| Objective:The abnormal discharge induced by over activation of ionic Glu receptor by Glu leads to epilepsy,which is an important cause of epilepsy.AMPA receptor and NMDA receptor,as the most important members of Glu receptor,belong to fast channel and slow channel respectively.Local current of AMPA receptor leads the opening of NMDA receptor.They cooperate with each other to accelerate the progress of the disease during the occurrence and spread of abnormal discharges in epilepsy.Previous inhibitors or drugs targeting NMDA receptor and AMPA receptor have some defects.In this study,EU1794-4,a preclinical drug targeting NMDA receptor,originally developed by Emory University,and perampanel,a newly marketed drug targeting AMPA receptor,were deeply explored in terms of receptor inhibition,residual current,receptor channel gating and calcium permeability,so as to explore their significant advantages.It provides a theoretical basis for further understanding of Glu receptor,drug regulation mechanism and the combination of amap/NMDA receptor antagonists.Methods:The c DNA plasmids of NMDA receptor glun1,Glu N2A?2D,AMPA receptor Glu A1 wild type and its mutants Glu-L497Y-S831A-S845A,Glu-L497Y-S831E-S845A were constructed;RNA was synthesized in vitro and microinjected into frog eggs;the inhibitory effects of NMDA receptor antagonist EU1794-4 and AMPA receptor antagonist were evaluated by bipolar voltage clamp;the c DNA was transfected into HEK293T cell line,and the residual current method was used The membrane conductance,channel opening time,opening probability and calcium ion permeability were verified by analyzing,single channel patch clamp and current voltage curve.The molecular mechanisms of action of the two inhibitors on receptors were systematically reviewed.Results:In the first part,the IC50 values of EU1794-4 for all the combinations of Glun1/Glun2 were in the range of 0.28-3.1?m,and the maximum inhibition residual current was 27%to 55%of the control group;the single channel results showed that the membrane conductance of Glu N1/Glu N2A receptor was decomposed from 61ps and 42ps to60ps,47ps and 23ps,and the opening time of low conductance was prolonged The conductance structure inhibitory activity relationship of eu1794 was evaluated by whole cell current residual analysis.In the second part,the membrane uptake of wild-type and mutant Glu A1 was almost the same,and the mutant showed a slight functional gain compared with wild-type Glu A1.The EC50 of Glu A1-LYSS,Glu A1-LYEA and Glu A1-LYAA were 18.84?m,13.13?m and 14.03?m,respectively.In HEK cells,the membrane conductance and opening probability of perampanel to three Glu A1 receptors decreased with the increase of concentration The IC50 of Glu A1 LYSS subunit,Glu A1LYAA subunit and Glu A1 LYEA subunit were 2.15um,1.82um and1.95um,respectively,P<0.01;the difference of membrane conductance was the largest between the two receptors.The results of calcium permeability were 1.78±0.18 in Glu A1-LYEA subunit control group and 1.44±0.78 in perampanel group(P>0.05,unpaired t test);1.67±0.37 in Glu A1 LYAA subunit control group and 2.10±0.37 in perampanel group 60(P>0.05,no paired t-test),there was no significant difference and no statistical significance.Conclusions:(1)Eu1794-4,a novel NMDA receptor targeting compound containing thiophene iminothiazolidone ring,is a preclinical new drug originally developed by Emory University.It can reduce the macro current by reducing the membrane conductance,retain the residual current at the dose,and reduce the side effects.It has stronger targeting for epileptic lesions.(2)Eu1794-4 can significantly reduce the calcium ion permeability of Glu N1/Glu A2,reduce the current Ca2~+component,and has potential long-term neuroprotective effect,so it has a broad application prospect in the long term.(3)Perampanel can reduce the macro current by reducing the membrane conductivity and the gate opening probability.(4)However,perampanel did not show better neuroprotective effect than current drop rate. |
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