Abstract
ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca2+ and facilitate voltage-sensitive Ca2+ entry in excitable cells. To study Ca2+signaling by P2XRs and its dependence on voltage-sensitive Ca2+ influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca2+]i. P2XR subtypes differed in the peak amplitude of [Ca2+]i response independently of the level of receptor expression, with the following order: P2X1R < P2X3R < P2X4R < P2X2bR < P2X2aR < P2X7R. During prolonged agonist stimulation, Ca2+ signals desensitized with different rates: P2X3R > P2X1R > P2X2bR > P2X4R ≫ P2X2aR ≫ P2X7R. The pattern of [Ca2+]iresponse for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca2+]i. The P2X1R, P2X3R, and P2X4R-derived [Ca2+]i signals were predominantly dependent on activation of voltage-sensitive Ca2+ influx, both voltage-sensitive and -insensitive Ca2+ entry pathways equally contributed to [Ca2+]i responses in P2X2aR- and P2X2bR-expressing cells, and P2X7R operated as a nonselective pore capable of conducting larger amounts of Ca2+ independently on the status of voltage-gated Ca2+ channels. Thus, Ca2+signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca2+]i patterns in response to a common agonist.
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 P2XxR; 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 laevisoocytes 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
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, P2X2aR, and P2X2bR.
For stable transfection, cDNAs with the full-length coding sequence of P2X1R, P2X2aR, and P2X2bR 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+]imeasurements 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 andEcoRV (P2X1R), BspEI (P2X2aR, P2X2bR, and P2X7R), or XhoI (P2X3R), gel-purified, and ligated to the corresponding sites of the expression constructs.
Protein samples for Western blot were prepared from GT1 cells 24 h after transient transfection. Cells in a 100-mm dish were rinsed with ice-cold PBS and collected in TE buffer (50 mM Tris·HCl, pH 7.4, containing 5 mM EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride). Cells were homogenized on ice and crude membrane fractions were collected by centrifugation at 10,000g for 30 min at 4°C. The resultant pellet (about 1 mg protein) was resuspended in the same buffer for SDS-polyacrylamide gel electrophoresis (PAGE) (10 μg protein/lane). After electrophoresis, size-fractionated proteins were transferred to polyvinylidene fluoride membranes by electroblotting. The blots were incubated in 3% bovine serum albumin in TBS (10 mM Tris-Cl, pH 7.5, and 150 mM NaCl), followed by monoclonal anti-HA antibody (BAbCo, Richmond, CA) at a dilution of 1:3,500. After that, positive signals of individual blots were visualized by incubating the membrane with peroxidase-conjugated anti-mouse antibody (1: 5,000) and subsequent treatment with enhanced chemiluminescence Western blotting detection reagents (Amersham-Pharmacia, Arlington Heights, IL). Protein concentrations in the samples for Western blot were determined using a Pierce BCA protein assay kit (Pierce, Rockford, IL).
Electrophysiological Recordings.
Ionic currents and membrane potential (Vm) 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 MΩ) 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 MΩ was reached 10 min following the formation of a GΩ seal (seal resistance > 5 GΩ) 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 Vm.
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 Vm. 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 Vmvalues 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
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 P2X2aR-expressing cells, followed by P2X2bR-, 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.
Concentration-dependent studies revealed that no further increase in [Ca2+]i was observed by increasing ATP concentration above 100 μM in cells expressing P2X1R, P2X2R, P2X3R, and P2X4R. That was not the case with P2X7R-expressing cells in which a maximum [Ca2+]iresponse was observed when stimulated with 1 mM ATP. Because of their low sensitivity to the native agonist, 3′O-(4-benzoyl)benzoyl-ATP (BzATP), a potent agonist for these channels, was also used in experiments. Furthermore, preincubation of cultures with apyrase, an ectoATPase, was necessary to detect changes in [Ca2+]iin P2X1R- and P2X3R-expresssing cells, but not in cells expressing other P2XR subtypes. These results suggest that P2X1R and P2X3R are endogenously desensitized (i.e., that ATP concentrations spontaneously released by GT1 cells are sufficient to desensitize these but not other P2XRs). In all additional studies with these two channels, cells were incubated in apyrase-containing medium, and [Ca2+]i and electrophysiological recordings were also done in the presence of this compound.
