The Cell Biology Of ATP Signalling At Intracellular P2X Receptors

ATP is an energy source used inside every cell in our bodies but is also released by cells allowing cells to communicate with one another. ATP released from cells binds to and opens receptors on the cell surface, called P2X receptors. Once open these receptors allow sodium and calcium to flow into the cell resulting in the activation of further processes. In animals, P2X receptors are present on the cell surface on nerves, immune cells and muscle, they consequently are involved in sensing pain, inflammation and the control of blood flow. It is the overall aim of our research to understand P2X receptors better in order to develop pain relieving and anti-inflammatory therapies in humans. We recently identified a new P2X receptor, called DdP2X, in a species of amoeba. This was the first time a P2X receptor had been found in a single celled organism. In contrast to animal P2X receptors DdP2X was found inside the cell and not at the cell surface. DdP2X was present on a specialised organelle inside the amoeba called a vacuole. The vacuole is responsible for pumping out excess water entering the cell, which allows the amoeba to regulate its volume. In order to understand the role of DdP2X, we generated amoeba lacking DdP2X by removing the gene that makes it. Amoeba lacking DdP2X could not pump water out as effectively and consequently swelled when placed in water. This observation suggested that DdP2X is required for the vacuole to function correctly. One of the major goals of this proposal is too understand how DdP2X regulates vacuole function in amoeba. This findings of this research may inform us of how P2X receptors may regulate vacuoles in human cells. We will identify whether calcium moving through DdP2X is essential for correct vacuole function and excretion of water. We will also identity other proteins that are associated with DdP2X. This will help us to understand how the receptor is localised to the inside of the cell and identify the molecules inside the cell that regulate or are regulated by DdP2X. The second major focus of this study is to understand how P2X receptors work and their evolution. We have now identified several other primitive P2X receptors in singled celled organisms like green algae. Primitive P2X receptors are useful tools for understand how receptor structure relates to function. This is because primitive P2X receptors share only few similarities with human, mouse and rat receptors and therefore comparing and manipulating them will reveal the critical parts that are required for a P2X receptor to work as an ATP-binding ion channel. We ultimately want to understand the parts of P2X receptors that bind pain relieving drugs. In summary, we will us amoeba as a model cell to investigate the functions that P2X receptors play inside the cell. Experiments using P2X receptors from single celled organisms will be used to inform us of how human P2X receptors function in health and disease.

The molecular evolution and cell biology of P2X receptors in simple organisms is currently unclear. Investigation into this area would inform us greatly on the molecular operation and physiology of P2X receptors and ATP signalling. We recently identified the first P2X receptor from a single celled organism, the amoeba D. discoideum. In contrast to cell surface P2X receptors of animal cells the amoeba P2X receptor was intracellular, located on the contractile vacuole, an osmoregulatory organelle. We demonstrated that amoeba lacking this receptor were void of a regulatory cell volume decrease in response to hypotonic stress, demonstrating a role for P2X receptors in vacuolar function. We propose to investigate the mechanism of P2X receptor-dependent control of vacuolar function in D. discoideum, in order to understand the potential role that P2X receptors may play in the control of animal vacuole and cell function. We will address several fundamental questions: Is P2X-dependent vacuolar membrane Ca2+ flux required for correct vacuole function? Does compartmentalised ATP release inside the cell regulate vacuolar P2X function? What are the determinants of intracellular P2X receptor targeting? What are the molecular assemblies that mediate P2X-dependent vacuolar regulation? We will use vacuolar-specific Ca2+ and ATP biosensors to assay vacuolar flux in wild-type and P2X null amoeba. We will use protein chemistry, genetics and immunofluorescence to determine protein partners and cellular location of wild-type and mutant receptors. To better understand the molecular operation, pharmacology and evolution of P2X receptors we will compare the function of human and low homology primitive counterpart receptors from single celled organisms that we have identified, using mutagenesis to test hypothesise. Comparisons will be made to understand structure-function relationship with the ultimate aim of improving analgesic and anti-inflammation drug development in man.