Introduction

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Extracellular ATP acts as a signaling molecule within the central, peripheral, and enteric nervous systems; elsewhere at neuroeffector junctions of the autonomic nervous system; and locally as a paracrine or autocrine humoral agent (Ralevic and Burnstock, 1998). Irrespective of its sites of action, ATP exerts its effects through two main classes of P2 receptorsthe P2X and P2Y families. For the P2X receptor class, seven subunits (P2X_1-7) have been identified thus far (Burnstock and King, 1996). P2X subunit proteins have intracellular N and C termini with two membrane-spanning domains connected by a large cysteine-rich extracellular loop. Three, possibly four, P2X subunits assemble to form homomeric complexes that function as nonselective cation channels gated by extracellular ATP (Kim et al., 1997; Nicke et al., 1998). Different P2X receptor subunits can also coassemble to form heteromeric complexes with pharmacological and operational profiles distinct from the homomeric P2X receptors made by the constituent subunits. So far, five functional P2X heteromers (P2X_1/2, P2X_1/5, P2X_2/3, P2X_2/6, and P2X_4/6) have been characterized (Brown et al., 2002), of a series of 11 heteromers predicted by immunoprecipitation studies (Torres et al., 1999). The formation of heteromeric P2X receptors has helped explain in part, but not totally, some pharmacological aspects of purinergic signaling at endogenous P2X receptors in whole tissues.

The P2X₅ receptor was first cloned from rat celiac ganglia (Collo et al., 1996). Homomeric P2X₅ was viewed as a slowly desensitizing receptor sensitive to ATP and 2MeSATP, but insensitive to ,-meATP and many other nucleotides. Agonist responses were noted to be exceedingly small (by a factor of 100) compared with the responses of other recombinant P2X receptors tested under similar conditions, which raised questions over the functional role of homomeric P2X₅ receptors in vivo (Collo et al., 1996). Also, P2X₅ transcripts and protein have a restricted distribution, with expression mainly in the trigeminal mesencephalic nucleus of the brainstem; a lesser presence in sensory neurons, cervical spinal cord, and some blood vessels; and a limited presence in kidney, heart, skeletal muscle, adrenal gland, retina, and assorted epithelial cell types (Collo et al., 1996; Garcia-Guzman et al., 1996; Brändle et al., 1998; Phillips et al., 1998; Gröschel-Stewart et al., 1999; Phillips and Hill, 1999; Taylor et al., 1999; Gitterman and Evans, 2000; Ryten et al., 2001). However, many of these tissues and cell types contain mRNA for other P2X isoforms and, on occasion, this includes transcripts for the P2X₁ subunit. P2X₁ receptors are abundant in smooth muscle yet also occur in heart, sensory neurons, spinal cord, duodenal submucosa, blood platelets, and megakaryocytes. Functionally, homomeric P2X₁ receptors are viewed as rapidly desensitizing, ,-meATP-sensitive P2X receptors that are blocked by suramin (Khakh et al., 2001). Interestingly, a novel P2X_1/5 receptor can form when P2X₁ and P2X₅ subunits are coexpressed in the same cell (Torres et al., 1998; Haines et al., 1999; Lê et al., 1999; Surprenant et al., 2000). This observation, together with proposed assemblies of P2X_2-4 subunits with the P2X₅ subunit (Torres et al., 1999), has lent weight to the suggestion that the main physiological role of the P2X₅ subunit probably rests with the construction of heteromeric P2X receptors.

Earlier histochemical investigations have revealed P2X₅-like immunoreactivity (P2X₅-ir), without the colocalization of other P2X subunit proteins, in the apical border of epithelial cells lining the collecting duct of the rat nephron (Chan et al., 1998; Bailey et al., 2000; Schwiebert and Kishore, 2001). This site is also the locus of a pernicious disease, called polycystic kidney disease (PKD), in which a prominent up-regulation of P2X₅-ir has been observed in human and rat PKD cell cultures (Schwiebert, 2001; Schwiebert and Kishore, 2001). In a broader context, dense P2X₅-ir has also been associated with the proliferating and differentiating cell layers of rat squamous epithelial tissues (Gröschel-Stewart et al., 1999) and with developing (E15-18) rat embryo skeletal myotubes (Ryten et al., 2001). Thus, we have taken a fresh look at recombinant P2X₅ receptors to see under what circumstances homomeric P2X₅ assemblies might be able to function. The positive outcome of such experiments then led us to compare homomeric P2X₅ receptors with homomeric P2X₁, because the possibility exists that multiple P2X subtypes of close pharmacological profile, yet dissimilar operational properties, are present elsewhere in rat kidneyspecifically the intrarenal vasculature (van der Giet et al., 1999). Part of this study has been presented elsewhere (Wildman et al., 2002).

