A Structural and Functional analysis of Purinergic receptors formed from P2X4 and P2X7

Lead Research Organisation: University of Cambridge
Department Name: Pharmacology


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.


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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