Mapping "missing" conformations of ATP-gated P2X receptor ion channels

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Cells within the body communicate with one-another through the release of chemicals recognised by specific cell surface receptors. One example of such a chemical is ATP that binds to P2X receptors (P2XRs) and activates them. In humans there are at least 13 different types of P2XR (e.g P2X1R and P2X7R) that vary in their properties, for example how long they are able to be "turned-on/activated". P2XRs play an important role in a range of normal bodily functions e.g. in control of blood clotting and taste sensation, as well being drug targets for the treatment of pain and neurodegenerative diseases e.g. Alzheimer's disease. P2XRs are membrane proteins with parts on the outside of the cell (extracellular) that recognize the ATP molecule, a channel region that passes through the cell wall (a valve/tap that regulates the movement of positively charged ions) and a region inside the cell (intracellular) that regulates how long the receptor is "ON" for (dependent on the receptor type). In the absence of ATP the P2XR channel is closed and "OFF". ATP binding to the extracellular region leads to a change in the shape of the receptor; an "ON" signal opening the channel (movement of positive ions through it excites the cell). Recent studies have shown the 3D structure of a P2XR in the "OFF" and "ATP-ON" states and this has provided a major advance in our understanding of how this novel family of receptors works. However, these are only two snapshots of the receptor, and it is clear that additional movements in the 3D shape are important that result in distinct forms of the receptor with special properties. This proposal aims to gain 3D structural information on three distinct "missing" conformations/states of the receptor. (i) A "relaxed-OFF" form of the receptor in the absence of ATP that may be important for understanding of how drugs work to block/stop the receptor being turned on. (ii) An "ATP-CLOSED" desensitized receptor, where after opening following ATP binding the channel closes (i.e. turns off); a feature of the P2X1R involved in blood clotting. (iii) An "ATP-EXTRA-ON" state where the channel region gets larger and allows large molecules to enter the cell; this is particularly associated with the P2X7R and its role in inflammation and cell death. Information on these additional "missing" structures is essential to understand the fundamental mechanisms associated with the activity of this distinct family of receptors. P2XRs are made up of different amino acid "building blocks" and these can be individually changed. Of particular use for this is mutating an amino acid to cysteine; this is a chemically unique amino acid that can be targeted with a wide range of cysteine-specific compounds. These bind to the cysteine residue and change the chemical properties/size. In normal P2XRs there are no cysteine residues available for modification and cysteine-specific compounds have no effect. Therefore we can introduce cysteine mutations at defined parts of the receptor and determine the effects of cysteine reactive compounds to investigate the structure. This will be carried out in the absence of ATP (relaxed-OFF), and in the presence of ATP at desensitizing P2XRs (ATP-CLOSED) and at the ATP-EXTRA-ON P2X7R. We will test whether an introduced residue is accessible (on the receptor surface or in the channel), and by varying the size of the cysteine reactive compound measure the dimensions around that residue as well as test the effects of the modification on ATP evoked responses. These results will give molecular dimensions that will then be used in computer based studies to map the molecular changes in the receptor and provide validated 3D models of the missing receptor structures. This will provide a fundamental insight into how P2XRs work at the molecular level, understanding variations between receptors, and why genetic mutations affect receptor properties that can lead to imbalances in signalling and disease.