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

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.

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.