Issue 21, 2021

Vibrational analysis of acetylcholine binding to the M2 receptor


The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm−1 as the C[double bond, length as m-dash]O and C–O stretches of ACh, respectively. The C[double bond, length as m-dash]O stretch of ACh in M2R is close to that in aqueous solution (1736 cm−1), and much lower in frequency than the free C[double bond, length as m-dash]O stretch (1778–1794 cm−1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.

Graphical abstract: Vibrational analysis of acetylcholine binding to the M2 receptor

Supplementary files

Article information

Article type
07 Feb 2021
13 Mar 2021
First published
07 Apr 2021
This article is Open Access
Creative Commons BY license

RSC Adv., 2021,11, 12559-12567

Vibrational analysis of acetylcholine binding to the M2 receptor

K. Suzuki, K. Katayama, Y. Sumii, T. Nakagita, R. Suno, H. Tsujimoto, S. Iwata, T. Kobayashi, N. Shibata and H. Kandori, RSC Adv., 2021, 11, 12559 DOI: 10.1039/D1RA01030A

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