Interactions of the nicotinic acetylcholine receptor transmembrane segments with the lipid bilayer in native receptor-rich membranes.
Proper ion channel function of the nicotinic acetylcholine receptor (nAChR) requires the interaction of the protein with distinct lipid species present in the receptor's membrane microenvironment. Two classes of lipid binding sites present at the protein-membrane interface have been postulated: annular binding sites primarily occupied by phospholipids and non-annular binding sites mainly occupied by cholesterol [Jones & McNamee (1988) Biochemistry 27, 2364-2374]. We investigated the binding of these lipids to the nAChR and potential dynamics of these interactions during events associated with signal transduction by electron spin resonance spectroscopy (ESR) using spin-labeled analogues of phospholipids, androstane, and stearic acid. Protein-lipid interactions were characterized in receptor-rich membranes prepared from Torpedo californica electric tissue preserving the native lipid environment of the nAChR. We found a strong preference of the receptor for the phosphatidylserine (PS) analogue as compared to the other probes. Up to 57% of PS were perturbed by the membrane protein, while the fraction of motionally restricted lipid for the other analogues was on the order of 30%. After removal of the extramembrane portions of the membrane-bound receptor, we observed a loss of binding sites for the spin-labeled analogue of androstane and for stearic acid, but not for phospholipids and sphingomyelin analogues. Our results demonstrate the existence of topologically distinct lipid binding sites for different lipid species. In the case of cholesterol, extramembrane portions of the receptor are involved, whereas the transmembrane segments meet the requirements for the binding of phospholipids. Tyrosine phosphorylation of the nAChR did not affect protein-lipid interactions in samples of intact nAChR. Similarly, no significant changes were observed in the presence of carbamoylcholine at concentrations that caused rapid and quantitative desensitization of the nAChR.
Dreger
M
M
FU Berlin, Institut für Biochemie, Germany.
Krauss
M
M
Herrmann
A
A
Hucho
F
F
D000818:Animals; D001665:Binding Sites; D002217:Carbachol; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D019286:Endopeptidase K; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D013113:Spin Labels; D014101:Torpedo; D014443:Tyrosine / Q000737:chemistry
D007473:Ion Channels; D008051:Lipid Bilayers; D010743:Phospholipids; D011978:Receptors, Nicotinic; D013113:Spin Labels; D014443:Tyrosine; D002217:Carbachol; D002784:Cholesterol; D019286:Endopeptidase K