Abstract
The tetrameric capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) in mammals has evolved the capability to integrate pain signal arising from harmful temperature and chemical irritants. The four repetitions of TRPV1 subunits result in an ion channel with excellent pain sensitivity, allowing this ionotropic receptor to differentiate graded injuries. We manipulated the stoichiometry and relative steric coordination of capsaicin-bound structures at the molecular level to determine the rules by which the receptor codes pain across a broad range of intensities. By introducing capsaicin-insensitive S512F mutant subunits into the TRPV1 channel, we found that binding of the first ligand results in low but clear channel activation. Maximal agonist-induced activation is already apparent in tetramers harboring two or three wild-type TRPV1 subunits, which display comparable activity to wild-type tetramer. The non-vanilloid agonist 2-aminoethoxydiphenyl borate (2-APB) differs from that of capsaicin in the TRPV1 channel opening mechanism activating all S512F-mutated TRPV1 channels. Two or more wild-type TRPV1 subunits are also required for full anandamide-induced channel activation, a cannabinoid that shares overlapping binding-pocket to capsaicin. Our results demonstrate that the stoichiometry of TRPV1 activation is conserved for two types of agonists.
Highlights
Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeating channel assembled from four identical subunits that surround the ion-conducting pore
We determined if the stimulatory effects of capsaicin on transient receptor potential vanilloid 1 (TRPV1) and its ion permeability can be monitored by changes in Fura fluorescence within Human embryonic kidney 293T (HEK293T) cells
TRPV1 activation by 30 μM capsaicin resulted in large-scale Ca2+ influx, with a 1.9-fold increase as measured by Mega-Fura2 dye, an 8-fold increase by Fura4F, and a 10-fold increase by Fura-2 (Figure 1A)
Summary
Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeating channel assembled from four identical subunits that surround the ion-conducting pore. Capsaicin is the best known vanilloid ligand for TRPV1, a chemical in chili peppers that can induce cation influx and primary sensory neuronal action potentials, typically resulting in a burning sensation in mammals (Wood et al, 1988; Caterina et al, 1997). Mutational and molecular modeling studies have shown that capsaicin can interact with residues Y511, S512, T550, and E570 on the intracellular side of rat TRPV1, resulting in channel opening (Yang and Zheng, 2017). We mixed capsaicininsensitive S512F mutants and wild-type TRPV1 subunits to tune the capacity for ligand-induced channel opening. While TRPV1 receptor activation depends on compositional stoichiometry, it can be further complicated by heterogeneity of arrangements of mutant and wild-type subunits. Our findings reveal that multiple capsaicin-bound subunits are required to drive maximal channel opening
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