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COMPARISON OF THE ACTIVATION KINETICS OF THE M3‐ACh‐RECEPTOR AND A CONSTITUTIVE ACTIVE MUTANT RECEPTOR IN LIVING CELLS

The activation of G protein‐coupled receptors (GPCRs) is the first step of the signaling cascade which is triggered by the binding of an agonist to a GPCR. Here we compare the kinetics of receptor activation of the M3 acetylcholine receptor (M3‐AChR) in comparison to that of a constitutively active mutant receptor M3‐AChR‐N514Y. M3‐AChR constructs that report receptor activation by changes in the FRET‐signal were created for wild‐type and M3‐AChR‐N514Y receptors. We investigated the receptor activation for both constructs and observed a leftward shift in the concentration dependent FRET‐response for acetylcholine and carbachol at the M3‐AChR‐N514Y compared to the M3‐AChR. At maximal agonist concentrations the kinetics of receptor activation were identical, whereas at sub‐maximal agonist concentrations activation kinetics of the M3‐AChR‐N514Y were significantly faster. This observation could be due to higher agonist affinity for the M3‐AChR‐N514Y, since receptor activation times were indistinguishable when kinetic data for similar receptor occupancy ratios were compared. In conclusion, our data give further insight receptor activation and may help in understanding constitutive activity in more detail.