Pain is among the most common reasons people seek medical care, yet the treatment options are limited, and the current opioid epidemic especially highlights the urgent need of new safe and effective therapeutics. Thermal TRP channels are a group of temperature sensitive ion channels recently discovered in peripheral sensory neurons and keratinocytes where they mediate the first step of thermal sensation and nociception, and have emerged as attractive targets for creation of novel analgesics. The goal of this proposal is to fully characterize thermal sensing in these channels and distinguish it from agonist sensing in the same protein. Current studies of the channels rely on patch clamp which is limited to detection of pore opening, while properties of stimulus sensing, which is allosterically linked to gating, can only be inferred from influences on gating. Moreover, patch responses, commonly resolved with slow temperature ramps, are analyzed assuming equilibrium gating that recent studies indicate is not valid. These fundamental limitations cause an uncertainty about temperature sensitivity of channels and interpretations of mechanisms and mutagenesis results. This application will expand the span of the studies with a new methodology of calorimetry to directly probe temperature sensing in channels and a unique laser heating approach to time-resolve temperature activation. These novel tools will allow us to separate sensing and gating and tackle non-equilibrium dynamics and thus enable an in-depth mechanistic analysis of channels. We will exploit these approaches, in conjunction with biophysical modeling and functional reconstitution of purified proteins in liposomes, to address the central question of how these channels obtain their strong temperature sensitivity. Our Aim 1 will exploit calorimetry to directly detect thermal transitions of heat sensing in reconstituted TRPV1, a prototypical heat-sensitive TRP channel, and will combine that measurement with patch recordings to derive a complete and rigorous analysis of heat sensitivity of the channel. Aim 2 will combine electrophysiology and calorimetry with mutagenesis to determine whether there are subdomains of the channel protein that dictate the energetics of heat sensing and are thus acting like heat sensors. Aim 3 will address the polymodal TRPV1 activation mechanisms by testing several prominent models that are difficult to differentiate by patch clamp alone. The experiments will elucidate whether heat sensitivity is localized within the channel and fundamentally separable from agonists and other stimuli. Overall, the application will transform our ability to study thermal channels, and the findings can guide the design of analgesics with specificity to particular stimuli.