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Biophysical Journal 86:2261-2272 (2004)
© 2004 The Biophysical Society

Cholesterol Modulates the Organization of the {gamma}M4 Transmembrane Domain of the Muscle Nicotinic Acetylcholine Receptor

Rodrigo F. M. de Almeida *, Luís M. S. Loura * {dagger}, Manuel Prieto *, Anthony Watts {ddagger}, Aleksandre Fedorov * and Francisco J. Barrantes §

* Centro de Química-Física Molecular, Instituto Superior Técnico, Lisboa, Portugal; {dagger} Departamento de Química, Universidade de Évora, Évora, Portugal; {ddagger} Biochemistry Department, Oxford University, Oxford, United Kingdom; and § UNESCO Chair of Biophysics & Molecular Neurobiology and Instituto de Investigaciones Bioquímicas de Bahía Blanca, Argentina

Correspondence: Address reprint requests to Francisco J. Barrantes, UNESCO Chair of Biophysics & Molecular Neurobiology and Instituto de Investigaciones Bioquímicas de Bahía Blanca, C.C. 857, Camino La Carrindanga km 7, B8000FWB, Argentina. E-mail: rtfjb1{at}criba.edu.ar.

A 28-mer {gamma}M4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor {gamma}-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The {gamma}M4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in {gamma}M4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Förster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the {gamma}M4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the {gamma}M4 peptide occupies ~30% of the patch area.




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