Regulated expression of the rat recombinant P2X3 receptor in stably transfected CHO-K1 tTA cells

Journal of the Autonomic Nervous System 81 (2000) 75–81 www.elsevier.com / locate / jans

Regulated expression of the rat recombinant P2X 3 receptor in stably transfected CHO-K1 tTA cells Wilhelm G. Lachnit a , Ian B. Oglesby a , Joel R. Gever a , Marina Gever a , Chiao-Chain Huang b , b b a a, Xing Cheng Li , Hong Jin , Joseph G. McGivern , Anthony P.D.W. Ford * a

Neurobiology Unit, Center for Biological Research, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, CA 94304, USA b CLONTECH Laboratories, Inc., Palo Alto, CA 94304, USA

Abstract In this report, the regulatible expression by tetracycline of the rat recombinant P2X 3 receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X 3 -receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 mM ATP evoked inward currents of 2.961.6 nA in transfected cells grown in the absence of tetracycline (tet2 ). The P2X 3 receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [ 35 S]ATPgS yielded a pKd of 8.260.1 and a Bmax of 31.963.5 pmol / mg protein in tet2 cell membranes and a pKd of 8.160.1 and a Bmax of 5.860.8 pmol / mg protein in tet1 cell membranes. The agonist ligands 2MeSATP and abMeATP displaced the binding of [ 35 S]ATPgS in tet2 cell membranes with very high affinity, yielding pIC 50 values of 9.460.2 and 7.560.2, respectively. In tet1 cell membrane, displacement of [ 35 S]ATPgS by 2MeSATP and abMeATP was of much lower affinity (pIC 50 values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [ 35 S]ATPgS binding in tet2 and tet1 cell membranes. In other experiments, cytosolic Ca 21 was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca 21 were elicited by 100 nM abMeATP in tet2 cells while no increases in cytosolic Ca 21 were detected below 100 mM abMeATP in either tet1 cells or untransfected cells. These calcium responses to abMeATP had a pEC 50 of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E / [A] curves to abMeATP yielding a pKB of 5.6. PPADS produced non-parallel, dextral shifts of E / [A] curves to abMeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X 3 receptor which is under regulated expression in a stably transfected mammalian cell line.  2000 Elsevier Science B.V. All rights reserved. Keywords: Rat P2X 3 receptor; [ 35 S]ATPgS; abMeATP; FLIPR; Nucleotide receptor

1. Introduction ATP has long been known as a ubiquitous intracellular source of energy in metabolism. Yet it was not until 1972 that Burnstock proposed that ATP was an active substance in the autonomic nervous system (Burnstock, 1972). Since then, ATP has been established as a neurotransmitter (Burnstock, 1976; Burnstock, 1978). Extracellular ATP has been shown to activate P2 receptors which are divided into two families according to their structure and transduction mechanism: P2Y receptors which are metabotropic and are coupled to G-proteins (review: King et al., 1998); *Corresponding author. Tel.: 11-650-3547-794; fax: 11-650-3547400. E-mail address: [email protected] (W.G. Lachnit)

and P2X receptors which are cation-selective ligand-gated ion channels (Abbracchio and Burnstock, 1994). Currently, seven P2X subunits, P2X 1 through P2X 7 , have been isolated from the rat (review: Buell et al., 1996). The deduced rat proteins, 379–595 amino acids long, share 35–59% identity with each other. They are predicted to contain two transmembrane domains, M1 and M2, connected by a large extracellular loop of |270 amino acids that contain 10 conserved cysteine residues, and intracellular amino and carboxyl termini (North, 1996). cDNA sequences for each of these subunits form functional ATPgated, cation-selective channels with electrophysiological and pharmacological profiles similar to that of P2X receptors in native tissues when heterologously expressed in cells such as Xenopus oocytes, HEK 293 cells and CHO-K1 cells (review: Buell et al., 1996). The P2X 3

