Biophysical Society Thematic Meeting

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

20-POS

Board 20

The N-Terminus Fine-Tunes Multiple Ligand-Binding of the Pain Sensing TRPV1 Channel

to Adjust Its Response to Capsaicin

Wei-Bin Lai

, Dennis Chang

, Huai-Hu Chuang

Academia Sinica, Taipei, Taiwan,

University of California, Los Angeles, CA, USA.

Transient receptor potential vanilloid type 1 (TRPV1) assembles into tetrameric non-selective

cation channels to serve as a polymodal sensor for noxious stimuli including capsaicin, acidic pH

and painful heat. We exploited tandem repeat tetrameric TRPV1 with all or some of the

capsaicin binding pockets replaced by the capsaicin insensitive S512F mutation, to elucidate the

mechanism with which TRPV1 differentiates the severity of noxious stimuli through graded

ligand occupancy. We created all 16 different wild type-mutant binding permutations to assess

the efficacy of capsaicin as an agonist. Binding of one capsaicin molecule is sufficient to open

TRPV1, but only partially. Further inclusion of wild type subunits in tandem repeat tetramers

promotes ligand induced channel opening. The gating effect of capsaicin is asymmetric, far more

pronounced when the only ligand-binding site was introduced into the first repeat of the tandem

TRPV1 possessing a single wild-type subunit. A flexible N-terminal domain can more

effectively propagate the binding event to the rest of tetrameric complex to open the channel

pore. Oppositely, adding a lipid-anchoring motif at the N terminus of the S-F-F-F tandem

tetramer retards channel response to capsaicin. We applied ribosomal skipping strategy by

introducing the 2A peptide between the first and second subunits, to release the N terminus at the

second position of the F

S-F-F tandem receptor. This drastically enhances the responsiveness of

the F

S-F-F to capsaicin when compared to F-S-F-F. In fact, the channel formed by F

S-F-F is

indistinguishable from the S-F-F-F tandem tetramer. These results highlighted the crucial role of

an N-terminus with a full range of mobility for binding the first ligand in the holo-receptor and

reveal the design used by this pain sensing receptor to avoid transmission of spurious noxious

information.