Identification Of A Molecular Mechanism For Endocannabinoids Activation Of TRPV1 Ion Channel

Objective:Transient receptor potential vanilloid type-1（TRPV1）is a non-selective cation channel on the cell membrane,which can be activated by extracellular protons,noxious heat,and various animal toxins and other harmful factors.It plays an important role in pain production and transmission,inflammatory response,and immunological stress.The molecular mechanism of TRPV1 activation by capsaicin,a specific agonist of TRPV1,has been verified in a series of experiments,which laid a solid foundation for studying the ligand-gating mechanism of TRPV1 and exploring the mystery of pain response.Previous studies have found that in normal bod y with constant body temperature and p H as well as free of harmful stimuli such as capsaicin and animal toxins,endocannabinoids（Anandamide（AEA）,N-arachidonoyl dopamine（NADA）,N-Oleoylethanolamine（OEA）,and N-Oleoyldopamine（O LDA））are involved in TRPV1pain regulation in vivo.TRPV1 is both a capsaicin receptor and an ionic cannabinoid receptor.Although the high-resolution three-dimensional structure of TRPV1 has been successfully resolved and the binding mode with the potent agonist capsaicin is clear,how endocannabinoids bind and activate the channel remains unclear.In view of this,the molecular mechanism of TRPV1 activation by endocannabinoids was studied in this study.Methods:In this study,computational structural modeling and molecular docking techniques were used to predict key amino acid residues in four endocannabinoids（AEA,NADA,OEA and OLDA）forming hydrogen bonds with mouse wild-type TRPV1channel（wild-type m TRPV1）.Then,the site-directed mutagenesis of the channel were prepared by using molecular biological methods to target the amino acid residues producing hydrogen bonds between the TRPV1 and endocannabinoids.The activation processes of wild-type TRPV1 and mutants were recorded on HEK293T cells by patch clamp technique（whole-cell mode）for functional verification.Then according to the results from patch clamp experiment,the coupling energies between ligands and channels were calculated,and the molecular mec hanisms of the interaction between these four endocannabinoids and TRPV1 channel were finally determined.Results:（1）After finishing whole-cell patch clamp recordings,the EC₅₀ values of four endocannabinoids for wild-type m TRPV1 were derived from fittings Hill equation to the concentration-response relationship,AEA:EC₅₀=6.02±0.43μM,n=8;NADA:EC₅₀=0.08±0.02μM,n=7;OEA:EC₅₀=0.35±0.03μM,n=5;OLDA:EC₅₀=0.29±0.03μM,n=6.（2）The activations of TRPV1 by four endocannabinoids were eliminated by mutating Y512 residues into non-polar amino acids using single-point mutation technique.However,when S513 residue was mutated into non-polar amino acid to break hydrogen bond,the EC50 value was not significantly different from that of the wild-type.（3）Endocannabinoids were applied to T551 and E571,the key sites that capsaicin activates wild-type m TRPV1,the EC₅₀ values of T551V activated by AEA,NADA,O EA and O LDA were not significantly different from those of wild-type.E571A was activated by NADA and showed a significantly increased EC₅₀（0.22±0.02μM,n=5）and a decreased Po＿max（0.42±0.03,n=5）compared with wild-type m TRPV1（0.83±0.02,n=7）.（4）By fitting the concentration-response curves of TRPV1 mutants,which may produce hydrogen bonding with AEA and NADA,and using the thermodynamic mutant cycle analysis,the K_d value,L value and coupling energy were calculated.As a result,we found that the coupling energies of Y512F and E571S mutants were greater than 1.5 k T.（5）Combined with the results of patch clamp experiment,thermodynamic mutation cycle analysis and the molecular docking we found that four endocannabinoids were similar to capsaicin,which were bound in the same pocket,but the binding details were significantly different.Conclusion:（1）The four endocannabinoids,AEA,NADA,O EA and OLDA share similar structures with capsaicin such as a polar‘Head’with hydroxyl group（s）,an amide‘Neck’and a‘Tail’made of aliphatic chain..They are partial agonists of TRPV1 channel,and their activation potencies are significantly lower than that of TRPV1 agonist 2-APB and capsaicin.（2）Residual Y512 is an important site in the interaction between four endogenous cannabinoids and TRPV1.Residuals Y512 and E571 are particularly important for the binding of the"head"of AEA and NADA to TRPV1.（3）The four endocannabinoids are the same as capsaicin,all of which bind to the vanilloid binding pocket（VBP）formed by S3,S4 transmembrane helices and S4-S5 linker with a‘tail-up,head-down’configuration.（4）The binding mechanism of AEA and O EA is similar,and the hydroxyl group in the"head"of AEA may form hydrogen bond with residue E571.The benzene ring group in the"head"of NADA may form a hydrogen bond with the residue E571.OLDA’s"head"is bound deeper inside the VBP.