XB-ART-46297PLoS One January 1, 2012; 7 (11): e50487.
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Cloning and characterization of a P2X receptor expressed in the central nervous system of Lymnaea stagnalis.
P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC(50) 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αβmeATP was a weak agonist. BzATP was a partial agonist with an EC(50) of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC(50) values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn(2+) and Cu(2+) ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function.
PubMed ID: 23209755
PMC ID: PMC3510196
Article link: PLoS One
Species referenced: Xenopus
Genes referenced: hk1 p2rx1 p2rx2 p2rx4 tff3.7
Article Images: [+] show captions
|Figure 2. Alignment of the predicted LymP2X amino acid sequence with mammalian and lower organism P2X receptors.The LymP2X amino acid sequence (LymP2X) was aligned with human P2X1-6 (P51575, Q9UBL9, P56373, Q99571, Q93086, O15547), rat P2X2 (P49653), rat P2X4 (P51577), Schistosoma mansoni P2X (smP2X) (Q65A65) and Hypsibius dujardini P2X (HdP2X) (ACL14328) using CLUSTALW software. Equivalent residues in the two transmembrane domains (TM1 and TM2) of the zP2X4 crystal structure  are indicated as horizontal blue bars. Highlighted conserved residues are numbered according to the hP2X1 sequence. Residues involved in ATP binding  are highlighted in red. Ten conserved cysteine residues which form five disulphide bonds  are in grey with yellow text and the consensus protein kinase C phosphorylation site (T18) in blue. Black arrows with light blue shaded amino acid sequence indicate the locations of CODHOP PCR primer pairs 1 and 2 used to identify LymP2X (Table 1). Rat P2X4 residues thought to be involved in the interaction with ivermectin (Q36, L40, V43, V47 W50, N338, G342, L346, A349 and I356)  are highlighted in purple with yellow text. Residues shown to be involved in the actions of metal ions are shaded orange where human P2X2 H204, H209 and H330 control access of zinc to its binding site , H120 and H213 in rat P2X2 are involved in the formation of an intersubunit zinc binding site – and D138, H140 and C132 in rat P2X4 are involved in the inhibitory modulation by metal ions . A cluster of four positively charged residues thought to be involved in the actions of suramin and NF449 at hP2X1 (K136, K138, R139 and K140) ,  are shaded light green with yellow text whilst a lysine residue thought to be involved in PPADS action at P2X1 and P2X2  is shaded dark green with white text (K249).|
|Figure 3. The LymP2X gene encodes an ATP gated ion channel.Membrane currents were recorded by two-electrode voltage clamp in Xenopus oocytes expressing LymP2X receptors. (A) Example sequential current traces in response to a 2 second application (solid black bar) of 100 µM ATP showed a marked run down in peak current amplitude with a 5 minute recovery period between applications. (B) Sequential application of ATP (100 µM for 2 seconds) with a 15 minute recovery period between applications showed no rundown in amplitude after the second ATP application (n = 5). (C) A 5 minute recovery period between sequential application of 10 µM ATP (black bars) resulted in reproducible responses after two initial applications of 100 µM ATP.|
|Figure 4. Properties of ATP evoked currents.(A) Concentration response curves for ATP in LymP2X expressing oocytes. Mean peak currents (± s.e.m) were normalized to responses evoked by 10 µM ATP, EC50 = 6.2 µM for LymP2X (n = 10 oocytes) and 5.8 µM for clone 11 (n = 5). B) Example LymP2X current traces in response to different concentrations of ATP (black bar). (C) Current voltage relationship of LymP2X. The reversal potential of ATP mediated currents was determined by recording ATP (10 µM, indicated by bar) induced currents at holding potentials ranging from –60 mV to +40 mV with a 5 minute interval between applications. Currents obtained in different oocytes were expressed as a negative percentage of the maximum current for each individual cell (n = 7). D. Example currents for the plot depicted in C.|
