谷氨酸转运体4b-4c环在转运底物过程中的构象变化及功能分析

我们前期已经证明发夹结构HP2是真核生物谷氨酸转运体(EAATs)细胞外侧闸门，揭示了细胞内侧闸门HP1向胞浆开放的构象变化。然而EAATs转运机制还没有完全阐明，结构区段4b-4c环在转运底物过程中的构象变化及功能仍不清楚。我们进一步研究发现4b-4c环在转运底物过程中构象发生了较大改变，而且与底物转运密切相关。据此我们推测：4b-4c环对底物的转运具有一定的功能。4b-4c环支撑HP1、HP2、TM7构象变异完成底物转运，同时其部分位点可能参与了转运通道的组成。为验证推测本项目拟用定点突变、半胱氨酸突变对交联、圆二色光谱、体积排阻色谱等方法对4b-4c环进行研究，剖析4b-4c环在转运底物过程中的构象变化，揭示4b-4c环位点与底物转运通道的关系，阐明4b-4c环在底物转运中的功能，阐述原核与真核谷氨酸转运体转运机制差异的分子结构基础。对完全阐明EAATs的转运机制提供一定的理论基础。

We already illuminated that helical hairpin(HP)2 acts as outer gate for excitatory amino acid transporters (EAATs). The conformational changes of HP1 during glutamate transporters opening to cytoplasm were revealed by us. However the transport mechanism of EAATs and the conformational changes and function of 4b-4c loop during the transport cycle is not completely clear until now. In our current study we found that the conformation of 4b-4c loop undergoes great changes during the transport cycle, and 4b-4c loop is correlated to the substrate transport. So we hypothesize that 4b-4c loop has certain function for the substrate transport. The conformation of 4b-4c loop undergoes great changes during the transport cycle, which supporting HP1, HP2, TM7 conformation change to execute substrate transport. Meanwhile some amino acid residue sites may be involved in the transport pathway. We are going to verify this hypothesis using the site-directed mutagenesis, cysteines cross linking, circular dichroism (CD) and size exclusion chromatography (SEC). From these researches we will try to analyze the conformational changes of 4b-4c loop during the transport cycle, reveal the relationship between the amino acid residue sites of 4b-4c loop and transport pathway, clarify the function of 4b-4c loop during the transport cycle, identify molecular determinants of the differences of the transport mechanism between prokaryotes and eukaryotes glutamate transports. The finding of this project may provide theoretical basis to illuminate the transport mechanism of EAATs.