The acetylcholine binding protein of Lymnaea stagnalis as a biosensor and model for ligand gated ion channel proteins

Abstract

The structural similarity of the Acetylcholine Binding Protein (AChBP) from Lymnaea stagnalis to the extracellular ligand binding domain of Ligand Gated Ion Channel (LGIC) receptors suggests that the AChBP could be used to mimic cys-loop ligand-gated ion channel (LGIC) receptors. An LGIC mimic could be used as a sensor molecule in a range of biotechnology applications including high throughput drug screening as well as in vivo and in vitro sensing of biologically active compounds. It could also be used as a lead molecule for engineering novel proteins with binding characteristics similar to non-acetylcholine receptor LGIC's. The soluble AChBP is easily expressed and purified and can be produced in reasonably large amounts. This thesis explores the potential for using the AChBP and related proteins as biosensors by evaluating their action on three key medium and high throughput systems: Scintillation Proximity Assay (SPA), Surface Plasmon Resonance (SPR), and Microcantilevers (MC). As a preliminary step to developing a 5-HT₃R-ligand binding protein, by altering the ligand specificity of the AChBP, the interaction of 5-HT₃R ligands with the AChBP is also evaluated. The work presented in this thesis contributes to improved methods of drug design and testing, and to a better understanding of LGIC structure.