Towards the dynamic changes of the structure and interaction of ion channels and receptors, -Analysis by Fluorescence Resonance Energy Transfer method-

离子通道和受体在各个方面发挥着重要的作用。结构-功能关系的阐明是生理学和生物生理学的一个有趣的主题。本研究旨在用荧光共振能量转移(FRET)方法分析代谢性谷氨酸受体(MGluR1)的动态结构重排。到目前为止，我们观察到mGluR1不仅可以被谷氨酸激活，还可以被多价阳离子激活，如Ca^&lt；2+&gt；和Gd^&lt；3+&gt；，并且敏感性因表达系统的不同而不同，包括非洲爪哇卵母细胞和HEK293细胞。我们假设这种差异是由于磷酸化、糖基化或聚集的不同所致。众所周知，mGluR1是以同源二聚体的形式组成的，这种激活是通过亚基间相互作用平衡的改变而发生的。我们的目的是测量mGluR1亚基间相互作用的波动，并用FRET方法估计簇状分子和多价阳离子的存在对mGluR1的影响。我们将ECFP或EYFP连接到羧基末端，并测量了两个亚基之间的FRET。由于记录的FRET效率不够高，我们插入了一个带有连接子的新结构，并成功地提高了效率。我们将继续这项研究，以检查多价阳离子和聚集分子对亚单位间相互作用的影响，估计为FRET效率。

Ion channels and receptors play important roles in various aspects. The elucidation of structure-function relationship is a physiologically and biophysically interesting theme. In this study, we aimed to analyze the dynamic structural rearrangement of metabotropic glutamate receptor (mGluR1) by FRET (Fluorescence Resonance Energy Transfer) method. We observed so far that mGluR1 can be activated not only by glutamate but also by polyvalent cations, such as Ca^<2+> and Gd^<3+>, and that the sensitivities differ depending on the expression systems including Xenopus oocytes and HEK293 cells. We hypothesized that the difference is due to the difference of phosphorylation, glycosylation, or clustering. As it is known that mGluR1 is composed as homodimers, and that activation occurs by the shift of the equilibrium of inter subunit interaction. We aimed to measure the fluctuation of the inter-subunit interaction of mGluR1 and to estimate the effect of the presence of clustering molecules and polyvalent cations by FRET method. We attached ECFP or EYFP to the carboxy teminal end, and measured FRET between the two subunits. As the recorded FRET efficiency was not high enough, we inserted a new construct with a linker and succeeded in incresing the efficiency. We will continue this study to examine the effect of polyvalent cations and clustering molecules on the inter subunit interaction estimated as FRET efficiency.