Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

41-POS Board 41 Probing the Molecular Details of TRPM8 Ligand Gating

Jacob K. Hilton 1,2,3 , Parthasarathi Rath 1,2,3 , Melanie L. Aprahamian 4 , Steffen Lindert 4 , Wade D. Van Horn 1,2,3 . 1 Arizona State University, Tempe, AZ, USA, 2 Biodesign Institute, Tempe, AZ, USA, 3 Arizona State University, Tempe, AZ, USA, 4 Ohio State University, Columbus, OH, USA. The human TRPM8 ion channel is a polymodally gated ion channel that is activated by diverse stimuli, including cold temperature, small molecules such as menthol, phosphatidylinositol 4,5- bisphosphate, and regulatory subunit proteins. This channel has garnered increasing interest in recent years due to emerging discoveries of its physiological roles in pain, metabolism and temperature regulation, migraines, and cancer. This makes TRPM8 a potentially attractive therapeutic target; however, the molecular level details of ligand binding and activation are still under investigation. Previous work in our lab has shown that the TRPM8 agonist menthol binds to the S1-S4 ligand-sensing domain. Nuclear magnetic resonance (NMR) and microscale thermophoresis (MST) binding data show that Y745H and R842H mutants, which previous electrophysiology studies had implicated in menthol binding, retain the ability to directly bind menthol with similar affinity as the WT domain. To follow up on these studies, we use a combination of computational modeling techniques and whole-cell patch-clamp electrophysiology to probe channel-ligand interactions. A Rosetta membrane model of the transmembrane domain of human TRPM8 was constructed based on homology to cryo-EM structures of the TRPV1 ion channel, and TRPM8 agonist docking experiments were performed to identify potential binding sites. These results were used to guide the design of TRPM8 mutants which were functionally tested for menthol sensitivity in electrophysiology experiments and eventually subjected to direct binding studies by NMR.

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