FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors

Bioorg Med Chem. 2011 Feb 1;19(3):1048-54. doi: 10.1016/j.bmc.2010.07.060. Epub 2010 Jul 30.


Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes*
  • Green Fluorescent Proteins*
  • HEK293 Cells
  • Humans
  • Kinetics
  • Ligands
  • Microscopy, Confocal
  • Muscarinic Agonists / metabolism
  • Muscarinic Agonists / pharmacology
  • Oxotremorine / analogs & derivatives
  • Oxotremorine / metabolism
  • Oxotremorine / pharmacology
  • Protein Conformation
  • Receptor, Muscarinic M1 / chemistry
  • Receptor, Muscarinic M1 / metabolism*
  • Receptor, Muscarinic M3 / chemistry
  • Receptor, Muscarinic M3 / metabolism*
  • Receptor, Muscarinic M5 / chemistry
  • Receptor, Muscarinic M5 / metabolism*
  • Signal Transduction
  • Stereoisomerism


  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Ligands
  • Muscarinic Agonists
  • Receptor, Muscarinic M1
  • Receptor, Muscarinic M3
  • Receptor, Muscarinic M5
  • Green Fluorescent Proteins
  • Oxotremorine
  • oxotremorine M