	Materials and Methods

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Oocyte Preparation. Xenopus laevis frogs were anesthetized in tricaine (0.4% w/v), killed by decapitation, and their ovarian lobes were removed surgically. Oocytes (stages V and VI) were defolliculated by a 2-step process involving collagenase treatment (Type IA, 2 mg/ml in a Ca²⁺-free Ringer solution, for 2 h) followed by a mechanical stripping away of the follicular layer with fine forceps (in double-strength Ca²⁺-free Ringer solution). Defolliculated oocytes were stored mainly in a (Ca²⁺-free) Ba²⁺-containing Ringer solution, pH 7.5 at 4°C, containing 110 mM NaCl, 2.5 mM KCl, 5 mM HEPES, and 1.8 mM BaCl₂. In a few experiments, BaCl₂ was replaced with equimolar CaCl₂. Gentamicin sulfate was not present in storage solutions. Defolliculated oocytes were injected cytosolically with either rat P2X₅ or rat P2X₁ cRNA (40 nl, 1 µg/µl), incubated for 24 to 48 h at 18°C in a Ba²⁺-containing Ringer solution then kept at 4°C for up to 7 days until used in electrophysiological experiments. Only those P2X₅-injected oocytes with maximal ATP-evoked whole-cell currents of 20 nA and greater were used in this investigation.

Electrophysiological Recordings. Agonist-activated membrane currents (V_h = 95 mV) were recorded from cRNA-injected oocytes using a twin-electrode voltage-clamp amplifier (Axoclamp 2A; Axon Instruments, Union City, CA). The voltage-recording and current-recording microelectrodes (1-5 M tip resistance) were filled with 3.0 M KCl. Oocytes were superfused with a Ba²⁺-Ringer solution (10 ml/min, pH 7.5, at 18°C). In some cases, MgCl₂ or CaCl₂ (1.8 mM) was added to the superfusate, either in place of or in addition to BaCl₂, to investigate the effects of different divalent cations in the superfusate. Where stated, the pH of the bathing solution was adjusted using either 1.0 N HCl or NaOH to achieve the desired level. Electrophysiological data were stored on a computer using a MP100 WSW interface (Biopac Systems, Goleta, CA) and analyzed using the software package Acqknowledge III (Biopac Systems).

Data Analysis. Agonists were prepared in a Ca²⁺-containing Ringer solution (1.8 mM Ca²⁺) and superfused (12 ml/min) by a gravity-fed continuous flow system that allowed rapid addition and washout. In some cases, agonists were prepared in a Ba²⁺-containing Ringer solution (1.8 mM Ba²⁺) to investigate P2X₅ receptor rundown. Agonists were added for 90 s or until the current reached a peak, then washed for a period of 15 and 20 min for rP2X₅ and rP2X₁ receptors, respectively. For concentration-response (C/R) curves, data were normalized to the maximum current (I_max) evoked by ATP (100 µM). The agonist concentration that evoked 50% of the maximum response (EC₅₀) was taken from Hill plots of the transform, log (I/I_max I), where I is the current evoked by each concentration of agonist. The Hill coefficient (n_H) was taken from the slope of the Hill plots. The activity of P2 antagonists was tested by adding each antagonist in cumulative concentrations (as mentioned in the text), each concentration applied 15 min or 20 min (rP2X₅ and rP2X₁, respectively) before the addition of a submaximal concentration of ATP (~EC₇₀ concentration, 1 µM ATP). The antagonist concentration that reduced ATP-responses by 50% (IC₅₀) was taken from inhibition curves. The reversibility of receptor blockade was tested after prolonged washout of antagonists.

