Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Abstract

Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the q218ft subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels.The nicotinic acetylcholine receptor from Torpedo electroplax is a chemically gated ion channel consisting of a pentameric complex of the five subunits a2fy8S (for reviews, see refs. 1 and 2). It is reproducibly purified and, when reconstituted into small liposomes or planar lipid bilayers, it exhibits the basic agonistinduced cation transport properties anticipated from electrophysiological measurements in vivo (3-10). By applying recently developed methods (11, 12) for patch-recording from large liposomes containing reconstituted ion channels, we have now measured, with high resolution, single-channel properties of the purified and reconstituted Torpedo acetylcholine receptor. We report here that this pentameric complex showed an agonist-independent main conductance level, a subconductance state, saturation of the single-channel conductance at high Na+ concentration, slow inactivation and burst-kinetics at desensitizing concentrations of agonists, and open channel lifetime distributions that are not described by single exponentials. The similarity of these properties with those reported for other vertebrate acetylcholine receptor species recorded in situ provides further evidence that the purified receptor is fully functional and that the well-characterized biochemical properties associated with this isolated receptor complex may apply to a wide class of nicotinic receptor ion channels. MATERIALS AND METHODSPreparation of Liposomes Containing Reconstituted Acetylcholine Receptor. Acetylcholine receptor was isolated, purified, and reconstituted as described (4). Briefly, postsynaptic membranes rich in acetylcholine receptor were prepared from freshly dissected electric organ of Torpedo Californica. The receptor was then purified from a Na cholate extract of the postsynaptic membranes by affinity chromatography on a choline carboxymethyl affinity gel as described by Huganir and Racker (4), except that the loaded column was washed with 20 column. vol of "wash buffer" before elution. The purified and solubilized receptor was incorporated into asolectin (Associated Concentrates, Woodside, NY) small unilamellar vesicles by the detergent dialysis technique. The ...

Monoclonal antibodies specific to the beta and gamma subunits of the Torpedo acetylcholine receptor inhibit single-channel activity

“…A few mAbs block the function of ACh receptor when studied by the Na+ flux assay in reconstituted vesicles (Lindstrom et al, 198 1 b, 1983;Wan and Lindstrom, 1985). The detailed identification of the inhibitory effects of mAbs on the ACh receptor channel can be further investigated in reconstituted planar lipid bilayers, where the ion conduction and gating characteristics of the purified ACh receptor channel are readily measurable (Boheim et al, 198 1;Labarca et al, 1984a;Nelson et al, 1980;Schindler et al, 1984;Tank et al, 1983). This is illustrated in the left-hand panel of Figure arrows.…”

“…All four ACh receptor subunits are transmembrane polypeptides (Froehner, 198 1;Strader and Rafferty, 1980;Strader et al, 1979;Tarrab-Hazdai et al, 1978;Wennogle and Changeux, 1980) and the complete amino acid sequences of all four subunits have been deduced from the nucleotide sequences of cDNA clones (Claudio et al, 1983;Devillers-Thiery et al, 1983;Noda et al, 1982Noda et al, , 1983a. Reconstitution of the purified ACh receptor in lipid vesicles (Anholt et al, 1981(Anholt et al, , 1982Changeux et al, 1979;Gonzalez-Ros et al, 1980;Huganir et al, 1979;Lindstrom et al, 1980;Wu and Raftery, 1979) and in lipid bilayers (Boheim et al, 1981;Labarca et al, 1984a, b;Nelson et al, 1980;Schindler et al, 1984;Tank et al, 1983) demonstrated that the a,&6 structure is sufjicient to express the transduction of ligand binding into the opening of a cation-specific channel. Expression of functional ACh receptor in Xenopus oocytes directed by the cloned cDNAs encoding the four subunits of the ACh receptor indicated that all four subunits are necessary to exhibit the cholinergic response (Mishina et al, 1984).…”

Commentary on the Pleasures of Solving Impossible Problems of Experimental Physiology

“…We worked out a way to bulge multilayer lipid membrane aggregates called Bangasomes by osmotic stress to expose an area of lipid single bilayer to patch clamp them. The results showed that the ACh channel does consist of the hypothesized assembly of transmembrane subunits alone and that they display the same set of open and closed conductance states and switching kinetics that had been seen in the synapse membranes of cells (49). This experiment appeared to quiet the controversy.…”

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

“…Boheim et al [9] report the successful fusion of vesicles containing purified AcChRs into planar steroyl-myristoyl-phosphatidylcholine (SMPC) bilayers; here, too, the observed channel openings probably arose from the desensitized receptor state. Another group found that AcChR channels vanish shortly after the formation of a vesicle patch; this observation was also referred to receptor desensitization [14]. In contrast, when the vesicle/planar bilayer fusion method [9,10] is used, the first channel events are detected from several minutes up to half an hour after formation.…”

“…Most of the data from reconstituted nAcChRs do not fit into this physiological data frame: the patch data from reconstituted vesicles have too high a conductance for the presence of 2 mM divalent cations [14]. The channel data of Schindler and Quast [7] show a linear dependence on NaCl concentration.…”