Biophysical Society Thematic Meeting - June 28-July 1, 2015

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 1 , Dennis Chang 2 , Huai-Hu Chuang 1 . 1 Academia Sinica, Taipei, Taiwan, 2 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 2A S-F-F tandem receptor. This drastically enhances the responsiveness of the F 2A S-F-F to capsaicin when compared to F-S-F-F. In fact, the channel formed by F 2A 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.

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