Biochemical Society Transactions zyxwvuts 776 Role of cholesterol as a structural and functional effector of the nicotinic acetylcholine receptor Gregorio Fernandez-Ballester*, Jose Castresanat, Asia M. Fernandez*, Jose-Luis R. Arrondot, Jose A . Ferragut* and Jose M. Gonzalez-Ros*$ *Department of Neurochemistry and Institute of Neurosciences, University of Alicante, 03080 Alicante, Spain and tDepartrnent of Biochemistry, Faculty of Sciences, University of the Basque Country, 48080 Bilbao, Spain zy The nicotinic acetylcholine receptor (AcChR) from zyxwvu Torpedo is a transmembrane glycoprotein com- posed of four different polypeptide subunits zyxwvuts (a, zyxwvu p, y and 6) in a 2: 1 : 1 : 1 stoichiometry [ 1-31, Binding of cholinergic agonists to sites on extracellular domains of the AcChR elicits the formation of a transient cation channel within the protein, which is responsible for the initiation of postsynaptic mem- brane depolarization. On continuous exposure to the agonist, however, the channel opening response becomes blocked and the affinity for the agonists increases, a process known as desensitization [4]. Keconstitution of the purified AcChR protein into artificial liposomes of defined composition has shown that the presence of certain lipids in the reconstituted samples, namely cholesterol and acidic phospholipids, is important in preserving the ability of the reconstituted AcChR to exhibit an optimal cation channel activity [ 1,s-lo]. Neverthe- less, in spite of the existing information on the lipid dependence of AcChR function, very little is known about the molecular basis of such phenomena. In this paper, we have used the conformational sensi- tivity of the amide I band in the i.r. spectrum of the purified AcChR protein reconstituted into lipid vesicles to explore the possibility that cholesterol causes changes in the protein structure that might be responsible for the observed alteration in AcChR function. Fourier-transform i.r. (F.t.i.r.) spectro- scopic methods have shown great potential for detecting structural differences between the various possible conformers of complex membrane pro- teins [ 11-14], including the AcChR [15-201. This information is summarized in Table 1, along with rapid kinetics data of cholinergic agonist-induced cation translocation, to illustrate the functional status of the AcChR in the different reconstituted hilayers. Effects of cholesterol on the i.r. amide I band of the AcChR A previous attempt to use i.r. methods to study the effects of cholesterol on AcChR structure was based Abbreviations used: AcChK, acetylcholine receptor; Pc, phosphatidylcholine. $To whom correspondence should be addressed. on monitoring i.r. bands in the so-called skeletal region (ranging 1000-900 cm- ') of the protein spectrum [21] and yielded uncertain results, partly because of the low intensity of the spectroscopic signal and the weakly established correlation of the selected vibrational modes to specific protein struc- tures. In contrast, the strong 1600-1700 cm- ' amide I band used in this work (Figure la) results primarily from stretching vibrations of C=O groups in peptide bonds [22], the exact frequencies of which depend on the particular secondary structure adopted by the protein. Resolution-enhancing, band-narrowing techniques [23,24], such as Fourier self-deconvolution or Fourier derivation, show that the amide I region exhibits maxima at approximately 160.5, 1615, 1636, 1656, 1672, 1680 and 1690cm-' (Figure lb). Whereas the 160.5 and 1615 cm ~ ' components correspond to amino acid Table I Stopped-flow kinetics of carbamylcholine-activated TI+ influx in reconstituted AcChR vesicles made from different lipid mixtures Reconstituted AcChR vesicles were prepared as described in [ 191 Vesicles in 10 mM Hepes buffer, 200 mM NaNO, pH 7 4, were rapidly mixed with an equal volume of IOmM Hepes buffer, 170 mM NaNO,, 30 mM TINO,, pH 7 4 containing increasing concentrations of carbamylcholine ranging from 0 to 250 yM (final concentration), in a Hi-Tech SF-5 I stopped-flow instrument Influx of TI+ into the reconstituted AcChR vesicles was recorded as a time-dependent quenching of the fluor- escence of pyrene I ,3,6&tetrasulphonate entrapped into the vesicles by externally added TI + The apparent maximum rate constant for TI+ influx zyxwv (k,,, , ) and the dissociation constant (K,) were determined as in [25] NA, no activity Lipid matrix ~ ~~ Whole asolectin lipids 131 zyx f5 5.16f0.5 Phospholipids from asolectin 59f I6 4.33 k 0.42 + 10% cholesterol 65 3.14 + 20% cholesterol I05 4.37 + 40% cholesterol I32 f 34 7.58 zy f 0.22 Egg phosphatidylcholine NA NA + 40% cholesterol NA NA