Mechanism Underlying The Desensitization Of ASIC3 Channels

Acid-sensing ion channel(ASIC)is one of subfamily of the superfamily of Degenerin/Epithelial Sodium Channels(DEG/ENa C).ASIC channels can be directly activated by extracellular acidosis and other nonproton ligands,such as a few small molecules,lipids and peptide toxins.This type of ion channel is implicated in diverse physiological and pathological processes.In recent years,more and more crystal or cryo-EM structures of ASIC channels have been determined,which advances the understanding of the gating mechanism of this type of ion channels.But still,due to the limited resolution of transmembrane(TM)region and the truncated intracellular region in those structures,details about the conformational changes of TM region during channel gating,especially the desensitization process,are still unclear.The postgraduate thesis here is mainly to study conformational changes of the TM domain during the ASIC3 desensitization,and explore key residues/domains that played a crucial role in this process.The author hopes to finally unveil the mechanism underlying the whole process of ASIC3 desensitization.Main results are as follows:(1)Covalent ligands NBM and NPM can be used as molecular probes to study the ASIC desensitization.In whole-cell recordings,NBM and NPM are capable of inducing distinct desensitizations in cells expressing ASIC3^E79C mutant.Single channel recording showed that the conductances of unitary currents of these two covalent ligands are similar,and both of which was?10 PS.In previous studies,author and his colleagues have demonstrated that the number of binding molecules is not contributing to the difference in channel desensitizations produced by these two compounds.Similarily,their ion selectivities to Na⁺and K⁺are also similar,and thus the author believes that these two covalent ligands could be used as molecular probes to study the channel desensitization of ASIC3.(2)The author performed mutagenesis screening for residues located at the upper and lower parts of the TM regions,and identified 2 interaction pairs:R64...L437 and A49...L456,which participate in the channel desensitization of ASIC3.They formed two‘clip'-likeds structural elements to clamp the GAS-belt in the middle,which may contribute to regulate the channel open-to-desensitization processed through modulating the allosteric changes the upper and lower parts of TM domain.(3)Using p H 5.0,Ca²⁺-free,GMQ,and desensitization agents SGI,AE848,the author screened key amino acids in the TM domain of WT channels.The author found that R64 at the upper part of the first TM domain(TM1)plays a crucial role in the channel desensitization of ASIC3.R64 mutations could significantly modulate the sustained activations induced by factors mentioned above.Mutagenesis carried out at the position of R64 also displayed effect on channel desensitization of ASIC3,which is consistent with the observation obtained by mutagenesis screening for sustained or transient activations induced by NBM and NPM.It is possibly that R64 interacts with its surrounding residues to coordinate the allostery of the TM domain during the process of desensitization.In summary,R64 plays an important role in the channel desensitization of ASIC3,and it also laid a foundation for the future discovery of endogenous and exogenous small molecules that could regulate the ASIC3 desensitization,and to further explore the biological function correlated to the channel desensitization of ASIC3.In addition,it could provide accurate site information about knocking on some mutations that could change the desensitization of ASIC3,using the CRISPR/Cas9 approach,which will facilitate the study the biological functions mediated by fast and slow desensitization of ASIC channels.