JBC PeproTech; Our Business is Cytokines!

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M408203200 on August 3, 2004

J. Biol. Chem., Vol. 279, Issue 40, 41650-41657, October 1, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/40/41650    most recent
M408203200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Agboh, Kelvin. C.
Right arrow Articles by Ennion, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Agboh, Kelvin. C.
Right arrow Articles by Ennion, S. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Functional Characterization of a P2X Receptor from Schistosoma mansoni*

Kelvin. C. Agboh{ddagger}, Tania E. Webb§, Richard J. Evans{ddagger}, and Steven J. Ennion{ddagger}

From the {ddagger}Department of Cell Physiology and Pharmacology, University of Leicester, P. O. Box 138, Leicester LE1 9HN, United Kingdom and the §Cell Signalling Laboratory, Leicester School of Pharmacy, The Hawthorn Building, De Montfort University, Leicester LE1 9BH, United Kingdom

Received for publication, July 20, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cloning and characterization of a P2X receptor (schP2X) from the parasitic blood fluke Schistosoma mansoni provides the first example of a non-vertebrate ATP-gated ion channel. A number of functionally important amino acid residues conserved throughout vertebrate P2X receptors, including 10 extracellular cysteines, aromatic and positively charged residues involved in ATP recognition, and a consensus protein kinase C site in the amino-terminal tail, are also present in schP2X. Overall, the amino acid sequence identity of schP2X with human P2X1–7 receptors ranges from 25.8 to 36.6%. ATP evoked concentration-dependent currents at schP2X channels expressed in Xenopus oocytes with an EC50 of 22.1 µM. 2',3'-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) was a partial agonist (maximum response 75.4 ± 4.4% that of ATP) with a higher potency (EC50 of 3.6 µM) than ATP. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid blocked schP2X responses to 100 µM ATP with IC50 values of 9.6 and 0.5 µM, respectively. Ivermectin (10 µM) potentiated currents to both ATP and Bz-ATP by ~60% with a minimal effect on potency (EC50 of 18.2 and 1.6 µM, respectively). The relative permeability of schP2X expressed in HEK293 cells to various cations was determined under bi-ionic conditions. schP2X has a relatively high calcium permeability (PCa/PNa = 3.80 ± 0.29) and an estimated minimum pore diameter similar to that of vertebrate P2X receptors. SchP2X provides a useful comparative model for the better understanding of human P2X receptor function and may also provide an alternative drug target for treatment of schistosomiasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
P2X receptors comprise a family of cation-selective ion channels gated by extracellular ATP. Mammalian species possess seven distinct P2X channel subtypes (P2X1–7), each encoded by a separate gene. These subunits assemble as functional homotrimeric or heterotrimeric channels and play an important role in a wide array of physiological processes including neurotransmission, smooth muscle contraction, immune cell function, and platelet aggregation (for a recent review see Ref. 1). This remarkably diverse range of physiological roles for P2X receptors has contributed to the notion that ATP is a "primitive" extracellular signaling molecule (2, 3). Functional evidence suggests that ATP-gated ion channels exist in some lower organisms including Tetrahymena thermophilia (4), leech (5), and Amoeba proteus (6), supporting the view that the development of P2X receptors for ATP occurred relatively early in the evolution of eukaryotic organisms. However, to date, definitive molecular identification of P2X channels is restricted to vertebrate species including fish (7), amphibians (8, 9), birds (10), and mammals (1).

With the recent expansion in the range of species for which genomic and EST1 sequence databases are available, it is now possible to use a bioinformatics approach to screen a wide range of lower organisms for P2X receptor-like proteins. Using such a strategy, we have identified a P2X channel from the trematode worm Schistosoma mansoni. This human blood fluke is one of three main species of blood parasite that cause the disease schistosomiasis in humans. Approximately 200 million people are infected with schistosomes worldwide, and the disease is endemic in countries throughout Africa, South America, and the Middle East (11). Schistosomiasis is an insidious disease, often not diagnosed until the pathology is well advanced and impacting most on childhood development (12). The World Health Organization estimates 0.2 million deaths per year from schistosomiasis with an annual morbidity impact of 1.76 million disability-adjusted life years, making this disease a substantial global health problem.

