Mechanistic Insights Into Activation Of Temperature-Sensitive TRPV3 Channel By Natural Molecule Carvacrol

Ion channels are the basis of life activities,and its most basic function is to produce cellular bioelectrical activity,which is closely related to the physiological and pathological process of human body.Drugs targeting ion channels account for about 18%of global drugs,second to G protein-coupled receptors（GPCRs）.However,the pharmacological action or molecular mechanism of some drugs and compounds is still in doubt.Transient receptor potential vanilloid 3（TRPV3）,robustly expressed in the skin,is a nonselective calcium-permeable cation channel activated by warm temperature,voltage,and certain chemicals.Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel.However,how carvacrol activates TRPV3 mechanistically remains to be understood.Here,we describe the molecular mechanism for chemical activation of TRPV3 by the agonist carvacrol.Objective:The purpose of this study was identified the molecular mechanism of TRPV3channel activation by natural molecule carvacrol.Methods:Whole-cell patch clamp recording is used to confirm the EC₅₀ of TRPV3 channel activated by carvacrol,and single channel recording further recorded the unit conductance,open probability and open frequency of single TRPV3 channel.By molecular docking combined with site-directed mutagenesis,we confirmed the key binding pocket S2-S3linker of TRPV3 channel activated by carvacrol.The key amino acid residues in the pocket were identified by further electrophysiology and site-directed mutagenesis.Results:1.Carvacrol activated the whole-cell currents of TRPV3 channels in a concentration-dependent manner with an EC₅₀ value of 215.0±47.9μM.2.Carvacrol caused a voltage-dependent activation of TRPV3 single-channels with a unitary conductance of 182.0±6.4 p S.3.Carvacrol can significantly increase the opening probability and opening frequency of TRPV3 channels at the single channel currents level.4.Through molecular docking and site-directed mutagenesis,it was found that the S2-S3 linker is an important domain for carvacrol to activate the TRPV3 channel.5.The key amino acids Ile505,Leu508,Arg509 and Asp512 in the S2-S3 linker binding pocket mediate the activation of TRPV3 channels by carvacrol.Conclusions:Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3.Carvacrol is not only a molecular tool to study the mechanism of TRPV3 channel activation,but also provides an idea for the design of new regulators targeting on TRPV3.Significance:In this study,natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel.In addition,TRPV3channel is a potential therapeutic target for skin diseases such as skin itching,inflammation and alopecia,while TRPV3 channel currently lacks specific molecular tools.Therefore,it is necessary to understand the mechanistic insights into activation of TRPV3 channel by carvacrol and to further design and synthesize new compounds.We found that the mechanistic insights into activation of TRPV3 channel by carvacrol and propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3modulators for the further understanding of TRPV3 channel pharmacology.