Distribution, subunit composition and regulatory mechanisms controlling the activity of P2X receptors
I am interested in studying the distribution, subunit composition and regulatory mechanisms controlling the activity of P2X receptors, the ligand-gated ion channels activated by ATP. These receptors have many important physiological and pathophysiological roles in the brain and the immune system.
Aims of Project
My research uses a number of techniques to study receptors in native and transfected cells (in situ hybridisation, Western blotting, immunocytochemistry coupled to confocal and scanning electron microscopy, yeast two-hybrid assays) and to manipulate the composition of the receptor protein (PCR, subcloning, construction of tagged receptors, point mutations).
P2X receptor distribution
Knowledge of receptor distribution is essential for a clearer understanding of receptor function and studying the developmental regulation of receptor expression can reveal hitherto unidentified roles for particular receptors. I am therefore examining the distribution of several of the seven identified P2X receptors in the rat nervous system at various developmental stages.
P2X receptor regulation
The regulation of receptor numbers at the cell surface is an essential mechanism which allows cells to control their responses to agonists and maintain homeostasis. Very little is known about P2X receptor regulation by endogenous and exogenous drugs and the importance of this phenomenon for cell function. My work is concentrating on two P2X receptor types, P2X3 and P2X7 , which mediate the nociceptive effects of ATP and its cytotoxicity in the immune system, respectively. I am using an extracellular P2X7 receptor antibody to study receptor expression in human monocytes (THP-1 cells). Figure 1 shows a confocal micrograph of the punctate distribution of P2X7 receptors in these cells with only a subpopulation of cells expressing the receptor, a not unexpected finding for a potential 'suicide receptor'. At a higher magnification in the scanning electron microscope, a diffuse distribution of P2X7 receptors labelled with silver-enhanced gold particles is also seen (Figure 2). I am now studying the effects of P2X receptor agonists on P2X7 receptor distribution and number in native and transfected cells. High levels of P2X3 receptors are found in dorsal root ganglia (DRG) neurones. Having raised an antibody against an extracellular region of the P2X3 receptor, I will use it to examine receptor regulation in primary cultures of DRG neurones. Further information on P2X3 receptor regulation will be obtained using cells transfected with a GFP-P2X3 receptor construct.
P2X receptor heteropolymerisation
The number of subunits required to form functional P2X receptors is unknown and I will use a variety of techniques (immunoprecipitation, receptor colocalisation and yeast two-hydrid technology) to study this further. P2X7 receptors may be associated with an accessory protein and this will be investigated by comparing receptor regulation in different immune cells.