Yeast Based Functional Assays for Identification of Activators/Inhibitors of TRPV1 and Structural Elements Involved in Thermosensing

Abstract

Transient receptor potential (TRP) channels form a huge family of cation channels first described from Drosophila as a putative integral membrane protein required for phototransduction (1). Ion channels of this family display a wide range of selectivity and gating mechanisms and are essential factors in sensing of various external stimuli, such as temperature, mechanical forces or noxious chemicals.We expressed the TRPV1 channel from Rattus norvegicus in the bakers yeast, Saccharomyces cerevisiae, and established assays to study channel function in this heterologous expression system. On standard agar plates, activation of TRPV1 by capsaicin had little effect on growth of yeast, but selective conditions could be identified where TRPV1 activity was toxic to yeast. Simple growth based assays will be presented which might be useful to screen for activators and inhibitors of TRPV1. The suitability of this assay is demonstrated with the activators capsaicin, pseudocapsaicin and natural extracts from various spices (chili, black pepper) as well as temperature activation in various C-terminal mutants of TRPV1. Growth data are compared with electrophysiological recordings from yeast.(1) Montell, C., Rubin, G.M. (1989) Neuron.,2, 1313-1323.