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.
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)
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)