| Research Abstract |
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.
|
