PROJECT SUMMARY The detection and rapid avoidance of harmful external stimuli via pain perception is crucial for survival. The sensors of pain pathways are nociceptors, specialized peripheral sensory neurons. Nociceptors detect external extreme stimuli such as temperature, pressure and chemicals associated with injury via sensory ion channels expressed on the plasma membrane. In response to such stimuli, they fire action potentials and convey the information to the brain, thus alerting us to potential injury and allowing us to take proper actions. Transient receptor potential melastatin 3 (TRPM3) is one of three molecular pain sensors that, together with TRPV1 and TRPA1, detect noxious heat. TRPM3 is a calcium-permeable nonselective cation channel belonging to the melastatin subfamily of the TRP superfamily. It is expressed in a large subset of somatosensory neurons and is activated by heat and by a variety of chemical ligands, including the endogenous neurosteroid pregnenolone sulfate (PS). Both heat and PS-induced activation of TRPM3 evoke pain. TRPM3-deficient mice show a deficit in the development of inflammatory thermal hyperalgesia. Inhibition of TRPM3 by low doses of primidone in PS- and heat-induced pain models showed analgesic effects in mice. The activity of TRPM3 in somatosensory neurons is also negatively modulated by Gβγ protein which is released upon activation of several G-protein- coupled receptors, thereby reducing TRPM3-mediated pain. Because pain is a major unresolved medical problem and because pharmacotherapies targeting GPCR have many and severe unwanted effects, pharmacological targeting of the downstream TRPM3 may have great potential to influence several signaling pathways relevant in pain sensation and in the development of new and alternative analgesic drugs. TRPM3 is also highly expressed in pancreatic beta cells and has application in glucose-induced insulin release. Moreover, TRPM3 is implicated in developmental and epileptic encephalopathies which are a heterogeneous group of disorders characterized by epilepsy with comorbid intellectual disability. At present, the physiological and pathological properties of TRPM3 are still poorly understood. The focus of this proposal is to determine the first TRPM3 structures in different functional states at high-resolution, and to understand the molecular architecture, gating mechanism, and pharmacology of TRPM3 by combining single-particle cryo electron microscopy (cryo-EM) and patch-clamp electrophysiology. We will also initiate the purification of TRPM3 from native tissues using monoclonal antibodies that recognize three-dimensional epitopes of TRPM3 as well as knocked-in affinity tag to TRPM3 gene in mice as there is growing evidence that proteins purified from native sources may display different assembly, complex composition, and functional properties relative to proteins from recombinant systems. The strong preliminary data we provide will ensure the success of this proposal, and the outcome of this proposal will pave a solid foundation for the development of novel analgesics.

PROJECT NARRATIVE TRPM3, a calcium-permeable nonselective cation channel, plays a key role in pain perception in somatosensory neurons, and inhibition of TRPM3 in pain models showed analgesic effects in mice, highlighting TRPM3 as a potential target for novel pain medications. This research project will focus on determining the first molecular structures of TRPM3 and elucidating the gating mechanism and pharmacology, taking advantage of single- particle cryo-EM and electrophysiological recording. The proposed research is relevant to public health and NIH’s mission, because the work will provide a deep understanding and knowledge of the relationships between atomic structure, function, and pharmacology of the nociceptor channel TRPM3, as well as a foundation for developing new drugs for the treatment of chronic pain.