Immunohistochemistry. Defolliculated oocytes were injected with either cRNA for rP2X₅ or sterile H₂O (control), stored in either a Ba²⁺- or Ca²⁺-containing Ringer solution (18°C, for 48 h) and then prepared for immunohistochemistry. All reactions were performed on free-floating whole oocytes. Injected and control oocytes were fixed for 2 min in paraformaldehyde [4% (w/v) in a 0.1 M phosphate buffer] and washed in phosphate-buffered saline containing 0.1% Triton X-100 (PBST). Oocytes were preincubated for 30 min at room temperature with 10% normal rabbit serum in phosphate-buffered saline (with 1% Triton X-100 added), followed by overnight incubation at 4°C with a goat polyclonal antibody for P2X₅ (Santa Cruz, CA) diluted 1:100 in PBST and containing 1% normal rabbit serum. Oocytes were washed in PBST and incubated for 1 h at room temperature with biotinylated rabbit anti-goat IgG (Vector Laboratories, Peterborough, UK) diluted 1:200 in PBST containing 1% normal rabbit serum and 1% bovine serum albumin. After washing with PBST, oocytes were incubated for 1 h at room temperature with a preformed avidin-biotinylated alkaline phosphatase complex (Vector Labs) diluted according to kit instructions with PBST. A Vector Red alkaline phosphatase substrate kit was used to visualize immunoreactivity. Stained oocytes were washed in distilled water, placed onto slides and covered in Histotec, a permanent aqueous mountant (Serotec Ltd., Oxford UK). Fluorescent immunostaining was viewed with a Leica DMR microscope fitted with a Rhodamine filter (515-560 nm). Digital pictures were taken with a Nikon CoolPix 990 digital camera; fluorescence images were converted from color to grayscale, and then inverted (white-on-black to black-on-white) using Photoshop v5.5 (Adobe Systems Inc., San José, CA).

Drugs. ATP, related nucleotides, and other drugs, including tricaine, were obtained from Sigma-Aldrich (Poole, Dorset, UK). Suramin was a gift from Bayer plc (Newbury, Berkshire, UK), Ip₅I was a gift from Dr. J. Pintor (Universidad Complutense, Madrid, Spain) and TNP-ATP was obtained from Molecular Probes (Eugene, OR). All reagents were AnalaR grade from Sigma-Aldrich. Contaminating ATP was removed from ADP stocks by adding hexokinase (50 U/ml) and glucose (25 mM) for 2 h before assay as described in Bogdanov et al. (1998).

DNA Constructs. The rP2X₅ receptor cDNA was a kind gift from Dr. Xuenong Bo and Professor Alan North (University of Sheffield, South Yorkshire, UK) and the rP2X₁ receptor cDNA from Dr. Gary Buell (Ares Sereno, Geneva, Switzerland). The rP2X₅ DNA was resequenced by MWG Biotech Ltd (Milton Keynes, Buckinghamshire, UK). Using BLAST analysis of a comprehensive database (957,033 sequences), the sequence exactly matched the rat P2X₅ purinoceptor (GenBank accession number X92069).

	Results

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Whole-Cell Inward Currents. ATP (10 and 1 µM) elicited maximal inward membrane currents in defolliculated oocytes expressing rP2X₅ and rP2X₁ receptors, respectively. At the rP2X₅ receptor, the evoked inward current was slowly desensitizing with a maximum amplitude of the order of 75nA (at 95 mV), whereas, at the P2X₁ receptor, the evoked inward current was rapidly desensitizing with a maximum amplitude of approximately 1 µA at the same holding potential (Fig. 1A). At the rP2X₅ receptor, successive ATP-activated inward currents (I_ATP) showed nearly complete and irreversible rundown (92 ± 8% of the initial ATP response) over a period of 45 to 60 min when oocytes were exposed to a Ca²⁺-containing Ringer solution (Ca²⁺, 1.8 mM) (Fig. 1B). However, rundown could be avoided if a Ba²⁺-containing Ringer solution (Ba²⁺, 1.8 mM) was used during washout periods and Ca²⁺ ions (1.8 mM) returned to the superfusate (with Ba²⁺ present in the background; 1.8 mM) during the period of agonist application (Fig. 1B). If Ca²⁺ was omitted during agonist application and only Ba²⁺ was present, subsequent agonist responses were significantly decreased in amplitude (by 10-fold) although there was no further rundown of ATP responses (Fig. 1C). There was no significant difference between the amplitude of agonist responses evoked in the presence of either Ba²⁺ and Ca²⁺ together or Ca²⁺ alone (Fig. 1C). Similar results were obtained when Ba²⁺ was substituted for Mg²⁺ ions (data not shown). At the rP2X₁ receptor, substitution of Ca²⁺ with equimolar Ba²⁺ in the Ringer solution caused no significant difference in the amplitude of agonist responses or rundown of ATP responses. Additionally, using a Ba²⁺-Ringer superfusate during interagonist periods and Ba²⁺/Ca²⁺-Ringer solution during agonist application (90-s conditioning Ca²⁺-pulse), no significant difference was observed in the amplitude of agonist responses at P2X₁ (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Activation of homomeric P2X receptors. A, whole cell inward currents activated by near saturating concentrations of ATP at homomeric rP2X5 and rP2X1 receptors [holding potential (Vh), 95 mV]. B, rundown of successive ATP responses at the rP2X5 receptor bathed in a Ringer solution containing Ca2+ ions (1.8 mM) (upper row), and consistent-amplitude ATP responses where a 90-s conditioning Ca2+-pulse (1.8 mM) was added to a superfusate containing Ba2+ ions (1.8 mM) (lower row). C, maximum ATP responses at the rP2X5 receptor in either Ba2+-Ringer, or Ba2+-Ringer and 90-s conditioning Ca2+-pulse, or Ca2+-Ringer. D, mean amplitude of maximum ATP-evoked responses (n = 4) at the rP2X5 receptor after conditioning Ca2+-pulses of various durations before agonist application. E, current-voltage relationship ATP evoked inward currents at the rP2X5 receptor after a 90-s conditioning Ca2+-pulse (n = 3).

