Structural Studies of the Cold and Menthol Sensing Ion Channel TRPM8
The ability to sense and avoid detrimental harm from the environment is key to survival of organisms. In mammals, nociceptive neurons detect potentially damaging stimuli from the external environment, convert them into electrical signals, and elicit pain sensations in higher nerve center inside bodies. The detection of noxious stimuli at the beginning of this sensory circuit is mediated by receptors or ion channels expressed at peripheral nerve endings, which respond to specific chemical, thermal, or mechanical stimuli and trigger ion fluxes that lead to firing of action potentials for signal transduction. Understanding the mechanisms by which these receptor and ion channels transduce noxious signal for nociception would advance the development of analgesics for pain treatment.
A large group of such nociceptive transducers belongs to the transient receptor potential (TRP) channels superfamily. Several members of TRP channels have been found expressed in subsets of nociceptors and specialized in sensing diverse noxious chemical and thermal stimuli. The TRP melastatin subfamily member 8 (TRPM8) has been characterized as the principal molecular sensor in humans for detecting innocuous to noxious cold temperatures. It is also activated by the naturally occurring cooling compound menthol which induces cooling sensation. TRPM8 function requires the important signaling phospholipid phosphatidylinositol 4,5- bisphosphate (PIP2) as a cofactor, which allosterically couples with cooling agonists for channel activation. Since the cloning of trpm8 gene in 2002, substantial amount of functional studies has dedicated to understanding the molecular basis of ligand recognitions in TRPM8 and the underlying mechanisms of ligand- and temperature-dependent channel gating. However, a major barrier to achieve these goals has been the dearth of structural information of TRPM8, which would allow for direct visualization of ligand-channel interactions and for dissection of conformational pathways in channel gating.
We have attempted to address these gaps in knowledge from a structural biology standpoint. We employed single-particle cryo-electron microscopy (cryo-EM) and reported the first structure for a full-length TRPM8 channel form the collared flycatcher (Ficedula albicollis), which was resolved to an overall resolution of ~4.1 ångström. Our structure revealed a three-layered architecture for the homotetrameric TRPM8 channel and identified distinct features in channel assembly and interfacial contacts between subdomains. Combined with previous mutagenesis studies, we proposed the putative binding sites for menthol and PIP2. Our initial study provided a structural glimpse for the design principle of TRPM8 channel as the cold- and menthol-sensor. Next we examined the molecular basis of ligand recognition in the channel by determining additional cryo-EM structures of TRPM8 in complex with cooling compounds and PIP2. On the basis of our structural data and functional characterizations, we revealed the binding sites for cooling agonists and PIP2, and identified essential ligand-channel interactions, which corroborated with predictions from previous mutagenesis studies. More importantly, in comparison with other TRP channel structures, our TRPM8 complexes provided the structural basis of synergistic binding between cooling agonists and PIP2, which enables allosteric coupling between these two modulators for TRPM8 activation. Taken together, our study provided a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents. It offered structural insights for development of novel analgesics targeting TRPM8 for pain treatment. Our study may also facilitate the sensory biology field to progress one step forward towards dissecting polymodal gating mechanisms of TRPM8 and other nociceptive TRP channels as well.
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