Analysis Of Ion Channel Gating Regulation Mechanism Based On Cryo-EM

Ion channels are widely distributed in various tissues of human body and play an extremely important role in many physiological processes.Ion channels can be divided into voltage gated ion channels,ligand gated ion channels and mechanical gated ion channels.The activity of ion channels is regulated by many factors.Apart from the inherent factors such as potential changes,ligand molecules or surface tension,small chemical molecules and peptides can also regulate the activity of ion channels.The activity regulation of ion channels is one of the basic scientific problems in the field of ion channels,which is of great significance for us to understand the gating mechanism of ion channels.During my doctoral study,I chose voltage gated ion channel KAT1 and ligand gated ion channel ASIC1a as the research objects,and studied the gated regulation mechanism of the above ion channels with the help of structural biology technology and patch-clamp electrophysiology technology.In the first part of this paper,we studied the hyperpolarization activation mechanism of KAT1.KAT1 is a hyperpolarized activated inward rectifying potassium channel found in Arabidopsis thaliana.We determined the cryo-electron microscopy structure of KAT1 at a resolution of 3.2 A.This is the first electron microscopic structure of a voltage-gated potassium channel in plants.We found that KAT1 is a homologous tetramer and acts as a "non-domain swap" channel.Each monomer contains six helical transmembrane domains,C-linker domain extending into the intracellular region,and CNBD domain at the C-terminal.KAT1 has a typical potassium selective filter structure and can selectively permeate potassium ions.Through structural analysis,we found that the pore area of KAT1 channel was closed.We analyzed the positively charged amino acids on the S4 helix of KAT1 and found that these positively charged amino acid residues are important for voltage sensing.Although the KAT1 structure contains the CNBD domain,both structural analysis and functional experiments showed that cGMP could not regulate the channel activity of KAT1.In KAT1 channel,S4-S5 linker is a short loop,and the interaction between this linker region and C-linker plays an important role in opening or closing the channel.If the interaction between these two domains is weakened,the channels will not open properly.This indicated that the interaction between S4-S5 linker and C-linker is the key to KAT1 channel gating regulation.In the second part of this paper,we investigated how peptide toxin binds to and regulate the activity of human acid-sensitive ion channel ASIC1a.Human acid sensing ion channel ASIC1a(hASIC1a)is mainly distributed in the central nervous system and peripheral nervous system.ASIC1a channel is mainly involved in pain perception,learning and memory and other physiological processes,and is a potential drug target.The activity of the hASIC1a channel is regulated by proton concentration and can be modulated by various factors,such as the polypeptide toxins.The African black mamba snake venom Mamba1 inhibits the activity of hASICla.After the multiple optimization steps of protein expression purification and the selection of structure analysis methods,we finally determined the apo state of hASIC1a and the structure of Mamba1-hASIC1a complex by cryo-electron microscopy,with the resolutions of 3.56 A and 3.9 A,respectively.Compared with the determined chicken ASIC1(cASIC1)structure,the hASIC1a channel in the resting state has a similar structure to cASIC1 channel,which is a homotrimer,and its gating mechanism is similar to cASICl channel.Mambal toxin mainly binds to the extracellular domain of hASIC1a.When the toxin binds to the channel,local conformational changes occur in the channel.Mambal locks hASICla in the closed conformation.We found that although Mambal had similar affinity for hASIC1a and cASIC1,the inhibition of hASIC1a by the toxin was stronger than that of the cASIC1 channel.Through sequence alignment analysis and structural comparison,we identified the key amino acid residues with different inhibitory effects on hASIC1a and cASIC1.Arg155 and Glu 102-Asp 165 pairs in hASICla(corresponding to Leu 156 and Arg103-Glu168 in cASIC1,respectively)cause hASIC1a and cASIC1 to react differently to Mamba1.