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receptor was originally cloned from a rat dorsal root ganglion cDNA library and, when expressed in Xenopus oocytes, is characterized by its sensitivity to abMeATP and its rapid rate of desensitization (Chen et al., 1995; Lewis et al., 1995). The mRNA for the P2X 3 receptor is reported to be expressed in sensory neurons from trigeminal, dorsal root, and nodose ganglia (Chen et al., 1995; Lewis et al., 1995; Collo et al., 1996). Furthermore, recent immunohistochemical observations clearly demonstrate that P2X 3 is principally expressed by small- or mediumsized neurons in sensory ganglia (Vulchanova et al., 1997; Bradbury et al., 1998; Novakovic et al., 1999). These observations have led to the suggestion that this receptor plays a role in nociception (see Kennedy and Leff, 1995; Burnstock, 1996; Burnstock and Wood, 1996). Indeed, recently, Cook et al. (1997) reported ATP-evoked action potentials in nociceptive (tooth-pulp afferents) rat sensory neurons which had P2X 3 immunoreactivity in their sensory endings and cell bodies. The pharmacological characterization of these receptors in native systems has been difficult because of the lack of suitable pharmacological probes. Therefore, expression of P2X receptors in heterologous expression systems in high density is critical for functional characterization of these receptors and the identification of novel ligands. To this end, we sought a method to prepare a cell line in which the P2X 3 receptor would be stably transfected and could be expressed at high levels without introducing selection pressure / resistance to expression that would delimit the usefulness of such a recombinant cell line. In order to achieve this, we used a tetracycline-controlled gene expression system developed by Gossen and Bujard (1992) which has been demonstrated to give a higher level of gene expression than wild-type CMV-promoter / enhancer systems (Yin et al., 1996). Here, we report the regulated expression by tetracycline of the rat recombinant P2X 3 receptor in stably transfected CHO-K1 tTA cells (transfected with the tetracycline-controlled transactivator) and pharmacological analysis by radioligand binding using [ 35 S]ATPgS which has been reported, in the absence of divalent cations, to specifically label the P2X receptor (Michel and Humphrey, 1996; Michel et al., 1996). Functional pharmacological characterization was also conducted by measuring changes in intracellular calcium.

2. Methods

2.1. Cloning and transfection Poly (A1) RNAs were extracted from dorsal root ganglia from L4–L5 spinal levels of Sprague–Dawley rats using a Micro-FastTrack Kit (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was carried out with 1 mg of rat dorsal root ganglia poly (A1) RNA, 10 pmol of oligo (dT), and 200 units of SuperScript II (GIBCO BRL,

Gaithersburg, MD, USA) followed by reverse transcription-coupled polymerase chain reaction (PCR) using oligos X3P1-2: 59-GACTCCGCGGCTGTGAGCAGTTTCTCAGTATG and X3P2-2: 59-TCATGAATTCTGCAGCCTAAGGGTGAGCATG. PCR was performed and the resulting PCR product contained the entire coding region of the rat P2X3 gene as well as 21 nucleotides from the 59 untranslated sequence and 42 nucleotides from the 39 untranslated sequence. This cDNA fragment was inserted into the pCRII vector (Invitrogen), fully sequenced and then digested with restriction enzymes SacII and EcoRI, respectively. The insert was isolated and directionally cloned into the mammalian expression vector pTRE (CLONTECH Labs., Palo Alto, CA, USA) at SacII and EcoRI sites. pTRE is a tetracycline (tet)-regulatable expression vector developed by Gossen and Bujard (1992). The resulting construct was used for transfection to generate stably transformed CHO cell lines. To establish stably transfected cell lines, 5.0 mg of P2X 3 -pTRE DNA and 1.0 mg of pTK-Hygromycin DNA were incubated with 5 mg of CLONfectin (CLONTECH Labs.) for 30 min and overlaid onto 10 6 CHO-K1 tTA cells for 1 h in serum-free medium. Cells were then selected in the presence of 200 mg / ml of hygromycin. Positive individual colonies were selected by PCR following propagation of cells in the absence of tetracycline. Following selection, the cells were maintained in Ham’s F-12 nutrient media supplemented with 10% fetal bovine serum, G418 (250 mg / ml), hygromycin (200 mg / ml) and 1 mg / ml tetracycline at 378C in 7% CO 2. Immunoblots were performed on cell membranes as previously reported by Oglesby et al. (1999).

