Dependence of Purinergic P2X Receptor Activity on Ectodomain Structure*

Mu-Lan He, Hana Zemkova, and Stanko S. StojilkovicDagger

From the Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510

Received for publication, September 5, 2002, and in revised form, January 7, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purinergic receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using receptors with variable sensitivity to ATP in order: P2X4R > P2X2aR = P2X2bR P2X7R. Chimeras having the ectodomain Ile66-Tyr310 sequence of P2X2R and Val61-Phe313 sequence of P2X7R in the backbone of P2X4R were expressed but were non-functioning channels. P2X2a + X4R and P2X2b + X4R chimeras having the Val66-Tyr315 ectodomain sequence of P2X4R in the backbones of P2X2aR and P2X2bR were functional and exhibited increased sensitivity to ligands as compared with both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X2bR and P2X2aR, chimeric P2X2b + X4R desensitized more rapidly than P2X2a + X4R, and the rate of desensitization of P2X2a+X4R increased by substituting its Arg371-Pro376 intracellular C-terminal sequence with the Glu376-Gly381 sequence of P2X4R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rate of receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purinergic receptors (P2XRs)1 are a family of ligand-gated receptor channels that open in response to the binding of extracellular ATP. Like other ligand-gated receptor channels, P2XRs also become refractory to the stimulus during the sustained agonist occupancy. This process, called desensitization, was initially characterized in acetylcholine receptors by Katz and Thesleff (1) and occurs because receptors enter stable desensitized states in which ion permeation is blocked or attenuated although ligand remains bound. Significant progress has been made recently in characterizing the ectodomain architecture of other ligand-gated receptor channels and the relationship between ligand-binding domain occupancy and receptor activity (2-4). However, the boundaries of the ectodomain and ATP-binding domain of P2XRs and the molecular mechanisms of transduction of information from ligand-binding domain to the pore of channels are largely unknown (5).

Modification at the triphosphate moiety of ATP served well in identification of native P2XR subtypes and provided useful information about the putative ligand-binding domain. For example, the substitution of bridging oxygen between alpha - and beta -phosphorus with a methylene group resulted in a ligand, called alpha beta -meATP, which is a high potency agonist for P2X1R and P2X3R, low potency agonist for P2X2R, and partial agonist for P2X4R (6). High sensitivity of P2X3R to alpha beta -meATP can be transferred to P2X2aR and P2X2bR subtypes by generating the extracellular chimeras having the ectodomain of the P2X3 subunit in the P2X2-based backbone (7). These chimeras also exhibited enhanced rates of desensitization (7, 8). Within this region, single residue mutation studies have identified positively charged Lys68, Lys70, and Lys309 of P2X1R and the corresponding Lys69 and Lys71 of P2X2R as contributing to the ATP-binding site and control of rate of receptor desensitization (9, 10). In general agreement with these observations, the N-terminal half of the P2X3 ectodomain, from Val60 to Arg180, is necessary for high alpha beta -meATP sensitivity of the receptor. The attempt to further narrow this region was obstructed, indicating that the ectodomain is sensitive to modification by site-directed mutagenesis (7).

P2XRs activate and desensitize in a receptor- and ligand-specific manner. When stimulated with ATP, P2X1R and P2X3R desensitize very rapidly (in an ms time scale), P2X4R and P2X6R desensitize with a moderate rate (within a few seconds), and P2X2R, P2X5R, and P2X7R show little or no desensitization (11, 12). Two main hypotheses emerged from previous work on desensitization of P2XRs, one based on the structure of channels and the other based on the actions of intracellular messengers. Heteromultimerization results in P2XRs that desensitize with different kinetics from those seen in cells expressing homomeric receptors (13-15). The site-directed mutagenesis experiments indicated the relevance of C-terminal structure on the desensitization of P2XR (15-19), as well as the relevance of a highly conserved N-terminal site for protein kinase C in functional desensitization of receptors (19-21). Phosphorylation of a protein kinase A site in C-terminal of P2X2aR may also participate in receptor desensitization (22). In parallel to other ligand-gated receptor channels (2, 23-28), the ligand-binding domain of P2XRs may also contribute to the control of rates of desensitization (7, 8).

