Integrated mutagenesis, bio-informatic and fluorescence approaches to characterize the molecular basis of antagonist action at P2X7 receptors for ATP

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Damage to cells leads to the release of the chemical ATP, this then acts as a danger signal that is sensed by P2X7 receptors. Binding of ATP to P2X7 receptors on the surface of immune cells triggers their stimulation and an inflammatory response that can contribute to a range of clinical conditions. Blocking P2X7 receptor stimulation can reduce the symptoms of pain, arthritis, Crohn's disease, high blood pressure, as well as kidney injury and damage to the heart following a heart attack. Drugs that block the action of ATP at P2X7 receptors (antagonists) therefore have considerable potential for treating disease and some of these are undergoing clinical trials. However we do not understand where on the receptor these drugs bind. In this study we aim to determine how four chemically distinct types of P2X7 receptor antagonist bind to the receptor. We have constructed a 3D model of the P2X7 receptor based on the known structure of a related P2X receptor. We then used computer based simulations, with the chemical structure of the drugs and the receptor, to make predictions on where the antagonists could bind to the receptor. These suggested several different solutions for binding that were clustered into two regions on the receptor surface. We therefore propose to test these predictions by generating P2X7 receptors that have been modified by systematically replacing parts of the P2X7 receptor predicted to bind antagonists with the corresponding region from an antagonist insensitive P2X1 receptor. We can inject the cDNA for the mutated P2X receptors into unfertilized frog eggs that then synthesise the receptors, and make electrical recordings from the eggs to measure responses to added ATP and whether this is blocked by the antagonists. If the region that is replaced is important we would expect the mutant P2X7 receptor to have reduced sensitivity to the antagonist, if there is no change in sensitivity this would indicate the variant region that was swapped did not contribute to drug action. Therefore we can use this replacement strategy to identify regions of the receptor that are important for antagonist binding. P2X7 receptors are made up from amino acid building blocks (there are 20 different types of these that vary in their chemical properties and thus interaction with drugs). In a second round of mutagenesis studies we will determine which amino acids within a region shown to affect antagonist action are involved directly in drug binding. To do this we will systematically mutate individual amino acids to a cysteine residue and characterize receptor properties in frog eggs. If a cysteine mutation reduces antagonist inhibition that will identify an important residue for drug action. The advantage of cysteine is that this amino acid has unique properties in that it can be chemically modified and fluorescently labelled. We can use this fluorescent labelling to measure directly how accessible the introduced cysteine residue is. To do this we will take oocytes expressing the cysteine mutant and treat them with the fluorescent label. We will then determine whether this level of "control" fluorescence is different from that where the receptors have also been treated with the antagonist. If a cysteine residue is part of the drug binding site we expect the antagonist to block accessibility to the cysteine and so reduce fluorescent labelling. These fluorescent methods will thus show which residues directly line the antagonist binding pocket. We will then undertake additional computer modelling and mutagenesis with other amino acid substitutions to determine the exact nature of the chemical interaction between the drug and the P2X7 receptor. This information will help in the improvement of P2X7 receptor drugs and be useful to develop drugs to block the related P2X4 receptor that is involved in pain but for which there are currently no effective blockers.