Localization of the Temperature Sensors to the Pore Domain of the TRPV1 Channel

Temperature detection is key to animal survival, and thermo-TRP channels play important roles in sensing environmental temperature in mammals. The TRPV1 channel is a classic thermo-TRP channel that is also a detector of noxious stimuli, including acidosis, vanilloid compounds, venom toxins, membrane lipids and heat. Despite extensive investigation, the mechanisms of temperature sensing and the location of the temperature sensor in TRPV1 channels remains enigmatic. One candidate for housing the temperature sensor is the outer pore of TRPV1, as mutants in this region, as well as occupancy by toxins and sodium ions, have been shown to alter temperature-dependent activation. Here we engineered chimaeras between TRPV1 and the Shaker Kv channel using available cryo-EM and X-ray structures to see if functional chimeras could be generated. Our biochemical analysis shows that a chimera containing the pore domain of TRPV1 in the background of the Shaker Kv channel can be robustly expressed in oocytes and traffic to the membrane surface. Electrophysiological recordings show that these chimeric channels can be activated by a TRPV1-selective pore targeting double-knot toxin and are permeable to calcium ions similar to TRPV1. Remarkably, these chimeric channels are highly temperature sensitive, being activated by temperatures above 40°C, similar to the WT TRPV1 channel. Taken together, our results suggest that the pore domain of TRPV1 houses the sensory apparatus underlying steep temperature-dependent activation.