Structural Basis of Nociceptor Channel TRPM3 gating and pharmacology - 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.