Immunohistochemistry. Relative amounts of membrane bound P2X₅-like material were assessed in rP2X₅ cRNA-injected oocytes stored for 48 h in either a Ba²⁺- or Ca²⁺-containing Ringer solution (Fig. 2). A dense and even layer of P2X₅-like immunoreactive material was observed in the plasmalemma of whole cRNA-injected oocytes that had been kept in a Ba²⁺-containing Ringer solution (Fig. 2A). In contrast, immunostaining was much less dense and markedly punctate for cRNA-injected oocytes stored in a Ca²⁺-containing Ringer solution (Fig. 2C). Little, if any, immunostaining was observed in control (H₂O-injected) oocytes (Fig. 2E), and where the anti-P2X₅ antibody was omitted, very little alkaline phosphatase surface activity was seen in either cRNA-injected or control oocytes (Fig. 2, B, D, and F).

View larger version (75K): [in this window] [in a new window]   Fig. 2.   Immunostaining in P2X5 cRNA-injected oocytes. A, P2X5-like immunoreactivity in a cRNA-injected oocyte stored for 48 h in a Ba2+-containing bathing solution before immunohistochemical processing. B, the primary antibody (anti-P2X5) was omitted. Experiments were repeated for cRNA-injected oocytes stored for 48 h in Ca2+-containing bathing solution (C, D) and for H2O-injected control oocytes for 48 h in a Ba2+-containing bathing solution (E, F). The plasmalemmal accumulation of rP2X5-like material in cRNA-injected oocytes was affected by prolonged exposure to extracellular Ca2+ ions, resulting in reduced immunohistochemical staining and formation of punctate islands of P2X5-like protein. Scale bar, 50 µm.

Comparison of I_ATP at Homomeric P2X Receptors. All agonist responses at rP2X₅ receptors were evoked after a 90-s conditioning Ca²⁺-pulse. The maximum ATP-evoked inward current was 74 ± 22 nA at the rP2X₅ homomer (n = 15), compared with 950 ± 260 nA at the rP2X₁ receptor (n = 15) (Fig. 3A). Increasing the concentration of injected rP2X₅ cRNA 5-fold (from 1 to 5 µg/µl) did not result in larger whole-cell currents for maximum ATP responses (data not shown). The area-under-the-curve (I × t = Q, or charge transfer) for the rP2X₅ receptor was 583 ± 212 × 10⁹ Q, compared with 2667 ± 209 × 10⁹ Q for the rP2X₁ receptor (Fig. 3B). At the rP2X₅ receptor, the time for I_ATP to activate and deactivate (20-80%) was 4.1 ± 0.9 s and 11.8 ± 3.9 s, respectively (n = 15). At the rP2X₁, the activation time (20-80%) was 0.7 ± 0.2 s, and inactivation time (20-80%) was 13.3 ± 1.6 s (n = 15) (Fig. 3C, 3D). Maximal amplitude, area-under-the-curve, and activation times were statistically different (p < 0.05) for inward currents evoked by the two P2X receptor subtypes.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   ATP responses at homomeric P2X receptors. The maximum amplitude (A), area-under-the-curve (I × t = Q, charge transfer) (B), activation-time (20-80%) (C), and inactivation-time (20-80%) (D) of ATP responses (10 or 1 µM, respectively) at homomeric rP2X5 and rP2X1 receptors. Data are expressed as mean ± S.E.M., n = 15.

