Exploring functional heteromeric interactions of the human P2X4 receptor

Fortuny Gomez, Anna (2023) Exploring functional heteromeric interactions of the human P2X4 receptor. Doctoral thesis, University of East Anglia.

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P2X receptors are non-selective ATP-gated ion channels. ATP can act as a neurotransmitter and activate P2X receptors, allowing the passage of small cations into the cell, causing membrane depolarisation and ultimately increasing intracellular calcium levels. Functional P2X receptors are trimeric, formed by the association of three subunits around a common pore. Seven pore-forming subunits have been cloned and characterised in mammals, named P2X1-P2X7. Amongst them, the P2X4 receptor subtype is involved in numerous biological responses, contributing to ATP-mediated neurotransmission, neuroinflammation, and other inflammatory responses. P2X4 receptors can also modulate normal function and disease development in various organs, namely the heart, lungs, kidneys, and liver. Hence, P2X4 receptors have remarkable therapeutic potential for the treatment of neuropathic pain and cardiovascular disease, for example. Over the past fifteen years, there has been a growing interest in identifying possible heteromeric P2X subunit arrangements, combining different P2X subunits to constitute a functional channel pore. While the existence of heteromeric P2X2/3 receptors is well established, the composition of other P2X heteromers and the interaction between distinct trimeric receptors remains controversial.

This project aims to gain biochemical and functional evidence of human P2X4 subunit interactions. To do so, we developed a novel experimental strategy that combined functional calcium mobilisation assays to assess the effects of human P2X ‘dead receptor’ tools (i.e., double lysine-to-alanine mutants that generate a non-functional human P2X subunit) on the human P2X4 receptor activity, and Western blotting and co-immunoprecipitation assays to verify physical protein-protein interactions.

Our data revealed that human P2X1, P2X5, and P2X6 dead receptor tools exert a dominant negative effect on the human P2X4 receptor ATP-evoked calcium responses, compromising the ATP binding pocket and indicating that functional subunit interactions occurred. Analysis of protein expression and co-immunoprecipitation assays also suggested the formation of functional interactions between human P2X4 subunits and P2X1, P2X5, and P2X6 subunits. These data revealed that functional human P2X1/4, P2X4/5, and P2X4/6 heteromeric receptors can exist in this heterologous system. Human P2X2 dead receptor tools did not affect the human P2X4 ATP-evoked calcium responses, yet protein data showed a positive interaction between P2X2 and P2X4 subunits. These results supported interaction between homomeric P2X2 and P2X4 receptors rather than between subunits. Human P2X3 dead receptor tools did not affect the human P2X4 receptor ATP-evoked calcium responses and did not co-immunoprecipitate with P2X4 subunits, indicating that no functional heteromeric interactions occurred and that a heteromeric P2X3/4 receptor is unlikely. Human P2X4 receptor ATP-evoked calcium responses were affected when co-expressed with human P2X7 dead receptor tools due to a reduction in P2X4 receptor cell surface expression. Furthermore, human P2X4 and P2X7 subunits did not co-immunoprecipitate. These results indicate that a functional human P2X4/7 heteromer is unlikely, although their expression patterns seem to be intertwined.

This thesis has contributed to the understanding of novel functional interactions of the human P2X4 receptor and critically interpreted previous and current P2X4 heteromerisation studies. However, further research is required to decipher whether human P2X4 subunits form functional interactions between subunits and/or trimeric receptors. Unravelling the multimeric organisation of human P2X4 receptors will deepen our understanding of ATP signalling in health and disease and open new routes for the development of more selective therapeutics.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Nicola Veasy
Date Deposited: 22 Jun 2023 14:16
Last Modified: 22 Jun 2023 14:16
URI: https://ueaeprints.uea.ac.uk/id/eprint/92480


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