Critical dependence of the solubilization of lipid vesicles by the detergent CHAPS on the lipid composition. Functional reconstitution of the nicotinic acetylcholine receptor into preformed vesicles above the critical micellization concentration

The critical concentration of free detergent [D-w](c), which is necessary for lipid vesicle solubilization, is widely believed to be near to the critical micellization concentration (CMC) of the detergent. Here it is shown that [D-w](c) and the critical detergent/lipid ratio R(c)(M) in the mixed micelles strongly depend on the lipid composition. In agreement with the concept of packing constraints, phospholipids with a large head-group were solubilized by the detergent CHAPS at low CHAPS concentrations, for example [D-w](c) = 0.27 mM for phosphatidyl-inositol and [D-w](c) = 2.3 mM for phosphatidylcholine, T = 293 K (20 degrees C). In contrast, phospholipids (PL) with a small head-group required larger [CHAPS] values for (mixed) micelle formation: [D-w](c) = 3.2 mM for phosphatic acid (PA) and [D-w](c) = 5.2 mM for a mixture of palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), POPC:PE:PG = 30:60:10 (mol.-%). Values obtained for the partition coefficient K = R(c)(M)/[D-w](c) ranged from K = 0.12 mM(-1) for dimyristoylphosphatidylcholine to K = 1.1 mM(-1) for (soy) phosphatidylcholine:phosphatidylglycerol (50:50). After addition of 30 mol.-% cholesterol (Ch) or with dipalmitoylphosphatidylcholine below T = 308 K (35 degrees C), [D-w](c) is > 10 mM, which is far above the CMC = 4 mM. This indicates that CHAPS micelles are formed before vesicle solubilization begins. The analysis of vesicle solubilization was a prerequisite for the controlled reconstitution of protein membrane proteins. AChR proteins reconstituted into preformed PL/Ch-vesicles at [D-w] > CMC had a 4-5 time higher value of Li-influx compared to reconstitutions from completely solubilized lipid and proteins indicating a higher efficiency of right-side out AChR incorporation.