Structural studies of the acetylcholine receptor in the membrane environment

The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of ligand-gated ion channels. All members of this superfamily mediate fast intercellular communication in response to endogenous neurotransmitters. Here I review a series of biophysical studies on the AChR protein with particular focus on the interactions of the macromolecules with its lipid membrane microenvironment. Spectroscopic studies of the micro-to-millisecond translational and rotational mobility of the AChR in the native membrane and synthetic lipid systems provide information on the dynamics of this protein in a two-dimensional lattice, the lipid bilayer. Motional regimes in the nanosecond time-scale can be explored with other techniques, such as electron spin resonance. Application of the latter technique led to the discovery of a lipid fraction in direct contact with the AChR, with rotational dynamics 50-fold slower than those of the bulk lipids. The lipid belt-region around the AChR molecule has since become the focus of a variety of investigations aimed at defining its possible role in the modulation of AChR function. The polarity and molecular dynamics of solvent dipoles - mainly water - in the vicinity of the lipids in the AChR membrane have been studied with Laurdan extrinsic fluorescence, and Forster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET has been used to discriminate between the bulk lipid and the lipid beft-region in the vicinity of the protein. The AChR-vicinal lipid is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipid and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. The topography of the AChR membrane-embedded domains has recently been explored with fluorescence methods using whole Torpedo AChR protein and transmembrane peptides. The location of pyrene maleimide-labeled Cys residues in transmembrane domains alpha M1, alpha M4, gamma M1 and gamma M4 was determined by differential fluorescence quenching with spin-labeled derivatives of various lipids. Pyrene-labeled Cys residues were found to lie in a shallow position. For M4 segments, this is in agreement with a predicted linear alpha -helix; for M1, it is necessary to postulate a substantial amount of nonhelical structure, and/or of kinks, to rationalize the superficial location of Cys residues. Three evolutionarily conserved proline residues in M1 may account for such structure. Proline residues appear to be conserved in transmembrane segments of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.