Similarly as in vertebrate species, the control of intracellular calcium is fundamental in S. mansoni cell physiology, particularly in the function of adult muscle, which shows morphological and biochemical similarities with vertebrate smooth muscle (13). Praziquantel, the only currently available drug used for the effective treatment of schistosomiasis, acts by an unknown mechanism, possibly through the modulation of calcium channel {beta}-subunit function (14), to cause a rapid influx of calcium resulting in muscle contraction and paralysis. However, praziquantel-resistant strains of schistosomes are now emerging (15), and there is an acute need for alternative drugs and targets that will allow the alternation of treatment regimes to prevent the development of further resistant strains (16). P2X channels are known to have a significant calcium permeability (17) and to play a central role in mammalian smooth muscle contraction (1). The functional characterization of a S. mansoni P2X channel is therefore important from a therapeutic standpoint, as this receptor may represent a novel target for the development of new drugs to treat schistosomiasis or as an antigenic target for vaccines against this parasite. Furthermore, a better understanding of P2X receptors in a simple invertebrate system may shed new light on structure-function aspects of the receptor and provide insights into new signaling pathways for P2X receptors that could be extrapolated to better understand human P2X receptor function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of the S. mansoni P2X Receptor—A S. mansoni EST sequence (accession number CD081583 [GenBank] ) showing sequence homology to mammalian P2X receptors was identified by BLAST searches of the GenBankTM EST data base. The corresponding clone for this EST sequence (SmAE 609847.1) appeared to contain a partial cDNA consisting of ~130 carboxyl-terminal amino acids and the 3'-untranslated region of a P2X-like gene. To obtain a full-length cDNA for this gene, PCR was performed on a S. mansoni cDNA library obtained from Dr. A. Agnew, University of Leeds, Leeds, United Kingdom (18). The forward primer (5'-GTGGTAACAACGCAGAGTA-3') corresponded to the polylinker sequence in the library vector (Clontech SMART system), whereas the reverse primer (5'-AGTGAAGATTGAAGGAAACG-3') corresponded to a 3'-untranslated region sequence from the CD081583 [GenBank] EST. A one-fiftieth dilution of the cDNA library was used as the template in a PCR reaction containing 200 µM dNTP, 1.5 mM MgCl2, 25 pmol of each primer, 1x Opti-Buffer (Bioline, London, United Kingdom), and 2.5 units of Bio-X-Act TaqDNA polymerase (Bioline). Thermal cycling consisted of 28 repetitions at 94 °C for 1 min, 48 °C for 1 min, and 68 °C for 1.5 min. This was followed by incubation at 68 °C for 7 min. The PCR product was separated on a 1% agarose gel, recovered, and cloned into a pcDNA3.1 (Invitrogen) plasmid that had been modified to contain a poly(A) tail adjacent to the 3'-untranslated region of the cloned cDNA. This cDNA insert was subsequently recloned by PCR to generate a construct (schP2X) containing a consensus Kozak sequence and the 5'-untranslated region of the human P2X1 receptor. This modification was necessary to obtain expression in Xenopus oocytes and did not change the amino acid sequence from the original non-Kozak-containing clone. Cloned inserts were sequenced on both strands using vector and insert-specific primers (Automated ABI Sequencing Service, University of Leicester, Leicester, United Kingdom).

Electrophysiological Recordings in Xenopus Oocytes—Sense strand cRNA was generated from the schP2X plasmid using the T7 mMessage mMachineTM kit (Ambion) according to the manufacturer's instructions. cRNA was quantified by spectrophotometry and dissolved in nuclease-free water at a concentration of 1 µg/µl. Manually defolliculated stage V-VI Xenopus oocytes were injected with 50 nl (50 ng) of cRNA using an Inject+Matic microinjector (J. Alejandro Gaby, Geneva, Switzerland) and stored at 18 °C in ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM sodium pyruvate, and 5 mM HEPES, pH 7.6) prior to recording 3–7 days later.

Two-electrode voltage clamp recordings were made from oocytes using a Turbo TEC 10C amplifier (NPI Electronic Instruments, Tamm, Germany) with a Digidata 1200 analogue to digital converter (Axon Instruments) and WinWCP acquisition software (Dr. J. Dempster, University of Strathclyde, Strathclyde, Scotland). Microelectrodes were filled with 3 M KCl, and the external solution consisted of ND96 buffer with 1.8 mM BaCl2 replacing the 1.8 mM CaCl2 to prevent activation of endogenous calcium-activated chloride channels (19). Membrane currents were recorded at a holding potential of –60mV. The agonists, ATP (magnesium salt) (Sigma) and Bz-ATP (Sigma) were applied from a U-tube perfusion system (20), whereas ivermectin and antagonists (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin (Sigma)) were bath-perfused in addition to being present at the appropriate concentration in the U-tube with the agonist. Concentration-response curves were constructed using a 5-min recovery period between applications and by normalizing data points to two applications of 100 µM ATP (one preceding and one following the data point concentration). Non-injected and water-injected oocytes tested from at least seven separate batches of oocytes gave no detectable currents in response to ATP or Bz-ATP application (range 100 µM to 1 mM).

Data are presented as mean ± S.E. Differences between means were tested using Student's paired t test. Concentration response data were fitted with the equation Y = ((X)H·M)/((X)H + (EC50)H), where Y is response, X is agonist concentration, H is the Hill coefficient, M is maximum response, and EC50 is the concentration of agonist evoking 50% of the maximum response. pEC50 is the –log10 of the EC50 value.

Permeability Studies of schP2X Expressed in Human Embryonic Kidney (HEK) Cells—HEK293 cells were cultured in six-well plates with Optimem1 plus Glutamax (Invitrogen). Cells were transiently transfected with a mixture of schP2X plasmid (3.6 µg) and pEGFP-C1 (Clontech) (40 ng) using LipofectAMINE according to the manufacturer's instructions (Invitrogen). The green fluorescent protein (GFP) plasmid (pEGFP-C1) was included to allow visualization of transfected cells. For whole cell patch clamp recordings, cells were superfused at 2 ml/min with an external solution consisting of either 154 mM NaCl, 154 mM Tris-Cl, 154 mM methylamine-HCl, 154 mM dimethylamine-HCl, or 154 mM triethylammonium chloride, 2.5 mM HEPES, and 20 mM L-histidine (pH 7.3). Electrodes (2–5 megaohms) were filled with 154 mM NaCl, 10 mM EGTA, and 10 mM HEPES (pH 7.3). Currents were recorded at room temperature using an Axopatch 200B amplifier, and data were collected using pClamp8.2 software (Axon Instruments). ATP was applied rapidly using a U-tube perfusion system (20). Liquid junction potentials had been measured previously, and no correction was made for the small offset. The reversal potential (Vrev) values for currents in different test cation solutions were obtained using a dual ramp protocol that stepped from a holding potential of –60mV to 60mV for 0.5 s followed by a ramp-down to –100mV over 1.6 s and a ramp-up to 60mv over 1.6 s before a step-back to –60mV. This ramp protocol was applied in the absence and presence of ATP (100 µM), and the reversal potential was taken as the points where the current traces crossed on the ramps (Fig. 6A). Recordings were made from single GFP-expressing cells following at least 5 min of whole cell recording to allow for dialysis of the intracellular solution. The difference between the two crossover points obtained for each cell (up-ramp and down-ramp) was typically <1 mV, and the mean of the two values was taken as the reversal potential of the cell. Relative permeability ratios for monovalent cations were calculated from the equation Px/PNa = exp({Delta}Vrev F/RT), where F is Faraday's constant, R is the universal gas constant, and T is the absolute temperature.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 6.
Relative permeability of cations. Reversal potentials in various external cation solutions were determined for schP2X receptors expressed in HEK293 cells by applying a ramp protocol in the presence and absence of ATP. A, example current traces in 154 mM Tris-Cl. The reversal potential was taken as the mean value of the two crossover points between the current in the absence of ATP and the current in the presence of ATP (100 µM). The voltage ramp protocol is indicated below the current traces. B, permeability ratios (square root) plotted against the median diameter of the external cation. TEA, triethylammonium. C, high external calcium (112 mM) inhibits the current evoked by 100 µM ATP. This inhibition was overcome by increasing the concentration of ATP to 1 mM.

