Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

ATP-gated purinergic receptor-channels (P2XRs) are expressed in the central and peripheral nervous systems and in neuroendocrine and endocrine cells (Ralevic and Burnstock, 1998), including those of the pituitary gland (Koshimizu et al., 1998a,b). Activation of P2XRs evokes an inward current that is associated with membrane depolarization and an elevation in intracellular free calcium concentration ([Ca²⁺]_i). In excitable cells, Ca²⁺ influx through the pore of these channels accounts in part for the increase in [Ca²⁺]_i, the rest occurring indirectly through voltage-gated Ca²⁺ channels. The elevation of [Ca²⁺]_i by P2XR participates in the control of a number of cellular functions, such as neurotransmission and hormone secretion (Brake and Julius, 1996; North, 1996). Expression, cloning, and homology screening of cDNA for P2XRs revealed the presence of a family of seven subunits (denoted P2X_xR; x = 1 to 7) and several spliced variants (Brake et al., 1994; Bo et al., 1995; Chen et al., 1995; Lewis et al., 1995; Collo et al., 1996; Garcia-Guzman et al., 1996; Soto et al., 1996; Brandle et al., 1997; Rassendren et al., 1997). They have little primary sequence homology to other ligand-gated channels (Buell et al., 1996). Each subunit is proposed to have two transmembrane domains connected with a large extracellular loop, with both the amino- and carboxyl-termini located in the cytoplasm. The subunits have 35 to 59% amino acid homology and all of them can form cation-permeable pores when expressed in Xenopus laevis oocytes or in mammalian cells (Buell et al., 1996).

The temporal pattern and extent of Ca²⁺ entry plays a key role in the course of [Ca²⁺]_i signaling generated by P2XRs. However, relative contribution of the voltage-sensitive Ca²⁺ influx to the P2XR-induced Ca²⁺ signaling has been incompletely characterized. Some P2XRs are more permeable to Ca²⁺ ions than they are to monovalent cations, with a relative permeability of Ca²⁺ to Na⁺ of about 4:1 (Evans et al., 1996). This finding suggests that the Ca²⁺-conducting capability of P2XRs, like that of NMDA-type glutamate receptors, is more important, as are their roles in controlling electrical activity and voltage-sensitive Ca²⁺ influx (Hille, 1991). Such a hypothesis also explains how P2XRs generate Ca²⁺ signals when expressed in nonexcitable cells. On the other hand, in physiological conditions only a fraction of the total current through P2XR pores (between 6 and 15%) is carried by Ca²⁺, because extracellular Ca²⁺ is lower than Na⁺ (Brake and Julius, 1996; North, 1996). This suggests that the capacity of these receptors to directly generate Ca²⁺ signals is limited. Furthermore, activation of P2XRs is frequently accompanied with their desensitization, the rate of which is intrinsic to the composition of channel subunits. Fast desensitization effectively abolishes current response within seconds of exposure to ATP (Chen et al., 1995; Lewis et al., 1995; North, 1996). The impact of the receptor desensitization to the voltage-insensitive and voltage-sensitive Ca²⁺ influx has not been studied extensively in a subtype specific manner. This emphasized the need for a study on current and Ca²⁺ signaling by recombinant P2XRs expressed in excitable mammalian cells.

