Abstract
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 P2X(1-7) receptors ranges from 25.8 to 36.6%. ATP evoked concentration-dependent currents at schP2X channels expressed in Xenopus oocytes with an EC(50) of 22.1 microM. 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 (EC(50) of 3.6 microM) than ATP. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid blocked schP2X responses to 100 microm ATP with IC(50) values of 9.6 and 0.5 microM, respectively. Ivermectin (10 microM) potentiated currents to both ATP and Bz-ATP by approximately 60% with a minimal effect on potency (EC(50) of 18.2 and 1.6 microM, 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 (P(Ca)/P(Na) = 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.
Highlights
The cloning and characterization of a P2X receptor 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 present in schP2X
This study describes the first example of a non-vertebrate P2X receptor and provides definitive molecular evidence that the range of species known to utilize ATP as an extracellular signaling molecule extends beyond vertebrates
Summary
Cloning of the S. mansoni P2X Receptor—A S. mansoni EST sequence (accession number CD081583) showing sequence homology to mammalian P2X receptors was identified by BLAST searches of the GenBankTM EST data base. This cDNA insert was subsequently recloned by PCR to generate a construct (schP2X) containing a consensus Kozak sequence and the 5Ј-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. 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 Ϫ60mV to 60mV for 0.5 s followed by a ramp-down to Ϫ100mV over 1.6 s and a ramp-up to 60mv over 1.6 s before a step-back to Ϫ60mV This ramp protocol was applied in the absence and presence of ATP (100 M), and the reversal potential was taken as the points where the current traces crossed on the ramps (Fig. 6A). The diameter of the ions was taken as the median value for measurements taken in the x, y, and z axes
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