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Eur J Pharmacol
2011 Mar 05;6542:155-9. doi: 10.1016/j.ejphar.2010.11.039.
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Contribution of the intracellular C terminal domain to regulation of human P2X1 receptors for ATP by phorbol ester and Gq coupled mGlu(1α) receptors.
Wen H
,
Evans RJ
.
???displayArticle.abstract??? P2X1 receptors are expressed in arteries and blood platelets, play an important role in the cardiovascular system, and their activity can be potentiated following stimulation of Gq coupled receptors or phorbol ester treatment. The contribution of the intracellular carboxy terminus of the P2X1 receptor to this regulation was determined using over-expression of the C terminus and a mutagenesis based approach on recombinant receptors expressed in Xenopus oocytes. PMA induced potentiation of P2X1 receptor currents (~125% above control) was abolished following over-expression of the intracellular carboxy terminus of the P2X1 receptor. To determine the molecular basis of regulation by the carboxy terminus a series of individual cysteine point mutations between His(355) and Tyr(370) was characterized. PMA potentiation was abolished for the P2X1 receptor mutants H355C, P358C, Y363C, K367C, F368C, K369C and Y370C. When these mutations were introduced into the carboxy terminus fragment the inhibitory effect was absent only for P358C, K367C and Y370C mutants. These results suggest that residues Pro(358), Lys(367) and Tyr(370) are involved in the sequestering effect of the carboxy terminal fragment and indicate they are directly involved in modulation of the receptor by binding to a regulatory factor. The other mutants that abolished the PMA effect when introduced into the P2X1 receptor are likely to be involved in transduction of the regulatory event. These studies highlight the importance of the carboxy terminus in determining the properties and regulation of the P2X1 receptor and suggest that the intracellular terminal regions of the receptor close to the transmembrane segments interact.
Fig. 1. PMA and mGlu1α receptor evoked potentiation of P2X1 receptors is blocked by over-expression of the intracellular C terminus. The carboxy terminus of the P2X1 receptor was co-expressed with wild type P2X1 and mGlu1α receptors in Xenopus oocytes. (A) Upper left panels show representative currents evoked by a maximal concentration of ATP (100 μM, indicated by bar) at control oocytes (wild type P2X1) and those following 10 min incubation with PMA (100 nM). Right upper panels show the effects of over-expression of the interacellular carboxy terminus on the effects of PMA. The bar chart shows summary data, n = 5â15. (B) Upper panels show sample traces for a given oocyte co-expressing P2X1 and mGlu1α receptors (left) or P2X1 receptors, mGlu1α receptors and the P2X1 receptor carboxy terminal fragment (right traces). Responses to a maximal concentration of ATP (100 μM, indicated by bar) are shown before and after the application of glutamate (100 μM). Glutamate evoked an inward calcium activated chloride current and potentiated subsequent ATP evoked responses. This potentiation was reduced by co-expression of the P2X1 receptor C terminal mini-gene. The bar chart shows a summary of the data, n = 5â7. *** P < 0.001.
Fig. 2. Properties of individual cysteine point mutations of the C terminus. (A) ATP (100 μM, application period indicated by bar) evoked rapidly desensitising currents at wild type P2X1 receptors. Cysteine mutation had no effect at the Q365C mutant but responses were reduced in amplitude by ~ 50% for R360C and by > 98% for Y363C. (B) Peak current amplitudes of ATP (100 μM) evoked currents from wild type (WT) and cysteine mutant P2X1 receptors, * P < 0.05, *** p < 0.001. (n = 4â18). (C) Total and surface expression levels of wild type and mutant P2X1 receptors with reduced peak current amplitudes are shown.