Agonist Potency at Homomeric P2X Receptors. A broad range of nucleotides was tested for agonist activity at homomeric rP2X₅ and rP2X₁ receptors. C/R curves for both P2X subtypes were determined under conditions of a Ba²⁺-containing superfusate during interagonist periods and Ba^2+/Ca²⁺-containing solution just before (90-s conditioning Ca²⁺-pulse) and during agonist application. For ATP, the EC₅₀ (and n_H) values were 441 ± 49 nM (1.4 ± 0.2) and 104 ± 65 nM (0.8 ± 0.3) at rP2X₅ and rP2X₁ receptors, respectively (n = 12). For other agonists, the potency order at the rP2X₅ was (by comparison of EC₅₀ values): ATP = 2MeSATP = ATPS > ,-meATP = BzATP > ADP > UTP > ,-meATP > GTP > CTP. Here, ,-meATP, BzATP, ,-meATP, ADP, and UTP were partial agonists and ITP and UDP were inactive (Fig. 4A, Table 1). At the rP2X₁ receptor, the agonist potency order was: ATP = 2MeSATP > ATPS > ,-meATP > ,-meATP > BzATP > CTP > UTP. Here, ATPS, ,-meATP, CTP, ,-meATP, and UTP were partial agonists and GTP, ADP, ITP, and UDP were inactive (Fig. 4B, Table 1). EC₅₀ values for most agonists were within 1 log unit of concentration for the two P2X subtypes, except for GTP (which activated only rP2X₅) and BzATP and UTP (which were significantly more potent at rP2X₅). Of these three agents, only GTP was a full agonist at homomeric rP2X₅ receptors.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Agonism of homomeric P2X receptors. C/R curves for nucleotide agonists of homomeric rP2X5 receptors (A and C) and rP2X1 receptors (B and D) at pH 7.5. Data were normalized to the amplitude of maximum ATP responses (10 and 1 µM for rP2X5 and rP2X1, respectively). EC50 and nH values are given in Table 1. Data are expressed as mean ± S.E.M. (n = 4 or more).

Antagonism of Homomeric P2X Receptors. A series of known P2 receptor antagonists was tested against I_ATP evoked by homomeric rP2X₅ and rP2X₁ receptors. At the rP2X₅ receptor, the potency order for five antagonists was: PPADS > TNP-ATP > suramin > RB-2 Ip₅I (Fig. 5A, Table 1). Notably, PPADS was active at submicromolar concentrations (mean IC₅₀, 200 nM), whereas TNP-ATP, suramin, and RB-2 were 3-, 8-, and 90-fold less potent. Ip₅I was a weak antagonist (15 ± 7% inhibition at 30 µM) and, because of limited availability, was not tested at higher concentrations. At the rP2X₁ receptor, the potency order was: TNP-ATP = Ip₅I > PPADS > suramin = RB-2 (Fig. 5B, Table 1). Both TNP-ATP and Ip₅I were active in the nanomolar concentration range and virtually equipotent. Relative to these two compounds, PPADS was about 10-fold less potent, whereas suramin and RB-2 were about 1000-fold less potent. Where data for homomeric rP2X₅ and rP2X₁ receptors were compared, TNP-ATP and Ip₅I are relatively selective for rP2X₁ receptors. PPADS and suramin were equally effective at the two P2X subtypes. RB-2 was about 9-fold more potent at rP2X₁ than rP2X₅ receptors. The blocking actions of TNP-ATP, Ip₅I, and suramin at both P2X receptors subtypes were reversible with washout periods of about 1 h, whereas blockade by either RB-2 or PPADS was only partially reversible after 1-h washout.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Antagonism of homomeric P2X receptors. Inhibition curves for antagonists of homomeric rP2X5 receptors (A) and rP2X1 receptors (B). Data were normalized to the amplitude of control ATP responses (at 1 µM, approximately EC70 at pH 7.5). IC50 and nH values are given in Table 1. Data are expressed as mean ± S.E.M. (n = 4).

