, LaPointe LD, and Hume RI. The role of histidine residues in modulation of the rat P2X2 purinoreceptor by zinc and pH. J Physiol 539: 347–359, 2002.[Abstract/Free Full Text]

10. Ding S and Sachs F. Single channel properties of P2X2 purinoreceptors. J Gen Physiol 113: 695–719, 1999.[Abstract/Free Full Text]

11. Evans RJ. Single channel properties of ATP-gated cation channels (P2X receptors) heterologously expressed in Chinese hamster ovary cells. Neurosci Lett 212: 212–214, 1996.[ISI][Medline]

12. Evans RJ, Lewis C, Virginio C, Lundstrom K, Buell G, Surprenant A, and North RA. Ionic permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X receptors) expressed in mammalian cells. J Physiol 497: 413–422, 1996.[Abstract]

13. Garcia-Guzman M, Soto F, Gomez-Hernandez JM, Lund PE, and Stuhmer W. Characterization of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue. Mol Pharmacol 51: 109–118, 1997.[Abstract/Free Full Text]

14. Haines WR, Migita K, Cox JA, Egan TM, and Voigt MM. The first transmembrane domain of the P2X receptor subunit participates in the agonist-induced gating of the channel. J Biol Chem 276: 32793–32798, 2001.[Abstract/Free Full Text]

15. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[ISI][Medline]

16. He ML, Koshimizu T, Tomic M, and Stojilkovic SS. Purinergic P2X2 receptor desensitization depends on coupling between ectodomain and C-terminal domain. Mol Pharmacol 62: 1187–1197, 2002.[Abstract/Free Full Text]

17. He ML, Zemkova H, and Stojilkovic SS. Dependence of purinergic P2X receptor activity on ectodomain structure. J Biol Chem 278: 10182–10188, 2003.[Abstract/Free Full Text]

18. King BF, Wildman SS, Zinanshina LE, Pintor J, and Burnstock G. Effects of extracellular pH on agonism and antagonism at recombinant P2X2 receptor. Br J Physiol 121: 1445–1453, 1997.

19. Koshimizu T, Koshimizu M, and Stojilkovic SS. Contributions of the C-terminal domain to the control of P2X receptor desensitization. J Biol Chem 274: 37651–37657, 1999.[Abstract/Free Full Text]

20. Koshimizu T, Tomic M, Koshimizu M, and Stojilkovic SS. Identification of amino acid residues contributing to desensitization of the P2X2 receptor channel. J Biol Chem 273: 12853–12857, 1998.[Abstract/Free Full Text]

21. Koshimizu T, Tomic M, Van Goor F, and Stojilkovic SS. Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization. Mol Endocrinol 12: 901–913, 1998.[Abstract/Free Full Text]

22. Koshimizu T, Tomic M, Wong AOL, Zivadinovic D, and Stojilkovic SS. Characterization of purinergic receptors and receptor-channels expressed in anterior pituitary cells. Endocrinology 141: 4091–4099, 2000.[Abstract/Free Full Text]

23. Koshimizu T, Ueno S, Tanoue A, Yanagihara N, Stojilkovic SS, and Tsujimoto G. Heteromultimerization modulates P2X receptor functions through participating extracellular and C-terminal subdomains. J Biol Chem 277: 46891–46899, 2002.[Abstract/Free Full Text]

24. Koshimizu T, Van Goor F, Tomic M, Wong AOL, Tanoue A, Tsujimoto G, and Stojilkovic SS. Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells. Mol Pharmacol 58: 936–945, 2000.[Abstract/Free Full Text]

25. Krsmanovic LZ, Mores N, Navarro C, Arora KK, and Catt KJ. An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion. Proc Natl Acad Sci USA 100: 2969–2974, 2003.[Abstract/Free Full Text]

26. Le KT, Boue-Grabot E, Archambault V, and Seguela P. Functional and biochemical evidence for heteromeric ATP-gated channels composed of P2X1 and P2X5 subunits. J Biol Chem 274: 15415–15419, 1999.[Abstract/Free Full Text]

27. Lewis C, Neidhart S, Holy C, North RA, Buell G, and Surprenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature 377: 432–435, 1995.[ISI][Medline]

