New Strategies To Regulate Polymodal Activations Of TRPV1 Channels

Transient receptor potential vanilloid 1（TRPV1）channels,are ligand-gating cation channels widely distributed in mammalian peripheral sensory neurons and central nervous system.These polymodal channels are sensitive to noxious heat,protons,and other endogenous and exogenous compounds.Thus,TRPV1 channels are implicated in many physiological and pathological processes in mammals,such as the maintenance and regulation of normal body temperature.The disorders of TRPV1 will cause abnormal pain sensations,such as the neuropathic pain,hyperalgesia and spontaneous burning pain,as well as acute and chronic diseases,like the inflammatory bowel disease,asthma and cardiac pain,which makes this ion channel becoming the novel and potential target in the pain relief.In recent years,the development of new analgesics and finding new therapeutic strategies based on TRPV1 channels have attracted wide attentions.However,distinct with other ligand-gated ion channels that can only sense one type of stimuli,the polymodal activations and more complicated gating process of TRPV1 led to altered the maintenance and regulation of normal body temperature of the human after TRPV1 drug candidate administrations,and the final fail of most of drug candidates in the clinical trials.It is urgent to further explore and develop new strategies in the drug developments.In 2013,the structural determinations of rat TRPV1 channel at both the resting and open states have driven the researches of TRPV1 ion channels into the post-structure era at atomic level.Subsequently,more protein structures of other subfamily of TRP channels were determined,providing more information for structural and functional studies of TRPV1 channels,and the discovery of new compounds targeting these channels in future.Nevertheless,the cryo-electron microscopy（Cryo-EM）and single-particle analysis techniques have a milestone contribution to determine the protein structures.But it is still hard able to determine the detailed structural information for some flexible regions.Here,we suggest that the pore turret loop（PTL）located near the entrance of ion transduction pathway of TRPV1 channel,being missing in the TRPV1 structures,is very important in maintaining the conformation of the pore region during the gating progress of TRPV1 channel.Through a combination of using homologous modeling,engineered disulfide crosslink and Western blotting,we demonstrated that the PTL adopts an upward conformation and is located far away from the pore region of the channel.In addition,an important regulatory site in proton-mediated potentiation of TRPV1 channel,E600,not only interacts with the PTL,but also forms a structural machinery behaving with regulation function in the activity of TRPV1 channels.This structural machinery plays a negative role during the gating progress of TRPV1 channel.Additionally,this negative structural machinery,consisting of the site E600 and the PTL,is an inherent regulation component of TRPV1 rather than being directly implicated in the gating process of TRPV1 in responses to p H,hot and other endogenous/exogenous ligands.This finding will advance our understanding in the polymodal activation of TRPV1 channels.In the studies of TRPV1 channel activation process,scientists noticed that administration of high or repeat does of capsaicin induce pain sensations at first then is followed by analgesia when extracellular Ca²⁺exists.The underlying mechanism of capsaicin-mediated analgesia is based on that extracellular calcium ions can trigger desensitization of the TRPV1 channel.Thus,both the agonists and antagonists of TRPV1 channel could act as the potential analgesics and anesthetic agents,and capsaicin has been used as analgesics for clinical treatment many years ago.However,these compounds may simultaneously interfere a variety of other activation processes of TRPV1 channels,especially the acid-and heat-mediated activations,which will finally affect the normal human body temperature and noxious heat perception and thus caused their limited clinical applications.As we know,the binding site of capsaicin is deeply buried into the interface between the cell membrane and the transmembrane（TM）domains of TRPV1 channel,distinct with other ligand-gated ion channels whose ligand-binding sites are generally located at the extracellular region of the channels.How capsaicin binds to this buried site remains unclear.Here,we uncover a possible binding/unbinding pathway of capsaicin in TRPV1 channel.Metadynamics and the lowest free energy path plotted onto the free energy surface showed a hydrophobic tunnel,surrounding by TRPV1 channel and the cell membrane,specifically for capsaicin’s accessing to its buried site.Mutagenesis carried out in the residues of this pathway,especially the residue at the entrance of tunnel,remarkedly delayed the binding and unbinding of capsaicin,but have no influence on the acidosis-,heat-or 2-APB-evoked TRPV1 activation.Through a comparison between inside-out and out-side out single channel recordings,we confirmed the specific role of residues of the pathway in the binding of capsaicin,rather than directly affecting the TRPV1 gating.Based on this finding,we have designed and screened small molecule compounds and discovered novel small molecule that could disturb capsaicin’s accessibility by occupying the entrance of this hydrophobic pathway,providing a promising strategy in developing new analgesics that would not interfere body temperature.