Cationic Modulation of Homomeric P2X Receptors. The potency and efficacy of ATP were re-examined with different divalent cations present in the bathing solution. Four extracellular solutions, containing either Ba²⁺, Ca²⁺, or Mg²⁺ ions (1.8 mM) or using a conditioning Ca²⁺-pulse (1.8 mM, for 90 s) with background presence of Ba²⁺ ions (1.8 mM) were examined at homomeric rP2X₅ and rP2X₁ receptors.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Modulators of homomeric P2X receptors. A and B, C/R curves for ATP (at pH 7.5), in the presence of either extracellular Ba2+, Ca2+, or Mg2+ ions (each 1.8 mM) or in the presence of Ba2+ ions and with a conditioning Ca2+-pulse (Ba2+/Ca2+-pulse, each 1.8 mM) at homomeric rP2X5 and rP2X1 receptors. In C and D, C/R curves for ATP at four levels of extracellular pH (pHe) (8.0, 7.5, 6.5, 5.5) at homomeric rP2X5 and rP2X1 receptors. In E and F, C/R curves for modulation of agonist responses by extracellular Zn2+ ions (0.01-1000 µM) at homomeric rP2X5 and rP2X1 receptors activated by 300 nM ATP, pH 7.5. Zn2+ ions were added to the superfusate and applied either simultaneously with ATP (0 min preincubation), or before and during application of ATP (15 or 20 min preincubation). Data in A are amplitudes of measured currents. Data in B, C, and D are normalized to the maximum ATP response at pH 7.5. Data in E and F are normalized to the control ATP response at pH 7.5. EC50 and nH values are given in Table 2. Data expressed as mean ± S.E.M. (n = 4).

Proton Modulation of Homomeric P2X Receptors. The potency and efficacy of ATP was reassessed at homomeric rP2X₅ and rP2X₁ receptors at four different levels of extracellular pH (pH 8.0, 7.5, 6.5 and 5.5). At rP2X₅, alkaline conditions (pH 8.0) had no effect on ATP activity, whereas acidic conditions (pH 6.5 and 5.5) caused a progressive reduction of ATP efficacy (Fig. 6C, Table 2). A 3- to 4-fold reduction of ATP potency was observed at pH 5.5 (Table 2). At rP2X₁, alkaline conditions also had no effect on ATP activity, whereas acidic conditions caused a progressive reduction of ATP potency without affecting efficacy (Fig. 6D, Table 2).

Zinc Modulation of Homomeric P2X Receptors. The modulatory effect of extracellular Zn²⁺ ions (0.01-1000 µM) on ATP responses was examined at homomeric rP2X₅ and rP2X₁ receptors. At rP2X₅, Zn²⁺ ions (1-100 µM; EC₅₀, 42.6 ± 23.9 µM) potentiated membrane currents to ATP, by approximately 2-fold, when coapplied with the agonist (Fig. 6E). Higher concentrations (300-1000 µM) inhibited ATP responses without full blockade at the higher concentration tested. When applied 15 min before the agonist, Zn²⁺ caused the same concentration-dependent effects. The modulatory effects of Zn²⁺ were reversed after washout. At rP2X₁, Zn²⁺ ions (0.01-300 µM) inhibited ATP responses in a concentration-dependent and reversible manner (Fig. 6F). The inhibitory effect of Zn²⁺ was also dependent on preincubation time: IC₅₀, 9.3 ± 0.6 µM (0 min preincubation) and 0.8 ± 0.1 µM (20 min preincubation).

	Discussion

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In the present study, it was shown that homomeric rP2X₅ receptors could produce small-amplitude slowly desensitizing inward currents when activated by ATP. In the past, investigators have found it difficult to generate functional P2X₅ receptorsfor example, where this subtype was studied originally by Collo et al. (1996), and in the control experiments of investigators studying heteromeric P2X_1/5 receptors (Torres et al., 1998; Haines et al., 1999; Lê et al., 1999; Surprenant et al., 2000). If there has been any prior success with homomeric P2X₅, the resultant agonist-evoked responses have been consistently small [<100 pA in HEK293 cells at 70mV (Collo et al., 1996; Garcia-Guzman et al., 1996), and <100 nA in X. laevis oocytes at 100mV (Lê et al., 1999)]. We tried to increase receptor levels and agonist responses by injecting greater amounts of rP2X₅ transcripts into oocytes. This procedure failed here, although it has been successful for us in the past when studying other P2X subtypes (Liu et al., 2001).

