Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.

Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.:Index of contents Introductory remarks „Wissenschaftlicher Anteil des Promovenden an der Publikation“ „Bibliographische Beschreibung“ I. Introduction Pain as a sensory quality Neuronal circuitry for pain processing and sensation in the PNS and CNS Transformation of thermal, mechanical and chemical stimuli into electrical activity by nociceptors; nociceptor-targeted therapeutic approaches Release mechanisms for nucleotides and their fate in the extracellular space Nucleotide receptor-types ATP-sensitive P2 receptors and pain-sensation References II. Scientific background and aims of my thesis ATP binding-sites of P2X3 receptors; subunit composition of P2X2/3 and P2X2/6 heteromeric receptors The aims of the present work III. Publications IV. Summary and conclusions Amino acid residues constituting the agonist binding site of the human P2X3 receptor ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors „Eigenständigkeitserklärung“ Curriculum vitae Acknowledgements

Orientador: Maria Cláudia Gonçalves de Oliveira Fusaro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-24T04:40:35Z (GMT). No. of bitstreams: 1 Schiavuzzo_JalileGarcia_M.pdf: 1205741 bytes, checksum: b599c1bee7cda79ed3554fcb789eea9d (MD5) Previous issue date: 2013 / Resumo: Existem evidências do envolvimento do ATP via ativação do receptor P2X3 na dor muscular. Portanto, o objetivo deste estudo foi verificar se a ativação do receptor P2X3 no músculo gastrocnêmio de ratos induz hiperalgesia mecânica, e em caso afirmativo, analisar os mecanismos inflamatórios pelo qual os receptores P2x3 induzem hiperalgesia mecânica. O Antagonista não seletivo para o receptor P2X3 ?,?meATP foi administrado no músculo gastrocnêmio de ratos, induzindo hiperalgesia, a qual foi significativamente reduzida pelo antagonista seletivo do receptor P2X3 e P2X2/3 - A-317491. A hiperalgesia mecânica induzida pelo ?,?meATP foi reduzida pelo inibidor de ciclooxigenase Indometacina, pelo antagonista seletivo do receptor de Bradicinina B1 e B2- Dalbk e Bradyzide, respectivamente, antagonista dos adrenoceptores ?1 e ?2 - Atenolol e ICI 118,551 respectivamente, e inibidor não específico de selectinas Fucoidan. O ?,?meATP também induziu o aumento da concentração local de citocinas pro inflamatórias TNF-?, IL-1?, IL-6 e CIN e migração de neutrófilos. Juntos estes achados sugerem que o ?,?meATP induz hiperalgesia mecânica no músculo gastrocnêmio via ativação de receptor periférico P2X3, o qual envolve bradicinina, prostaglandinas e aminas simpatomiméticas e migração de neutrófilos. Portanto, nós sugerimos que os receptores P2X3 sejam um importante alvo no controle da dor muscular / Abstract: There is evidence of the involvement of endogenous ATP via activation of P2X3 in muscle pain. Therefore, the aim of this study was to verify whether the activation of P2X3 receptors in the gastrocnêmio muscle of rats induces mechanical hyperalgesia and, if so, to analyze the inflammatory mechanisms by which P2X3 receptors induce mechanical hyperalgesia. Intramuscular administration of the non-selective P2X3 receptor agonist ?,?-meATP in the gastrocnemius muscle of rats induced mechanical hyperalgesia, which was significantly reduced by the selective P2X3 and P2X2/3 receptors antagonist A-317491. The ?,?-meATP-induced mechanical hyperalgesia was prevented by the indomethacin cyclooxygenase inhibitor, the selective bradykinin B1- or B2- receptor antagonist DALBK and bradyzide, respectively, the ?1- or ?2-adrenoceptor antagonist atenolol and ICI 118,551, respectively, and the nonspecific selectin inhibitor fucoidan. ?,?-meATP also induced increase in the local concentration of the pro-inflammatory cytokines TNF-?, IL-1?, IL-6 and CINC-1 and the neutrophil migration. Together, these findings suggest that ?,?-meATP induced mechanical VIII hyperalgesia in the gastrocnemius muscle of rats via activation of peripheral P2X3 receptors, which involves bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines and neutrophil migration. Therefore, we suggest that P2X3 receptors are important targets to control muscle inflammatory pain / Mestrado / Biodinâmica do Movimento Humano e Esporte / Mestra em Ciências da Nutrição e do Esporte e Metabolismo