About: Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains

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About: Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains Goto Sponge NotDistinct Permalink

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rdf:type	skos:Concept http://linked.opendata.cz/ontology/domain/vavai/Vysledek
rdfs:seeAlso	http://dx.doi.org/10.1371/journal.pone.0112902
Description	Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio) adenosine 5'-triphosphate, adenosine 5'-(gamma-thio) triphosphate, 2'(3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate, and alpha,beta-methyleneadenosine 5'-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state. Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio) adenosine 5'-triphosphate, adenosine 5'-(gamma-thio) triphosphate, 2'(3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate, and alpha,beta-methyleneadenosine 5'-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state. (en)
Title	Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains (en)
skos:prefLabel	Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains (en)
skos:notation	RIV/00216208:11310/14:10285187!RIV15-MSM-11310___
http://linked.open...avai/riv/aktivita	I P S
http://linked.open...avai/riv/aktivity	I, P(ED1.1.00/02.0109), P(EE2.3.30.0025), P(GBP304/12/G069), S
http://linked.open...iv/cisloPeriodika	11
http://linked.open...vai/riv/dodaniDat	2015
http://linked.open...aciTvurceVysledku	Tvrdoňová, Vendula
http://linked.open.../riv/druhVysledku	J - Článek v odborném periodiku
http://linked.open...iv/duvernostUdaju	S - Úplné a pravdivé údaje nepodléhající ochraně podle zvláštních právních předpisů
http://linked.open...titaPredkladatele	Univerzita Karlova v Praze / Přírodovědecká fakulta
http://linked.open...dnocenehoVysledku	20282
http://linked.open...ai/riv/idVysledku	RIV/00216208:11310/14:10285187
http://linked.open...riv/jazykVysledku	eng - angličtina
http://linked.open.../riv/klicovaSlova	ivermectin; desensitization; activation; kinase-c; cation permeability; transmembrane segments; agonist binding; atp-binding-site; gated ion channels; human p2x(1) receptors (en)
http://linked.open.../riv/klicoveSlovo	activation gated ion channels ivermectin desensitization agonist binding atp-binding-site cation permeability human p2x(1) receptors kinase-c transmembrane segments
http://linked.open...odStatuVydavatele	US - Spojené státy americké
http://linked.open...ontrolniKodProRIV	[08E20B5E657E]
http://linked.open...i/riv/nazevZdroje	PLoS ONE
http://linked.open...in/vavai/riv/obor	ED
http://linked.open...ichTvurcuVysledku	1 (xsd:int)
http://linked.open...cetTvurcuVysledku	4 (xsd:int)
http://linked.open...vavai/riv/projekt	Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University Project of excellence in the field of neuroscience Center of Biomedical Research
http://linked.open...UplatneniVysledku	2014
http://linked.open...v/svazekPeriodika	9
http://linked.open...iv/tvurceVysledku	Stojilkovic, Stanko S. Tvrdoňová, Vendula Zemkova, Hana Rokic, Milos B.
http://linked.open...ain/vavai/riv/wos	000345558500107
issn	1932-6203
number of pages	12 (xsd:int)
http://bibframe.org/vocab/doi	10.1371/journal.pone.0112902
http://localhost/t...ganizacniJednotka	11310

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