MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2004; 42: 148–154 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1326 The aM1 transmembrane segment of the nicotinic acetylcholine receptor interacts strongly with model membranes Maurits R. R. de Planque, 1† Dirk T. S. Rijkers, 2 Rob M. J. Liskamp 2 and Frances Separovic 1* 1 School of Chemistry, University of Melbourne, Melbourne, Victoria 3010, Australia 2 Department of Medicinal Chemistry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Received 31 March 2003; Revised 12 August 2003; Accepted 27 August 2003 The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) plays a role in the regulation of the activity of this important ligand-gated ion channel. The lipid composition of the host membrane affects conformational equilibria of the nAChR and several classes of inhibitors, most notably anaesthetics, interact directly or indirectly with the four transmembrane M-segments, M1 – M4, of the nAChR subunits. It has proven difficult to gain insight into structure–function relationships of the M-segments in the context of the entire receptor and the biomembrane environment. However, model membrane systems are well suited to obtain detailed information about protein–lipid interactions. In this solid-state NMR study, we characterized interactions between a synthetic aM1 segment of the T. californica nAChR and model membranes of different phosphatidylcholine (PC) lipids. The results indicate that aM1 interacts strongly with PC bilayers: the peptide orders the lipid acyl chains and induces the formation of small vesicles, possibly through modification of the lateral pressure profile in the bilayer. The multilamellar vesicle morphology was stabilized by the presence of cholesterol, implying that either the rigidity or the bilayer thickness is a relevant parameter for aM1–membrane interactions, which also has been suggested for the entire nAChR. Our results suggest that the model systems are to a certain extent sensitive to peptide–bilayer hydrophobic matching requirements, but that the lipid response to hydrophobic mismatch alone is not the explanation. The effect of aM1 on different PC bilayers may indicate that the peptide is conformationally flexible, which in turn would support a membrane-mediated modulation of the conformation of transmembrane segments of the nAChR. Copyright  2004 John Wiley & Sons, Ltd. KEYWORDS: NMR; solid-state NMR; 31 P NMR; 2 H NMR; nicotinic acetylcholine receptor; ˛M1 segment; peptide – lipid interactions; phosphatidylcholine model membranes; hydrophobic matching INTRODUCTION A prominent superfamily of membrane proteins is formed by the ligand-gated ion channels that act as neurotransmitter receptors, a prime example of which is the nicotinic acetylcholine receptor (nAChR). 1,2 The nAChR from the electric organ of the electric ray Torpedo is a large pentameric integral membrane protein that consists of four different subunits of about 470 amino acids each. One ˇ,  and υ and two ˛ subunits are arranged around a central axis perpendicular to the postsynaptic membrane, forming a L Correspondence to: Frances Separovic, School of Chemistry, University of Melbourne, Melbourne, Victoria 3010, Australia. E-mail: fs@unimelb.edu.au † Present address: Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Contract/grant sponsor: The Netherlands Organization for Scientific Research (NWO); Contract/grant number: S81-683. Contract/grant sponsor: Melbourne Research Development Grants Scheme (MRDGS). cation-selective ion channel which is essential for nerve impulse transduction over synaptic clefts. 3,4 Each subunit traverses the membrane with four polypeptide segments, M1–M4, and significant sequence homology exists between analogous M-segments of the ˛, ˇ,  and υ chains. The five M2 segments line the aqueous channel, whereas M1, M3 and M4 are exposed to the lipids that make up the host membrane. 1,4 The transmembrane domains of the nAChR are important for the regulation of its activity. For example, conformational equilibria of the receptor are modulated by the lipid composition of the membrane, 5,6 and effectors such as local anaesthetics inhibit ion channel activity by targeting the transmembrane segments, either by direct binding or indirectly by altering membrane properties. 7,8 Despite their functional relevance, little is known with certainty about the structure of the transmembrane M-segments and about the mechanisms by which membrane constituents and anaesthetics exert their influence. In the case of a large integral membrane protein such as the nAChR, it is especially complicated to obtain structural information and Copyright  2004 John Wiley & Sons, Ltd.