Here, we extended investigations on the relevance of the ectodomain structure on ligand selectivity and the pattern of P2XR desensitization. Experiments were done with wild-type P2X2aR, P2X2bR, and P2X4R and chimeric P2X2a + X4R and P2X2b + X4R containing the ectodomain sequence of P2X4R instead of the corresponding P2X2R sequence. The reverse P2X4 + X2R chimera was also constructed, as well as the P2X4 + X7R chimera containing the ectodomain sequence of P2X7R. To clarify the possible interaction between the ectodomain and C-terminal domain in control of agonist specificity and receptor activity, we also constructed P2X2a-6aa + X4R mutant chimera containing the C-terminal 6-residue sequence of P2X4R instead of the corresponding sequence of P2X2a + X4R. Both whole-cell patch clamp current recordings and calcium measurements were used to estimate the activity of receptors in response to ATP, the native agonist for P2XRs, and two agonist analogs, BzATP and alpha beta -meATP. The results of these investigations clearly indicate the C-terminal-independent influence of the ectodomain structure on agonist potency and rate of P2XR desensitization.

    MATERIALS AND METHODS
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DNA Constructs, Cell Culture, and Transfection-- The coding sequences of the rat P2X2a, P2X2b, P2X4 subunits were isolated by reverse transcription-PCR (15) and subcloned into the biscistronic enhanced fluorescent protein expression vector, pIRES2-EGFP (Clontech), at the restriction enzyme sites of XhoI/PstI for P2X2aR and P2X2bR and XhoI/EcoRI for P2X4R. Chimera P2X2a + X4R, P2X2b + X4R, P2X4 + X2R, and P2X4 + X7R were directly constructed by overlap extension PCR using the corresponding wild-type P2XRs cDNA as templates. Mutagenesis primers were pairs of chimeric sense and antisense that were 36-mer long with the joint sites positioned at the center. The constructed P2X2a + X4R, P2X2b + X4R chimeric subunits replace Ile66-Tyr310 with Val66-Tyr315 extracellular domain of P2X4R, whereas P2X4 + X2R and P2X4 + X7R contain the Ile66-Tyr310 and Val61-Phe313 sequence of P2X2R and P2X7R, respectively, instead of the native Val66-Tyr315 of P2X4R. We also constructed a mutant of chimera P2X2a + X4R, termed P2X2a-6aa + X4R, by overlap extension PCR, as described previously (15). This mutant contains the Glu376-Gly381 sequence of P2X4R instead of the corresponding Arg371-Pro376 sequence of P2X2a + X4R. These chimeric P2XRs were subcloned into GFP expression vector pIRES2-EGFP. The identity of all constructs was verified by dye terminator cycle sequencing (PerkinElmer Life Sciences), performed by the Laboratory of Molecular Technology (NCI, National Institutes of Health, Frederick, MD). The large scale plasmid DNAs for transfection were prepared using a Qiagen Plasmid Maxi kit (Qiagen).

Mouse immortalized gonadotropin-releasing hormone-secreting cells (hereafter GT1 cells) and human embryonic kidney cells (hereafter HEK293 cells) were used in functional studies of wild-type and mutant P2XRs, as described previously (15). GT1 cells were routinely maintained in Dulbecco's modified Eagle's medium/Ham's F12 medium (1:1) containing 10% (v/v) fetal bovine serum and 100 µg/ml gentamicin (Invitrogen) in a water-saturated atmosphere of 5% CO2 and 95% air at 37 °C. HEK293 cells were cultured in minimum Eagle's medium 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% w/v; Sigma)-coated coverslips at a density of 0.75-1 × 105 cells/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, the transfection mixture was replaced with normal culture medium. Cells were subjected to experiments 24-48 h after transfection.

Calcium Measurements-- Transfected GT1 cells were preloaded with 1 µM Fura-2 acetoxymethyl ester (Fura-2/AM; Molecular Probes, Eugene, OR) for 60 min at room temperature in modified Krebs-Ringer buffer: 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, and 15 mM HEPES plus 1.8 g/liter glucose (pH 7.4). After dye loading, cells were incubated in Modified Krebs-Ringer buffer 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). Cells were stimulated with various doses of agonists, the dynamic changes of [Ca2+]i 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. GFP was used as a marker for cells with P2XR expression as described previously (15). Cells expressing GFP were optically detected by an emission signal at 520 nm when excited by a 488-nm ultraviolet light. Experiments were done in cells with comparable GFP fluorescence signals (about 60 arbitrary units), and no repetitive stimulation was done to avoid the possible impact of desensitization on the amplitude and pattern of [Ca2+]i signals.

