The need to study novel protein targets for insecticide research is paramount as insecticide-resistant pests are putting global food production at risk. Acetylcholine (ACh) is a major neurotransmitter in the insect’s central nervous system. Proteins in the ACh signalling pathway, such as the nicotinic acetylcholine receptor and acetylcholinesterase, have been heavily exploited as insecticide targets. The vesicular acetylcholine transporter (VAChT) is an integral membrane protein (IMP) belonging to the Major Facilitator Superfamily (MFS) and is an unexplored target in the ACh pathway. The aim of the project was to study VAChT structurally using X-ray crystallography, and to biochemically characterise the protein using ligand binding and transport assays.
The production of VAChT in high enough quantities and purity for these studies was a significant challenge. Therefore, VAChT genes of various species have been expressed in yeast, bacterial and insect cell systems to increase the possibility of success. Yeast membrane containing VAChT was extracted from Saccharomyces cerevisiae and had the ability to bind a ligand, as confirmed by a radioligand binding assay. VAChT from Tribolium castaneum & Acyrthosiphon pisum displayed a high affinity towards the insecticidal compound – SYN01, with Kd values of 6 nM & 2.4 nM respectively. However, the purification of VAChT proved to be difficult and degradation was observed at various stages of the process. To overcome this issue, several methods to improve the stability of this membrane protein were investigated, including fusion partner proteins, transport deficient mutations and the use of high affinity ligands.