 
Permeability measurements of the divalent cation calcium were made with an external solution with 112 mM calcium of the composition 110 mM CaCl2, 1.8 mM Ca(OH)2, 10 mM HEPES, and 12 mM glucose (pH 7.3). ATP was applied at 1 mM, and the reversal potential was determined as described above. PCa/PNa was calculated from the equation PCa/PNa = [Na+]iexp(Vrev F/RT)(1 + exp(VrevF/RT))/4[Ca2+]o.

Ion activities were corrected for using values of 0.75 for sodium and all other monovalent cations and 0.25 for calcium at 112 mM (21). Atomic dimensions of the ions used were obtained by Prof. Alan North (University of Manchester, Manchester, United Kingdom) from spacefilling models (van der Waals radii) of energy-minimized conformations using Desktop Molecular Modeler software. The diameter of the ions was taken as the median value for measurements taken in the x, y, and z axes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of the schP2X cDNA—PCR on the S. mansoni cDNA library using a vector polylinker forward primer and a S. mansoni-specific reverse primer yielded a product of 1492 bp that contained an open reading frame of 1314 bp. When cRNA prepared from this construct was injected into Xenopus oocytes, no functional P2X channels were detected. This original PCR clone lacked a consensus vertebrate Kozak sequence at the initiation codon of the open reading frame. One possible explanation for the lack of functional expression was an inability of Xenopus oocytes to recognize an invertebrate translation initiation codon. We therefore recloned the Schistosoma P2X PCR product, introducing a consensus Kozak sequence at the initiation codon and substituting the 5'-untranslated region of the Schistosoma gene with the 5'-untranslated region of the human P2X1 gene. The introduction of these two modifications resulted in the expression of functional P2X channels when cRNA was injected into Xenopus oocytes. Neither modification changed the amino acid sequence from the original non-Kozak-containing PCR clone. The nucleotide sequence of the cDNA clone amplified from the S. mansoni cDNA library is available in the EMBL data base under the accession number AJ783803 [GenBank] . This sequence appears to correspond to the same gene represented by the partial EST clone SmAE 609847.1 (22), because the overlapping sequence between the two clones (373 bp) is identical.

The predicted amino acid sequence of schP2X (437 amino acids) shows homology to the human P2X receptor family ranging from 25.8% (P2X7) to 36.6% identity (P2X4). Prediction of the membrane topology using the TopPred algorithm (23) suggests a typical P2X topology with intracellular carboxyl and amino termini, two transmembrane domains, and a large extracellular loop region (Fig. 1). Six Asn-Xaa-Ser/Thr sequons (Asn-71, Asn-170, Asn-187, Asn-223, Asn-275, and Asn-305) are present in the putative extracellular loop region of the schP2X receptor, two of which (Asn-187 and Asn-305) are conserved in some mammalian P2X receptors and have been shown to be glycosylated in the rat P2X1 receptor (24).



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 1.
Alignment of the schP2X amino acid sequence with human P2X receptors. The predicted amino acid sequence of the schP2X receptor is shown aligned with the human P2X1–6 sequences. Predicted transmembrane regions (TopPred algorithm) (23) are shaded gray. Positively charged residues (lysine and arginine), cysteine, and a conserved consensus protein kinase C phosphorylation site (T18), which are conserved throughout the whole family of P2X isoforms, are shaded black. Numbering shown above the sequence corresponds to the human P2X1 sequence. Swiss-Prot accession numbers for sequences in the alignment are P51575 [GenBank] , Q9UBL9, P56373 [GenBank] , Q99571 [GenBank] , and O15547 [GenBank] for human P2X1–4 and P2X6, respectively. The sequence used for human P2X5 corresponds to Q93086 [GenBank] with exon 10 inserted as described by (38).

 
Nucleotide-evoked Currents in Xenopus Oocytes Expressing schP2X—ATP evoked inward currents at recombinant schP2X receptors expressed in Xenopus oocytes clamped at –60mV (Fig. 2A). The first currents in response to 100 µM ATP (mean peak amplitude was –5.86 ± 1.36 µA) decayed in the continued presence of agonist with a T-50% (time for current to decay to 50% of peak response) time of 6.16 ± 0.052 s (n = 9). After 5 min of recovery time between applications, the second responses had a lower peak amplitude (–3.73 ± 1.25 µA, n = 9; p < 0.01) and longer T-50% time (14.6 ± 0.07 s, n = 9; p < 0.01). Subsequent responses with 5 min between applications did not differ significantly from the second response.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2.
Concentration response data for ATP and Bz-ATP induced currents in schP2X-expressing oocytes. Two-electrode voltage clamp recordings at a holding membrane potential of –60mV were made from oocytes expressing schP2X receptors. A, schP2X receptor currents recorded in response to ATP, ADP, UTP, and AMP-CPP ({alpha},{beta}-me-ATP) (100 µM; indicated by bar). Only the application of ATP resulted in an inward current, with the second response to ATP showing a lower amplitude and slower desensitization (applications 5 min apart). B, example currents recorded in response to ATP and Bz-ATP (concentrations in µM; agonist application indicated by bar). C, concentration-response curves for ATP and Bz-ATP. Mean currents were normalized to the response given by 100 µM ATP (n = 6–7 oocytes).

