Reconstituted nicotinic acetylcholine receptors ( nAChR ) exhibit significant gain-of-function upon addition of cholesterol to reconstitution mixtures, and experimentally observed clustering of nAChRs within membranes is cholesterol-sensitive, leading to a common expectation that nAChRs will partition into cholesterol-rich liquid ordered ("raft" ) domains . We use coarse-grained molecular dynamics simulations to observe spontaneous interactions of cholesterol, saturated lipids, and polyunsaturated  lipids with nAChRs. In binary  DPPC:CHOL  mixtures, both cholesterol and DPPC acyl chains were observed spontaneously entering deep "non-annular" cavities in the nAChR TMD, particularly at the subunit interface and the \beta subunit center, facilitated by the low amino acid density in the cryo-EM structure of nAChR in a native membrane. Cholesterol was highly enriched in the annulus around the TMD, but this was a short range effect that extended over (at most) 5-10\AA. In  ternary mixtures containing polyunsaturated fatty acids , the presence of a single receptor did not significantly affect the likelihood of domain formation , and nAChR partitioned to the cholesterol-poor liquid-disordered domain whenever one was present. This was independent of whether the \l_{do domain lipids had PC or PE headgroups , but saturated lipids with PE headgroups were less depleted from the boundary than those with PC headgroups . Enrichment of unsaturated phospholipids among boundary lipids was positively correlated with their propensity for demixing from cholesterol-rich phases. Long n-3 chains (tested here with Docosahexaenoic Acid  ) were highly enriched in annular and non-annular embedded sites, partially displacing cholesterol and completely displacing DPPC, and occupying sites even deeper within the bundle. Shorter n-6 chains (Linoleic acid) were far less effective at displacing cholesterol from non-annular sites.
Reconstituted nicotinic acetylcholine receptors ( nAChRs ) exhibit significant gain-of-function upon addition of cholesterol to reconstitution mixtures, and cholesterol URLanization of nAChRs within domain-forming membranes, but whether nAChR partitions to cholesterol-rich liquid-ordered ("raft" or l_o) domains or cholesterol-poor liquid-disordered (l_{do . We use coarse-grained molecular dynamics simulations to observe spontaneous interactions of cholesterol, saturated lipids, and polyunsaturated (PUFA) lipids with nAChRs. In binary Dipalmitoylphosphatidylcholine:Cholesterol ( DPPC:CHOL ) mixtures, both CHOL and DPPC acyl chains were observed spontaneously entering deep "non-annular" cavities in the nAChR TMD, particularly at the subunit interface and the \beta subunit center, facilitated by the low amino acid density in the cryo-EM structure of nAChR in a native membrane. Cholesterol was highly enriched in the annulus around the TMD, but this effect extended over (at most) 5-10\AA. In domain-forming ternary mixtures containing PUFAs , the presence of a single receptor did not significantly affect the likelihood of domain formation . nAChR partitioned to any cholesterol-poor l_{do of whether the l_{do domain lipids had PC or PE headgroups . Enrichment of PUFAs among boundary lipids was positively correlated with their propensity for demixing from cholesterol-rich phases. Long n - 3 chains (tested here with Docosahexaenoic Acid , DHA ) were highly enriched in annular and non-annular embedded sites, partially displacing cholesterol and completely displacing DPPC, and occupying sites even deeper within the bundle. Shorter n - 6 chains were far less effective at displacing cholesterol from non-annular sites.
R
nAChR
nAChRs
R
experimentally observed clustering
cholesterol URLanization
R
membranes is cholesterol-sensitive, leading to a common expectation that nAChRs will partition into
domain-forming membranes, but whether nAChR partitions to
R
liquid ordered
liquid-ordered
R
) domains
or l_o) domains or cholesterol-poor liquid-disordered (l_{do
A
(PUFA)
A
Dipalmitoylphosphatidylcholine:Cholesterol (
A
)
R
cholesterol
CHOL
R
was a short range effect that
effect
A
domain-forming
R
polyunsaturated fatty acids
PUFAs
R
, and
.
R
the
any
R
liquid-disordered domain whenever one was present. This was independent
l_{do
R
\l_{do
l_{do
D
, but saturated lipids with PE headgroups were less depleted from the boundary than those with PC headgroups
R
unsaturated phospholipids
PUFAs
R
n-3
n - 3
A
, DHA
R
n-6 chains (Linoleic acid)
n - 6 chains