Embedded cholesterol in the nicotinic acetylcholine receptor

Abstract

The nicotinic acetylcholine receptor (nAChR) is a cation-selective channel central to both neuronal and muscular processes and is considered the prototype for ligand-gated ion channels, motivating a structural determination effort that spanned several decades [Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J Mol Biol 346:967-989]. Purified nAChR must be reconstituted in a mixture containing cholesterol to function. Proposed modes of interaction between cholesterol and the protein range from specific binding to indirect membranemediated mechanisms. However, the underlying cause of nAChR sensitivity to cholesterol remains controversial, in part because the vast majority of functional studies were conducted before a medium resolution structure was reported. We show that the nAChR contains internal sites capable of containing cholesterol, whose occupation stabilizes the protein structure. We detect sites at the protein-lipid interface as conventionally predicted from functional data, as well as deeply buried sites that are not usually considered. Molecular dynamics simulations reveal that occupation of both superficial and deeply buried sites most effectively preserves the experimental structure; the structure collapses in the absence of bound cholesterol. In particular, we find that bound cholesterol directly supports contacts between the agonist-binding domain and the pore that are thought to be essential for activation of the receptor. These results likely apply to those other ion channels within the Cys-loop superfamily that depend on cholesterol, such as the GABA receptor.protein-lipid interaction ͉ ligand-gated ion channel ͉ cys loop receptor C ritical for memory and cognition, the nAChR is implicated in neurological disorders (1), addiction (2, 3), and the mechanism of anesthetics (4). Early efforts to reconstitute the nAChR noted that cholesterol as well as charged lipids (such as phosphatidic acid) are required for proper function (5). Proposed mechanisms for the role of cholesterol in nAChR function fall into two general types: indirect and direct. Indirect mechanisms are those in which a cholesterol-induced change in fluid or elastic properties of the lipid bilayer modulates the protein behavior. Although early studies (6, 7) indicated that current flow through the channel is insensitive to bilayer fluid properties, more recent studies (8, 9) have proposed that this result depends on the choice of order parameters. The direct mechanism requires cholesterol binding to the nAChR and effecting structural and/or dynamic changes. Proponents of this theory have determined that (i) cholesterol molecules in proximity to the protein exchange very slowly with the bulk lipid bilayer (10) (electron spin resonance difference spectroscopy); (ii) cholesterol accesses sites on the protein that cannot be occupied by phospholipids (11) (fluorescence quenching); and (iii) some cholesterol bound to the nAChR interacts with the phospholipids in the surrounding bilayer (12) (substit...

A Lipid-dependent Uncoupled Conformation of the Acetylcholine Receptor

“…Indeed, the identification of a steroid-binding site between M4 and M1-M3 in the mouse GABA A receptor, as well as a similar modulatory site in the rat ␣7 nAChR, appear to support this (38,39). Simulations have also hinted that a similar site might exist for cholesterol in the Torpedo nAChR (28).…”

“…One possibility is that lipids disrupt these inter-domain interactions by altering the packing of transmembrane helices. Transmembrane helix packing could ultimately dictate the conformation of the M2-M3 and ␤10-M1 linkers, thereby influencing crucial interactions at the interface between the transmembrane pore and agonist-binding domains (28).…”

Cholesterol Modulates the Rate and Mechanism of Acetylcholine Receptor Internalization

“…Several studies have indicated the need for Chol to maintain AChR conformation and function (10). Recent dynamic in silico molecular simulations of AChR suggest that the structure collapses in the absence of Chol (52). Hence, Chol depletion could also be associated with changes in AChR conformation, making it more sensitive to internalization.…”

Anionic Lipids Allosterically Modulate Multiple Nicotinic Acetylcholine Receptor Conformational Equilibria

“…Some studies have concluded that neutral and anionic lipids are both essential, with considerable research focusing on the role of Chol (6,8,37). It has been suggested that Chol and anionic lipids influence nAChR secondary (38,39) or tertiary/quaternary structure (26,40). The implication is that lipids are required for the nAChR to "fold" into a native conformation.…”