A Structural and Functional analysis of Purinergic receptors formed from P2X4 and P2X7
Lead Research Organisation:
University of Cambridge
Department Name: Pharmacology
Abstract
Improving therapeutic strategies for the treatment of pain and chronic inflammation requires an understanding of the chemical mediators and receptors that are involved in these processes. Only then can new, selective drugs be developed. One of these chemicals is ATP, present in all cells and released into the external environment whenever there is tissue damage or inflammation. The sensors for extracellular ATP on the surface of cells are the purinergic receptors. Two members of this family that are expressed in immune cells and have been shown to be involved in pain pathology are P2X4 and P2X7. They have a trimeric structure and form ion channels that allow cations to move into and out of the cell. These two receptors have many different functional properties, not least their sensitivity to ATP; P2X4 is activated by much lower ATP levels than is required to activate P2X7 receptors. In addition, we have recently shown that these two receptors can also associate with each other to form mixed, heteromeric, receptors with properties that distinguish them from either parent type. This diversity of receptors increases the complexity of ATP signalling. Our aim is to determine the subunit composition of these receptors to reveal the structural diversity of receptors expressed, and to understand what role these different receptors play in transmitting the ATP signals in immune cells.
Technical Summary
ATP acting via P2X purinergic receptors has many effects on macrophages and microglia. It evokes inward currents, an increase in intracellular calcium, activation of mitogen activated protein kinase, cytokine release and cell death. The predominant P2X receptor subtypes expressed are P2X4 and P2X7, and both of these have been shown to play a key role in neuropathic pain. To develop therapeutic strategies for the treatment of pain we need to understand which receptors transmit the ATP signal. The P2X4 and P2X7 subtypes can function as homomers and their properties differ from each other in many key respects. Diversity is increased further by their ability to form heteromeric complexes. We have recently obtained persuasive evidence for the formation of P2X4/7 heteromers with properties distinguishable from the parent homomers. In addition P2X7 has recently been shown to form a signalling complex with the hemichannel pannexin-1. Our aims are to define the subunit identity of the physiological receptors and elucidate mechanisms for how P2X4 and P2X7 regulate the cellular response to ATP. Our specific aims are as follows: 1. To use atomic force microscopy to visualize the composition of receptors complexes formed by recombinant P2X4 and P2X7 and to determine the stoichiometry of the interaction with pannexin-1. 2. To characterize the electrophysiological and pharmacological properties of the recombinant P2X4/7 receptors. We will use mutant subunit, which function as heteromers but not homomers, to isolate the heteromeric component of the whole current. 3. To analyse P2X receptor currents in primary microglia and macrophages and identify a component with properties resembling the heteromers, and to look at the effects of knocking down P2X4 expression on these currents. 4. To compare the coupling of heteromeric and homomeric P2X4/7 receptors to downstream effectors using fluorescence based assays and confocal microscopy.
Organisations
- University of Cambridge (Lead Research Organisation)
- Max Planck Society (Collaboration)
- European Cooperation in Science and Technology (COST) (Collaboration)
- University of Portsmouth (Collaboration)
- François Rabelais University or University of Tours (Collaboration)
- UNIVERSITY OF CAMBRIDGE (Collaboration)
Publications
Masin M
(2012)
Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts.
in British journal of pharmacology
Antonio LS
(2011)
P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers.
in British journal of pharmacology
Xu XJ
(2012)
Splice variants of the P2X7 receptor reveal differential agonist dependence and functional coupling with pannexin-1.
in Journal of cell science
Murrell-Lagnado RD
(2022)
Methods for Studying Cholesterol-Dependent Regulation of P2X7 Receptors.
in Methods in molecular biology (Clifton, N.J.)
