Investigating structure and ligand binding in pentameric ligand gated ion channels

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences


Pentameric ligand gated ion channels (pLGICs), such as GABAA and nicotinic acetylcholine receptors, control the balance between excitatory and inhibitory signalling in the central nervous system (CNS) by reacting to specific neurotransmitter molecules. In humans they represent targets for anti-convulsants, anxiolytics and anaesthetics and in insect species they are also targets for insecticides.

The focus is on the pLGIC GABAA receptor called Resistance to Dieldrin (RDL), which is widely expressed across insect species and represents a key target for insecticides such as dieldrin and fipronil. However, spontaneous mutations in these receptors have rendered many species of insect resistant to numerous commercial insecticides. While homology models and electrophysiology have suggested RDL possesses similar functional properties and structural arrangements to human GABAA receptors, there are several key differences and a lack of direct information on the structural and ligand binding aspects of RDL.

Therefore, it is aimed to obtain purified recombinant RDL for biophysical structural and ligand binding studies, so to investigate the modes of action for insecticide binding and how mutations impact this. A series of expression constructs have been explored in insect and mammalian cells and conditions will be developed for purification. For structural studies the latest advances in cryogenic electron microscopy will be taken advantage of, but attempts will also be made to obtain crystals for X-ray diffraction. Binding properties will be defined with biolayer interferometry and surface plasmon resonance, with electrophysiology as an orthogonal assay for functional characterisation. This research could potentially enable the development of more effective novel insecticides for the control of disease carrying vectors and agricultural pests. Moreover, the specifics of RDL may illuminate details of mammalian GABAA receptors, in particular the class subunits.

Complementary research has involved using acetylcholine binding protein (AChBP) as a surrogate for characterising the binding of novel epibatidine and cytisine derivatives to nicotinic acetylcholine receptors. These ligands have potential uses as anti-nociceptive and smoking cessation agents. Binding properties have been characterised using a variety of biophysical approaches, including isothermal titration calorimetry, tryptophan fluorescence quenching and biolayer interferometry. To elucidate the structure-activity relationships (SARs) of the ligand interactions with AChBP, X-ray crystallography has been utilised. Experience has been obtained in setting up crystallisation experiments, diffraction of crystals, processing and refinement of diffraction data and the analysis of modelled structures to ascertain SARs.

Key Words and Skills:
Construct design, molecular biology, recombinant protein production and purification, crystallography, electrophysiology, cryo-EM, biophysical binding assays.


1. Explain interdisciplinary interface: The project explores aspects of ion channel function exploiting different molecular biology and biophysical approaches, exploiting the technology of organic chemistry synthetic methods by collaborators, electrophysiology and early stage drug discovery.

2. Does project require significant amount of quantitative skills? YES

3. Does project require significant amount of whole organism physiology skills? NO (delete as appropriate)


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Description Characterisation of novel cytisine derivatives with acetycholine binding protein. 
Organisation University of Bristol
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have characterised the binding of novel derivatives of the smoking cessation drug cytisine to acetylcholine binding protein, a surrogate of the nicotinic acetylcholine receptor binding site, by isothermal titration calorimetry. It has also been attempted to crystallise the derivatives with acetylcholine to elucidate the binding interactions, with one crystallising successfully and diffracting to high resolution. This structure and the associated binding data has been published in a peer reviewed journal.
Collaborator Contribution Design and supply of novel cytisine derivatives for us to characterise with AChBP.
Impact Davis, S., Rego Campello, H., Gallagher, T., and Hunter, W.N. (2020). The thermodynamic profile and molecular interactions of a C(9)-cytisine derivative-binding acetylcholine-binding protein from Aplysia californica. Acta Crystallogr. Sect. F Struct. Biol. Commun. 76: 74-80.
Start Year 2018