Two reasons may underlie the observed differences in the amplitudes of [Ca2+]i responses to high ATP concentration in cells expressing recombinant receptor-channels: the transfection and expression efficiency of these channels in common host cells, and/or the capability of these channels to promote Ca2+ influx. To analyze the potential impact of transfection and expression efficiency on the pattern of ATP-induced Ca2+ signaling, several different experimental approaches were employed. Initially, we estimated the amount of receptor protein in membrane fractions by Western blot analysis using a common anti-HA antibody, and compared it with the amplitude of [Ca2+]i response. Adding an HA tag at the C-terminus did not alter receptor activity estimated in current and Ca2+ measurements (data not shown). The level of P2XR expression in GT1 cells was variable (Fig.2A), indicating that each subunit shows a particular rate of protein production/degradation, even when expressed under the same transcriptional force by a CMV-promoter-based expression vector. Notably, the apparent amplitudes of averaged [Ca2+]i response estimated in single cell [Ca2+]i measurements (Fig. 1B) did not correlate with the protein amounts of subunits. For example, although the expression of P2X3R was the most effective, the averaged peak amplitude of [Ca2+]i response was the lowest among channels compared. Furthermore, the expression of P2X2aR, P2X2bR, and P2X7R was comparable (Fig. 2A), but not their [Ca2+]i responses when stimulated with 100 μM ATP (Fig. 1B). These findings indicate that the relative amount of receptors cannot account for the entire difference of the averaged peak amplitudes in [Ca2+]i responses among P2XR types.
In further studies, we compared the relative transfection efficiency of P2XRs construct in single cells by coexpressing GFP and P2XRs and analyzing the intensity of fluorescence signals as a measure of transfection efficiency. About 90% of GFP-expressing cells also responded to ATP, whereas only about 10% of GFP-negative cells responded to ATP. In the presence of fixed amount of the expression constructs, the percentage of GFP-ATP positive cells varied between 35 and 85%. It was independent on the channel type expressed and the method used for coexpression (see under Experimental Procedures). The GFP intensity varied among the cells in the same field, as well as the amplitude of ATP-induced [Ca2+]i response. As illustrated in Fig. 2B, there was a linear correlation between the expression of GFP and the peak amplitude of [Ca2+]i response in cells transiently transfected with P2X3R. Such variation was not unique for P2X3R, but was also observed in cells transfected with other channels. However, when the average GFP fluorescence was comparable for each set of cells (about 60 arbitrary units) (i.e., under similar transfection efficiency, the mean amplitude of [Ca2+]i(F340/F380) response to 100 μM ATP (peak response − baseline) were 0.51 ± 0.04, 0.69 ± 0.08, 1.43 ± 0.16, 2.18 ± 0.21, and 2.29 ± 0.18 for P2X1R, P2X3R, P2X4R, P2X2aR, and P2X2bR, respectively. These results again indicate that variations in the maximum [Ca2+]i response were determined by the number of functional channels expressed in single cells, as well as by the capacity of channels to elevate [Ca2+]i when stimulated with supramaximal agonist concentration.
We also generated cells stably expressing P2X1R, P2X2bR, or P2X2aR and analyzed their [Ca2+]iresponses. As shown in Fig. 3A, cell lines generated from single colonies exhibited variations in the peak [Ca2+]i responses when stimulated with 100 μM ATP, but those were of a smaller magnitude than those observed in transiently transfected cells (Fig. 3A versus Fig. 4). The averaged peak amplitudes of [Ca2+]i responses among these three receptors were significantly different (Fig. 4C) and comparable with those observed in cells coexpressing GFP and P2XRs (see above). We also examined the pattern of Ca2+signaling in four different cells stably expressing P2X1R. The peak [Ca2+]i responses by these individual clones were comparable and were about 30% of that observed in cells expressing P2X2bR and P2X2aR (data not shown). These results indicate that the coexpression of P2XRs and GFP does not affect the ability of these channels to elevate [Ca2+]i when activated and confirm the finding that Ca2+ signaling pattern by P2XRs is an intrinsic characteristic of these receptor-channels.
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. P2X2bR- 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 P2X2aR and P2X7R. The P2X2aR-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).
In contrast to the amplitude of [Ca2+]i response, the rate of receptor desensitization was independent on the levels of receptor expression. The numbers below the traces shown in Fig. 4indicate the rates of receptor desensitization derived from the fitted curves (full lines) in cells exhibiting different level of P2XR expression. When stimulated with 100 μM ATP, the mean rates of desensitization were (in s−1) 0.041 ± 0.004 (n = 54) for P2X3R; 0.034 ± 0.004 (n = 43) for P2X1R; 0.022 ± 0.006 (n = 57) P2X2bR; 0.017 ± 0.001 (n = 34) for P2X4R; and 0.006 ± 0.0004 (n = 58) for P2X2aR. Because P2X7R-expressing cells showed no obvious desensitization of [Ca2+]i response when stimulated with 100 μM BzATP, the rates of desensitization were not calculated.
