NMDA受体亚基NR_1的体外表达及功能检测

In vitro expression and functional dection of one subunit (NR 1) of NMDA receptor

目的 :体外表达NMDA受体亚基NR1并了解其电生理学特性 ,探讨体外表达受体亚基的意义。方法 :将克隆的NMDA受体亚基NR1质粒DNA经体外转录成RNA ,显微注射到爪蟾卵母细胞后进行受体表达 ,采用双电极电压钳位技术记录表达的NR1亚基的激活电流。结果 :注射NR1亚基RNA的爪蟾卵母细胞能表达出具有电生理学功能的受体 ,其激活电流能被D AP5阻断等特性 ,说明该表达的NR1亚基具有NMDA受体的功能。结论 :爪蟾卵母细胞表达的NR1亚基具有NMDA受体的生物学功能 。

Objective:It was designed to execute in vitro expression and functional dection of one subunit (NR 1) of NMDA receptor and to investigate NR 1′s electrophysiology characteristics. Methods:Primitive NR 1 plasmid DNA from Nakanishi (Japan) was transformed into DH 5α and identified by enzyme Not I cut. Then large scale plasmid DNA was prepared and transcripted into RNA. The obtained RNA was microinjected into matured Xenopus oocytes (50 nl, 1 ng/nl) for receptor expression over 24 h incubation in modified Bath′s solution at a temperature of -19 ℃. The functions of these expressed NR 1 subunit were detected via two electrode voltage clamp technique. Results:An inward current was induced by 10 -4 mol/L NMDA in Xenopus oocytes microinjected with the obtained RNA transcripted from NR 1 plasmid DNA. The equibrilium potential of the current was -22 mV, which is close to those of chloride ions. This current could be completely inhibited by the specific antagoinst D AP 5 of NMDA receptor and was dependent on glycine (10 -4 mol/L). This current was also blocked by the presence of Mg 2+ , which was inhibited by the replacement of CaCl 2 with MgCl 2 in Ringer′s solution. Conclusion:The NR 1 subunit of NMDA receptor is functionally in vitro expressed in Xenopus oocytes. Its physiological and pharmacological characteristic is similar to those of whole NMDA receptor.