P2X receptors for ATP: using the model organism Dictyostelium discoideum to understand their regulation and roles

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

Adenosine 5 -triphosphate (ATP) was discovered in muscles in 1929, and it was soon realised to be the key for energy production inside cells. But, as is often the case, the same molecule has an important role outside cells and is used to signal between cells. The cell surface receptors for ATP, known as P2X receptors, are proteins with a hole down the middle: they acts as gates that allow small ions to cross the membrane when ATP binds to the receptor. This in turn leads to changes in cell behaviour. In fact, it is now known that these responses to ATP regulate diverse physiological processes in mammals, including taste, bladder emptying, oxygen sensation, inflammation and pain. ATP signaling by P2X receptors therefore represents a novel target for disease involving pain and inflammation.

To understand how P2X receptors work, it is important to understand how they are regulated, and to discover the further effects of P2X receptor activity. But such studies have been difficult to perform because P2X receptors had not been found in simple model organisms suitable for laboratory studies. Recently, we discovered that P2X receptors are present in the social amoebae, Dictyostelium discoideum. Dictyostelium is used to study many processes in cell and developmental biology, due to its relative simplicity and the ease with which genetic and biochemical studies can be carried out. We were surprised to find that one Dictyostelium P2X receptor actually regulates responses to ATP inside cells, rather than at the cell surface. But we believe that this action is not related to energy metabolism: rather, the receptors are required for the cells to adapt to the stress of being submerged in water. As Dictyostelium cells normally live in the soil, this is likely an important adaptation for their survival.

Most importantly, these findings will allow us to address how P2X function is regulated. Firstly, we will use cutting edge genetic and biochemical techniques to identify other proteins that are required to regulate receptor activity in Dictyostelium. Secondly, we will determine the role of the other four P2X receptors and compare the ATP responses of these receptors to those seen with mammalian P2X receptors.

Since the basic mechanism of operation of P2X receptors is conserved from amoeba to man, we will be able to advance our understanding of P2X receptor function, regulation and structure in all animal species.

Technical Summary

Abstract of research

P2X receptors were discovered as cell surface proteins that responded to extracellular ATP. But we recently found that in the microbe Dictyostelium discoideum they can also function on internal membranes, where they respond to intracellular ATP. This is important because: (1) It draws our attention to possible key roles in intracellular organelles such as phagosomes and endoplasmic reticulum, where P2X receptors have been observed but their functions not understood. (2) It provides a unique opportunity to investigate P2X receptor function and regulation using genetics, biochemistry and cell biological approaches, as Dictyostelium is a model system of the post genomic era. (3) The limited sequence relatedness between P2X receptors of protists and mammals, compared with the similarities of function as ATP-gated ion channels, can be used to greatly improve our understanding of molecular function and structure.

Specifically we propose to:

Identify novel P2XA regulators
A cell line will be generated in which P2XA-TAP will replace the endogenous P2XA gene. P2XA interacting proteins will be purified from Dictyostelium cells and identified by mass spectroscopy. Knockout mutants in interacting proteins will be generated. Genes will be studied to define function in Dictyostelium and mammalian cells.

Techniques: Dictyostelium molecular genetics/cell culture, TAP-tag protein purification, Mass spectroscopy, Dictyostelium mutant and overexpression strain generation, Mammalian cell culture and transfection, Biophysical recording from mammalian cells (whole cell and single channel), Live cell fluorescence microscopy, Antibody staining of fixed cells

Characterise additional P2X-like genes in Dictyostelium
The expression pattern and subcellular distribution of four additional Dictyostelium P2X-like proteins will be determined. Knockout mutants and overexpression lines will be generated. Proteins will be humanized and expressed in HEK293 cells. Agonists and antagonists of each receptor will be defined and compared. Techniques: Dictyostelium molecular genetics/cell culture, Quantitative PCR, Live cell fluorescence microscopy, Biophysical recording from mammalian cells (whole cell and single channel)

Publications

10 25 50
 
Description BBSRC CASE STudentship
Amount £60,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 09/2018
 
Description Biomedical Resource
Amount £498,592 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2014 
End 04/2017
 
Title Dictyostelium strains and plasmids 
Description Dictyostelium strains and plasmids which will be used to study P2X receptor function 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact New ways to think about P2X receptor regulataion 
 
Description Baylor college of medicine 
Organisation Baylor College of Medicine
Department Department of Molecular and Human Genetics
Country United States 
Sector Academic/University 
PI Contribution Provision of materials
Collaborator Contribution Advice and expertise on next generation sequencing and bioinformatics
Impact Publications, training
 
Description BOLTON SCHOOL 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Poster Presentation
Geographic Reach Local
Primary Audience Schools
Results and Impact BOLTON SCHOOL PUPIL WORKED IN LAB FOR 6 WEEKS

SCHOOL ASKED TO CONTINUE PLACEMENTS. RESEARCH WAS HIGHLIGHTED TO 6TH FORM STUDENTS THROUGH TALK AND POSTER
Year(s) Of Engagement Activity 2013
 
Description Radio 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Radio interview with questions on the topic

further write ups in press
Year(s) Of Engagement Activity 2010
 
Description Wellcome Trust 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interviwe for Wellcome trust website. Description of research activities

asked to podcast research
Year(s) Of Engagement Activity 2012
 
Description Why am I similar to slime mould? 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Children felt Developmental Biology was interesting from feedback received

Feedback that wanted to do another lab visit next year
Year(s) Of Engagement Activity 2014