Here, we examined the pattern of Ca²⁺ signaling evoked by all P2XRs in the presence and absence of voltage-gated Ca²⁺ channel activity. To do this, we expressed recombinant P2XRs in immortalized gonadotropin-releasing hormone-secreting GT1-7 neurons (hereafter GT1 cells). Like many neuroendocrine and endocrine cells, GT1 cells exhibits spontaneous action potential-driven Ca²⁺ entry through T- and L-type voltage-gated calcium channels (Van Goor et al., 1999a,b). Furthermore, neither P2XRs nor the calcium-mobilizing P2Y receptors are native to GT1 cells (Koshimizu et al., 1998b), in contrast to many other immortalized cells that are commonly used for transfection studies. Therefore, these cells are an excellent mammalian model system to analyze Ca²⁺ signaling by P2XRs and its dependence on voltage-sensitive Ca²⁺ influx. Our study focuses on the comparison of current and Ca²⁺ signaling by different homomeric P2XRs in GT1 cells and on the dependence of Ca²⁺ signaling on the voltage-sensitive Ca²⁺ influx.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Cell Cultures and Transfection. GT1 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F12 medium (1:1) containing 10% (v/v) fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. Procedures for transient transfection in GT1 cells and expression constructs used were described (Koshimizu et al., 1999) with minor modifications. Briefly, cells were plated on coverslips coated with poly-L-lysine at a density of 0.5 to 1.0 × 10⁵ cell/35-mm dish and allowed to grow for 24 h. On the following day, 1.2 to 2.5 µg of expression constructs encoding P2XRs was mixed with 8 µl of Lipofectamine in 1.2 ml of Opti-MEM (both from Life Technologies, Rockville, MD) for 15 to 20 min at ambient temperature. The DNA mixture was then applied to cells for 3 to 6 h and replaced by normal culture medium. Cells were subjected to experiments 24 to 48 h after transfection.

Stable Transfection of P2X₁R, P2X_2aR, and P2X_2bR. For stable transfection, cDNAs with the full-length coding sequence of P2X₁R, P2X_2aR, and P2X_2bR were subcloned into the eukaryotic expression vector pcDNA 3.1/Zeo (Invitrogen, Carlsbad, CA). After transfection with Lipofectamine, stably transfected cell colonies were isolated by zeocin selection (400 µg/ml; Invitrogen) for at least 6 weeks. Reverse transcription-polymerase chain reaction, using primers flanking the coding sequence of respective receptors, verified expression of P2XR mRNAs in these cells. Functional expression of P2XRs were also confirmed by increases in [Ca²⁺]_i after challenging the cells with 100 µM ATP. In these studies, GT1 cells transfected with an empty vector without insert were used as the negative control and did not exhibit any [Ca²⁺]_i responses after ATP stimulation.

[Ca²⁺]_i Measurements. Cells were incubated at 37°C for 60 min with 1 µM fura-2 AM in phenol red- and ATP-free DMEM and subsequently washed with assay buffer containing 137 mM NaCl, 5 mM KCl, 1.2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose. Cultures were kept for at least 30 min in this medium before single-cell [Ca²⁺]_i measurements. Apyrase (grade I) was purchased from Sigma (St. Louis, MO) and used at 20 µg/ml throughout the incubation process indicated. 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). [Ca²⁺]_i responses were examined under a 40× oil immersion objective during exposure to alternating 340 and 380 nm light beams, and the intensity of light emission at 520 nm was measured. The ratio of light intensities, F₃₄₀/F₃₈₀, which reflects changes in [Ca²⁺]_i, was simultaneously followed in several single cells.

Use of GFP as a Marker for Cells with P2XR Expression. To characterize the transfection efficiency in single cells and to analyze the dependence of the pattern of Ca²⁺ response on expression efficiency, transfected GT1 cells with P2XR were identified from total cell population using green fluorescence protein (GFP) as a marker. Plasmid constructs for the simultaneous expression of GFP and P2XR in transfected cells was achieved either by subcloning the coding sequence of P2XR into a bicistronic GFP-expression vector pIRES₂-EGFP (Clontech, Palo Alto, CA), or by cotransfection of P2XR expression vector with a GFP-expression vector pEGFP-C₁ (Clontech) at ratio of 5:1. Cells expressing fluorescence protein were optically detected by an emission signal at 520 nm when excited by 488-nm light, and were not detectable by 340- or 380-nm excitations. We thus considered the emission signal from fluorescence protein by 340- or 380-nm excitations being within our background level for fura-2 measurements. The GFP intensity was recorded before [Ca²⁺]_i measurements and was expressed in arbitrary units (0 to 100). About 90% of GFP-positive cells responded with a rise in [Ca²⁺]_i after addition of 100 µM ATP.

