Changes of the membrane lipid organization characterized by means of a new cholesterol-pyrene probe.
We synthesized 3beta-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): K(DOPC) >> K(POPC) >> K(DMPC) > K(DMPC/15 mol % Chol) > K(DMPC/30 mol % Chol). This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I(1)-I(5)) and excimer (I(E)) emission bands. In model membranes, I(1)/I(3) and I(E)/I(3) ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse delta-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse delta-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.
Le Guyader
Laurent
L
Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.
Le Roux
Christophe
C
Mazères
Serge
S
Gaspard-Iloughmane
Hafida
H
Gornitzka
Heinz
H
Millot
Claire
C
Mingotaud
Christophe
C
Lopez
André
A
D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D008051:Lipid Bilayers / Q000737:chemistry*; D008560:Membrane Fluidity; D015336:Molecular Probe Techniques; D015335:Molecular Probes; D011721:Pyrenes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008051:Lipid Bilayers; D015335:Molecular Probes; D011721:Pyrenes; D002784:Cholesterol