 
ATP evoked concentration-dependent responses with an EC50 value of 22.1 µM (pEC50 4.68 ± 0.06, n = 7) and a Hill slope of 1.2 ± 0.1. (Fig. 2C). The ATP analogue Bz-ATP produced similar currents to those of ATP. However, Bz-ATP was a partial agonist (maximum response of Bz-ATP was 75.4 ± 4.4% that of the maximum response produced by ATP) with a higher (p < 0.01) potency (EC50 of 3.6 µM and pEC50 of 5.54 ± 0.06, n = 6) than ATP. AMP-CPP, ADP, UTP, UDP, CTP, and ITP (100 µM) did not evoke currents at the schP2X receptor.

Effects of the Antagonists PPADS and Suramin—The P2 receptor antagonist surinam reduced schP2X responses evoked by ATP (100 mM) in a concentration-dependent manner with an IC50 of 9.6 µM (pIC50 of 5.09 ± 0.13 µM, n = 5) (Fig. 3). At lower concentrations (0.1–10 µM), PPADS was more effective than suramin at blocking responses to 100 µM ATP at the schP2X receptor (IC50 of 0.5 µM; pIC50 of 6.81 ± 0.61 µM, n = 7). However, there was a PPADS-resistant component to the schP2X current (~20% of the maximum current) that persisted at concentrations of up to 100 µM PPADS. This PPADS-resistant component was largely blocked when 100 µM ATP plus 100 µM suramin was applied to oocytes previously equilibrated with 100 µM PPADS (mean current in 100 µM PPADS was 22.11 ± 1.96% with no antagonist response (n = 8); mean current in 100 µM PPADS plus 100 µM suramin was 7.02 ± 1.18% with no antagonist response (n = 6)). The current produced by 100 µM ATP in the presence of both 100 µM suramin and 100 µM PPADS was higher (p < 0.01) than the current produced in the presence of 100 µM suramin (4.34 ± 0.25%, 100 µM ATP-only current, n = 12).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3.
Antagonism of schP2X currents by PPADS and suramin. The sensitivity of schP2X receptors to the antagonists PPADS and suramin was determined in Xenopus oocytes at a holding membrane potential of –60mV. A, example currents in response to 100 µM ATP in the presence of varying concentrations of PPADS and suramin (concentrations shown in µM). B, mean responses to 100 µM ATP in the presence of PPADS (closed circles) and suramin (open circles). PPADS was a more effective antagonist than suramin in the range of 0.1–30 µM. However, at 100 µM PPADS there was a PPADS-resistant component of the schP2X current. C, bar chart of mean currents (percentage of 100 µM ATP response) in the presence and absence of PPADS and suramin. Note the mean current obtained when suramin and PPADS (100 µM each) were applied to oocytes preincubated in 100 µM PPADS, showing that suramin could antagonize the PPADS-resistant component of the schP2X current.

 
Potentiation of schP2X Currents by Ivermectin—Ivermectin, a derivative of fermentation products of Streptomyces avermitilis, is a broad spectrum, anti-parasite medication that has been shown previously to potentiate currents in human and rat P2X4 channels (25, 26). We therefore tested the effects of ivermectin on schP2X currents. Ivermectin potentiated schP2X currents evoked by both ATP and Bz-ATP. The maximum effect of ivermectin was seen at 10 µM (effects of 0.1, 1, 10, and 100 µM ivermectin were tested at an EC50 concentration of ATP (20 µM); data not shown). At this concentration, ivermectin increased the response to 100 µM ATP by 66 ± 24% (p < 0.05, n = 6) (example traces are shown in Fig. 4A). The onset of the potentiation by ivermectin was relatively rapid (<5 min exposure for maximum effect). However, after ivermectin washout, currents remained potentiated, taking 30 min to return to their pre-ivermectin exposure amplitude (Fig. 4A). Concentration response curves for both ATP and Bz-ATP in the presence of 10 µM ivermectin showed similar EC50 values to those obtained in the absence of ivermectin (ATP had an EC50 of 18.2 µM, a pEC50 of 4.84 ± 0.15, and a Hill slope of 1.1 ± 0.19, n = 6; Bz-ATP had an EC50 of 1.6 µM, a pEC50 of 5.83 ± 0.10, and a Hill slope of 2.2 ± 0.74, n = 5) (Fig. 4B). However, the maximum response for both ATP and Bz-ATP was increased in the presence of 10 µM ivermectin. For ATP the top of the dose-response curve was 179 ± 29% of the response to 100 µM ATP in the presence of 10 µM ivermectin (versus 120 ± 7% in the absence of ivermectin), and for Bz-ATP the top of the dose response curve was 120 ± 14% of the response to 100 µM ATP in the presence of 10 mM ivermectin (versus 92 ± 6% in the absence of ivermectin).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 4.
Effects of ivermectin on schP2X currents. Two-electrode voltage clamp recordings from oocytes expressing schP2X receptors. A, representative currents to 100 µM ATP in the presence and absence of 10 µM ivermectin (ATP and ivermectin applications are indicated by bars). B, concentration-response curves for ATP (closed squares) and Bz-ATP (closed circles) in the presence of 10 µM ivermectin. For comparison, concentration-response curves in the absence of ivermectin (data presented in Fig. 2) are shown in gray. Mean currents were normalized to the response produced by 100 µM ATP in the absence of ivermectin (n = 5–6 oocytes). Holding membrane potential was –60mV.