Immunological Detection of C-Terminally Tagged P2XRs. Hemagglutinin epitope (HA; YPYDVPDYA) was inserted after a glycine residue next to the last amino acid of each receptor polypeptide by polymerase chain reaction. The 3'-primer sequence contained six bases of EcoRI site, three bases for stop codon, 27 bases encoding the nine amino acid HA-peptide sequence, three bases for glycine, and 21 bases encoding the seven amino acids next to the stop codon. The 5'-primer sites were chosen from the receptor coding sequences. The resulting HA-tagged P2XR fragments were subcloned into pBluescript II (Stratagene, La Jolla, CA) for sequencing. The correctly tagged C-terminal fragment was digested with EcoRI and EcoRV (P2X₁R), BspEI (P2X_2aR, P2X_2bR, and P2X₇R), or XhoI (P2X₃R), gel-purified, and ligated to the corresponding sites of the expression constructs.

Electrophysiological Recordings. Ionic currents and membrane potential (V_m) were measured using the perforated-patch recording technique (Rae et al., 1991). Current- and voltage-clamp recordings were performed at room temperature using an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Foster City, CA) and were low-pass filtered at 2 kHz. Patch pipette tips (3-5 M) were briefly immersed in amphotericin B-free solution and then back-filled with amphotericin B (240 µg/ml)-containing solution. Before seal formation, liquid junction potentials were canceled. An average series resistance of 17 ± 1 M was reached 10 min following the formation of a G seal (seal resistance > 5 G) and remained stable for up to 1 h. Data acquisition and analysis were performed using a PC equipped with a Digidata 1200 A/D interface in conjunction with Clampex 8 (Axon Instruments). Unless otherwise stated, all ATP-induced currents were evoked in cells held at 90 mV.

Simultaneous Measurement of [Ca²⁺]_i and Current or V_m. GT1 neurons were incubated for 30 min at 37°C in phenol red-free medium 199 containing Hanks' salts, 20 mM sodium bicarbonate, 20 mM HEPES and 0.5 µM indo-1 AM (Molecular Probes, Eugene, OR). Coverslips with cells were then washed twice with modified Krebs-Ringer's solution containing 120 mM NaCl, 4.7 mM KCl, 2.6 mM CaCl₂, 2 mM MgCl₂, 0.7 mM MgSO₄, 10 mM HEPES, 10 mM glucose (pH adjusted to 7.4 with NaOH) and mounted on the stage of an inverted epifluorescence microscope (Nikon, Tokyo, Japan). A photon counter system (Nikon) was used to simultaneously measure the intensity of light emitted at 405 nm and at 480 nm after excitation at 340 nm. Background intensity at each emission wavelength was corrected. Perforated patch recording techniques (see above) were used to monitor current or V_m. The data were digitized at 4 kHz using a PC equipped with the Clampex 8-software package in conjunction with a Digidata 1200 A/D converter (Axon Instruments).

Solutions. Unless otherwise specified, the bath solution contained modified Krebs-Ringer salts and the pipette solution contained 70 mM KCl, 70 mM K-aspartate, 1 mM MgCl₂, and 10 mM HEPES (pH adjusted to 7.2 with KOH). Because of the inhibitory actions of divalent cations on P2X₇R, their activation by ATP was recorded in the presence of modified Krebs-Ringer salts without the addition of MgCl₂ and with 0.5 mM CaCl₂. All reported V_m values were corrected for a liquid junction potential between the pipette and a bath solution of +10 mV (Barry, 1994). The bath contained < 500 µl of saline and was continuously perifused at a rate of 2 ml/min using a gravity-driven perfusion system. The outflow was placed near the cell, resulting in complete solution exchange around the cell within 2 s. A solid Ag/AgCl reference electrode was connected to the bath via a 3 M KCl agar bridge.

