Roberts JA et al. (2009), Contribution of the region Glu181 to Val200 of ...

XB-ART-40083

J Neurochem 2009 May 01;1094:1042-52. doi: 10.1111/j.1471-4159.2009.06035.x.

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Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis.

Roberts JA , Valente M , Allsopp RC , Watt D , Evans RJ .

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At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrPhiPhixLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by approximately 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced. The ATP sensitivity of the K190C mutant was rescued towards WT levels by positively charged (2-aminoethyl)methanethiosulfonate hydrobromide and reduced by negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate. Both MTS reagents decreased ATP potency at the T186C mutant, and abolished responses at the F195C mutant. (32)P-2-azido ATP binding to the mutants T186C and K190C was sensitive to MTS reagents consistent with an effect on binding, however binding at F195C was unaffected indicating an effect on gating. The accessibility of the introduced cysteines was probed with (2-aminoethyl)methanethiosulfonate hydrobromide-biotin, this showed that the region Thr186-Ser192 is likely to form a beta sheet and that accessibility is blocked by ATP. Taken together these results suggest that Thr186, Phe188 and Lys190 are involved in ATP binding to the receptor and Phe185 and Phe195 contribute to agonist evoked conformational changes.

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Species referenced: Xenopus laevis
Genes referenced: p2rx1 p2rx2 p2rx7 tbx2

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	Fig. 1. Effects of cysteine point mutants of E181 to V200C on ATP potency at human P2X1 receptors. (a) Sequence line-up for the region corresponding to E181 to V200 of the P2X1 receptor for the seven human, Schistosoma mansoni (Schi) (Agboh et al. 2004), the green alga Ostreococcus tauri (Alga) (Fountain et al. 2008) and amoeba Dictyostelium discoidium (Dict) (Fountain et al. 2007) P2X receptors. Residues conserved in at least five of the human isoforms are shown in black bold, conservative substitutions are shown underlined in bold gray. (b) Concentration responses to ATP for oocytes expressing WT, T186C, F188C and K190C mutant P2X1 receptors. Application of ATP is indicated by bar. (c) Summary of concentration response data for WT and mutants T186C, F188C and K190C that showed significant decreases in ATP potency (n = 3–4).
	Fig. 2. Efficacy of the partial agonists Ap5A and BzATP. (a) Currents evoked by ATP and Ap5A application (both 100 μM) at WT, F185C, T186C and K190C mutant P2X1 receptors. Agonist application for 3 s is indicated by bar. (b) Summary of the efficacy of the partial agonist Ap5A (100 μM) expressed as a percentage of the response to a maximal concentration of ATP (100 μM). (c) Summary of the efficacy of the partial agonist BzATP (100 μM) expressed as a percentage of the response to a maximal concentration of ATP (100 μM). Significant reductions in efficacy are shown as black bars, p < 0.05, p < 0.01, **p < 0.001, (n = 3–5).
	Fig. 3. Effects of charged MTS reagents on ATP evoked P2X1 receptor currents. (a) Responses to an ∼ EC50 concentration of ATP are shown before (open symbol) and after the application of 1 mM MTSEA (filled symbol) for the mutants F185C, T186C and K190C. ATP application (3 s) is indicated by bar. (b) Summary of the effects of MTSEA on WT and mutant P2X1 receptors. (c) Responses to an ∼ EC50 concentration of ATP are shown before (open symbol) and after the application of 1 mM MTSES (filled symbol) for the mutants F185C, T186C and K190C. ATP application (3 s) is indicated by bar. (d) Summary of the effects of MTSES on WT and mutant P2X1 receptors. Data are expressed as % change (0% indicates no change), on MTS application, significant differences from WT are shown by black bars, p < 0.05, **p < 0.001, (n = 3–14).
	Fig. 4. Effects of MTS reagents on ATP concentration responses at P2X1 receptor mutants. Concentration response curves to ATP were determined under control conditions or following incubation with MTSEA (1 mM for 1 h followed by washout) or MTSES (1 mM for 3 h followed by washout). (a) At T186C the EC50 was significantly increased by both MTSEA and MTSES with no effect on the peak response. (b) At K190C MTSEA significantly increased whilst MTSES significantly decreased ATP potency. For MTSES peak current responses were also reduced. (c) MTSEA and MTSES treatment reduced responses at the F195C mutant receptor by > 90% even for a maximal concentration of ATP (1 mM).
	Fig. 5. Effects of MTS reagents on 32P-2-azido ATP cross-linking to WT and mutant P2X1 receptors. (a) Oocytes expressing WT and mutant P2X1 receptors were UV irradiated in the presence of 32P-2-azido ATP and P2X1 receptors were isolated by immunoprecipitation and run on a gel and exposed to X-ray film. The autoradiographs show the level of radioactivity associated with the P2X1 receptors under control conditions and the effects of MTSEA or MTSES (both 1 mM). (b) Summary data of the effects of MTSEA and MTSES on 2-azido ATP cross-linking to WT, T186C, K190C and F195C mutant P2X1 receptors. Data are expressed as % of control condition for each group of oocytes and corrected for background levels. (n = 4 batches of oocytes for each). p < 0.05, *p < 0.001.
	Fig. 6. MTSEA-biotinylation reveals the surface accessibility of the region E181-V200. Representative western blots show the equivalent total levels of WT and P2X1 receptor mutants expressed in the oocytes used for the biotinylation assay (left hand panel). To determine the surface accessibility of introduced cysteine residues oocytes were incubated with MTSEA-biotin, and biotinylated proteins isolated, run on a gel and blotted with an anti-P2X1 receptor antibody. Oocytes were either pre-treated with apyrase (to break down any endogenous nucleotides) or ATP (to activate the receptor). For WT receptors no biotinylation could be detected. Biotinylation was also below the limit of detection for E181C, E183C, L187C, I189C, N191C and I193C. At N184C and S194C weak biotinylation that was ATP sensitive was recorded. ATP sensitive biotinylation was detected at A182C, F185C, T186C, F188C, K190C, S192C and F195C. MTSEA-biotin was observed for P196C-V200C mutants either in the absence of presence of ATP. Blots are representative of those from three to eight separate batches of oocytes.
	Fig. 7. Model of the ATP binding site. Portions of the extracellular loop adjacent to either the first (in gray) or second (in black) transmembrane segment are shown for two adjacent P2X1 receptor subunits. Conserved residues that have been shown to contribute to ATP potency are shown (gray residues correspond to conserved aromatic residues in areas of high conservation). The dotted line corresponds to the region Glu181-Val200 that was characterized in the present study, the zig-zag line corresponds to the region of beta sheet as indicated by the pattern of MTSEA-biotinylation. Based on studies on the zinc and copper binding site of P2X7 receptors (Liu et al. 2007) it is possible that residues His62 and Asp197 (P2X7 receptor numbering, indicated by black balls) are close together (shown by arrow) as suggested for residues involved in the zinc binding site at P2X2 receptors (Nagaya et al. 2005). This would bring the residues Phe195, Lys190 and Thr186 close to the putative ATP binding site. If this is the case Lys190 could be close to the other conserved lysine residues and this could account for the charge dependent effects of MTS reagents on the K190C mutant. Asn184 is glycosylated (Roberts and Evans 2006).

References [+] :

Agboh, Characterisation of ATP analogues to cross-link and label P2X receptors. 2009, Pubmed