Difficulties in expressing rP2X₅ in oocytes were overcome by removing Ca²⁺ ions from the extracellular solution, yet Ca²⁺ was later found to be important when activating this homomeric receptor. So, extracellular Ca²⁺ had a dual roleor sensitizing and desensitizing actionsat rP2X₅ receptors. Extracellular Ca²⁺ ions had this effect, Ba²⁺ and Mg²⁺ did not, whereas Zn²⁺ acted somewhat like Ca²⁺ (see below). When the exposure time to extracellular Ca²⁺ was brief (5 min or less, before agonist addition), ATP routinely evoked consistent-amplitude responses. Exposure times to Ca²⁺ greater than 5 min caused a progressive rundown of agonist-evoked inward currents. Even when storing oocytes, the long-term presence of extracellular Ca²⁺ had a deleterious effect and later diminished the mean amplitude of ATP-activated currents at the rP2X₅ receptor. Immunostaining revealed that prolonged exposure to extracellular Ca²⁺ affected the density of P2X₅-like material in the membrane of injected oocytes and also led to the formation of punctate islands of P2X₅-like protein. The occurrence of puncta was reminiscent of ATP-induced P2X₂ receptor translocation and internalization in rP2X₂-GFP transfected rat embryonic hippocampal neurons (Khakh et al., 2001b). Movement of P2X₂ receptors and formation of puncta in hippocampal neurons was prevented by the T18A mutation of rP2X₂ subunits, thereby deleting an N terminus binding motif for Ca²⁺-dependent PKC (Khakh et al., 2001b). Our immunostaining results, showing lowered P2X₅-ir density and clumping in the presence of extracellular Ca²⁺, suggested a similar movement of P2X₅ receptors in oocytesa possibility that we are continuing to investigate.

The phenomenon of sensitization and desensitization by extracellular Ca²⁺ at rP2X₅ was also reminiscent of earlier findings at P2X₃ receptors. Short pulses of extracellular Ca²⁺ (10 mM, for 30-90 s) rapidly resensitized P2X₃-like receptors in sensory neurons and recombinant rP2X₃ receptors in HEK293 cells (Cook and McCleskey, 1997; Cook et al., 1998), although the mechanism for this phenomenon remains unexplained. On the other hand, influx of extracellular Ca²⁺ plays a role in the inactivation of rP2X₃ receptor (King et al., 1997). This Ca²⁺-dependent mechanism again may be due to the intracellular PKC site (TX[K/R]) conserved in the N terminus of all P2X_1-7 subunits and shown to be important in the inactivation of wild-type and mutant P2X receptors (Boué-Grabot et al., 2000; Khakh et al., 2001b). Our attempts to alter intracellular Ca²⁺ levels by exposing oocytes to BAPTA-AM (30 µM for 30 min) were largely inconclusive, although our paradigm exactly followed another oocyte study in which Ca²⁺/PKC-dependent potentiation of N-methyl-D-aspartate receptors was shown to be BAPTA-AM-sensitive (Skeberdis et al., 2001). More convincingly, the rate of desensitization was significantly reduced by lowering intracellular Ca²⁺ in HEK293 cells expressing chick P2X₅ receptors (Ruppelt et al., 2001). Thus, we believe that difficulties in recording inward currents from homomeric P2X₅ receptors may be due to the complex actions of Ca²⁺ ions at extracellular and intracellular loci on the P2X₅ subunit.

For homomeric rP2X₁ receptors, it was comparatively easier to evoke large-amplitude ATP responses, and the substitution of Ca²⁺ with either Ba²⁺ or Mg²⁺, or use of Ca²⁺-pulses in the presence of extracellular Ba²⁺, did not alter the position of the ATP C/R curve. Thus, a crucial difference between homomeric rP2X₅ and rP2X₁ receptors is the sensitization of the former by extracellular Ca²⁺ ions. We have previously investigated the modulatory effects of extracellular H⁺ and Zn²⁺ ions at the rP2X₁ receptor (Wildman et al., 1999) and repeated these experiments in the present study for both P2X receptor subtypes. For rP2X₁, reduction of extracellular pH (pH_e) reduced the potency but not efficacy of ATP, whereas, at the rP2X₅ receptor, lowering pH reduced both potency and efficacy. At the rP2X₁ receptor, agonist activity was inhibited in a concentration- and time-dependent manner by extracellular Zn²⁺ because of a reduction in agonist potency without a concomitant change in agonist efficacy (Wildman et al., 1999). At the rP2X₅ receptor, extracellular Zn²⁺ (0.1-100 µM) caused a potentiation of ATP responses and, at higher concentrations (100-1000 µM), a potent inhibition of agonist responses. The potentiating and inhibitory effects of Zn²⁺ at rP2X₅ superficially resembled the sensitizing and desensitizing effects of extracellular Ca²⁺. However, the effects of the former were time-independent, whereas the latter were time-dependent.