Calculations. The time course of [Ca²⁺]_i response to ATP was fitted to one or two exponential functions using GraphPad Prism (GraphPad Software, San Diego, CA). All values in the text are reported as mean ± S.E.M. Significant differences, with P < .05, were determined by Student's t test.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Ca²⁺ Signaling by Homomeric P2XRs. To examine Ca²⁺ signaling by recombinant P2XRs in excitable cells, the different P2XR subtypes were transiently expressed in GT1 neurons and the ATP-induced [Ca²⁺]_i response was monitored in single cells using fura-2 as a Ca²⁺ indicator. When the cells were stimulated with 100 µM ATP, all eight P2XR subtypes responded with a significant rise in [Ca²⁺]_i (Fig. 1A), whereas cells transfected with an empty vector did not respond (data not shown). The highest amplitude of [Ca²⁺]_i response to 100 µM ATP, expressed as mean values from 40 to 70 individual cells, was observed in P2X_2aR-expressing cells, followed by P2X_2bR-, P2X₄R-, P2X₇R-, P2X₃R-, and P2X₁R-expressing cells (Fig. 1B). [Ca²⁺]_i responses were also observed in P2X₅R- and P2X₆R-expressing cells, with a peak amplitude of 10 to 30% of that observed in P2X₂R-expressing cells. Because of their low and inconsistent [Ca²⁺]_i responses, P2X₅R and P2X₆R were not used in further studies.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Calcium signaling by homomeric P2XRs expressed in GT1 cells. A, representative tracings of 100 µM ATP-induced peak [Ca2+]i responses. In these and the following experiments, cells were loaded with fura-2 and recordings were done in Ca2+-containing Krebs-Ringer buffer, if not otherwise specified. B, averaged data of the peak [Ca2+]i responses to 100 µM ATP (means ± S.E.M.) generated from at least 40 records per group. [Ca2+]i was expressed as peak F340/F380 subtracted by basal levels.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Dependence of Ca2+ signaling by P2XRs on the efficiency of receptor expression. A, P2XRs having HA-tag at the C terminus were detected by Western blot analysis. Crude membrane fractions were prepared from transfected GT1 cells and used for SDS-PAGE as described under Experimental Procedures. Calculated molecular masses from blots were 58.3 kDa, 62.8 kDa, 51.8 kDa, 50.1 kDa, and 65.5 kDa for P2X1HA, P2X2aHA, P2X2bHA, P2X3HA, and P2X7HA, respectively. These numbers are larger than expected molecular masses from their amino acid sequences, indicating expressed receptor proteins were glycosylated in GT1 cells. B, the relationship between GFP intensity and [Ca2+]i response in cells expressing P2X3R. The results shown are from a single experiment with simultaneous measurements of [Ca2+]i in all cells expressing GFP, the intensity of which was recorded before [Ca2+]i measurements. Cells were stimulated with 100 µM ATP. r, coefficient of correlation. Similar results were observed in three additional experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Pattern of Ca2+ signaling by P2XR stably expressing in GT1 neurons. A, representative tracings (dotted lines) and fitted curves (solid lines) of 100 µM ATP-induced [Ca2+]i signals in P2X1R-, P2X2bR-, and P2X2aR-expressing cells. B and C, the calculated rates of receptor desensitization (B) and peak amplitude [Ca2+]i responses (C). The bars shown are means ± S.E.M. derived from at least 15 records. * P < .05 versus P2X1R; ** P < .05 versus P2X2bR.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Pattern of Ca2+ signals by P2XRs transiently expressed in GT1 neurons. The tracings shown by dotted lines are derived from the single recording for each receptor and are illustrative for the variations among cells expressing a particular receptor. Cells were stimulated with 100 µM ATP, or with 100 µM BzATP when P2X7Rs were analyzed. In all tracings, single exponential functions were sufficient to describe the desensitization (solid lines). The fitted function is extrapolated for clarity. The calculated desensitization constants are shown below the tracings. X and Y scales shown in the right bottom corner are common for all tracings shown.

