The Cryo-electron Microscopy Structure Study Of TRPML2 Ion Channel

Transient receptor potential mucolipin（TRPML）is a kind of non-selective cation channel on endosomal-lysosomal membrane.In mammals,TRPML family includes three members:TRPML1,TRPML2 and TRPML3.Unlike most TRP channel members,TRPMLs usually localize on intracellular organelles,such as early endosome,recycling endosome,lysosome and lysosome-related organelles.TRPMLs play an important role in endo-lysosomal membrane transport,autophagy,p H regulation,signal transduction and the maintenance of ion homeostasis.Studies have shown that TRPML ion channels are involved in the occurrence and development of many diseases,such as lysosomal storage disorder,metabolic and infectious diseases,and tumors^[1-8]。Compared with the other two TRPML family members,the function and structure of TRPML2 ion channel are less understood.In recent years,the physiological function of TRPML2 ion channel has been reported by more and more researchers at home and abroad.TRPML2 ion channel is mainly expressed in immune cells,which promotes the secretion of chemokines and the aggregation of macrophages in inflammatory sites,indicating that TRPML2 ion channel plays a key role in immune response;TRPML2 ion channel is also involved in the transport of virus in vivo.In addition,TRPML2 ion channel is also involved in the process of tumor growth and differentiation.Recent studies have found that TRPML2has the physiological characteristics of osmo/mechano sensitivity,which is significantly different from other TRPML family members.It can quickly adapt to osmotic changes during the formation of endo-lysosomal tubulation and vesiculation,and maintain the immune monitoring and immune clearance function of immune cells^[1].Therefore,in order to deeply understand the structure and function of the whole TRPML family ion channels,clarify their gating mechanism and pathogenesis of disease occurrence,it is important to obtain TRPML family ion channels with high-resolution structure.At present,the high-resolution structure of full-length TRPML2ion channel has not been reported.In this study,the full-length mouse（mm）TRPML2 protein was expressed by BacMam（Baculovirus translation of mammalian cells）expression system.We obtained the target protein with high expression and high purity.The construction of TRPML2-nanodiscs simulated the existence environment of membrane protein in the plasma membrane,and which then retained the physiological characteristics of the interaction between membrane protein and natural lipid.Here,we report the cryo-electron microscopy（Cryo-EM）structure of TRPML2-nanodiscs in the unliganded state（apo,p H 7.4）at 3.14（?）resolution.The cryo-EM structural analysis of homologous tetramer TRPML2 ion channel reveals that it has its own unique structural characteristics of extracytosolic-luminal domain（ELD）,voltage sensor-like domain（VSLD）and ion conduction channel.In order to verify that TRPML2 fused with maltose binding protein（MBP）purified tag has the same biological function as wild-type（WT）TRPML2,calcium imaging experiment was verified that both overexpressed MBP-TRPML2 and WT-TRPML2 proteins can be activated by a selective small molecule agonist ML2-SA1.The MBP tag does not affect the biological characteristics of TRPML2 itself.Western blot results showed that overexpression of MBP-TRPML2and WT-TRPML2 would not affect the expression level of endogenous calcium pump SERCA2 protein.Therefore,it was considered that the increase of Ca²⁺concentration shown by calcium imaging results was indeed caused by the activation of MBP-TRPML2 and WT-TRPML2 proteins by specific small molecule agonist ML2-SA1.By comparing TRPML2 ion channel with other TRP family members,the structural analysis provides more structural details for us to deeply understand the universality and diversity of TRPML ion channel family structure and function;the research on the structure of TRPML2 ion channel would provide a broader and comprehensive perspective for further clarifying the gating regulation mechanism of TRPML ion channel family at different p H values and with or without agonist binding,and also provide key structural information for the development of a small molecular compound targeting TRPML2 ion channel.