目的:探讨TM8区段在谷氨酸转运体GLT-1转运底物过程中所负功能.方法:将GLT-1的TM8区段的氨基酸残基(Thr460-Val483)分别突变为半胱氨酸,然后应用生物化学的方法检测突变体转运活性,巯基试剂MTSET对突变体转运活性的影响以及底物、抑制剂对MTSET抑制效应的影响.结果:1463C、V467C及A468C对MTSET非常敏感;L465C、L466、V469C及W471C对MTSET敏感性稍弱.底物L-glu及KCl对MTSET作用于V467C、A468C的抑制效应具有保护作用,而抑制剂TBOA则增强了MTSET的抑制效应;L-glu及KCl对MTSET作用于V469C、W471C的抑制效应具有保护作用,但TBOA对MTSET的抑制效应没有影响.结论:在GLT-1转运底物的过程中,TM8参与了底物转运通道的组成,TM8对底物的结合与转运发挥着重要的作用.%Objective GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. The function of TM8 during the transport cycle is not clear yet. We used cysteine mutagenesis with treatments with MTSET, to investigation the function of TM8. Methods Cysteine mutants were introduced from Thr460 to Val483 of TM8 respectively. We checked the activity of the mutants before and after treatments with MTSET, furthermore we also investigated the effect of the substrate and blocker on the inhibition of cysteine mutants by MTSET. Results A inhibition of transport by MTSET was observed in the mutants I463C, V467C, A468C, L465C, L466, V469C and W471C. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of V467C , A468C, V469C and W471C by MTSET, while TBOA increased the inhibition of transport activity of V467C and A468C by MTSET, but had no effect on the inhibition of transport activity of V469C and W471C by MTSET. Conclusions Our results suggest that TM8 is important for the substrate binding and transport, and the conformation of TM8 is altered during the transport cycle.
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