Characterization of Receptor Desensitization. We next compared the rates of receptor desensitization during sustained agonist stimulation and their dependence on the transfection and expression efficacy. Figure 4 illustrates typical tracings for P2XRs when recordings were done simultaneously in several cells in the field exhibiting different receptor expression. All P2XRs responded to ATP with a biphasic increase in [Ca²⁺]_i that was composed of an initial spike response, followed by sustained steady-state plateau phase. P2XR subtypes differed in the relative amplitude of the plateau response and in the time needed to reach it; the latter reflects the desensitization rate. In P2X₁R- and P2X₃R-expressing cells, ATP-induced [Ca²⁺]_i response reached the plateau phase within 1 to 2 min of stimulation. P2X_2bR- and P2X₄R-derived [Ca²⁺]_i responses also decreased exponentially, reaching the low steady-state level within 2 to 4 min. On the other hand, application of ATP induced a long-lasting [Ca²⁺]_i response in cells expressing P2X_2aR and P2X₇R. The P2X_2aR-induced [Ca²⁺]_i response decreased to one third of the maximum response within 10 min of stimulation. In the P2X₇R-expressing cells stimulated with 100 µM BzATP, no obvious desensitization was observed during the first 10 min of stimulation (Fig. 4).

1

Relationship between [Ca²⁺]_i and Current Responses in P2XR-Expressing Cells. In parallel to [Ca²⁺]_i measurements, ATP-induced currents were measured using perforated patch-recording techniques. For this purpose, cells expressing homomeric P2XRs were given 100 µM ATP, whereas P2X₇R-expressing cells received 500 µM ATP. To exclude the participation of voltage-sensitive Ca²⁺ influx to the ATP-induced [Ca²⁺]_i response, cells were clamped at a holding potential of 90 mV. Activation of all recombinant channels evoked an inward current, the pattern of which was unique to each receptor subtype with respect to its desensitization rate (Fig. 5). The rank orders of the current amplitude and desensitization rate were highly comparable with those observed in [Ca²⁺]_i measurements, suggesting that the pattern of Ca²⁺ signaling by recombinant receptors is determined by the pattern of depolarizing current. Thus, when expressed in an excitable cell, both current and [Ca²⁺]_i responses can be used to study receptor activation and desensitization.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   ATP-induced inward current in GT1 cells expressing recombinant P2XRs. The P2X7R-expressing cells were stimulated with 500 µM ATP, whereas all other receptor-expressing cells were stimulated with 100 µM ATP. Note the differences in the amplitudes of current responses (variable Y-axes). All recordings were done in cells clamped at 90 mV and bathed in Ca2+-containing medium.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   ATP-induced calcium and current signaling in P2X2bR- and P2X3R-expresssing cells. A, simultaneous measurements of current and [Ca2+]i in P2X3R-expresssing cells. B, pattern of ATP-induced current and [Ca2+]i response in GT1 cells expressing P2X2bR. The half-times for desensitization of current and [Ca2+]i responses were 11.90 ± 1.34 s (n = 5) and 54.92 ± 12.37 s (n = 5; P < .015). To exclude the participation of voltage-sensitive Ca2+ entry, the cells were clamped at 90 mV. In these experiments, cells were loaded with indo-1 and stimulated with 100 µM ATP. Note the difference in time-scales between the two experiments.

Dependence of [Ca²⁺]_i Response on Voltage-Sensitive and -Insensitive Ca²⁺ Influx. To examine the impact of voltage-sensitive Ca²⁺ entry on ATP-induced [Ca²⁺]_i response in more detail, GT1 neurons expressing P2X₁R, P2X₃R, P2X₄R, P2X_2aR, P2X_2bR, and P2X₇R were bathed in medium containing 1 µM nifedipine, a blocker of L-type calcium channels, and [Ca²⁺]_i was monitored in unpatched cells. As shown in Fig. 7A, GT1 neurons exhibit spontaneous firing of action potential that controls basal [Ca²⁺]_i. Addition of nifedipine abolished electrical activity and decreased [Ca²⁺]_i.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Nifedipine-sensitivity of ATP-induced [Ca2+]i responses. A, effects of nifedipine on spontaneous electrical activity and [Ca2+]i in GT1 neurons. Electrical activity and [Ca2+]i were recorded simultaneously in indo-1 loaded cells. B, averaged tracings of [Ca2+]i profile (n = 10) in controls (thick lines) and cells bathed in 1 µM nifedipine-containing medium (thin lines). The amplitudes of [Ca2+]i responses were normalized (Y axes). Dashed lines illustrate the baseline [Ca2+]i. The averaged data on the effects of nifedipine on the peak [Ca2+]i responses are shown in Table 1. [Ca2+]i was recorded in fura-2-loaded cells.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Calcium signaling by P2X7R. A, independence of agonist-induced [Ca2+]i response on Ca2+ influx through L-type voltage-gated calcium channels. Thick line illustrates the pattern of [Ca2+]i response in BzATP-stimulated cells bathed in medium without nifedipine. B, permeabilization of cells, indicated by the leak of fura-2. This effect was occasionally observed in BzATP-stimulated cells expressing P2X7R, but only during prolonged stimulation.

