Cholesterol interacts with transmembrane alpha-helices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: photolabeling studies using [3H]Azicholesterol.
The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.
Hamouda
Ayman K
AK
Department of Pharmacology and Neuroscience, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Chiara
David C
DC
Sauls
Daniel
D
Cohen
Jonathan B
JB
Blanton
Michael P
MP
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism*; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D010446:Peptide Fragments / Q000378:metabolism; D019920:Photoaffinity Labels; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium
D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D014316:Tritium; D002784:Cholesterol