Interaction Between The N-terminal Domains Of The AMPA Receptor Analyzed By Fluorescence Detection Methods

AMPA receptors mediate the fast excitatory synaptic transmission in the central nervous system. The majority of AMPA receptor is a GluR2-containing heterotetramer in hippocampus. The assembly of AMPA receptor might properly be regarded as a dimmer of dimmers, may occur sequentially: an initial formation of dimmers, which subscequently form a tetramer. Although the most N-terminal domain (NTD) is suggested to play important role in the initial assembly of iGluR subunits, it is unclear how this domain interacts in living cells and intact iGluRs. Here, we used fluorenscence detection methods including bimolecular fluorescence complementation (BiFC) and Fluorescence Resonance Energy Transfer (FRET) to address these issues. A fluorescent protein split at appropriate sites can fold and reconstitute the chromophore when the two halves are fused to interacting partners Firstly, we constructed mammalian expression vector, which can be used to detect protein interaction in the cytoplasm and endoplasmic reticulium (ER). When HEK293 and COS7 cell co-transfected with fluorescent protein fragment tagged GluRl-NTD, yellow fluorescence can be seen both in the cytoplasm or ER. These results demonstrated GluRl-NTD dimerization in living cells. To eliminate proteinsnon-specific interaction, the metabotropic glutamate receptor ligand binding domain (mGluRl-LBD) been chosen as negative control, very weak fluorescence can be seen, which is may be non-specific interaction.To detect GluRl-NTD interaction in intact iGluRs, we used FRET to explore their special distance. FRET is unique in providing signal sensitivity to intermolecular distances in the 1-1 Onm range. Thus, FRET is capable of resolving molecular interactions and comformationals with a spatial resolution far exceeding conventional optical microscopy, yet is also compatible with super-resolution techniques. Our results show GluRl-NTD approach to each other, form complex, may stablized receptor's structure. BiFC also detect the fluorescence complementation in intact iGluRs, suggest NTD may facilitate assembly of AMP A receptors.