Current Measurements-- Electrophysiological experiments were performed on HEK293 cells at room temperature using whole-cell patch clamp recording techniques (29). ATP-induced currents were recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments, Union City, CA) and were filtered at 2 kHz using a low pass Bessel filter. 40-70% 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 megaohms. All current records were captured and stored using the pClamp 8 software packages in conjunction with the Digidata 1322A A/D converter (Axon Instruments). Patch electrodes were filled with a solution containing 140 mM KCl, 0.5 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES; the pH was adjusted with 1 M KOH to 7.2. The osmolarity of the internal solutions was 282-287 mosM. The bath solution contained 142 mM NaCl, 3 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES; the pH was adjusted to 7.3 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, Westbury, NY). The application tip was routinely positioned about 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). The time between each ATP application was about 10 min to allow recovery from receptor desensitization.

Calculations-- The time course of the [Ca2+]i was fitted to a single exponential function (ae-ktb+b) using GraphPad Prism (GraphPad Software, San Diego, CA) to generate the rates of signaling desensitization (k) and half-times of decay (tau  = ln2/k). The time courses of currents evoked by sustained ATP stimulation were fitted to a single exponential function using the pClamp 8 program (Axon Instruments). Significant differences, with p < 0.05, were determined by one-way analysis of variance with Newman-Keuls multiple comparison test. Concentration-response relationships were fitted to a four-parameter logistic equation using a non-linear curve-fitting program, which derives 50% efficient concentrations (EC50) and 50% desensitizing concentrations (DC50) (Kaleidagraph, Synergy Software, Reading, PA).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Characterization of Wild-type and Chimeric P2XRs-- When expressed in GT1 cells under identical experimental conditions, parental receptors P2X2aR, P2X2bR, and P2X4R responded to ATP, BzATP, and alpha beta -meATP stimulation with a rapid rise in [Ca2+]i followed by a gradual decline toward steady plateau levels. In accordance with previously published data (8, 30), in all agonist concentrations studied, the peak [Ca2+]i responses were comparable in P2X2aR- and P2X2bR-expressing cells. Fig. 1A illustrates the concentration dependence of three agonists on the peak amplitude of [Ca2+]i responses for both receptor subtypes combined. The calculated EC50 values (Fig. 1, dotted vertical lines) were in general agreement with the data in the literature (6): ATP was the most potent agonist for P2X2Rs, followed by BzATP, whereas alpha beta -meATP acted as a low potency agonist.


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Fig. 1.   Concentration-dependent effects of agonists on peak calcium response in single cells expressing homomeric P2XRs. A-C, comparison of the effects of ATP and its two analogs, BzATP and alpha beta -meATP, on peak calcium response in cells expressing P2X2aR and P2X2bR (A), P2X4R (B), and P2X2a + X4R and P2X2b + X4R (C). Data shown are means ± S.E. derived from 3 to 11 experiments per dose, each done in at least 15 single cells. The results for P2X2aR and P2X2bR (A) and P2X2a + X4R and P2X2b + X4R (C) are combined because no differences in peak [Ca2 + ]i responses were observed between them. Dotted vertical lines indicate the calculated EC50 values for three agonists. P2X2 + X4R chimeras were constructed as described under "Materials and Methods."

On the other hand, the estimated EC50 values for ATP, BzATP, and alpha beta -meATP in GT1 cells expressing rat P2X4R (Fig. 1B) differed significantly from previously published data (reviewed in Ref. 6). In our experiments, ATP was a highly potent agonist for these receptors, with an EC50 of about 1 µM, and the calculated EC50 for BzATP was 2.5 µM as compared with >500 µM reported in other expression systems. At supramaximal concentrations, the peak amplitudes of ATP- and BzATP-induced [Ca2+]i responses in P2X4R-expressing cells were about 40% of those observed in P2X2R-expressing cells, whereas the peak amplitudes of current responses were comparable (Fig. 1). alpha beta -meATP-induced peak current/[Ca2+]i responses were 44% as compared with those in ATP- and BzATP-stimulated cells, consistent with the partial agonist action of this ATP analog observed in other expression systems (31), but the calculated EC50 was 4 µM (Fig. 1B).

