Involvement Of P2X4R Ectodomain In ATP Binding And Preliminary Study On Electrophysiological Properties Of Neurons Derived From IPSCs

P2X receptors are trimeric ATP-gated cation-permeable ion channels. Upon binding of ATP, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there is scant functional data corroborating the role of the dorsal fin in ligand-binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid or phenylalanine at L214of rat P2X4receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, D224, did not significantly alter ATP sensitivity. Exposure of L214C to MTSES-or MTSEA+in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES-was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist TNP-ATP, and the blockade was more rapidly reversible after washout. Certain L214mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data identify that position L214participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains. In2006, Shinya Yamanaka demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. iPSCs offer several advantages over embryonic stem cell (ESCs). Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling.Problems associated with analyses of nervous system disorders centered on1) Identifying cell biological or biochemical changes in the initial stages of the disease, before onset of symptoms, has been difficult given analyses conducted on postmortem brains.2) the animal or cell models did not necessarily reflect the human pathology. However, with the development of iPS cell technologies, it opened up new avenues for obtaining neuronal cells. A variety of disease-specific iPSCs have been used to study nervous system diseases.Identification experiments indicated that human iPSCs we obtained can maintain a pluripotent state which is the same as hESCs. These experiments include alkaline phosphatase (AP) staining; Karyotype analysis showed chromosome morphology is normal; Immunofluorescence used to test ESCs surface specific markers NANOG and TRA1-8.1; qPCR used to test endogenous Tg OCT4, Tg SOX2, Tg c-MYC, Tg KLF4and exogenous transgenes endo-OCT4, endo-SOX2, NANOG, REX1in iPSCs; Teratoma assay showed that iPSCs have the ability to differentiate into three germ layers, endoderm, mesoderm, and ectoderm.The iPSC-derived neurons exhibited normal electrophysiological activity. They have ability to generate action potentials, voltage-gated K+currents, voltage-gated Na+currents and give a typical postsynaptic activity. This part of the experiment laid a foundation for subsequent establishment of human nervous system disease model.