The receptor-specific desensitization rates were also observed in stably transfected cells. As shown in Fig. 3B, there was a significant difference in the rate of desensitization between P2X1R and P2X2aR/P2X2bR, as well as between P2X2aR and P2X2bR when cells were stimulated with 100 μM ATP. The average rates of receptor desensitization derived from these experiments were comparable with those derived from the transiently transfected cells. These results further indicate that the rate of P2XRs desensitization is an intrinsic and unique feature of a particular receptor and is not affected by the level of protein expression.
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+]imeasurements, 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+]iresponses can be used to study receptor activation and desensitization.
In further experiments, we simultaneously monitored ATP-induced current and [Ca2+]i responses to ATP in indo-1-loaded cells expressing P2X3R and P2X2bR. Activation of both receptors generated currents with profiles highly comparable with those observed in cells without dye (Fig. 6 versus Fig. 5). Thus, loading the cells with Ca2+ indicator dye does not affect the pattern of the current response. However, clamping the cells at −90 mV significantly altered the profile of the [Ca2+]i response. In clamped cells, only a low-amplitude monophasic [Ca2+]i response accompanied the ATP-induced current in P2X3R-expressing cells (Fig. 6A, upper tracing), in contrast, in unpatched cells expressing P2X3R, which responded to ATP with a biphasic [Ca2+]i profile (Fig. 3). These results indicate that clamping the cells at −90 mV effectively abolishes the spike [Ca2+]i response in P2X3R-expresssing cells. This suggests that voltage-sensitive Ca2+ influx is the major pathway contributing to the peak rise in [Ca2+]i in P2X3R-expressing cells.
In contrast to P2X3R-expresssing cells, P2X2bR-expressing cells responded to ATP when clamped at −90 mV with a pattern of [Ca2+]i response that was comparable with those observed in unpatched cells (Fig. 6B versus Figs.3 and 4). These results indicate that P2X2bR can generate biphasic Ca2+ signals when voltage-sensitive Ca2+ influx is blocked. Our measurements also confirmed that the time course for the activation and desensitization of ATP-induced current and [Ca2+]i responses significantly differ when measured simultaneously in the same cells (see Fig. 6 legend) [i.e., that desensitization of the [Ca2+]i response occurred with 43 ± 12 s delay (n = 5) compared with the current response (indicated by arrows in Fig. 6B)]. Because voltage-sensitive Ca2+ influx was blocked by clamping the cells at −90 mV, the Ca2+ handling mechanism of the cells seemed to be a major factor contributing to the delay in decrease of [Ca2+]i during continuous agonist stimulation.
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, P2X2aR, P2X2bR, 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.
In cells expressing P2X3R, ATP induced a small monophasic [Ca2+]iresponse in nifedipine-containing medium. The profile of [Ca2+]i response was similar to that observed in voltage-clamped cells (Figs. 6A and 7B, tracing b). Nifedipine also abolished the spike [Ca2+]i response in P2X1R- and P2X4R-expressing cells (tracings a and c) but not in P2X2aR- and P2X2bR-expressing cells. As shown in Fig. 7B, d and e, both spike and plateau responses were observed in nifedipine-treated cells, but the amplitudes of these responses were reduced.
The dependence of Ca2+ signaling by P2X7R on voltage-sensitive Ca2+ influx was more complex than that observed in P2X2R-expressing cells. A nifedipine-sensitive component in [Ca2+]iresponse was consistently observed, but only during the early phase of stimulation with lower concentrations of ATP. As shown in Fig. 7B, tracing f, the rise in [Ca2+]i in response to 100 μM ATP was delayed, but not reduced, in nifedipine-treated cells, and there was no difference in the amplitude of the plateau responses in controls and treated cells 200 s after the addition of ATP. Similarly, the rise in [Ca2+]i was delayed in nifedipine-treated cells stimulated with 500 μM ATP, but these cells frequently showed a secondary elevation in [Ca2+]i (Fig.8A, right tracing). The amplitude of [Ca2+]i response during the second phase reached that observed in cells stimulated with BzATP, a selective and highly potent agonist for P2X7R. The [Ca2+]i response to BzATP was nifedipine-insensitive (Fig. 8, left tracings). Finally, P2X7R-expressing cells responded to 1 mM ATP by a rise in [Ca2+]i with a pattern indistinguishable from that observed in 100 μM BzATP-stimulated cells and unaffected by nifedipine (data not shown). In some cells, we also observed the third phase in activation of P2X7R. GT1 neurons stimulated with 100 μM BzATP were permeabilized after prolonged (10 to 20 min) stimulation, in which [Ca2+]i measurements were affected by the leak of fura-2 from cells (Fig. 8B).
