The Acetylcholine Receptor Channel from Torpedo

We recorded single channel currents activated by agonist binding to purified acetylcholine receptors from the electric organ of Torpedo californica reconstituted in planar lipid bilayers. Analysis of single channel records indicate that the acetylcholine receptor channel displays two kinetically distinct open states which differ in their mean open times but have similar channel conductances. The nicotinic acetylcholine (ACh) receptor is currently the best characterized neurotransmitter receptor. The availability of specific neurotoxins that act on the ACh receptor and of a rich tissue source for the biochemical isolation of the receptor protein has facilitated the detailed characterization of the structure of the ACh receptor primarily from Torpedo electric organ (for a recent review, see Anholt et al., 1983). Recently, a number of laboratories succeeded in cloning the genes coding for the various receptor subunits, and their entire amino acid sequence has been elucidated (Noda et al., 1982, 1983; Claudio et al., 1983; Devillers-Thiery et al., 1983). On the other hand, detailed electrophysiological information on the channel of the muscle ACh receptor is becoming available through patch recording (Neher and Sakmann, 1976; Sakmann et al., 1980; Colquhoun and Sakmann, 1981; Jackson et al., 1982, 1983); still, little is known about the electrophysiological properties of the electric organ ACh receptor (Moreau and Changeux, 1976; Schindler and Quast, 1980). ’ We thank Jim Donovan for his generous assistance in developing the programs for the computer analysis, Myrta Montal for her dedicated collaboration in the experiments, and Robert Anholt, Peter Schoch, and Donald R. Fredkin for their perceptive reading of the manuscript. This research was supported by grants from the Office for Naval Research (Grant N00014-79-C-0978), the National Institutes of Health (Grants EY02084 and NS11323), the Muscular Dystrophy Association, and the Department of the Army Medical Research and Development Command (Grant 17-82-C2221). P. L. is a postdoctoral fellow of the Muscular Dystrophy Association. During this work M. M. was a John Simon Guggenheim Foundation Fellow. *To whom correspondence should be addressed, at University of California, San Diego, B-019, La Jolla, CA 92093. In recent years, the technology for the reconstitution of functional ACh receptors in lipid vesicles (Epstein and Racker, 1978; Changeux et al., 1979; Huganir et al., 1979; Lindstrom et al., 1980; Anholt et al., 1981, 1982; Walker et al., 1982) and in planar lipid bilayers (Nelson et al., 1980; Schindler and Quast, 1980; Boheim et al., 1981) has been developed in order to establish systems that allow investigation of structure-function correlates in the receptor molecule. These studies have contributed to our understanding of the molecular basis of postsynaptic transmission, by demonstrating that the (u&6 subunit structure of ACh receptor monomers contains the ACh-binding sites and the cation channel it regulates (Anholt et al., 1983). Purified ACh receptors, reconstituted in lipid vesicles and in planar bilayers, display activation and desensitization induced by cholinergic agonists (cf. Anholt et al., 1983). Here, we used the reconstituted membrane to study the gating kinetics of the purified Torpedo ACh receptor channel and found that it has two open states. Thus, single channel recordings of purified Torpedo ACh receptor provide new facets about the molecular mechanism of channel gating which, in principle, can be correlated with its molecular structure. Materials and Methods Experimental procedure Receptor preparation. Receptor from the electric organ of Torpedo californica (Pacific Bio-Marine Laboratories, Inc., Venice, CA) was solubilized, purified, and reconstituted in lipid vesicles as described in detail previously (Lindstrom et al., 1980; Anholt et al., 1981, 1982). The reconstituted soybean lipid vesicles (40 mg/ml) used in