|Figure 5. BzATP (2′, 3′-O-(4-Benzoylbenzoyl ATP)) is a partial agonist and αβmeATP a weak agonist at LymP2X.(A) Concentration response curves for BzATP (EC50 of 2.4 µM, n = 5−10) and αβmeATP (n = 6). Mean peak currents (± s.e.m) are normalized to responses evoked by 10 µM ATP. The concentration response curve for ATP is also plotted as grey dashed line for comparison. (B) Representative current traces for data plotted in A) (agonist concentrations in µM, 10 µM ATP traces in red). Neither UTP nor ADP (hexokinase treated) evoked membrane currents at LymP2X.|
|Figure 6. Actions of antagonists, pH and metal ions on LymP2X currents.The effects of the P2 receptor antagonists suramin and PPADS, pH and zinc and copper on ATP evoked LymP2X currents were determined in Xenopus oocytes. (A) Inhibition curves for mean responses to 10 µM ATP in the presence of suramin (open circles) and PPADS (closed circles) (n = 5−9 oocytes). Mean peak currents (± s.e.m) are normalized to responses evoked by 10 µM ATP in the absence of antagonist. (B) Representative current traces for data plotted in A) demonstrating the inhibitory effects of different concentrations of PPADS or suramin on the current response evoked by 10 µM ATP (black bar). Antagonists were bath perfused and also present in the ATP application at the appropriate concentration. (C) Concentration response curves for ATP in recording solutions of different pH. Alkaline pH 8.5 had no effect on ATP efficacy or potency whereas acidic pH 6.5 reduced current amplitudes (n = 6). Mean peak currents (± s.e.m) are normalized to responses evoked by 10 µM ATP at pH 7.5. (D) Biphasic effects of Zn2+ and Cu2+ on ATP (10 µM) evoked LymP2X currents. Mean peak currents (± s.e.m) are normalized to responses evoked by 10 µM ATP in the absence of metal ion. Both Zn2+ and Cu2+ potentiated ATP evoked current amplitude when present at 100 µM (p<0.05 (*) for Zn2+ and p<0.01 (**) for Cu2+) but inhibited ATP evoked currents when present at a concentration of 1 mM (p<0.01) (n = 5−8).|
|Figure 7. LymP2X is widely expressed in Lymnaea CNS.(A) RT-PCR using primers for LymP2X (primer pair 8 (Table 1) and β-tubulin (primer pair 9).+indicates PCR reactions on cDNA samples reverse transcribed from RNA prepared from the various ganglia indicated. – indicates negative control reactions where reverse transcriptase had been omitted from the cDNA synthesis reaction to control for genomic DNA contamination. M = molecular mass ladder (size in kb). (B) Quantitative PCR data showing the relative expression levels of LymP2X in various ganglia (normalised to pleural ganglia), n = 3 independent reactions for each sample.|
|Figure 8. In situ hybridization.Wax embedded sections of Lymnaea CNS were probed with Digoxigenin labelled LymP2X sense and antisense cRNA probes. Representative images of each ganglia are shown. Scale bars = 100 µm unless indicated otherwise. Bottom right hand panel shows a higher magnification image (scale bar = 25 µm) of neurones in the visceral ganglia, emphasising the diffuse cytoplasmic and punctate nuclear staining observed with the antisense LymP2X probe.|
|Figure 1. Overview of LymP2X cloning strategy.(A) Nested CODEHOP PCR was performed on cDNA prepared from Lymnaea CNS to generate a LymP2X gene amplification product. The sequence of this internal product could then be utilised to design gene specific primers to facilitate amplification of the 5′ end of the gene by 5′RACE and the 3′ end of the gene by PCR on a Lymnaea cDNA library. Once the 5′ and 3′ sequence of the gene had been determined, further gene specific primers could then be designed to amplify the full length LymP2X coding sequence from Lymnaea cDNA. Numbered arrows indicate primer pairs listed in Table 1. (B) Agarose gel showing separation of CODEHOP PCR products using primer pair 1 with cDNA as template (lane 1), primer pair 2 with PCR reaction 1 as template (lane 2) and control reactions using PCR reaction 1 as template with only the forward primer of primer pair 2 (lane 3) or only the reverse primer of primer pair 2 (lane 4). (C) Amplification of the 3′ end of LymP2X by PCR on a Lymnaea cDNA library using primer pair 5 (lane 1). (D) 5′RACE PCR using primer pair 4. (E) Amplification of the full length LymP2X coding sequence using primer pairs 6 (lane 2) or 7 (lane 3). Lane 1 shows no template negative control. M indicates molecular mass ladder (size in kb). (F) Nucleotide and predicted amino acid sequence of the 5′ end of the LymP2X transcript. The two potential start methionines are indicated in bold.|
References [+] :
Agboh, Functional characterization of a P2X receptor from Schistosoma mansoni. 2004, Pubmed, Xenbase