Award details

Pharmacology and biophysics of pannexins:a new family of ion channels

Reference	BB/F018770/1
Principal Investigator / Supervisor	Professor Alexej Verkhratsky
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	School of Biological Sciences
Funding type	Research
Value (£)	350,764
Status	Completed
Type	Research Grant
Start date	01/08/2008
End date	31/07/2011
Duration	36 months

Abstract

Pannexins are a three-membered family of mammalian membrane proteins (panx1,2,3) identified in 2003/2004 about which little is currently known. Panx1 has generated much interest in both the immunology and purine receptor biology field in the past year because of our work which identified panx1 as the long-sought dye-uptake pathway ('large pore') activated by the ATP-gated P2X7 purinergic receptor (P2X7R) and as a critical upstream activator of the caspase-1 cascade leading to the release of the pro-inflammatory cytokine, interleukin 1 beta, from activated macrophage. How endogenous panx1 acts is unknown but it seems possible panx1 forms a large-conductance plasma membrane hemichannel that acts as a conduit for molecules up to 900 d. This could account for its role as the P2X7R-induced 'large pore'. Heterologous expression of panx1 in oocytes or mammalian cells results in the appearance of a distinct depolarization-evoked current but similar currents have so far not been identified in cells natively expressing panx1. Moreover, although key features of this current (current-voltage relation and inhibition by carbenoxolone) are similar in oocytes or mammalian cells, apparent discrepancies may exist in other pharmacological properties and in single-channel currents which do not seem compatible with the hypothesis that panx1 acts as a large conductance hemichannel in mammalian cells. The proposed project will provide the first detailed biophysical and pharmacological profile of ectopically expressed panx1 in both oocytes and mammalian cells, will use high throughput dual-fluorescence screening to characterize putative panx1 inhibitors and to identify new panx1 agonists and antagonists, and will use macrophage from wildtype, P2X7R and panx1 knockout mice in order to establish whether, and how, endogenous panx1 may function as an ion channel in response to inflammatory stimuli.

Summary

Sequencing of the human genome identified each one of our genes, but this does not mean that we now know what each of the molecules encoded by each of these genes does. Indeed, there remain hundreds of proteins we don't know anything about - other than the gene (the DNA composition) that makes it. But having the DNA means that we literally can take one single gene into the lab, artificially put it into a cell and make that cell synthesize a large amount of its 'gene product' - the unknown protein. Then the fun begins: What is the protein? What does it do? Where is it found? Might it be altered in disease, or might it be manipulated with drugs so that it becomes a target for fighting disease? These are the questions that we want to answer in regard to a virtually unknown protein, pannexin-1 (panx1). We stumbled on panx1 18 months ago when we were looking for drugs that would block a known protein, the so-called P2X7 receptor (P2X7R), which we have studied for over a decade. The P2X7R is of much interest to basic researchers and pharmaceutical companies because it is responsible for causing the release of important inflammatory molecules (cytokines) from immune cells that have been recruited to areas of inflammation or injury, and blocking the P2X7R reduces pain caused by chronic inflammation. This has made P2X7R an attractive target for anti-inflammatory drug therapy. We found a drug that did not block the P2X7R itself but was even more effective in blocking the release of cytokines than was blocking P2X7R. It turned out that panx1 was physically associated with ('stuck to') P2X7R and was somehow turned on when P2X7R was stimulated and it was the panx1 protein that led to cytokine release. These findings made us realize we need to start working on panx1 by itself. The panx1 protein was just identified in 2003-4 and to date there have been only five publications by two different groups (other than ours) in which the functional properties of the panx1 protein alone have been studied. Panx was first found by labs who were searching the genome database for genes that might be responsible for making gap junctions, or bridges between cells. These bridges are formed by two half-junction proteins (called hemichannels) on the surface of adjoining cells fusing into a single protein that makes a physical link, or gap junction, through which intracellular constituents up to a certain size can readily cross. Initially panx1 was thought to be a new type of gap junction protein but it now seems that, although it can make true gap junctions under some circumstances, it is more likely to function as a solitary hemichannel. It is now important and timely to provide a detailed pharmacological and functional profile of this protein when it is expressed artificially so it can be studied 'in isolation' and also to see how panx1 functions in its native environment. To this end, we will use high-throughput drug screening technology to provide initial pharmacological characterization of artificially expressed panx1 and to screen for new compounds that activate or block panx1. We will then examine functional properties of the artificial panx1 hemichannel using high-resolution biophysical techniques (called patch-clamp techniques that can measure the tiny electric currents which flow when one or more ion channel opens) and see exactly how the compounds identified in our high throughput screen are acting. We do this by using molecular biology techniques to change one amino acid at a time in the panx1 protein and then see how this changes the function of the panx1 ion channel and its response to drugs. We will also use antibody technology to see where exactly where native panx1 protein is located in immune cells. Finally, using information obtained from this, we will make patch-clamp recordings from immune cells which normally express panx1 in order to find out how native panx1 acts as to alter immune cell function.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Immunology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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