Lipid-protein interactions in reconstituted membranes containing acetylcholine receptor

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Abstract
Functional membranes containing purified Torpedo californica acetylcholine receptor and dioleoylphosphatidylcholine (DOPC) were prepared by a cholate dialysis procedure with lipid to protein ratios of 100-400 to 1 (mol/mol). Spin-labeled lipids were incorporated into the reconstituted membranes and into native membranes prepared from Torpedo electroplax, and EPR spectra were recorded between 0.degree.-20.degree. C. The spin-labels included nitroxide derivatives of stearic acid (16-doxylstearic acid), androstane, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). The phospholipid spin-labels had 16-doxylstearic acid in the sn-2 position. All the spectra showed 2 components corresponding to a relatively mobile bilayer component and a motionally restricted protein-perturbed component. The relative amounts of mobile and perturbed components were quantitated by spectral subtraction and integration techniques. The mobile/perturbed ratio was somewhat temperature dependent, and the results are discussed in terms of exchange between mobile and perturbed environments. Plots of the mobile/perturbed ratios vs. lipid/protein ratios at 0.degree. C gave straight lines from which the relative binding affinity of each spin-label and the number of perturbed lipids per receptor protein could be calculated. All the spinlabels gave similar values for the number of perturbed lipids (40 .+-. 7), a number close to the number of lipids that will fit around the intramembranous perimeter of the receptor. The affinities of the spin-labeled lipids for the receptor relative to DOPC were androstane (K = 4.3) .simeq. 16-doxylstearic acid (4.1) > PA (2.7) > PE (1.1) .apprx. PC (1.0) .apprx. PS (0.7). The lipids having the highest affinity for the acetylcholine receptor were those with the largest effects on the ion flux functional properties of the receptor, and the results are discussed in terms of lipid effects on receptor function.

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