Murrell-Lagnado RD
(2008)
Assembly and trafficking of P2X purinergic receptors (Review).
in Molecular membrane biology
Boumechache M
(2009)
Analysis of assembly and trafficking of native P2X4 and P2X7 receptor complexes in rodent immune cells.
in The Journal of biological chemistry
Robinson LE
(2014)
Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization.
in The Journal of biological chemistry
Nicke A
(2009)
A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 knock-out mice.
in The Journal of biological chemistry
Srivats S
(2016)
Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1.
in The Journal of cell biology
Murrell-Lagnado R
(2009)
More cross-talk between purinergic receptors.
in The Journal of physiology
Description | Our research has focused on a family of receptors (P2X) that recognize the signaling molecule ATP and mediate its effects in the immune and nervous systems. These receptors are up-regulated in disease and play an important role in inflammation. The academic community and pharmaceutical industry are very interested in developing novel drugs that target these receptors to treat chronic inflammatory diseases such as arthritis, pain and also cancer. We have identified novel mechanisms that are important for controlling the functional properties of these receptors and in particular P2X4 and P2X7. One example is cholesterol - we have shown that cholesterol strongly inhibits the activity of the P2X7 receptor which suggests that changes in cholesterol that take place where there is inflammation, might be associated with up-regulating receptor activity. The identification of cholesterol as an important regulator of receptor activity will hopefully open up new avenues for the development of therapeutics. We have also identified variants of the receptor and characterized them in order to understand how they behave differently from the original forms of the receptors and in what tissues they are expressed. This work has been carried out on the mouse receptors. Much of our understanding of the role of P2X receptors in health and disease is based upon work carried out on mice, both wild type and those which have the receptors genetically deleted; so-called knock out mice. We have compared different knock-outs and shown that some are not true knock-outs but continue to express variants of the receptor. Our work has contributed to an understanding of the structure and function of these two important P2X receptors - P2X4 and P2X7, and will aid in their development as novel drug targets. |
Exploitation Route | There is considerable interest in developing therapeutics that target this receptor for treatment of inflammatory conditions and cancer. Our research has improved our understanding of the structure and function of P2X4 and P2X7 receptors and of the important differences that exist between the properties of the mouse and human receptors, which is of crucial importance if we are to utilize mice to investigate the role of these receptors in health and disease and to test new drugs. We have also identified one of the commonly used strain of mice that was thought to have P2X7 genetically deleted, as not being a true knock-out because of expression of different variants of the receptor. It is important that scientists working with these mice understand how expression has been altered in the different tissues. |
Sectors | Pharmaceuticals and Medical Biotechnology |
URL | http://www.sussex.ac.uk/lifesci/murrelllagnadolab/ |
Description | The findings from this project have been utilized by the research community who are working on purinergic signaling and who wish to understand the role of the purinergic receptors under normal physiological conditions and in diseases associated with inflammation. There has been considerable interest from pharmaceutical companies in developing new therapeutics targeting the P2X4 and P2X7 receptors and therefore our work highlighting differences in the structure and function of the human and mouse isoforms of the receptor is crucially important because the actions of these novel compounds are first tested in mice. Also our work highlighted that one of the two commonly used strain of mice in which P2X7 was deleted, was not a good knock-out model because other active variants of the receptors were still expressed. This has influenced the utilization of this particular strain of mice and the interpretations of results that have been obtained using it. |
First Year Of Impact | 2009 |
Sector | Pharmaceuticals and Medical Biotechnology |
Description | Project grant as coapplicant |
Amount | £226,712 (GBP) |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2014 |
End | 02/2017 |
Description | Analysis of expression of alternatively spliced transcripts of the P2X7 receptor |
Organisation | Max Planck Society |
Department | Max Planck Institute for Brain Research |
Country | Germany |
Sector | Academic/University |
PI Contribution | We identified protein expression of a variant of the P2X7 receptor in certain tissues of mouse in which the receptor was thought to have been genetically deleted. |
Collaborator Contribution | Identification of an alternatively spliced variant of the P2X7 receptor transcript that encoded a channel with an altered N-terminus that escaped inactivation in one of the commercially available strain of P2X7 knock out mice. |
Impact | The publication Nicke et al (2009) that is listed. |
Start Year | 2008 |