2.2. Electrophysiology ATP-gated currents were recorded from single cells using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Cells were clamped using an Axopatch 200A patch-clamp amplifier (Axon Instruments, Burlingame, CA, USA) and voltage-clamp protocols were generated by an IBM-compatible computer running PClamp software (version 6.0.3; Axon Instruments) and controlling a Digidata 1200 Interface (Axon Instruments). Recording pipettes were constructed from type 7052 borosilicate glass (Garner Glass Company, Claremont, CA, USA) to have tip diameters of about 2 mm and resistances of about 2 MV after fire polishing and filling. Following attainment of the whole-cell configuration, cells were held at a membrane potential (Vhold ) of 270 mV and ATP-gated currents were evoked by ‘‘Picopump’’ (Model PV830; World Precision Instruments, Sarasota, FL, USA) application of ATP-containing extracellular solution. Recordings began 400 ms before a 1 s application of ATP. Whole-cell currents were low-pass filtered (1 kHz, fourpole Bessel), digitized (5 kHz) and stored on magnetic

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medium for off-line analysis using AxoGraph software (version 2.0; Axon Instruments, Foster City, CA, USA). All recordings were made at room temperature (208C). The following solutions were used to record ATP-gated currents in patch-clamp experiments: extracellular (in mM), NaCl (147), KCl (2), HEPES (10), D-glucose (12), CaCl 2 (2), MgCl 2 (1), adjusted to pH 7.3 with NaOH; intracellular (in mM), K-aspartate (140), NaCl (20), HEPES (5), EGTA (10), adjusted to pH 7.3 with KOH. Extracellular solution containing 100 mM ATP was used to evoke the currents.

2.3. Radioligand binding Transfected and untransfected CHO-K1 tTA cell membranes were prepared as previously reported (Oglesby et al., 1999). Saturation binding isotherms were conducted by incubating membranes (5–10 mg protein) at room temperature for 90 min with 0.05–30 nM [ 35 S]ATPgS in the absence and presence of 10 mM ATPgS (200–500 ml) in 50 mM Tris, 1 mM EDTA buffer (pH 7.4). For competition experiments, displacement of 0.25 nM [ 35 S]ATPgS by various nucleotides as well as suramin, PPADS and reactive blue-2 was determined over a range of concentrations. The assays were terminated by vacuum filtration through wet, 20 mM Na 4 P2 O 7 pretreated GF / B glass fiber filters and washed for 10 s with 48C buffer. Equilibrium binding data were fitted to a one-site or two-site model and competition data was fitted to a four- or fiveparameter logistic equation using Graphpad Prism software (GraphPad Software, San Diego, CA, USA).

2.4. Calcium measurements For changes in cytosolic calcium measurements, P2X transfected and untransfected CHO-K1 tTA cells were plated onto 96-well viewplates (Costar, Cambridge, MA, USA) at a density of 50 000 cells / well for 24 h. The cells were then washed with 20 mM HEPES buffered-HBSS and incubated with 5 mM Fluo 3-AM in 20 mM HEPES buffered-HBSS buffer at 378C for 60 min. Extracellular dye was then washed with the same buffer. Cells were then incubated with vehicle or antagonist for 30 min and placed in a Fluorometric Imaging Plate Reader (Molecular Devices, Sunnyvale, CA, USA) and changes in fluorescence were monitored at 1 Hz. Baseline fluorescence was recorded for 10 s prior to the addition of abMeATP. Peak changes in fluorescence following the addition of abMeATP were measured. E / [A] curves were constructed by measuring peak changes in fluorescence using the four-parameter logistic Hill equation in GraphPad Prism. Data presented are means6standard error of the mean (S.E.M.). Single curves are represented in the figures which are representative of at least three independent experiments.

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2.5. Materials Ham’s F-12 Nutrient Media, Hank’s Balanced Salt Solution, phosphate-buffered saline, geneticin (G418), fetal bovine serum and versene (EDTA) were obtained from Gibco (Gaithersburg, MD, USA). 2MeSATP, abMeATP, bgMeATP, suramin and PPADS (chemicalstructure?) were obtained from Research Biochemicals (Natick, MA, USA). Hygromycin was obtained from Calbiochem (La Jolla, CA, USA). Fluo 3-AM was obtained from Molecular Probes (Eugene, OR, USA). Tetracycline, reactive blue-2, ATP, ADP, AMP, ATPgS, adenosine, bulk chemicals, and reagents were obtained from Sigma (St. Louis, MO, USA). Solutions were prepared in deionized water or dimethylsulfoxide.