The effects of nifedipine on [Ca2+]i responses by recombinant receptors are summarized in Table1. The peak [Ca2+]i response was significantly affected in P2X1R-, P2X3R-, P2X4R-, P2X2aR-, and P2X2bR-expresssing cells, but not in P2X7R-expresssing cells. The nifedipine-insensitive [Ca2+]i response was 7, 8, 13, 46, 47, and 96% in cells expressing P2X1R, P2X3R, P2X4R, P2X2aR, P2X2bR, and P2X7R, respectively. Thus, Ca2+ signaling by all P2XRs was to some extent dependent on both voltage-insensitive and -sensitive Ca2+ influx. Calcium signaling by P2X1R, P2X3R, and P2X4R was critically dependent on voltage-sensitive Ca2+ influx, whereas both pathways equally contributed to [Ca2+]i responses in P2X2aR- and P2X2bR-expressing cells. Activation of P2X7R also led to the facilitated voltage-sensitive Ca2+ influx, but only transiently and when cells were stimulated with lower ATP concentrations. The inefficacy of nifedipine at supramaximal concentrations and the high amplitude of current/[Ca2+]i responses indicated that this channel operates as a nonselective pore capable of conducting larger amounts of Ca2+ independently on the status of voltage-gated Ca2+ channels.
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 Vm 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 Vm were recorded simultaneously during the initial stimulation and measurement of current was performed during the second ATP stimulation.
Figure 9A illustrates the Vm response measured in two unloaded GT1 cells expressing P2X2bR, one responding to ATP with a small (15 pA) peak amplitude current (left tracings) and the other responding to ATP with higher (180 pA) peak amplitude current (right tracings). In both cells, the peak current response coincided with the highest frequency of firing, and the desensitization rate of current response paralleled the normalization of firing frequency. The average frequency of spiking was higher in the cell generating current of higher amplitude. A similar dependence of the pattern of firing on the size of the depolarizing current and the rate of current desensitization was observed in indo-1-loaded cells (Fig. 9B). Simultaneous measurements of Vm and [Ca2+]i responses also revealed that the frequency of spiking encodes ATP-induced voltage-gated Ca2+ influx.
To examine whether voltage-sensitive Ca2+ influx can alter the desensitization rate of ATP-induced [Ca2+]i responses, P2X2aR- and P2X2bR-expressing cells bathed in medium with or without nifedipine were stimulated with 100 μM ATP. The tracings shown in Fig. 10 (dotted lines) are representative of 15 records for both receptors and each group (with and without nifedipine). The peak amplitude of [Ca2+]i response was significantly reduced in the presence of nifedipine, but the rate of Ca2+ signal desensitization was not affected, as indicated by the parallelism in the fitted curves (full lines) and the mean values for the calculated rates of desensitization, shown on right. These results indicate that despite amplifying ATP-induced Ca2+ signals, voltage-sensitive Ca2+ influx does not affect the rate of receptor desensitization.
Discussion
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 P2X2aR and P2X2bR. 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 P2X2aR and P2X2bR current desensitization were prolonged compared with P2X1R, P2X3R, and P2X4R, a time-frame sufficient to generate high amplitude [Ca2+]isignals 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+]isignals 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 > P2X2bR > P2X4R ≫ P2X2aR ≫ P2X7R. Similar order was observed in current measurements in X. laevisoocytes 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 P2X2bR-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 P2X2aR, P2X2bR, 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+]iresponses, 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.
Footnotes
- Received February 4, 2000.
- Accepted July 17, 2000.
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Send reprint requests to: Dr. Stanko Stojilkovic, Section on Cellular Signaling, ERRB/NICHD/NIH, Bldg. 49, Room 6A-36, 49 Convent Drive, Bethesda, MD 20892. E-mail:stankos{at}helix.nih.gov
Abbreviations
- P2XRs
- purinergic receptor-channels
- GT1 cells
- gonadotropin-releasing hormone-secreting neurons
- GFP
- green fluorescence protein
- HA
- hemagglutinin
- BzATP
- 3′-O-(4-benzoyl)benzoyl-ATP
- The American Society for Pharmacology and Experimental Therapeutics