Optimizing TRP channels for use as stable thermosensors

This PhD-project is part of the trans-national training network BioInspireSensing (www.bioinspiresensing.eu), a Horizon 2020 International Training Network (H2020-MSCA-ITN-2020). The overall aim of the BioInspireSensing project is to train students in the development of a new generation of bioinspired implantable sensors of pressure, temperature and acidity. In particular, naturally occurring ion channels sensitive to these stimuli will be optimized for sensitivity, selectivity and stability, and incorporated into bioresorbable membranes for in vivo monitoring of these medically relevant parameters. The specific aim of this PhD-project is to develop biological sensors for temperature with optimization of their properties, such as sensitivity, sustained response, selectivity and stability. This will be achieved by site-directed mutagenesis of naturally occurring temperature-sensitive ion channel proteins of the transient receptor potential (TRP) superfamily, such as the cold and menthol sensor TRPM8 and the heat and vanilloid sensor TRPV1. Based on literature, molecular modelling and structural information, site-directed mutagenesis will be performed to produce mutant channels with optimized thermal sensitivity and stability, and minimal interference by other biological parameters such as pH or circulating endogenous agonists. Mutants will be expressed in HEK293 cells and characterized using patch-clamp electrophysiology and calcium microfluorimetry. Optimized channel constructs will be expressed in cells for purification and insertion into lipid bilayers. The results of these experiments will not only constitute an important step in the development of novel bioinspired and bioresorbable sensors, but also provide important novel insights into the structural determinants of temperature and ligand sensitivity of TRP channels, which may aid the development of novel drugs targeting these channels to treat pain and related pathologies.