Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipets.
The channel of the purified acetylcholine receptor from Torpedo californica electric organ reconstituted in lipid vesicles was assayed by direct electrical recording using patch-clamp pipets. High-resistance seals were obtained by gentle suction of vesicles into the pipet or after the formation of lipid bilayers from monolayers at the tip of the pipet. Single-channel currents were activated by three cholinergic ligands: acetylcholine, carbamylcholine, and suberyldicholine. The single-channel conductance, gamma, was 40 +/- 5 pS in 0.5 M NaCl, irrespective of the agonist used. The distributions of channel open times were fitted by a sum of two exponentials. The lifetimes of the two exponential components were a factor of 2 longer for suberyldicholine than for acetylcholine or carbamylcholine. At desensitizing concentrations of agonists the single events appeared in paroxysms of channel activity followed by quiescent periods. These results suggest that the full cycle of solubilization, purification, and reconstitution of this membrane receptor can be achieved without impairment of channel function.
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002794:Choline / Q000031:analogs & derivatives / Q000494:pharmacology; D004553:Electric Conductivity; D004557:Electric Organ / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism; D008051:Lipid Bilayers; D008722:Methods; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo
D007473:Ion Channels; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic; C009335:subecholine; D002217:Carbachol; D002794:Choline; D000109:Acetylcholine