Description | COST Action BM1406: Ion Channels and Immune Response |
Organisation | European Cooperation in Science and Technology (COST) |
Department | COST Action |
Country | Belgium |
Sector | Public |
PI Contribution | Partner of COST Action BM1406: Ion Channels and Immune Response 'Toward a global understanding of immune cell physiology and for new therapeutic approaches' My work on P2X4 and P2X7 purinergic receptors funded by this BBSRC award provided the basis for me to be invited to join this European grouping involving ~40 groups across Europe. The COST award provides funds for travel, student exhange, meetings and workshops. |
Collaborator Contribution | All members of the COST Action are contributing towards meetings and workshops. The chair of this COST action is Dr. Florence VELGE ROUSSEL, from Université F. Rabelais - UFR Médecine. |
Impact | I have formed new collaborations with members of the COST Action, including Dr Colin Adrain, to look at the regulation of P2X7 receptor trafficking. |
Start Year | 2015 |
Description | Imaging P2X4-7 complexes by atomic force microscopy |
Organisation | University of Cambridge |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Generation of epitope tagged constructs to express and purify P2X4 and P2X7 receptors in a heterologous system. |
Collaborator Contribution | Imaging of purified P2X receptor complexes by atomic force microscopy to determine their stoichiometry. |
Impact | The paper Antonio et al. (2011) was from this collaboration and is listed in the publications list. Also with this collaboration we moved on to use atomic force microscopy to image the Orai1-STIM1 Ca2+ channels and there are two publications from this: Srivats et al., 2016 and Balasuriya et al. 2014. |
Start Year | 2007 |
Description | The role of P2X4 in breast cancer cell invasiveness |
Organisation | François Rabelais University or University of Tours |
Country | France |
Sector | Academic/University |
PI Contribution | We are looking at the role of P2X4 and P2X7 receptors in breast cancer cell invasiveness and are currently carrying out a series of in vitro experiments which show that knockdown of P2X4 in highly invasive breast cancer cells reduces invasiveness stimulated by ATP and mediated by P2X7. |
Collaborator Contribution | Dr Sebastian Roger is an expert in cancer cell biology and the role of P2X7 in cancer cell invasiveness. He has provided various cancer cell lines to us and we are currently writing grant applications to take this work forwards into in vivo mouse studies to look at the role of P2X4 in metastases formation. |
Impact | We will shortly write a collaborative paper on the role of P2X4 in breast cancer cell invasion and we are currently seeking joint funding. |
Start Year | 2016 |
Description | This collaboration provided tissue from two strains of P2X7 knock-out mice and carried out some RT-PCR reactions to identify alternatively spliced variants of the P2X7 receptor transcript. |
Organisation | University of Portsmouth |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | During the course of the project we embarked upon a collaboration with School of Pharmacy and Biomedical Sciences, University of Portsmouth, to provide us with two strains of the P2X7 knock-down mice, and to carry out an analysis of P2X7 transcripts in wild type and knock-down strains. |
Start Year | 2008 |
Description | Biannual Purine conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This is the main international meeting for researchers in the purine field and it was held in Spain, Japan and Germany in 2010, 2012, 2014 respectively. I was invited to speak at one of the sessions for each of these meeting and present our work on P2X4 and P2X7 receptors. |
Year(s) Of Engagement Activity | 2010,2012,2014 |
Description | Cost Action workshop in Warsaw, Poland |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | Workshop meetings are held every 6 months from April 2015 and members of the COST action from different European Countries present work, participate in round table discussions with the the aim of forming partnerships and collaborations. I have given two talks, chaired the round table discussion and organized the workshops. |
Year(s) Of Engagement Activity | 2015,2016,2017 |
Description | Ion Transport Conference Sandbjerg, Denmark |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Conference on different membrane transport systems provided the opportunity to make new collaborations with researchers into breast cancer working in Copenhagen. Gave a talk and lead discussions. |
Year(s) Of Engagement Activity | 2016 |
Description | Open Day University of Sussex |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Undergraduate students |
Results and Impact | Open days provide the opportunity for A level students to attend the University and consider whether or not they would like to embark upon their undergraduate degree course here at Sussex. Faculty give short talks about their research to the students. |
Year(s) Of Engagement Activity | 2017 |
Description | UK Purine Club |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | The UK purine club runs an annual one day symposium which provides the opportunity for researchers in the UK within this field to get together and talk about their work and network. I have been invited to talk at three of these and in 2013 I organized the symposium at Jesus College, Cambridge. |
Year(s) Of Engagement Activity | 2009,2011,2013,2015 |