The P2X2a + X4R and P2X2b + X4R chimeras having the ectodomain of P2X4R in the backbone of P2X2aR and P2X2bR, respectively, were functional and responded to three agonists in a concentration-dependent manner with highly comparable peak amplitudes. Fig. 1C illustrates the combined results for both receptors. Chimeric receptors differed from parental receptors in two respects. First, although the structure of the pore was not altered, the peak amplitudes of [Ca2+]i responses in chimeric receptors were 80% of those observed in P2X2aR- and P2X2bR-expressing cells (p < 0.01 in the 10-1000 µM concentration range of ATP). Second, chimeric receptors exhibited higher sensitivity to ATP as compared with both parental receptors, and alpha beta -meATP exhibited the full agonistic action as compared with partial agonistic action in P2X4R- expressing cells.

The reverse P2X4 + X2R chimera, having the ectodomain of P2X2R inserted into the backbone of P2X4R, was expressed at the levels comparable with those observed in experiments with P2X2a + X4R and P2X2b + X4R, as estimated by GFP fluorescence intensity. However, the receptor did not respond to ATP in the 1-1000 µM concentration range. The lack of effects of ATP was not due to the endogenous desensitization of receptors because ATP was also ineffective in cells cultured in the presence of apyrase, an ectoATPase. Also, the P2X4 + X7R chimera having the ectodomain of P2X7R instead of P2X4R was expressed but was not functional in the presence or absence of apyrase. All together, experiments with chimeras suggested that although the ectodomain sequences we selected contained several residues critical for ligand binding, they were not sufficient to preserve intact ligand-binding domains. The P2X4R-specific ligand potency was enhanced, whereas the P2X2R- and P2X7R-specific ligand potency was abolished in chimeric channels, indicating the interaction between the ectodomain and nearby residues.

Receptor-specific Desensitization Pattern-- To characterize the pattern of receptor desensitization and its impact on calcium signaling, both current and calcium measurements were used. Fig. 2A illustrates typical profiles of ATP (100 µM)-induced current responses in cells expressing wild-type P2X2aR, P2X2bR, and P2X4R. The peak amplitudes of current responses were in high pA (P2X4R) to low nA (P2X2aR and P2X2bR) range. Consistent with previously published data (16), P2X2aR desensitized slowly and incompletely, reaching the steady levels with a tau  of 22 s, whereas P2X2bR desensitized rapidly with a calculated tau  of about 4 s. P2X4R desensitized to the steady levels comparable with P2X2bR but with a tau  of about 9 s.


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Fig. 2.   Receptor-specific desensitization pattern of P2XRs. A and B, comparison of the effects of ATP on the pattern of current (A) and calcium (B) responses in cells expressing wild-type P2X2aR (left traces), P2X4R (central traces), and P2X2bR (right traces). In this and following figures, experimental records for current responses are shown by solid lines and are representative from at least 20 traces per receptor, whereas experimental records for calcium response are shown by open circles (mean values from at least 15 traces in representative experiments). Horizontal bars indicate the time of exposure to 100 µM ATP. The current traces shown are from cells clamped at -50 mV.

The significance of receptor-specific desensitization pattern on [Ca2+]i response is illustrated in Fig. 2B. Three receptors generated calcium signals, which differed in profiles. The rates of signal desensitization (expressed as tau ) were: 96 s in P2X2aR-expressing cells, 18 s in P2X2bR-expressing cells, and 45 s in P2X4R-expressing cells. Differences in the calculated tau  values in current and [Ca2+]i measurements illustrate the impact of calcium-handling mechanisms of the cells used in experiments on the rate of receptor desensitization. On the other hand, the relative ratios in the rates of P2X2aR, P2X2bR, and P2X4R desensitization estimated from two measurements were highly comparable. This clearly indicates that [Ca2+]i measurements not only provide information about the physiological relevance of receptor activation and desensitization but also could be used as valuable parameters in characterizing the nature of P2XR desensitization, at least for slower desensitizing receptors.

