The Mechanism Underlying RhTx2 Regulating Nociceptor TRPV1 Ion Channel

Objective: Transient receptor potential vanilloid 1 ion channel(TRPV1)is an important receptor in the pain pathway.It is widely distributed in the central and peripheral nervous systems of mammals.It is involved in the pain caused by a variety of physical and chemical factors such and inflammatory pain,therefore is actively regarded as the target of analgesics.Due to the important role of TRPV1 in thermoregulation,many small molecule inhibitors have failed in clinical trials due to the abnormal body temperature caused by TRPV1.Polypeptide toxins are important tools for ion channel research and have been widely developed as drugs due to their high affinity and selectivity.TRPV1 has been shown to be a target for several toxins.We identified a polypeptide toxin from Chinese red-headed centipede and named it red-headed centipede toxin 2(RhTx2),which can selectively act on TRPV1.We hope to provide new ideas for the research of pain and the development of new analgesics by studying the structure and functional characteristics of RhTx2.Methods: 1.We firstly isolated and purified the toxin polypeptide from the venom of Chinese red-headed centipede by gel chromatography(GC)combined with reversed-phase high performance liquid chromatography(HPLC),and obtained its cDNA sequence by protein sequencing.2.A linear toxin polypeptide with a purity of 95% was synthesized from a biological company,the polypeptide was refolded by REDOX method,and further purification and lyophilization are then required.3.Functional verification and result analysis were carried out at the cellular level by using electrophysiological experimental techniques and whole-cell combined with single-channel recording mode.4.Finally,Rosetta was used to calculate the structural biological method to simulate the binding conformation of peptides on the channel,and to explore the structural mechanism of toxin binding and desensitization.Results: 1.In this study,we have identified a variant of RhTx from the venom of the Chinese red-headed centipede,which contains four more residues at the N terminus.We named this 31-residue peptide toxin red-headed centipede toxin 2(RhTx2).2.Structural simulation shows that RhTx2 has a larger positive surface,and hydrogen bonding is formed between "head and tail" to increase protein stability.3.RhTx2 can selectively activate TRPV1 channel and quickly desensitize,and the desensitization is relatively thorough and difficult to recover.4.RhTx2 activates TRPV1 in extracellular and could not be inhibited by Capsazepine.5.Compared with RhTx,the activation curve of RhTx2 significantly shifted to the right,and EC50 increased by nearly 100 times.6.Single-channel recording shows that the current desensitization of TRPV1 caused by RhTx2 involves two mechanisms: simultaneously reducing both the open probability of the channel and the single-channel conductance.7.Molecular docking experiments showed that the binding position of RhTx2 on TRPV1 changed with the opening of the channel.When the channel was closed,it bound to the edge of the outer pore area,and moved to the center of the selective filter when the channel was open.Conclusion: Our results suggest that the interaction between RhTx2 and TRPV1 is a unique mechanism.RhTx2 is not only a tool to study the mechanism of TRPV1 channel activation and desensitization,but also provides an idea for the study of new analgesics targeting on TRPV1.Significance: In this study,we found a small peptide toxin RhTx2 in Chinese red-headed centipede.Its properties of rapid activation and rapid desensitization on TRPV1 play a positive role in the study of the activation and desensitization mechanism of TRPV1.In addition,TRPV1 ion channel is a multimodal receptor in response to a variety of pain stimuli,which is an important target of analgesics.However,because TRPV1 plays a key role in thermoregulation,some small-molecule inhibitors of the channel have failed in clinical trials because they often cause serious side effects,such as changing a patient’s body temperature and thermal sensation,resulting in an abnormally high body temperature.Therefore,in order to develop new molecules targeting TRPV1 as analgesics,alternative strategies besides inhibiting TRPV1 activity are needed.Our polypeptide toxin RhTx2 can rapidly desensitize the activation of TRPV1,which is expected to explore whether RhTx2 has analgesic effects in animal models.