The normal development and function of the human nervous system is critically dependent upon the function of neurotransmitter- or ligand-gated ion channels, molecular machines which facilitate the communication between one nerve cell and another. Equally important, these ion channels are the targets of many therapeutic agents, from sedatives and anesthetics to anticonvulsants, and they are implicated or associated with a broad range of devastating diseases including Alzheimer's and Parkinson's diseases as well as epilepsy. Two major and important families of neurotransmitter-gated ion channels are the ATP- sensitive P2X receptors and the pentameric Cys-loop receptors, the latter of which subsumes a super family of receptors that includes those for 3-amino butyric acid (GABA), glycine, serotonin and, in invertebrates, for glutamate. Unfortunately there are no high resolution, atomic views of any of these receptors in complexes with their cognate neurotransmitters or agonists. Furthermore, in the case of Cys- loop receptors, there is not yet even a high resolution structure of a eukaryotic receptor or a published method for production of receptor suitable for high resolution structural studies. Without this information, we are unable to understand how these important neurotransmitter-gated ion channels 'work'and, most significantly, we do not have molecular maps of the binding sites for neurotransmitters on the one hand, and molecules that inhibit neurotransmitter activity, i.e. antagonists, on the other hand. Because neurotransmitter-gated ion channels are multimeric integral membrane proteins they are particularly difficult to isolate and crystallize.
The aim of the work proposed in this application is to develop new methods to screen and prepare neurotransmitter-gated ion channels for crystallographic studies, to design and implement new crystallization screening conditions, and to apply these methods to study P2X and Cys-loop receptors. Most specifically, we aim to solve high resolution x-ray crystal structures of P2X and Cys-loop receptors bound to their cognate neurotransmitter and to competitive antagonists, to test the veracity of the mapped sites by site-directed mutagenesis and ligand-binding assays, and to develop molecular mechanisms for the action of agonists and antagonists in these receptors. Taken together, our studies will provide the first atomic resolution views of these crucial receptors in their active and inhibited states, thus not only providing fundamental insight into their biological function, but also laying the foundation for the rational design of new therapeutic agents.
Neurotransmitter-gated ion channel proteins are eukaryotic, multimeric integral membrane proteins that have proven especially challenging to isolate and crystallize.
The aim of the work proposed in this application is to develop new methods for the screening, expression and crystallization of these crucial molecular machines and to apply the methods to two classes of crucial neurotransmitter-gated ion channels, ATP-gated P2X receptors and Cys-loop receptors. Completion of the proposed work will provide new insights into molecular mechanism and will yield atomic-resolution 'blueprints'of neurotransmitter and competitive antagonist binding sites, information which in turn will spur the development of novel therapeutic agents.
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