Reconstitution of the Torpedo californica nicotinic acetylcholine receptor into planar lipid bilayers

Detergent-solubilized acetylcholine receptor (nAcChR) proteins can be purified by affinity chromatography and reconstituted into lipid vesicles and afterwards into planar lipid bilayer membranes via, in principle, two methods: fusion or assembly from two vesicle-spread monolayers. In the presence of agonists (carbamoylcholine, suberoyldicholine) different kinds of channel openings are recorded: fast single channels, bursts and long openings with short closures in between. Similar results have been obtained with reconstituted membrane fragments rich in nAcChR. In addition, Torpedo californica nAcChR proteins give rise to fuzzy channels and less defined events of conductivity, which “reemerge” again all the time. Frequently the channel events have conductance levels of about 200 to 300 pS, obviously simultaneous openings of several aggregated receptors. Under these conditions 40 pS conductance events occur also. It appears that the conductance of the channels measured is a multiple of 6.3 pS. Often, with the same sample, no channel openings are seen. Contrary to patch-clamp investigations on whole cells, AcChR-channel openings in reconstituted systems occur only several minutes after agonist application and not immediately. It is not clear whether the “reconstituted channels” reflect rapid activation or whether they result from desensitized receptor states only. Although a clear-cut correlation of channel event and channel protein unit is only possible by reconstitution of the biochemically characterized protein, e.g. monomer, dimer or higher oligomers, the reconstitution technique is still in its infancy.