Relationship between Voltage-Sensitive and Voltage-Insensitive Ca²⁺ Influx. To further analyze the relationship between voltage-sensitive and voltage-insensitive Ca²⁺ influx in activated receptors, we chose P2X₂R-expressing cells, because of the significant contribution of both pathways to the ATP-induced [Ca²⁺]_i response. The dependence of voltage-sensitive Ca²⁺ influx on the pattern of current response was studied in unloaded and indo-1-loaded cells. In both experiments, we initially recorded the pattern of spontaneous V_m before and during ATP stimulation in the current-clamp recording mode. The cells were then washed for 10 min to allow for recovery from desensitization (Koshimizu et al., 1998b) and clamped to 90 mV in the voltage-clamp recording mode, and restimulated with 100 µM ATP to measure the current response. In indo-1-loaded cells, the [Ca²⁺]_i and V_m were recorded simultaneously during the initial stimulation and measurement of current was performed during the second ATP stimulation.

View larger version (41K): [in this window] [in a new window]   Fig. 9.   Modulation of electrical activity by activated P2X2bR in spontaneously firing GT1 neurons. A, two examples of ATP-induced changes in the firing pattern in GT1 cells. Notice the difference in the amplitude of inward currents on the left and right tracings. B, simultaneous measurement of Vm and [Ca2+]i, in ATP-stimulated cells. In A and B, cells expressing P2X2bR were initially stimulated with 100 µM ATP for Vm recordings, and then washed for 10 min and restimulated with 100 µM ATP to measure the current amplitude in cells clamped at 90 mV.

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Independence of receptor desensitization on voltage-sensitive calcium influx. Desensitization of P2X2aR (A) and P2X2bR (B) derived from [Ca2+]i responses in controls and cells bathed in the presence of nifedipine. In both experiments, a single exponential function was sufficient to describe the desensitization (solid lines). The fitted function is extrapolated for clarity. The calculated desensitization constants are shown on the right, with n = 15 for all groups.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In this study, we compared the ability of recombinant P2XRs to initiate and sustain current and Ca²⁺ signaling. For this purpose, we used immortalized GT1 neurons, which do not express ATP-gated receptors or receptor-channels (Koshimizu et al., 1998b), in contrast to many other immortalized cells commonly used in transfection studies. GT1 neurons express T- and L-type voltage-gated Ca²⁺ channels and spontaneously fire nifedipine-sensitive action potentials (Van Goor et al., 1999a,b). This makes them an excellent cell model for studying the participation of voltage-gated Ca²⁺ influx in response to activation of P2XRs. This is of the physiological relevance, because P2XRs are expressed in many excitable cell types and participate in the control of synaptic transmission and neurosecretion (Brake and Julius, 1996).

When expressed individually in GT1 cells, all eight types of P2XRs studied responded to ATP with a significant rise in [Ca²⁺]_i, but with variable patterns of calcium signaling. Ca²⁺ influx by P2X₁R, P2X₃R, and P2X₄R was predominantly mediated through depolarization of cells and activation of voltage-sensitive Ca²⁺ influx, whereas both Ca²⁺ influx through the pore of channels and voltage-sensitive Ca²⁺ influx participated in the [Ca²⁺]_i responses of P2X_2aR and P2X_2bR. Activation of P2X₇R also led to facilitation of voltage-sensitive Ca²⁺ influx, but only transiently. These novel observations suggest that all P2XR studied operate as bifunctional molecules when expressed in excitable cells; they conduct Ca²⁺ and promote voltage-sensitive Ca²⁺ influx.