Fig. 3. Effects of cysteine mutations in the carboxy terminus of P2X1 receptors on PMA potentiation. (A) Representative traces of ATP evoked currents (100 μM, application indicated by bar) from oocytes under control conditions and following treatment with PMA (100 nM) for wild type (WT) and mutants H355C, I356C and K367C. (B) Summary of the percentage change in peak current amplitude to ATP following PMA treatment for P2X1 receptor cysteine mutants (n = 4â18). (C) Effects of cysteine mutations that reduced PMA potentiation when introduced into the P2X1 receptor when introduced into the C terminal fragment. Cysteine mutations P358C, K367C and Y370C abolished the inhibitory effect of the C terminal fragment on PMA regulation. (n = 5â15).
Fig. 4. Model of the amino and carboxy termini of the P2X1 receptor showing residues involved in PMA regulation. The effects of cysteine mutants of residues Y16-G30 (from (Wen and Evans, 2009)) and H355-Y370 on PMA regulation are shown. Residues in back abolished PMA regulation when introduced into the P2X1 receptor and removed the inhibitory effect of the corresponding mini-gene (involved in sequestering effect of mini-gene and predicted interaction with regulatory protein). Residues in grey are those that when mutated in the P2X1 receptor abolished PMA regulation but did not reduce the inhibitory effect when introduced into the C terminal fragment (transduction effect). The transmembrane segments are shown as boxes (TM1 and TM2). Secondary structural predictions of a beta sheet in the amino terminus and an alpha helix in the carboxy terminus are shown, * indicates residues that are conserved throughout the P2X receptor family and # indicates where there is a conserved positive charge.
Ase,
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Ase,
Potentiation of P2X1 ATP-gated currents by 5-hydroxytryptamine 2A receptors involves diacylglycerol-dependent kinases and intracellular calcium.
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,
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Boué-Grabot,
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2000,
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,
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Brändle,
Desensitization of the P2X(2) receptor controlled by alternative splicing.
1997,
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,
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Chaumont,
Identification of a trafficking motif involved in the stabilization and polarization of P2X receptors.
2004,
Pubmed
Ennion,
The role of positively charged amino acids in ATP recognition by human P2X(1) receptors.
2000,
Pubmed
,
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Fountain,
A C-terminal lysine that controls human P2X4 receptor desensitization.
2006,
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Gachet,
Regulation of platelet functions by P2 receptors.
2006,
Pubmed
Hechler,
A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo.
2003,
Pubmed
Kawate,
Crystal structure of the ATP-gated P2X(4) ion channel in the closed state.
2009,
Pubmed
La,
Endothelin-1 enhances vasoconstrictor responses to exogenously administered and neurogenically released ATP in rabbit isolated perfused arteries.
1993,
Pubmed
Lalo,
P2X1 receptor mobility and trafficking; regulation by receptor insertion and activation.
2010,
Pubmed
MacKenzie,
Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets.
1996,
Pubmed
McGuffin,
The PSIPRED protein structure prediction server.
2000,
Pubmed
Mulryan,
Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors.
2000,
Pubmed
North,
Molecular physiology of P2X receptors.
2002,
Pubmed
Oury,
Overexpression of the platelet P2X1 ion channel in transgenic mice generates a novel prothrombotic phenotype.
2003,
Pubmed
Paukert,
Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit.
2001,
Pubmed
,
Xenbase
Ralevic,
Receptors for purines and pyrimidines.
1998,
Pubmed
Smith,
Identification of amino acids within the P2X2 receptor C-terminus that regulate desensitization.
1999,
Pubmed
,
Xenbase
Vial,
G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation.
2004,
Pubmed
,
Xenbase
Vial,
P2X(1) receptor-deficient mice establish the native P2X receptor and a P2Y6-like receptor in arteries.
2002,
Pubmed
Vial,
P2X receptor expression in mouse urinary bladder and the requirement of P2X(1) receptors for functional P2X receptor responses in the mouse urinary bladder smooth muscle.
2000,
Pubmed
Wen,
Regions of the amino terminus of the P2X receptor required for modification by phorbol ester and mGluR1alpha receptors.
2009,
Pubmed
,
Xenbase
Young,
P2X receptors: dawn of the post-structure era.
2010,
Pubmed
