Acetylcholine Receptor-Cholesterol Crosstalk: Structure meets Dynamics at the Lipidprotein Interface

The nicotinic acetylcholine receptor (nAChR) is a transmembrane protein that mediates fast intercellular communication in response to the endogenous neurotransmitter, acetylcholine. It is the archetypal molecule in the superfamily of pentameric ligand-gated ion channels.
One of our laboratory’s main interests is to understand how lipids modulate the topography and function of the nAChR and, conversely, how the nAChR influences its immediate lipid milieu. Recent cryo-EM studies have unveiled the presence of phospholipids and cholesterol on the nAChR transmembrane segments, confirming our experimental and in silico work on the occurrence of these sites. However, even in its atomic detail, the structural data does not suffice to understand receptor function in a living cell. Characterizing the receptor’s dynamics is a step forward, but is usually hampered by the limited temporal and spatial resolution of conventional methods. We have recently resorted to minimal fluorescence emission photon fluxes (MINFLUX) microscopy to interrogate the 2D translational mobility of the nAChR together with a fluorescent cholesterol analogue. Single-molecule co-tracking experiments enabled us to follow for minute-long periods -with nanometric precision and sub-millisecond time resolution- the motion of the protein macromolecule together with the neutral lipid at the cell surface of a live mammalian cell. This new information has provided a more comprehensive depiction of the mutual lipid-receptor crosstalk, made apparent the cholesterol sensitivity of the nAChR translational motion, and confirms our view that the receptor’s diffusional behaviour can be accounted for in terms of a two-state physical model: Brownian motion alternating with confinement sojourns.