3. Results and discussion In order to characterize the P2X 3 receptor fully, we sought an expression method that would produce the P2X 3 receptor at very high density in a stably transfected cell line. We chose the tetracycline-controlled transactivator (tTA) gene expression system as it has been reported previously that the maximal gene expression level is higher than that of the wild-type CMV promoter / enhancer-driven system (Yin et al., 1996). In this expression system, a modified CMV promoter with a tet-repressor binding element is regulated by a fusion of the tet-repressor and the herpes simplex virus VP-16, designated tTA (Gossen and Bujard, 1992), which is constitutively expressed in CHOK1 tTA cells. Therefore, this strategy also allows controlled regulation of gene expression by the addition or removal of tetracycline in the growth media. Following transfection with the P2X 3 receptor into the CHO-K1 tTA cell line, a wide range of clonal variation in P2X 3 receptor expression levels was observed (data not shown). However, several clonal cell lines that were under tet-regulation with very high expression levels were identified. These cells were continuously maintained in the presence of 0.1 mg / ml tetracycline to prevent the expression of the P2X 3 receptor. Fig. 1A shows that following the removal of tetracycline from the growth media, the P2X 3 receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Maximal [ 35 S]ATPgS binding density obtained from saturation binding isotherms using cell membrane preparations also increased in a time dependent manner and were much greater than P2X 3 transfected CHO-K1 tTA cells grown in the presence of tetracycline (Fig. 1B). From these initial results, further characterization of the P2X 3 transfected cell line was conducted using cells that were cultured for 192 h following the removal of tetracycline (tet2 ). Saturation binding isotherms using [ 35 S]ATPgS yielded

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transfected cell membranes (Table 1). The maximal binding densities (up to 40 pmol / mg protein) observed in this study were higher than expected for heterologous expression of a membrane receptor protein in a mammalian system. However, these binding densities were similar in P2X 3 and P2X 4 infected CHO cells using the Semliki forest virus (SFV) expression system previously reported (Michel et al., 1996; Michel et al., 1997). The SFV infection system has also been shown to generate very high levels of gene expression in mammalian cells (Lundstrom et al., 1994). However, unlike the regulatible gene expression system used in this study, the SFV expression system is a transient expression system. The agonist ligands 2MeSATP and abMeATP displaced the binding of 0.25 nM [ 35 S]ATPgS with high affinity, yielding pIC 50 values of 9.360.2 and 7.760.2, respectively (Table 1). In tet1 transfected cell membranes, displacement of 0.25 nM [ 35 S]ATPgS by 2MeSATP and abMeATP was of much lower affinity (pIC 50 values of 8.1 and 5.4, respectively) (Table 1). The relatively high affinity of abMeATP is consistent with its agonist activity at this receptor (Chen et al., 1995; Lewis et al., 1995). Other nucleotide agonists, such as ATP, ADP and UTP showed similar displacement of [ 35 S]ATPgS binding in tet2 and tet1 transfected cell membranes (Table 1). Although agonist ligand affinities using [ 35 S]ATPgS were consistently 5–10-fold lower than previously reported (Michel et al., 1996; Miller et al., 1996), the rank order of agonist affinities was consistent with the previous report. Moreover, the rank order of agonist potencies originally reported for this receptor (Chen et al., 1995; Lewis et al., 1995) are similar to those observed in competition binding studies. The affinity differences observed in this study and those previously reported by Michel et al., (1996), may be due to a number of factors such as experimental binding conditions which could lead to changes in the affinity state of the receptor. In whole cell patch clamp experiments, 100 mM ATP

Fig. 1. Regulatible expression of the P2X 3 receptor in P2X 3 -pTRE transfected CHO-K1 tTA cells. (A) Immunoblot analysis of P2X 3 transfected CHO-K1 tTA cell membranes grown in the presence (1) and 24 h (2), 48 h (3), 96 h (4) and 192 h (5) in the absence of 0.1 mg / ml tetracycline. Saturation binding isotherms of [ 35 S]ATPgS to P2X 3 transfected CHO-K1 tTA cell membranes grown in the presence (j) and 24 h (.), 48 h (m), 96 h (d), and 192 h (♦) in the absence of 0.1 mg / ml tetracycline. Representative values presented are from single experiment performed in duplicate.

a pKd of 8.260.1 and a Bmax of 31.963.5 pmol / mg protein in tet2 transfected cell membranes and a pKd of 8.160.1 and a Bmax of 5.860.8 pmol / mg protein in tet1

Table 1 Summary of binding studies at the recombinant rat P2X 3 receptor labeled with [ 35 S]ATPgS P2X 3 (tet2 ) Bmax (pmol / mg protein) ATPgS

ATP ATPgS abMeATP bgMeATP 2MeSATP ADP AMP UTP a

31.963.5

Mock (tet1 ) KD (nM) 8.260.1

Bmax (pmol / mg protein) 5.860.8

KD (nM) 8.160.1

pIC 50

nH

pIC 50

nH

8.5 8.4 7.7 / 4.9 a 6.5 9.3 / 5.0 a 6.9 ,4 4.0

1.1 0.8

8.2 8.5 5.4 7.0 8.1 7.3 ,4

0.8 0.9 0.5 1.0 0.7 1.0

Data fit to a five-parameter logistic equation.