 
Reversal Potential of schP2X Currents in Xenopus Oocytes—To investigate the current-voltage relationship of schP2X currents, we determined the reversal potential in Xenopus oocytes by applying a ramp protocol from –100mV to 100mV in the presence and absence of 100 µM ATP (Fig. 5A). The subtracted current (current in the presence of ATP – current in the absence of ATP) was essentially linear with a mean reversal potential of –7.2 mV ± 3.0, n = 12 (Fig. 5C). A similar result was also obtained when peak currents at different holding potentials were plotted (Fig. 5, D and E). Because it is not possible to control the internal ionic composition of Xenopus oocytes via a patch clamp pipette, further experiments investigating the relative permeabilities of various cations were conducted in the human HEK293 cell line.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 5.
Current voltage relationship of schP2X expressed in Xenopus oocytes. A, the reversal potential of ATP-mediated currents through the schP2X channel was determined by applying a ramp protocol from –100 mV to 100 mV in the presence of 100 µM ATP (indicated by bar). B, plot of the subtracted current (current in the presence of ATP – current in the absence of ATP) against voltage during the ramp for the current depicted in panel A. C, steady state determination of reversal potential. ATP-induced (100 µM, indicated by bar) currents were recorded at holding potentials ranging from –40 to 40 mV with a 5-min interval between applications. D, plot of current against voltage for the currents depicted in panel C.

 
Monovalent Cation Permeability in HEK293 Cells—To investigate the relative permeability of various cations, we expressed the schP2X channel in HEK293 cells. As HEK293 cell transfections contained 90 times more schP2X plasmid than GFP plasmid, we assumed that any GFP-positive cells would also be cotransfected with schP2X. However, we only observed schP2X currents in 53% of GFP positive cells, suggesting that expression of schP2X and/or correct trafficking to the cell membrane was less efficient in HEK293 cells compared with Xenopus oocytes (100% of injected oocytes showed schP2X currents). The reversal potentials of schP2X currents in HEK293 cells with different extracellular cations are shown in Table I. The permeability ratios (to sodium) of the monovalent cations tested roughly relate to the ionic size of the cation (Fig. 6B) and extrapolate to an estimated minimum pore diameter of 0.9 nM.


View this table:
[in this window]
[in a new window]
  		TABLE I
Reversal potentials and relative permeability ratios for external cations

Values are means of 4–8 cells for each cation ± S.E.

 
Calcium Permeability—The relative permeability to calcium was determined from the reversal potential of schP2X currents in 112 mM extracellular calcium solution as described previously (17, 27). In 112 mM external calcium, currents in response to 100 µM ATP were 90 ± 2.7% (p < 0.01) smaller than the equivalent currents in 154 mM external sodium solution. This current inhibition at high external calcium could be overcome by using a higher concentration of ATP (1 mM ATP in 112 mM calcium produced currents corresponding to 111 ± 9.4% of currents produced by 100 µM ATP in external 154 mM sodium solution) (Fig. 6C). The reversal potential to ATP (1 mM) in 112 mM Ca2+ external solution was 10.25 ± 1.37 mV (Table I). This value corresponds to a relative permeability for calcium to sodium of 3.80 ± 0.29. Given this high calcium permeability of schP2X and the fact that praziquantel, the only currently available drug for the treatment of schistosomiasis, is known to act via a mechanism involving a rise in intracellular calcium (14), we explored the possibility that some of the actions of praziquantel could be mediated via modulation of schP2X currents. Praziquantel (up to 100 µM), however, had no effect on schP2X channels expressed in Xenopus oocytes (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study describes the first example of a non-vertebrate P2X receptor and therefore provides definitive molecular evidence that the range of species known to utilize ATP as an extracellular signaling molecule extends beyond vertebrates. The presence of a P2X receptor in an invertebrate species supports the hypothesis that the emergence of P2X receptors for extracellular ATP occurred relatively early in eukaryotic evolution. However, it is interesting to note that no P2X-like protein sequences are present in the three invertebrate species for which complete genomic sequences are available (Anopheles gambiae, Caenorhabditis elegans, and Drosophila melanogaster). Therefore, either the three invertebrate species described above have lost their P2X gene(s) during evolution, or schP2X and vertebrate P2X receptors arose from a common ancestral non-P2X protein by two evolutionary distinct events that did not occur in A. gambiae, C. elegans, or D. melanogaster. Functional evidence for ATP-gated ion channels in T. thermophilia (4) and A. proteus (6) and the presence of P2X-like sequences in an EST data base from the social amoeba Dictyostelium discoideum (GenBankTM accession numbers AC116983 and AC116960 [GenBank] ) would appear to support the former of these two possibilities.