A comparison between the patterns of current responses in cells expressing wild-type P2X2aR and P2X2bR and chimeric receptors is shown in Fig. 3. The substitution of the native P2X2aR ectodomain with P2X4 ectodomain dramatically enhanced the rate of receptor desensitization (Fig. 3B). Significant differences were also observed between P2X2bR and P2X2b + X4R (Fig. 3A). Both chimeric receptors also desensitized more rapidly than P2X4R (Fig. 3 versus Fig. 2). The same conclusion was reached in experiments with single cell calcium measurements. As shown in Fig. 4A, calcium signals desensitized more rapidly in cells expressing chimeric receptors when stimulated with 10 µM ATP (upper panels) and 100 µM ATP (lower panels). In parallel with changes in the EC50 values for chimeric receptors (Fig. 1), calcium signals also desensitized with about one log unit leftward shift in the DC50 values as compared with parental P2X2Rs (Fig. 4B). The ratios between tau  values for current and calcium measurements in cells expressing two chimeric receptors were comparable with those observed in parental receptors. These results clearly indicate the relevance of the ectodomain structure on the kinetics of receptor desensitization and suggest that the ligand potency reflects on rate of receptor desensitization.


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Fig. 3.   Acceleration of P2X2aR and P2X2bR desensitization by substituting their common ectodomain with the P2X4R ectodomain (current recordings). The traces shown are representative for wild-type P2X2bR and chimeric P2X2b + X4R (A) and wild-type P2X2aR and chimeric P2X2a + X4R (B). The mean ± S.E. values are shown above traces, and the numbers in parentheses indicate the number of records for each receptor. Asterisks indicate significant differences (p < 0.05) between the pairs.


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Fig. 4.   Influence of ectodomain and C-terminal domain on acceleration of P2X2a + X4R and P2X2b + X4R desensitization (calcium recordings). A, representative traces of calcium responses in cells stimulated with 10 µM ATP (upper traces) and 100 µM ATP (bottom traces). In this and following figures, experimental records are shown by open circles (mean values from at least 15 traces in representative experiments), and fitted curves are shown by full lines. A single exponential function was sufficient to describe the desensitization rates. The fitted function is extrapolated for clarity. B, concentration dependence of ATP on the rate of P2XR desensitization. Asterisks indicate significant differences (p < 0.01) between the pairs. Vertical dotted lines indicate the calculated DC50 values, and horizontal arrows indicate a leftward shift in DC50 values for chimeric channels. C, comparison of 100 µM ATP-induced [Ca2+]i signals in GT1 cells expressing P2X2a + X4R, P2X2a-6aa + X4R, and P2X2b + X4R.

Current and calcium measurements showed that P2X2b+X4R desensitized more rapidly than P2X2a+X4R, consistent with the hypothesis that the C-terminal-specific desensitization pattern was preserved in chimeric channels. To test this hypothesis further, we generated P2X2a-6aa+X4R mutant chimera, having the Glu376-Gly381 C-terminal sequence of P2X4R instead of the Arg371-Pro376 sequence of P2X2a+X4R. Our earlier studies have shown the relevance of Arg371-Pro376 in slowing the rate of receptor desensitization (16). Substitution of this sequence with the Glu376-Gly381 sequence of P2X4R increased the rate of P2X2aR desensitization (15). In accordance with these observations, P2X2a-6aa + X4R mutant chimera showed an increased rate of desensitization as compared with P2X2a + X4R chimera (Fig. 4C), and the ratio between the rates of P2X2a + X4R, P2X2a-6aa + X4R, and P2X2b + X4R was comparable with that observed in cells expressing P2X2aR, P2X2a-6aaR, and P2X2bR (15). Thus, the increase in the potency of receptors for ATP increases the rates of receptor desensitization independently of the C-terminal-controlled desensitization.

