Functional characterization and potential applications for enhanced green fluorescent protein- and epitope-fused human M1 muscarinic receptors

Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), pot ential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the rece ptor C terminus, and, finally, a FLAG epitope was either fused at the recep tor N terminus or inserted into its shortened i3 loop. High expression leve ls and ligand binding properties similar to those of the wild-type hM1 rece ptor together with confocal microscopy imaging demonstrated that the recomb inant proteins were correctly folded and targeted to the plasma membrane, p rovided that a signal peptide was added to the N-terminal domain of the fus ion proteins. Their functional properties were examined through McN-A-343-e voked Ca2+ release, Despite the numerous modifications introduced within th e hM1 sequence, all receptors retained nearly normal abilities (EC50 values ) to mediate the Ca2+ response, although reduced amplitudes (E-max values) were obtained for the i3-shortened constructs: Owing to the bright intrinsi c fluorescence of the EGFP-fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.