Research On Function And Construction Of GluA1 And GluA2 Plasmids

Objectives:The AMPAR trafficking may represent a core mechanism underlying physiological forms of plasticity induction and the deregulation of AMPAR trafficking has been strongly implicated in neuronal damage and disease.In the future,the modulation of AMPAR will be supposed to be a possible strategy for therapeutic intervention in a number of mental and neurological diseases.The aim of this study was to construct Glu A1-GFP and Glu A2-His plasmids and express plasmids in HEK293 T cells,to verify the function of exogenous AMPAR,and to explore whether ketamine can act directly on AMPAR.Methods:1.Purify RNA from fresh samples and synthesis of first strand c DNA to obtain PCR template.The PCR method was used to synthesis the target gene and Glu A1 and Glu A2 subunits were all constructed in pc DNA3.1.PCR and enzyme digestion methods were used to identify positive clones.The expression of Glu A1-GFP and Glu A2-His were verified by Western Blot and immunohistochemistry methods in HEK293 T cells expressing exogenous gene.2.Whole cell patch clamp recordings were made from heteromer Glu A1-2–positive HEK293 T cells to explore the function of exogenous AMPAR.The ketamine(40μM)was applied to HEK293 T cells co-transfected with Glu A1 and Glu A2.Exogenous AMPA receptor-mediated excitatory currents were recorded before and after drugs added.Results:1.Our results showed that Glu A1-GFP and Glu A2-His were constructed successfully.Fluorescence microscopy showed strong membrane staining for Glu A2 subunit and the expression of green fluorescent protein for Glu A1 subunit,which confirmed the expression of Glu A1 and Glu A2 subunits.The results of immunoblotting also revealed the expression of Glu A2 in HEK293 T cells.2.We recorded the currents evoked by glutamic application(10 m M)in HEK293T cells expressing heteromer Glu A1–2 receptors by using whole cell patch clamp recording at a holding potential of-40 m V.The results showed that exogenous Glu A1-2 receptors can mediate excitatory currents and the currents can be specifically blocked by NBQX(10 μM).3.There was no significant difference in the amplitude and decay time of excitatory current mediated by exogenous AMPA receptors before and after application of ketamine(n=6),indicating that ketamine can not target on AMPAR directly.Conclusion:1.In this study,the target genes for the Glu A1 and Glu A2 subunits of the AMPA receptor were successfully synthesized,and Glu A1-GFP and Glu A2-His plasmids were constructed and normally expressed in cells.2.We used the whole-cell patch clamp technology to record the excitatory currents mediated by exogenous AMPA receptor on HEK293 T cells,confirming that the fusion of macro molecular fluorescent labeling protein with AMPA receptor does not affect its its channel properties.3.In HEK293 T cells,40 μM ketamine did not affect the excitatory currents mediated by AMPA receptor evoked by glutamic(10m M)and did not act directly on the AMPA receptor.