P2X receptors contain several amino acid residues that are highly conserved throughout all known vertebrate receptors. Such conservation suggests functional importance, and this probability has been confirmed in several cases by site-directed mutagenesis studies. Findings include amino acid residues thought to be involved in disulfide bond formation (28), agonist binding (29–31), and protein kinase C phosphorylation (32, 33). The 10 conserved cysteine residues present in the extracellular loop region of all vertebrate P2X receptors known to date are thought to be involved in disulfide bond formation (28). It is interesting to note that these 10 cysteine residues are also present in the schP2X sequence, suggesting a similar role for these residues in vertebrate and invertebrate receptors (Fig. 1). This perfect conservation of cysteine residues between evolutionary remote species seems somewhat surprising given that the removal of individual cysteines by mutation to alanine generally results in no significant change in receptor function (28). Ten conserved positively charged amino acids are also found throughout the vertebrate P2X family, four of which are involved in ATP recognition (30). These four residues are also present in the schP2X sequence as are the two aromatic residues thought to coordinate binding of the adenine ring of ATP (29), suggesting that the agonist binding site is similar between invertebrate and vertebrate P2X receptors. A conserved protein kinase C consensus phosphorylation site in the N-terminal tail of the receptor (Fig. 1), which has been shown to regulate the time course of responses in P2X1 (32) and P2X2 (33) receptors, is also present in schP2X.

schP2X currents were blocked in a concentration-dependent manner by both PPADS and suramin (Fig. 3B) with IC50 values in the range of those for antagonist-sensitive mammalian P2X receptors (34). Despite PPADS being slightly more effective than suramin at sub-micromolar concentrations, PPADS did not completely block schP2X currents at higher concentrations (up to 100 µM PPADS). The PPADS resistant current could, however, be further reduced by the application of 100 µM suramin to cells that had been pre-equilibrated to PPADS (Fig. 3C), suggesting that PPADS and suramin bind to different sites at the schP2X receptor.

Ivermectin has recently been shown to have two distinct sites of action at human P2X4 receptors; the binding of ivermectin to a higher affinity site causes a ~6-fold increase in maximum current, whereas the binding of ivermectin to a lower affinity site results in a ~10-fold slowing of current deactivation (26). Ivermectin also potentiated agonist-induced currents at the schP2X receptor, although to a lesser extent (~66% increase in current) than at the human P2X4 receptor. However, unlike human P2X4, ivermectin had no effect on the deactivation of schP2X currents (Fig. 4A). Thus, of the two ivermectin binding sites present in human P2X4, only the higher affinity site affecting current amplitude appears to be present in schP2X. Ivermectin is widely used as a broad spectrum antiparasitic agent and, given its facilitatory effect on schP2X currents, it might seem surprising that ivermectin treatment has no significant effect on S. mansoni infection in humans (35). However, the typical therapeutic dosage of ivermectin (150 mg/kg) only results in a peak plasma concentration of ~50 ng/ml (~60 nM) (26), and this would not be sufficient (at least in recombinant receptors expressed in Xenopus oocytes) to affect schP2X currents.

To compare the permeation properties of the schP2X pore with those of mammalian P2X receptors, we determined the relative ionic permeabilities of various cations by determining reversal potentials. Similarly as for human P2X1 and P2X2 receptors (17), the permeability ratios of monovalent cations roughly related to the ionic size of the cation (Fig. 6B). Although the extrapolation of such data to provide an estimate of pore size needs to be interpreted with caution (as discussed in Ref. 17), it is interesting to note that the estimated minimum pore diameter for schP2X (~0.9 nM) correlates well with that estimated for human P2X1 and P2X2 (~0.8 nM) (17, 27), suggesting that the ion permeation pathway and selectivity filter are also conserved between these evolutionary remote channels.

Given that the modulation of calcium entry is a proven target for the treatment of schistosomiasis, we sought to determine the calcium permeability of schP2X, as this is particularly relevant to the use of this channel as a drug target. schP2X had a relatively high calcium permeability (PCa/PNa = 3.80) comparable with the most permeant mammalian P2X subtypes, P2X1 (PCa/PNa = 3.9) (17) and P2X4 (PCa/PNa = 4.2) (36). Therefore, the future discovery of drugs that modulate schP2X currents could provide the basis of alternative treatment strategies for schistosomiasis.

Vertebrate homomeric P2X receptors can be divided into two main groups based on their rate of current desensitization, i.e. the rapidly desensitizing (millisecond range) P2X1 and P2X3 receptors and the more slowly desensitizing P2X2 and P2X4–7 receptors. Because of its relatively slow desensitization rate, schP2X appears to fall in the latter of these two groups. Similarly, the lack of sensitivity of schP2X to AMP-CPP would suggest that the receptor is not P2X1-, P2X3-, or P2X6-like (37). Sequence identity between schP2X and human P2X1–7 receptors ranges from 25.8% (P2X7) to 36.6% (P2X4). As schP2X shows the highest sequence identity to P2X4, it may seem logical to assign the Schistosoma P2X receptor described here as a P2X4 species homologue. The potentiation of schP2X currents by ivermectin would appear to support this assumption, as ivermectin also potentiates ATP-evoked currents in rat (25) and human (26) P2X4 receptors but has no effect on rat P2X2–4 and P2X7 (38) or on human P2X1 receptors.2 However, the relatively high sensitivity of schP2X to the antagonists PPADS and suramin does not mirror the much lower sensitivities of the human, rat, and mouse P2X4 receptors (36, 39, 40). Furthermore, to impose vertebrate P2X nomenclature on an invertebrate species may be inappropriate, because S. mansoni may only posses a single (or less than seven) P2X gene(s). As the percentage of sequence identity between schP2X and human P2X4 is less than the similarity between human P2X4 and human P2X1–3 and P2X5–7 (ranges of 38.1–47.9% identity), it seems more appropriate to consider schP2X as a distinct receptor subtype rather than a P2X4 species homologue.

