Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level

Abstract

Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic molecules. They are of particular interest in the develo pment of a sensor platform for G protein-coupled receptors (GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis and vectorial incorporation for achieving natural recepto r function. The vectorial insertion of OR5 into the planar membrane and their lateral distribution, their interactions a nd their mobility within the membrane are of great importance for ligand‐receptor interaction. We applied total internal re flection fluorescence (TIRF) microscopy and image analysis t o assess the insertion and the OR5 distribution as well as the l ateral mobility of these receptors at the single molecule le vel. The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation. With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about 0.02µm 2 min 1 . The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility of the incorporated receptors was measured with fluorescenc e correlation spectroscopy (FCS) and fluorescence recovery after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this class of lipid membranes.