Ligand-specific Desensitization Pattern-- In further experiments, we compared the patterns of calcium signals and rates of desensitization in response to ATP, BzATP, and alpha beta -meATP. Fig. 5 illustrates typical profiles of calcium signals in response to these three agonists in cells expressing parental receptors. In accordance with our previous study (8), the C-terminal-specific desensitization pattern of wild-type P2X2Rs observed in response to ATP (Fig. 5A, left traces) was partially mimicked by BzATP (central traces) but was lost in cells stimulated with alpha beta -meATP (right traces). The ligand specificity of receptor desensitization was also observed in cells expressing wild-type P2X4R (Fig. 5A, upper traces). The mean values of the rate of receptor desensitization in response to three agonists are shown in Fig. 5B. When stimulated with ATP, three receptors desensitized with significantly different rates (left panel). No differences in the rates of P2X2aR and P2X4R desensitization were observed in response to BzATP stimulation (central panel), and all three receptors desensitized with highly comparable kinetics when stimulated with alpha beta -meATP (right panel). The plateau [Ca2+]i levels in response to three agonists also differed (illustrated by dotted lines in Fig. 5A and quantified in Table I).


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Fig. 5.   Ligand-specific receptor desensitization pattern of wild-type P2XRs. A, representative traces of [Ca2+]i response in cells expressing P2X4R (upper traces), P2X2aR (central traces), and P2X2bR (bottom traces). Traces shown are representative from 3-5 independent experiments. Horizontal dotted lines indicate differences in the plateau [Ca2+]i. B, mean values of rates of receptor desensitization. Asterisks indicate significant differences (p < 0.01) between the pairs.


                              
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Table I
The level of [Ca2+]i signal desensitization in cells expressing parental and chimeric P2XRs

In further studies, we examined the ligand-specific desensitization pattern of chimeric receptors. Fig. 6 compares typical calcium signal profiles in cells expressing wild-type P2X2aR and chimeric P2X2a + X4R (Fig. 6A) and wild-type P2X2bR and chimeric P2X2b + X4R (Fig. 6B) during the prolonged stimulation with increasing BzATP concentrations. The C-terminal-specific desensitization patterns of P2X2aR and P2X2bR that are present in ATP-stimulated cells (Fig. 5), but are lost in BzATP-stimulated cells (Figs. 5 and 6), were reestablished in BzATP-stimulated cells expressing chimeric receptors. Fig. 6A (left panels) shows a typical pattern of receptor desensitization in wild-type and chimeric P2X2Rs, and bars (right panels) illustrate significant differences in the mean values for the rate of receptor desensitization, whereas Fig. 6B (left and right panels) illustrates more dramatic differences between wild-type and chimeric P2X2bR.


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Fig. 6.   Comparison of BzATP-induced calcium signals in cells expressing wild-type and chimeric receptors. A, concentration-dependent effects of BzATP on the pattern of calcium response in P2X2aR and P2X2a + X4R-expressing cells (A) and P2X2bR and P2X2b + X4R-expressing cells (B). In A and B, left panels illustrate representative traces, and right panels (bars) illustrate differences in the rates of calcium signal desensitization.

The establishment of C-terminal-specific desensitization pattern was also observed in chimeric receptors stimulated with alpha beta -meATP. Fig. 7A shows that wild-type P2X2Rs do not respond to 10 µM alpha beta -meATP, whereas chimeric receptors do. Fig. 7B illustrates the lack of receptor-specific desensitization pattern in wild-type P2X2Rs and a significant difference in the rates of chimeric receptor desensitization in response to 100 µM alpha beta -meATP. The same conclusions were also derived from cells stimulated with 500 µM alpha beta -meATP (Fig. 7C). Table I summarizes the level of calcium signal desensitization in wild-type and chimeric P2X2Rs. Thus, the introduction of the P2X4R ectodomain in P2X2aR and P2X2bR backbones had three obvious effects: increase of the rate of receptor desensitization for ATP without affecting the C-terminal-dependent desensitization pattern, reestablishment of the C-terminal-dependent receptor desensitization in response to BzATP and alpha beta -meATP stimulation, and comparable plateau levels in response to three agonists.


