Chemical modification of ion channels: development of a novel and fast binding assay for ion channel inhibitors

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Ligand-gated ion channels are proteins located at nerve terminals, also called synapses. They are responsible for the rapid transmission of nerve impulses from one nerve cell to the next one. Ligand-gated ion channels are transmembrane proteins and mostly comprise several subunits. The crucial physiological importance of ligand-gated ion channel becomes apparent when the channel function is impaired. In fact, numerous mutations in ligand-gated ion channel genes are known to cause neurological diseases. In addition, ligand-gated ion channels are the site of action of many therapeutic drugs as well as toxins from snakes, spiders and wasps.Small organic molecules (neurotransmitters) are released into the synaptic cleft from the pre-synaptic cell and they bind to the ligand-gated ion channel resulting in a conformational transition from a non-conducting closed state to a conducting open state. High ion flux across the biological membrane in the open channel state triggers further events in the post-synaptic cell and finally leads to the generation of a new action potential and transmission of the nerve impulse.There is evidence that small molecules which block channel function specifically may be useful for treatment of certain psychiatric disorders such as anxiety, drug-dependence, schizophrenia and cognitive dysfunction. Although there have been considerable achievements in the past ten years to solve the exact three-dimensional structure of these large proteins these structures are not accurate enough to allow rational structure-based drug design. As a result many different compounds or libraries of compounds are being synthesised and have to be tested against the ion channel receptors. Current screening methods are time consuming and some even rely on using radioactive compounds.The serotonin 5-HT3 ion channel receptor is the focus of this project. It functions as a pentamer of five identical subunits. Each subunit has an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain is predominantly located in the synaptic cleft and is responsible for forming the neurotransmitter binding site. This research proposal describes the development of a fast and efficient binding assay which is based on detection of fluorescence and which would be amenable for high-throughput screening. In the proposed project the serotonin 5-HT3 receptor, which can be expressed in mammalian cells using standard molecular biological techniques, will be chemically modified with a fluorescent dye near the binding site using a photochemical reaction and designed synthetic modifier compounds. Specially designed small organic molecules will be synthesised which will bind to the modified receptor and quench its fluorescence. Any new compound which has a higher binding affinity than the quencher compound will displace the quencher and as a result alter the fluorescence of the modified receptor. The restoration of fluorescence could be detected using an appropriate read-out. Hence increase in fluorescence upon addition of a new compound would mean it is binding to the ion channel receptor and accordingly represents a potential receptor inhibitor. Once set up the binding assay would be tested using known 5-HT3 receptor inhibitors in order to compare the results with data obtained by traditional analysis methods.Apart from being used in the proposed binding assay, undoubtedly, a fluorescent 5-HT3 receptor could prove a very useful tool to address fundamental questions of how these ligand-gated ion channels work and how the binding event is structurally linked to the channel opening event. Such improved knowledge about the function and structure would be very valuable for drug design and ultimately will lead to better and more selective compounds. Furthermore, if successfully developed, the same concept could be used to tackle more complex but pharmacologically more important ion channel receptors.