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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
79
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.