Relevance of CARC and CRAC Cholesterol-Recognition Motifs in the Nicotinic Acetylcholine Receptor and Other Membrane-Bound Receptors

Abstract

Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution.

Molecular Dynamics Simulations of Kir2.2 Interactions with an Ensemble of Cholesterol Molecules

“…Two general approaches were used in previous investigations to identify cholesterol-binding pockets on ion channels: identifying known cholesterol-binding motifs, such as CRAC and CARC in the sequence of the protein (2,51,52), or performing a docking of cholesterol to the channel structure unbiased to the presence of known motifs (11,53,54). The cholesterol-recognition amino acid consensus motif (CRAC) and its mirror image CARC have been identified as major elements involved in cholesterol binding in a variety of TM proteins (2,51,55). These motifs and their energetic requirements were explored in detail in nicotinic acetylcholine receptor (nAChR) (51).…”

Peripheral Myelin Protein 22 Preferentially Partitions into Ordered Phase Membrane Domains

“…While these motifs are loosely defined and are not always indicative of direct cholesterol interaction (45), there is substantial experimental and computational evidence supporting the notion that these motifs are sometimes directly involved in binding cholesterol (46,47).…”

Cholesterol recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: the case for TRKB

“…Increased cholesterol concentration enhances the plasticity and flexibility of 5HT1a dimers and adrenergic receptors (Prasanna et al 2014(Prasanna et al , 2016 . Given the opposed dispositions of CARC and CRAC motifs, it is possible to assume the co-existence of both in the same transmembrane domain and their potential interaction with two cholesterol molecules in a tail-to-tail configuration (Di Scala et al 2017) . However, none of the analyzed TMR of RTK family members in the present study displayed co-existing CARC and CRAC motifs.…”

“…According to in silico analysis of cholesterol interaction with CARC motifs, the Y to F or Y to W mutations should not interfere with the motif interaction with cholesterol. However, these analyses were performed in multipass or multi-subunits transmembrane receptors, such as nicotinic cholinergic (Di Scala et al 2017) or GPCR receptors (Prasanna et al 2014(Prasanna et al , 2016 . Given that TRKB and virtually all members of the RTK family possess a single transmembrane domain, small changes in this region may impact their function but remain undetected in the multipass proteins or in the direct interaction with cholesterol.…”