title: Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating.
abstract: P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify inter-subunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gating-induced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Charge-reversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.
authors.affiliation: Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Haertelstrasse 16-18, 04107, Leipzig, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Stephan
authors.affiliation: Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Haertelstrasse 16-18, 04107, Leipzig, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Kowalski-Jahn
authors.affiliation: Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Haertelstrasse 16-18, 04107, Leipzig, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Zens
authors.affiliation: Department of Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, 52074, Aachen, Germany.
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
authors.affiliation: Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Haertelstrasse 16-18, 04107, Leipzig, Germany. Peter.Illes@medizin.uni-leipzig.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Illes
authors.affiliation: Department of Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, 52074, Aachen, Germany. rhausmann@ukaachen.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hausmann
mesh_terms: D055785:Gene Knockdown Techniques; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D058477:Receptors, Purinergic P2X3 / Q000737:chemistry*
chemical_list: D058477:Receptors, Purinergic P2X3
keywords: ATP; Disulfide trapping; Gating; P2X3 receptor
references.citation: Biochem Pharmacol. 2013 Jan 15;85(2):153-62
references.pmid: 22975406
references.citation: J Biol Chem. 2001 May 4;276(18):14902-8
references.pmid: 11278888
references.citation: Mol Neurobiol. 2007 Oct;36(2):165-83
references.pmid: 17952660
references.citation: Mol Pharmacol. 2013 Apr;83(4):759-69
references.pmid: 23253448
references.citation: Nature. 2003 Jan 16;421(6920):272-5
references.pmid: 12529644
references.citation: Eur Biophys J. 2009 Mar;38(3):329-38
references.pmid: 18427801
references.citation: J Neurosci. 2003 Oct 1;23(26):8903-10
references.pmid: 14523092
references.citation: Mol Pharmacol. 2013 Jan;83(1):73-84
references.pmid: 23041661
references.citation: J Biol Chem. 2004 Dec 17;279(51):53109-15
references.pmid: 15475563
references.citation: J Mol Biol. 1995 Apr 28;248(2):478-86
references.pmid: 7739054
references.citation: J Biol Chem. 2013 Jul 12;288(28):20758-67
references.pmid: 23709221
references.citation: Proc Natl Acad Sci U S A. 2012 Mar 13;109(11):4140-5
references.pmid: 22378652
references.citation: Pharmacol Rev. 2001 Dec;53(4):553-68
references.pmid: 11734618
references.citation: Neuropharmacology. 2014 Apr;79:603-15
references.pmid: 24452010
references.citation: Front Cell Neurosci. 2013 Dec 30;7:273
references.pmid: 24415999
references.citation: J Biol Chem. 2012 Apr 20;287(17):13930-43
references.pmid: 22378790
references.citation: J Biol Chem. 2011 Jan 28;286(4):2739-49
references.pmid: 21098022
references.citation: Nature. 2009 Jul 30;460(7255):592-8
references.pmid: 19641588
references.citation: Science. 1995 Apr 14;268(5208):307-10
references.pmid: 7716527
references.citation: Naunyn Schmiedebergs Arch Pharmacol. 1999 Apr;359(4):339-44
references.pmid: 10344533
references.citation: Fold Des. 1996;1(6):R121-6
references.pmid: 9080186
references.citation: PLoS Biol. 2011 Mar;9(3):e1001027
references.pmid: 21423654
references.citation: Nature. 2014 Aug 21;512(7514):333-7
references.pmid: 25143115
references.citation: Proc Natl Acad Sci U S A. 2014 Jan 21;111(3):966-71
references.pmid: 24367074
references.citation: Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4663-7
references.pmid: 22393010
references.citation: J Biol Chem. 2010 May 21;285(21):15805-15
references.pmid: 20308075
references.citation: J Comput Chem. 2005 Dec;26(16):1781-802
references.pmid: 16222654
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1982-7
references.pmid: 16446432
references.citation: Trends Biochem Sci. 2013 Jan;38(1):20-9
references.pmid: 23206935
references.citation: J Biol Chem. 2014 Apr 4;289(14):9909-17
references.pmid: 24515105
references.citation: Br J Pharmacol. 2014 Nov;171(22):5093-112
references.pmid: 24989924
references.citation: J Biol Chem. 2005 Jul 15;280(28):25982-93
references.pmid: 15899882
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11396-401
references.pmid: 22745172
references.citation: J Mol Graph. 1996 Feb;14(1):33-8, 27-8
references.pmid: 8744570
references.citation: EMBO J. 2012 May 2;31(9):2134-43
references.pmid: 22473210
references.citation: Mol Pharmacol. 2012 Jul;82(1):80-9
references.pmid: 22498660
references.citation: Lancet. 2015 Mar 28;385(9974):1198-205
references.pmid: 25467586
references.citation: Purinergic Signal. 2012 Sep;8(3):375-417
references.pmid: 22547202
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13604-9
references.pmid: 18757734
references.citation: PLoS One. 2014;9(5):e97528
references.pmid: 24878662
references.citation: Nature. 2012 May 10;485(7397):207-12
references.pmid: 22535247
references.citation: Chembiochem. 2002 Jul 2;3(7):604-17
references.pmid: 12324994
references.citation: Front Cell Neurosci. 2013 Dec 19;7:267
references.pmid: 24391544
references.citation: Cell. 1984 Oct;38(3):835-40
references.pmid: 6488318
references.citation: Front Cell Neurosci. 2013 Dec 06;7:245
references.pmid: 24367291
references.citation: J Gen Physiol. 2011 Jun;137(6):579-90
references.pmid: 21624948
references.citation: Physiol Rev. 2002 Oct;82(4):1013-67
references.pmid: 12270951
references.citation: Neuron. 2012 Oct 4;76(1):51-69
references.pmid: 23040806
references.citation: Biochemistry. 1990 Oct 9;29(40):9343-52
references.pmid: 2248951
references.citation: Curr Med Chem. 2015;22(7):799-818
references.pmid: 25439586
references.citation: J Neurosci. 2007 Feb 7;27(6):1456-66
references.pmid: 17287520