0.8 1.0 1.0

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Fig. 2. Representative trace showing inward current in response to 100 mM ATP in (tet2) P2X 3 transfected CHO-K1 tTA cells.

evoked quickly rising, rapidly desensitizing, large inward currents ranging from 0.1 to 10 nA (2.961.6 nA; n57) in tet2 transfected cells (Fig. 2). No currents were observed in tet1 transfected cells or in untransfected cells (data not shown). These currents which desensitized rapidly were consistent with what has been reported by other laboratories using Xenopus oocyte expression systems (Chen et al., 1995; Lewis et al., 1995) as well as transient mammalian expression systems such as P2X 3 SFV infected CHO cells (Evans et al., 1995) and P2X 3 transfected HEK 293 cells (Stoop et al., 1997).

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abMeATP (0.1–100 mM) elicited concentration-dependent increases in fluorescence in fluo 3 loaded tet2 transfected cells yielding a p[A] 50 of 6.7 while no increases in cytosolic Ca 21 were detected below 100 mM abMeATP in either tet1 transfected cells or in untransfected cells (Fig. 3). The increase in fluorescence following the addition of abMeATP occurred rapidly (peaking within 3 to 4 s) and was transient, returning to baseline within 2–3 min (Fig. 3). In tet2 transfected cells, suramin (1–100 mM) produced concentration-dependent, parallel, dextral shifts of E / [A] curves to abMeATP without a change in maxima, consistent with simple competitive antagonism (Fig. 4A). Schild regression analysis yielded a pKB estimate of 5.6 with a slope that was not significantly different from unity. The functional affinity estimate for suramin is consistent with the affinity estimate obtained from competition studies with [ 35 S]ATPgS (data not shown). In contrast to suramin, PPADS (0.1–3 mM) produced dextral insurmountable concentration-dependent shifts in the E / [A] curves to abMeATP (Fig. 4B) and thereby appearing as pseudoirreversible as described previously (Evans et al., 1995). A functional agonist profile was not possible as CHO-K1 cells express an endogenous P2 receptor linked to increases in intracellular calcium that is activated by both ATP and UTP but not abMeATP (Iredale and Hill, 1993; Lachnit et al., unpublished observations). In summary, we have demonstrated that a high expression level of the P2X 3 receptor in a stably transfected mammalian cell line is achievable using a regulatible gene expression system and that the pharmacological properties of this homomeric receptor are consistent with what has been reported previously. Furthermore, this tetracycline

Fig. 3. Representative fluorescence traces showing intracellular calcium responses to abMeATP (0.01–10 mM) in untransfected (??????), tet1 P2X 3 transfected ( ), and tet2 P2X 3 transfected (———) CHO-K1 tTA cells. Arrows indicate addition of to abMeATP.

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Fig. 4. (A) The effect of suramin on intracellular calcium changes to abMeATP in tet2 P2X 3 transfected CHO-K1 tTA cells. A representative concentration–effect curve is shown for abMeATP which was repeated four to five times in the absence and presence of 1 mM (j), 3 mM (m), 10 mM (.) 30 mM (♦), and 100 mM (d) suramin. (B) The effect of PPADS on intracellular calcium changes to abMeATP in tet2 P2X 3 transfected CHO-K1 tTA cells. A representative concentration–effect curve is shown for abMeATP which was repeated four to five times in the absence and presence of 0.1 mM (j), 0.3 mM (m), 1 mM (.) and 3 mM (♦) PPADS. Values presented are expressed as change in fluorescence units.

controlled gene expression system has been shown to be a powerful tool in the quantitative analysis of P2X receptor function. Whether or not these gene expression systems can unrestrictedly generate mammalian cell lines with expression levels of this magnitude with other membrane receptors such as G-protein coupled receptors, remains to be investigated. If so, this system will be very useful in the critical and quantitative evaluation of such receptors.

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