28. North RA. Molecular physiology of P2X receptors. Physiol Rev 82: 1013–1067, 2002.[Abstract/Free Full Text]

29. Nowycky MC and Thomas AP. Intracellular calcium signaling. J Cell Sci 115: 3715–3716, 2002.[Free Full Text]

30. Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413–492, 1998.[Abstract/Free Full Text]

31. Schachter JB, Sromek SM, Nicholas RA, and Harden TK. HEK293 human embryonic kidney cells endogenously express the P2Y1 and P2Y2 receptors. Neuropharmacology 36: 1181–1187, 1997.[ISI][Medline]

32. Schilling WP, Sinkins WG, and Estacion M. Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblast. Am J Physiol Cell Physiol 277: C755–C765, 1999.[Abstract/Free Full Text]

33. Soto F, Garcia-Guzman M, Gomez-Hernandez JM, Hollmann M, Karschin C, and Stuhmer W. P2X4: An ATP-activated ionotropic receptor cloned from rat brain. Proc Natl Acad Sci USA 93: 3684–3688, 1996.[Abstract/Free Full Text]

34. Stoop R, Surprenant A, and North RA. Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors. J Neurophysiol 78: 1837–1840, 1997.[Abstract/Free Full Text]

35. Surprenant A, Rassendren F, Kawashima E, North RA, and Buell G. The cytosolic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272: 735–738, 1996.[Abstract]

36. Surprenant A, Schneidr DA, Wilson HL, Galligan JJ, and North RA. Functional properties of heteromeric P2X1/5 receptors expressed in HEK cells and excitatory junction potential in guinea-pig submucosal arterioles. J Auton Nerv Sistem 81: 249–263, 2000.

37. Tomic M, Jobin RM, Vergara LA, and Stojilkovic SS. Expression of purinergic receptor channels in their role in calcium signaling and hormone release in pituitary gonadotrophs. J Biol Chem 271: 21200–21208, 1996.[Abstract/Free Full Text]

38. Troadec JD, Thirion S, Nicaise G, Lemos JR, and Dayanithi G. ATP-evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysical terminals via P2X2 purino-receptor. J Physiol 511: 89–103, 1998.[Abstract/Free Full Text]

39. Valera S, Hussy N, Evans RJ, Adami N, North A, Surprenant A, and Buell G. A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP. Nature 371: 516–519, 1994.[ISI][Medline]

40. Van Goor F, Krsmanovic LZ, Catt KJ, and Stojilkovic SS. Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels. Mol Endocrinol 13: 587–603, 1999.[Abstract/Free Full Text]

41. Van Goor F, Krsmanovic LZ, Catt KJ, and Stojilkovic SS. Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion. Proc Natl Acad Sci USA 96: 4101–4106, 1999.[Abstract/Free Full Text]

42. Virginio G, North RA, and Suprenant A. Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurones. J Physiol 510: 22–35, 1998.

43. White SM, Imig JD, Kim TT, Hauschild BC, and Inscho EW. Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC. Am J Physiol Renal Physiol 280: F1054–F1061, 2001.[Abstract/Free Full Text]

44. Wildman SS, King BF, and Burnstock G. Modulation of ATP-responses at recombinant rP2X4 receptors by extracellular pH and zinc. Br J Pharmacol 126: 762–768, 1999.[Abstract/Free Full Text]

45. Xiong K, Peoples RW, Mntgomery JP, Chiang Y, Steward RR, Weight FF, and Li C. Differential modulation by copper and zinc of P2X2 and P2X4 receptor function. J Neurophysiol 81: 2088–2094, 1999.[Abstract/Free Full Text]

46. Zhou Z and Hume RI. Two mechanisms for inward rectification of current flow through the purinoreceptor P2X2 class of ATP-gated channels. J Physiol 507: 353–364, 1998.[Abstract/Free Full Text]

47. Zsembery A, Boyce AT, Liang L, Peti-Peterdi J, Bell PD, and Schwiebert EM. Sustained Ca2+ entry through P2X nucleotide receptor channels in human airway epithelial cells. J Biol Chem 278: 13398–13408, 2003.[Abstract/Free Full Text]