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Fig. 7.   Comparison of alpha beta -meATP-induced calcium responses in cells expressing wild-type (left panels) and chimeric (right panels) P2X2Rs. A, difference in the sensitivity of wild-type and chimeric channels to 10 µM alpha beta -meATP. B and C, comparison of the pattern of calcium signal desensitization in cells expressing wild-type and chimeric receptors during continuous stimulation with 100 µM alpha beta -meATP (B) and 500 µM alpha beta -meATP (C). Upper panels, representative traces from 3-8 independent experiments, each done in at least 15 cells. Bottom panels, mean values of rates of receptor desensitization.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we used the wild-type P2X2aR and P2X2bR because of their identical ectodomains and rapid activation properties, but distinct and well defined desensitization patterns in response to sustained stimulation with ATP (30, 32, 33). The P2X4R shares about 39% similarity with P2X2R and desensitizes with rates comparable with those observed in cells expressing P2X2bR, whereas P2X7R shares about 26% similarity with P2X2R and desensitizes in a manner more comparable with the P2X2aR subtype (6, 34). These four receptors also exhibit highly specific ligand potency profiles, including ATP, BzATP, and alpha beta -meATP. ATP is a highly potent agonist for P2X4R, a high to middle potency agonist for P2X2R, and a low potency agonist for P2X7R. BzATP is considered as a high potency agonist for P2X7R, a middle potency agonist for P2X2R, and a low potency agonist for P2X4R. alpha beta -meATP acts as a low potency agonist for P2X2R and a partial agonist for P2X4R, whereas P2X7R is insensitive to this agonist (6).

The sequences Ile66-Tyr310 of P2X2R, Val66-Tyr315 of P2X4R, and Val61-Phe313 of P2X7R used for construction of our chimeric channels contain the majority of residues relevant for ATP binding identified so far. They enclose the 10 conserved cysteine residues among all known P2XRs and three N-linked glycosylated sites (Asn182, Asn239, and Asn298 in rat P2X2R), which might be responsible for functionality of the channels (35-39). The ectodomain sequences we used also contain several conserved residues, which might contribute to the ATP-binding site, including Lys69 and Lys71 in rat P2X2 sequence and corresponding to Lys68 and Lys70 in human P2X1 (9, 10), Trp256 in rat P2X2 (40), and Lys309 in human P2X1 (9). Because those are common residues for all seven channels, it is obvious that other residues account for ligand specificity among receptors. By exchanging the ectodomain sequences, we hoped that the ligand selectivity profiles would be preserved and thus enabled us to examine the dependence of channel activity on the ligand-binding-specific domains. In accordance with this, our previously published data with P2X2 + X3R chimeras having the Val60-Phe301 ectodomain sequence of P2X3R instead of the native Ile66-Tyr310 sequence showed highly comparable ligand potency with parental P2X3R (7, 8).

However, the present data clearly indicate that the transfer of these ectodomains alters the native agonist selectivity and potency. First, the ATP potency for P2X2a + X4R and P2X2b + X4R chimeras was higher than that observed in both parental receptors. In parallel to that, two chimeras desensitized more rapidly than parental receptors. Second, alpha beta -meATP is a partial agonist for P2X4R (31) and a full and highly potent agonist for P2X2a + X4R and P2X2b + X4R chimeras. Third, P2X4 + X2R and P2X4 + X7R were expressed but were not functioning receptors. These results suggest the effects of transmembrane domain flanking sequences on ligand specificity and agonistic potency. We may speculate that these sequences act as "dominant-positive" and "dominant-negative" domains, depending on the structure of the main ectodomain sequence, to change the sensitivity of receptors for agonists. In accordance with this view, it has been reported recently that point mutations in the first transmembrane domain affect the ligand selectivity of rat P2X2R (41).

In this study, we also progressed in understanding the mechanism by which the putative ligand-binding domain may influence the rate of receptor desensitization and the relationship between the ectodomain and C-terminal domain in control of desensitization. The main conclusion that emerged from this work is that the P2XR desensitization pattern is receptor- and ligand-specific. The P2X2aR-, P2X2bR-, and P2X4-specific desensitization patterns were observed in response to ATP, the native agonist for these channels, but were less obvious when stimulated with BzATP and were lost when receptors were stimulated with alpha beta -meATP. The increase in EC50 values for all three agonists induced by substituting the ectodomains indicated that the potency of agonists reflects the ligand specificity of receptor desensitization, i.e. highly potent agonists trigger the subtype-specific desensitization pattern, whereas agonists with lower potency are less effective or are ineffective. Our effort to further establish this hypothesis by generating receptors with decreasing sensitivity for ATP was unsuccessful because P2X4 + X2R and P2X4 + X7R chimeras were nonfunctional.

