Screening for TRPV1 Temperature-Sensing Domains with Peptide Insertion

Abstract

As the prototype of temperature-activated thermoTRP channels, the vanilloid transient receptor potential channel TRPV1 is involved in acute noxious thermosensation and thermal hyperalgesia. Its temperature sensing mechanism remains mysterious and controversial. Multiple heat-sensing domains that spread across the protein have been proposed, such as the intracellular N-terminal segment between the ankyrin repeats domain and S1, the outer pore region, and the intracellular C terminus. To test the contribution of various channel regions to heat activation, we introduced short peptide insertions to uncouple peripheral domains from the pore-forming core domain. Effects on heat- and agonist-induced channel activation were tested with both calcium imaging and electrophysiological recordings. While disruption of covalent periphery-core interaction weakened desensitization upon prolonged capsaicin application, none of the tested insertions appeared to significantly alter heat activation. Proton activation and Mg2+ activation, which are mediated by the outer pore, were also not significantly affected. These observations are consistent with the transmembrane core domain being intrinsically temperature-sensitive.