The cloning and functional characterization of a P2X receptor from S. mansoni provides a useful comparative model for the better understanding of human P2X receptor function and a potential drug target for the treatment of schistosomiasis. Future studies aimed at elucidating the physiological roles of this receptor in S. mansoni may give an insight into new P2X-mediated signaling pathways that could be extrapolated to better understand human P2X function. A major question in this respect is the source of the ATP that normally activates the receptor. The parasitic nature of S. mansoni means that in addition to functional roles similar to those of vertebrate P2X receptors (in, for example, smooth muscle function where the activating ATP is of "internal" origin), there is also a possibility that schP2X receptors are located on the outer tegumental membrane (41) and are activated by ATP of "external" origin, e.g. from the blood of the host organism.

This study provides the first molecular evidence that the emergence of P2X receptors for extracellular ATP occurred relatively early in eukaryotic evolution. Whether schistosomes and other invertebrate species possess just a single or multiple subtypes of P2X receptors with different molecular properties remains to be determined.


   FOOTNOTES
 
* This work was supported by a project grant from the Biotechnology and Biological Sciences Research Council (BBSRC) (to S. J. E), a BBSRC Co-operative Awards in Science and Engineering (CASE) studentship (to K. C. A.), and a Wellcome trust program grant (to R. J. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ783803 [GenBank] . Back

To whom correspondence should be addressed. Tel.: 44-0116-252-3081; Fax: 44-0116-252-5045; E-mail: se15{at}le.ac.uk.

1 The abbreviations used are: EST, expressed sequence tag; AMP-CPP, 5'-({alpha},{beta}-methylene)triphosphate; Bz-ATP, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate; GFP, green fluorescent protein; HEK, human embryonic kidney; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid. Back