On the other hand, we were more successful in establishing that the ectodomain influences desensitization independently of the C-terminal domain. Earlier studies have indicated that the 6-residue receptor-specific sequences in C-terminal influence the rates of P2X2aR, P2X2bR, P2X3R, and P2X4R (15). In this study, current and calcium measurements indicated that chimeric P2X2a + X4R and P2X2b + X4R desensitized with different rates, i.e. a leftward shift in EC50 and DC50 for chimeric receptors proportionally affected both receptors. This suggests that the C-terminal-dependent desensitization pattern was not affected by substituting the ectodomains. To test this hypothesis further, we generated a mutant of P2X2a + X4R chimera in which we substituted the C-terminal Arg371-Pro376 sequence of P2X2a + X4R with Glu376-Gly381 sequence of P2X4R. In parallel to the desensitization patterns of P2X2aR and P2X2a-6aaR (15), the present data show that chimeric P2X2a-6aa + X4R desensitized more rapidly than P2X2a + X4R when stimulated with ATP.

At the present time, we cannot speculate about the possible molecular mechanism by which the ligand-binding domain influences receptor desensitization. The main limitation comes from the fact that the ligand-binding domain structure and the crystal structure of P2XRs have not been identified. The structural similarities of P2XRs with class II aminoacyl-tRNA synthases (42) provided a model for initial studies on the ATP-binding domain (9, 10, 40, 41), which could not give a rationale for the receptor behavior shown here. Based on studies with crystallization of glutamate channels, in a recently published study, Sun et al. (3) showed that desensitization of these receptors occurs through rearrangement of the dimmer interface, which disengages the agonist-induced conformation change in the binding core from the ion channel gate. To which extent this model provides a rationale for the observed effects with P2XRs is also difficult to discuss, especially with respect to data on the pattern of polymerization of these receptors (43).

The influence of the ectodomain structure on P2XR desensitization is a novel finding for this family of receptors but is reminiscent of those seen among subtypes of other ligand-gated receptor channels. For example, AMPA and glutamate maximally activate AMPA receptors, whereas kainite and domoate act as partial agonists and produce much less desensitization than glutamate (2, 23, 24, 44). A single amino acid mutation in the third loop of the binding pocket of the nicotinic acetylcholine receptor produces a right shift in the concentration-response curve but also significantly slows the rate of channel opening (25). The desensitization of this receptor is affected by amino acids near or within the agonist-binding domain, as well as by residues within M2 that line the pore (26, 27). A single residue substitution in GluR1 also gives a mutant channel that exhibits 6-20-fold lower EC50 values for kainate (28). Introduction of the alpha 4 residues in the agonist-binding site of nicotinic nicotinic acetylcholine receptors produces a 100-fold increase in the apparent agonist potency and desensitization of receptors and a 3-7-fold shift in the apparent affinity for activation (27).

In conclusion, our results show that wild-type P2X2aR, P2X2bR, and P2X4R desensitized in a receptor- and ligand-specific manner. The P2X4R-specific ligand potency and desensitization patterns were not only transferred but were enhanced in P2X2a + X4R and P2X2b + X4R, whereas the P2X2R and P2X7R ectodomains and the backbone of P2X4R generated nonfunctional channels. This suggests that flanking sequences around the transmembrane domains may act as modulatory regions. A parallelism in the leftward shift of EC50 and DC50 for ATP further suggests that the potency of agonists underlines the ligand-specific patterns of receptor desensitization.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: SCS/ERRB/NICHD, Bldg. 49, Rm. 6A-36, 49 Convent Dr., Bethesda, MD 20892-4510. Tel.: 301-496-1638; Fax: 301-594-7031; E-mail: stankos@helix.nih.gov.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M209094200

    ABBREVIATIONS

The abbreviations used are: P2XRs, purinergic receptor channels; alpha beta -meATP, alpha ,beta -methylene-ATP; BzATP, 3'-O-(4-benzoyl)benzoyl-ATP; GFP, green fluorescent protein; EGFP, enhanced GFP; AMPA, alpha -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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