Capsaicin- and heat-activated TRPV1 ion channel is a primary nociceptor for both chemical and thermal stimuli, hence an attractive target for pain medication. Despite the availability of cryo-EM structures of TRPV1 at up-to-2.9 resolutions, molecular mechanisms underlying TRPV1 activation remains unclear. A major limitation for obtaining mechanistic information from the cryo-EM structures is the general lack of resolution to determine side-chain orientation and the associated atomic interaction. We recently demonstrated that the limitation could be overcome by combining Rosetta structural prediction with site-specific functional tests such as thermodynamic mutant cycle analysis that serve to constrain, validate, and improve structure prediction. Using this iterative approach, in combination with site-specific fluorescence recordings including FRET and patch fluorometry, our study aims to identify functional interactions in the capsaicin-binding domain and the outer pore and, more importantly, to reveal dynamic changes of these interactions during capsaicin- and heat-induced activation. We will take particular advantage of the fluorescent unnatural amino acid (FUAA) incorporation method to introduce a small fluorophore to the channel?s moving parts. Our goal is to elucidate structural mechanisms for key molecular interactions that mediate chemical and thermal activation, thus providing a molecular framework to guide pharmaceutical intervention.
TRPV1 ion channels mediate nociception induced by both chemical and thermal stimuli. We will use a multidisciplinary approach to investigate the structural basis and molecular mechanism underlying TRPV1 activation by capsaicin and heat, aiming to provide information needed for pharmaceutical intervention of channel activation. !
| Yang, Fan; Xiao, Xian; Lee, Bo Hyun et al. (2018) The conformational wave in capsaicin activation of transient receptor potential vanilloid 1 ion channel. Nat Commun 9:2879 |