A greater range of nucleotides than previously realized could activate homomeric rP2X₅ receptors. In our hands, ATP, 2MeSATP, and ATPS were full and potent agonists. CTP and GTP also were full agonists although some 60-fold less potent than ATP. Of the remainder, ,-meATP, ,-meATP, BzATP, ADP, and UDP are partial agonists and of intermediate potency. ,-meATP activation of the rP2X₅ receptor was not seen in earlier studies (Collo et al., 1996). However, rather elaborate ionic conditions have allowed us to gain insight into the effects of this agonist, whereas earlier investigations may have been hampered by the Ca²⁺-dependent desensitization of P2X₅ receptors. Of the dinucleotides with three or more phosphates tested at rP2X₅, all were partial agonists and only Ap₄A was as potent as ATP.

The agonist properties of rP2X₁ receptors were reassessed in the hope of identifying selective agonists for rP2X₅ and rP2X₁ subtypes. When comparing EC₅₀ values, few compounds were wholly selective for homomeric rP2X₅. This led us to the conclusion that these two P2X receptor subtypes might be viewed as pharmacologically similar, although, structurally, their respective subunits are only 36% identical (Khakh et al., 2001a). Closer inspection of the agonist profiles revealed that GTP will activate rP2X₅ but not rP2X₁. However, there are no selective agonists that will activate rP2X₁ and not rP2X₅. Our attention turned to antagonists to identify similarities or differences in their activities at these two P2X subtypes. Differences were most obvious for TNP-ATP and Ip₅I, which blocked rP2X₁ receptors in the nanomolar range, whereas TNP-ATP was ~500 fold less potent at rP2X₅ and Ip₅I was relatively ineffective. In contrast, the potency of PPADS, suramin, and RB-2 was not vastly different at rP2X₅ and rP2X₁ receptors. Thus, if it was difficult to distinguish P2X₁ from P2X₅ receptors by agonist activity, it was easier to do so using TNP-ATP and Ip₅I as antagonists.

Setting aside the pharmacology of these two P2X receptor subtypes, it is important to consider whether or not homomeric P2X₅ receptors can exist naturally. From the Ca²⁺-sensitizing and desensitizing phenomena described here, it is difficult to see how homomeric P2X₅ receptors could be functional in the long term. P2X₅-ir occurs on the apical border of epithelial cells of the collecting duct in the rat kidney (Chan et al., 1998; Bailey et al., 2000; Schwiebert and Kishore, 2001); therefore, there may be circumstances in which the tubular fluid of the nephron may allow the resensitization of P2X₅ receptors, as shown here. We are in the process of determining the composition of tubular fluid in the collecting ducts of normal rats and in PKD models, in the hope of testing such extracellular fluids against the P2X₅ receptor. Others have shown that P2X₁ and other P2X subtypes are internalized after agonist activation and, thereafter, slowly externalized (Dutton et al., 2000; Li et al., 2000; Ennion and Evans, 2001; Khakh et al., 2001b; Bobanovic et al., 2002). Under such circumstances and with extracellular Ca²⁺ present, it may still be possible to activate freshly externalized P2X₅ receptors in the short term with ATP. This might be important where P2X₅ expression is up-regulated in developing and proliferating tissues. However, where P2X₅ subunits have been found in blood vessels, it seems likely that these subunits will be involved in heteromeric P2X_1/5 receptors. This heteromeric assembly, like homomeric P2X₁ receptors, is not believed to be sensitive to extracellular Ca²⁺ and differs from P2X₁ only by supporting a steady-state current in the continued presence of ATP. A last possibility is a post-translational modification of P2X₅ subunits to strip away Ca-desensitization, but there is no credible evidence yet for such a modification in mammalian kidney cells. Thus, the physiological role of the homomeric P2X₅ receptor remains a curious enigma, probably requiring P2X₅-knockout to unravel, although the present pharmacological data puts us in a better position to identify such assemblies wherever they might occur.