Functional Analysis of TRPV1 Channels with a Genetically Encoded Cross-Linker

Abstract

TRP channels constitute a family of non-selective, polymodal cation channels. They can sense different nature stimulus, i.e. chemical, electric, thermic and mechanic. The members of this family are homologous to the voltage-gated cation channels superfamily. Consequently, TRP channels have a tetrameric structure, composed by four monomers with six transmembrane domains (TMD) each, and substantial N- and C-terminal intracellular domains. The S1-S4 TMD region is considered the ligand binding domain, and the pore of the channel is comprised by the S5-S6 TMD and a pore loop between them. High-resolution structures are now available from multiple members of the TRPV subfamily. These structures reveal rich interaction networks between the N- and C-terminal domains, the intracellular loops and the pore forming helixes. For instance, the TRP domain, which is formed by an extended alpha-helix from the sixth TMD, is localized in the core of this interaction network, thus serving as a putative signaling integrator between modulatory signals and the channels gating mechanism(s). In order gather site- and conformation-specific information on TRPV1 function, we applied nonsense suppression to encode the non-canonical amino acid p-Benzoylphenylalanine (BPA) into conserved aromatic sites into TRPV1 in HEK cells. This aromatic amino acid is useful for functional perturbation analysis studies and in voltage-clamp UV dependent crosslinking assays. Specific encoding of BPA was achieved with an evolved E. coli. tyrosine synthetase and tRNA. Full length channel rescue at introduced TAG sites in the presence of BPA was first demonstrated by western blot of cell lysates. Functional expression was confirmed with whole-cell voltage clamp of TRPV1 channels in HEK293 cells, demonstrating that BPA containing channels could be gated by temperature and/or capsaicin induced activation. These data set the stage for site-specific cross-linking and the potential capture of transient gating states.