2 S. J. Ennion and R. J. Evans, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Gurparit Bachra for technical assistance in the cloning of schP2X and Dr. Alison Agnew (University of Leeds) for providing the S. mansoni cDNA library.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. North, R. A. (2002) Physiol. Rev. 82, 1013–1067[Abstract/Free Full Text]
  2. Burnstock, G. (2001) Drug Dev. Res. 53, 53–59[CrossRef]
  3. Burnstock, G. (2004) Curr. Opin. Pharmacol. 4, 47–52[CrossRef][Medline] [Order article via Infotrieve]
  4. Kim, M. Y., Kuruvilla, H. G., Raghu, S., and Hennessey, T. M. (1999) J. Exp. Biol. 202, 407–416[Abstract]
  5. Backus, K. H., Braum, S., Lohner, F., and Deitmer, J. W. (1994) J. Neurobiol. 25, 1283–1292[CrossRef][Medline] [Order article via Infotrieve]
  6. Pothier, F., Couillard, P., and Forget, J. (1984) J. Exp. Zool. 230, 211–218[CrossRef][Medline] [Order article via Infotrieve]
  7. Kucenas, S., Li, Z., Cox, J. A., Egan, T. M., and Voigt, M. M. (2003) Neuroscience 121, 935–945[CrossRef][Medline] [Order article via Infotrieve]
  8. Paukert, M., Hidayat, S., and Grunder, S. (2002) FEBS Lett. 513, 253–258[CrossRef][Medline] [Order article via Infotrieve]
  9. Jensik, P. J., Holbird, D., Collard, M. W., and Cox, T. C. (2001) Am. J. Physiol. 281, C954–C962
  10. Ruppelt, A., Ma, W., Borchardt, K., Silberberg, S. D., and Soto, F. (2001) J. Neurochem. 77, 1256–1265[CrossRef][Medline] [Order article via Infotrieve]
  11. Engels, D., Chitsulo, L., Montresor, A., and Savioli, L. (2002) Acta Trop. 82, 139–146[CrossRef][Medline] [Order article via Infotrieve]
  12. Awasthi, S., Bundy, D. A., and Savioli, L. (2003) BMJ 327, 431–433[Free Full Text]
  13. Noel, F., Cunha, V. M., Silva, C. L., and Mendonca-Silva, D. L. (2001) Mem. Inst. Oswaldo Cruz 96, (suppl.) 85–88[Medline] [Order article via Infotrieve]
  14. Kohn, A. B., Anderson, P. A., Roberts-Misterly, J. M., and Greenberg, R. M. (2001) J. Biol. Chem. 276, 36873–36876[Abstract/Free Full Text]
  15. Ismail, M., Botros, S., Metwally, A., William, S., Farghally, A., Tao, L. F., Day, T. A., and Bennett, J. L. (1999) Am. J. Trop. Med. Hyg. 60, 932–935[Abstract]
  16. Fenwick, A., Savioli, L., Engels, D., Bergquist, R. N., and Todd, M. H. (2003) Trends Parasitol. 19, 509–515[CrossRef][Medline] [Order article via Infotrieve]
  17. Evans, R. J., Lewis, C., Virginio, C., Lundstrom, K., Buell, G., Surprenant, A., and North, R. A. (1996) J. Physiol. 497, 413–422[Medline] [Order article via Infotrieve]
  18. Bentley, G. N., Jones, A. K., and Agnew, A. (2003) Gene 314, 103–112[CrossRef][Medline] [Order article via Infotrieve]
  19. Valera, S., Hussy, N., Evans, R. J., Adami, N., North, R. A., Surprenant, A., and Buell, G. (1994) Nature 371, 516–519[CrossRef][Medline] [Order article via Infotrieve]
  20. Fenwick, E. M., Marty, A., and Neher, E. (1982) J. Physiol. 331, 599–635[Abstract/Free Full Text]
  21. Benham, C. D., and Tsien, R. W. (1987) Nature 328, 275–278[CrossRef][Medline] [Order article via Infotrieve]
  22. Verjovski-Almeida, S., DeMarco, R., Martins, E. A., Guimaraes, P. E., Ojopi, E. P., Paquola, A. C., Piazza, J. P., Nishiyama, M. Y., Jr., Kitajima, J. P., Adamson, R. E., Ashton, P. D., Bonaldo, M. F., Coulson, P. S., Dillon, G. P., Farias, L. P., Gregorio, S. P., Ho, P. L., Leite, R. A., Malaquias, L. C., Marques, R. C., Miyasato, P. A., Nascimento, A. L., Ohlweiler, F. P., Reis, E. M., Ribeiro, M. A., Sa, R. G., Stukart, G. C., Soares, M. B., Gargioni, C., Kawano, T., Rodrigues, V., Madeira, A. M., Wilson, R. A., Menck, C. F., Setubal, J. C., Leite, L. C., and Dias-Neto, E. (2003) Nat. Genet. 35, 148–157[CrossRef][Medline] [Order article via Infotrieve]
  23. Claros, M. G., and von Heijne, G. (1994) Comput. Appl. Biosci. 10, 685–686[Free Full Text]
  24. Rettinger, J., Aschrafi, A., and Schmalzing, G. (2000) J. Biol. Chem. 275, 33542–33547[Abstract/Free Full Text]
  25. Khakh, B. S., Proctor, W. R., Dunwiddie, T. V., Labarca, C., and Lester, H. A. (1999) J. Neurosci. 19, 7289–7299[Abstract/Free Full Text]
  26. Priel, A., and Silberberg, S. D. (2004) J. Gen. Physiol. 123, 281–293[Abstract/Free Full Text]
  27. Lewis, C., Davies, N. W., and Evans, R. J. (2001) Drug Dev. Res. 52, 164–169[CrossRef]
  28. Ennion, S. J., and Evans, R. J. (2002) Mol. Pharmacol. 61, 303–311[Abstract/Free Full Text]
  29. Roberts, J. A., and Evans, R. J. (2004) J. Biol. Chem. 279, 9043–9055[Abstract/Free Full Text]
  30. Ennion, S., Hagan, S., and Evans, R. J. (2000) J. Biol. Chem. 275, 29361–29367[Abstract/Free Full Text]
  31. Jiang, L. H., Rassendren, F., Surprenant, A., and North, R. A. (2000) J. Biol. Chem. 275, 34190–34196[Abstract/Free Full Text]
  32. Ennion, S. J., and Evans, R. J. (2002) Biochem. Biophys. Res. Commun. 291, 611–616[CrossRef][Medline] [Order article via Infotrieve]
  33. Boue-Grabot, E., Archambault, V., and Seguela, P. (2000) J. Biol. Chem. 275, 10190–10195[Abstract/Free Full Text]
  34. Lambrecht, G. (2000) Naunyn Schmiedebergs Arch. Pharmacol. 362, 340–350[CrossRef][Medline] [Order article via Infotrieve]
  35. Whitworth, J. A., Morgan, D., Maude, G. H., McNicholas, A. M., and Taylor, D. W. (1991) Trans. R. Soc. Trop. Med. Hyg. 85, 232–234[CrossRef][Medline] [Order article via Infotrieve]
  36. Garcia-Guzman, M., Soto, F., Gomez-Hernandez, J. M., Lund, P. E., and Stuhmer, W. (1997) Mol. Pharmacol. 51, 109–118[Abstract/Free Full Text]
  37. Jones, C. A., Vial, C., Sellers, L. A., Humphrey, P. P., Evans, R. J., and Chessell, I. P. (2004) Mol. Pharmacol. 65, 979–985[Abstract/Free Full Text]
  38. Bo, X., Jiang, L. H., Wilson, H. L., Kim, M., Burnstock, G., Surprenant, A., and North, R. A. (2003) Mol. Pharmacol. 63, 1407–1416[Abstract/Free Full Text]
  39. Buell, G., Lewis, C., Collo, G., North, R. A., and Surprenant, A. (1996) EMBO J. 15, 55–62[Medline] [Order article via Infotrieve]
  40. Jones, C. A., Chessell, I. P., Simon, J., Barnard, E. A., Miller, K. J., Michel, A. D., and Humphrey, P. P. (2000) Br. J. Pharmacol. 129, 388–394[CrossRef][Medline] [Order article via Infotrieve]
  41. McLaren, D. J., and Hockley, D. J. (1977) Nature 269, 147–149[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Physiol.Home page
G. Burnstock
Unresolved issues and controversies in purinergic signalling
J. Physiol., July 15, 2008; 586(14): 3307 - 3312.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. J. Fountain, L. Cao, M. T. Young, and R. A. North
Permeation Properties of a P2X Receptor in the Green Algae Ostreococcus tauri
J. Biol. Chem., May 30, 2008; 283(22): 15122 - 15126.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
G. Burnstock
Physiology and Pathophysiology of Purinergic Neurotransmission
Physiol Rev, April 1, 2007; 87(2): 659 - 797.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
W. J. Wilkinson, L.-H. Jiang, A. Surprenant, and R. A. North
Role of Ectodomain Lysines in the Subunits of the Heteromeric P2X2/3 Receptor
Mol. Pharmacol., October 1, 2006; 70(4): 1159 - 1163.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
T. Nakano, H. Inoue, S. Fukuyama, K. Matsumoto, M. Matsumura, M. Tsuda, T. Matsumoto, H. Aizawa, and Y. Nakanishi
Niflumic Acid Suppresses Interleukin-13-induced Asthma Phenotypes
Am. J. Respir. Crit. Care Med., June 1, 2006; 173(11): 1216 - 1221.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
G. Burnstock
Pathophysiology and therapeutic potential of purinergic signaling.
Pharmacol. Rev., March 1, 2006; 58(1): 58 - 86.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/40/41650    most recent
M408203200v1
Right arrow Alert me when this article is cited
Right arrow