Both current amplitude and its rate of desensitization affect Ca²⁺ influx through the pore of recombinant receptors and through voltage-gated Ca²⁺ channels, doing the latter by encoding the frequency of action potential firing. ATP-induced current in P2X₁R-, P2X₃R-, and P2X₄R-expressing cells rapidly desensitized and the small amplitude [Ca²⁺]_i response observed in cells with blocked voltage-sensitive Ca²⁺ influx coincided with the steady-state current. On the other hand, the half-times of P2X_2aR and P2X_2bR current desensitization were prolonged compared with P2X₁R, P2X₃R, and P2X₄R, a time-frame sufficient to generate high amplitude [Ca²⁺]_i signals through the pore of receptors. Thus, although P2X₁R and P2X₂R exhibit similar conductivity for Ca²⁺ versus Na⁺ (Evans et al., 1996), the ability of the first receptor to generate Ca²⁺ signal in cells under a block of voltage-gated Ca²⁺ influx is limited by its rapid desensitization.

The pattern of [Ca²⁺]_i signals generated by homomeric P2XRs in cells without the blockade of voltage-sensitive Ca²⁺ influx resembled that of current signaling. Current and Ca²⁺ signals desensitized with rates characteristic to each receptor subtype: P2X₃R > P2X₁R > P2X_2bR > P2X₄R P2X_2aR P2X₇R. Similar order was observed in current measurements in X. laevis oocytes expressing homomeric P2XRs (Evans et al., 1997). However, times needed to reach the steady desensitized states for P2XRs were significantly longer in Ca²⁺ than in current measurements. Simultaneous [Ca²⁺]_i and current measurements in P2X_2bR-expressing cells suggest that a delay in Ca²⁺ signal desensitization reflects the slow kinetics of Ca²⁺ elimination from the cytoplasm. Thus, [Ca²⁺]_i recordings are of limited use for studies on dynamics of channel behavior, but they can be effectively employed as an indicator of receptor desensitization when expressed in an excitable cell.

The P2XRs also differed among themselves with respect to endogenous desensitization. P2X₁R and P2X₃R were able to initiate measurable Ca²⁺ signals, but only in the presence of apyrase. When expressed in GT1 neurons, as in other cell types, the activity of this receptor is critically dependent on the level of ectoATPase activity (Edwards et al., 1992; MacKenzie et al., 1996). The need for apyrase indicates that spontaneous release or pathological leakage of ATP is of sufficient magnitude to desensitize P2X₁R and P2X₃R though not other members of P2XRs. Consistent with this hypothesis, concentration-dependent studies revealed that lower concentrations of ATP were required to reach the peak [Ca²⁺]_i responses by P2X₁R and P2X₃R, compared with P2X_2aR, P2X_2bR, and P2X₄R.

In accordance with the literature in other cell types (Rassendren et al., 1997), P2X₇R expressed in GT1 cells exhibited several unique features compared with other P2XRs. This receptor is the least sensitive to ATP among P2XRs. At lower ATP concentrations, the channel conducts Ca²⁺ and activates voltage-sensitive Ca²⁺ influx. The lack of effects of nifedipine at supramaximal agonist concentrations and the sizes of current and [Ca²⁺]_i responses indicate that this channel also operates as a nonselective pore capable of providing a massive Ca²⁺ influx. Ultimately, prolonged agonist stimulation of P2X₇R-expressing cells leads to cell permeabilization. Thus, P2X₇R operates as a multifunctional receptor-channel. Because of the extremely high ATP concentrations needed to fully activate these channels, it is difficult to speculate on the physiological relevance of the transition from selective to nonselective pores and their permeability to larger molecules.

In conclusion, our results indicate that all recombinant P2XRs studied can generate Ca²⁺ signals when expressed as homomers in an excitable cell. The pattern of Ca²⁺ signaling is unique to each receptor with respect to the EC₅₀ for ATP, the amplitude and duration of current/[Ca²⁺]_i responses, and the dependence of [Ca²⁺]_i response on voltage-sensitive Ca²⁺ influx. Such specificity is of the potential physiological relevance because it provides an effective mechanism for generating variable [Ca²⁺]_i patterns in response to a common agonist.