Figure 1.

Bioluminiscence and fluorescence resonance energy transfer (BRET and FRET, respectively) techniques allow the demonstration of neurotransmitter receptor heteromers in the natural environment of the living cell. FRET can be obtained when two fluorescent proteins, one acting as a donor and the other acting as acceptor, are close enough (within 10 nm). Therefore, to demonstrate receptor dimerization, cDNA constructs of one receptor fused to the fluorescent donor and another receptor fused to the fluorescent acceptor are prepared and transfected in a heterologous cell system. Different versions of the green fluorescent protein (GFP) are currently used as donors whereas yellow fluorescent proteins (YFPs) are used as acceptors. If the two receptors are forming dimers, the acceptor fluorescent signal is detected after donor excitation. In BRET, instead of a fluorescent donor one of the receptors is fused to the luminescent protein, Renilla luciferase (Rluc), which upon addition of a substrate (coelenterazine or Deep Blue C) allows energy transfer and excitation of the fluorescent acceptor.