LIPID-PROTEIN INTERACI'IONS IN RECONSTITUTED MEMBRANES CONTAINING NICOTINIC ACETYLCHOLINE RECEPTOR. J.A. ENCINAR, A.M. FERNANDEZ, J.A POVEDA, & J.M. GONZALEZ-ROS. Department of NeurochemiStry and Institute of Neuroscience, University of Alicante, 03080 Alicante. Spain. The nicotlmc acetylcholine receptor (AcChR) from Torpedo is a large transmembrane glycoprotein composed of four different polypeptide subunits (a, 13, y and B) in a 2: I: 1:1 stoichiometry. Binding of cholinergic agonists to extracellular domains on the a subunits, causes the formation of a transient cation channel within the protein, responsible for the initiation of postsynaptic membrane depolarization. In this report, we have explored the possibility that the well known lipid dependence of AcChR ion channel function is partly due to the occurrence of specific phospholipid- induced modifications of the AcChR protein structure. To this end, purified AcChR protein has been reconstituted in vesicles made from lipid mixtures containing a constant amount of egg phosphatidylcholine (50% by mole) and cholesterol (25% by mole), plus a fixed mole percent (25%) of one of the following phospholipids whose effects on AcChR structure and function were to be tested: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidic acid (PA). These phospholipids were all derivatives of egg yolk: PC and therefore, have the same fatty acid composition. Use of such lipid mixtures in reconstituting the AcChR results in the formation of sealed unilamelar reconstituted vesicles, perfectly adequate for rapid ion flux measurements, as well as for Fourier-transform IR spectroscopic studies. To test the ability of the AcChR to rapidly increase the permeability to cations of the reconstituted membrane in response to binding of cholinergic agonists, we have used a rapid kinetics, "stopped-flow/fluorescence quenching" assay of TI+ influx. Our results show that the AcChR reconstituted in vesicles made exclusively from zwitterionic PC/cholesterol mixtures completely lacks the ability to activate the characteristic cation channel in response to cholinergic agonists. On the contrary, the presence of phospholipids other than PC in the reconstituted vesicles, partly retains AcChR activity to an extent which varies depending upon the phospholipid. Thus, the largest responses to cholinergic agonists correspond to samples containing PA (about one half of the maximal response seen in the samples reconstituted in whole asolectin lipids used as a reference for full functional reconstitution), followed by those containing PG, while the samples containing the zwitterionic PE exhibited a very low level of activity. The FTIR spectra of the above samples compared to that of the protein reconstituted in whole asolectin lipids, shows band-widening and other changes in the spectral shape 339 P. Carmona et al. (eels.), Spectroscopy of Biological Molecules: Modem Trends. 339-340. © 1997 Kluwer Academic Publishers.