AbstractChannels and transporters are essential proteins for the control, mediation and termination of neurotransmission. These are implicated in numerous pathological conditions, including epilepsy, Parkinson’s disease, neuropathic pain and nicotine addiction. However, the structural and ligand binding aspects of many of these channel and transporter proteins are poorly defined, which limits being able to design new molecules that can effectively target these conditions.
It was aimed to investigate structure and ligand binding at neurotransmitter channels and transporters. The first aims involved elucidating the binding modes and structure-activity relationships of novel ligands at nicotinic acetylcholine receptors (nAChRs), using the surrogate protein acetylcholine binding protein (AChBP). The ligands being characterised were of interest as potential anti-smoking agents and as research tools for studying nAChRs. Binding data and protein complex crystal structures were obtained for several of these novel ligands and it was possible to identify residues which were potentially responsible for their modes of action and affinity to AChBP, and henceforth likely to nAChRs. Knowledge of these interactions could assist in the future design of other ligands targeting nAChRs.
The second set of aims were associated with attempting to establish methodologies for the efficient recombinant production of complex eukaryotic ion channels and neurotransmitter sodium symporters. The initial objective was the insect ligand gated ion channel resistance to dieldrin (RDL), which is a target for insecticides. Sf9 insect cells proved unsuitable for production as only a small amount of the total protein could be extracted with non-ionic detergents and it was implied that the majority of the protein was likely in an un-folded state. Mammalian HEK293 cells were more successful as the protein could be efficiently solubilised, but ultimately the yields of purified protein were too low for this to be a feasible approach.
There was more success with producing the human GABA transporter 1 (GAT1). This terminates the actions of the inhibitory neurotransmitter GABA by removing it from the synapse and is a therapeutic target for the control of epilepsy. Using Sf9 cells and a conventional baculovirus system showed initial success, but there were ultimately problems with aggregation. Use of a recently described baculovirus system with an early Drosophila Hsp70 promoter however resolved these problems and led to high yields of purified GAT1. The obtained protein was suggested to be potentially suitable for future structural studies by single particle cryogenic electron microscopy (cryo-EM). Purified GAT1 was also used as a target to isolate recombinant nanobodies from a yeast library and these may be of assistance for increasing the size of the protein for cryo-EM studies.
|Date of Award||2022|
|Sponsors||Medical Research Council|
|Supervisor||Bill Hunter (Supervisor) & Tim Hales (Supervisor)|
- Resistance to Dieldrin
- GABA Transporter 1
- Acetylcholine Binding Protein
- Nicotinic Acetylcholine Receptors
- Ion Channels
- Transporter Proteins