The nucleotide sequence has been determined of a cDNA clone that codes for the 60,000-dalton y subunit of Torpedo californica acetylcholine receptor. The length of the cDNA clone is 2,010 base pairs. The 5' and 3' untranslated regions have respective lengths of 31 and 461 base pairs. Data suggest that the putative polyadenylylation consensus sequence A-A-T-A-A-A may not be required for polyadenylylation of the mRNA corresponding to the cDNA clone described in this study. From the DNA sequence data, the amino acid sequence of the y subunit was deduced. The subunit is composed of 489 amino acids giving a molecular mass of 56,600 daltons. The deduced amino acid sequence data also indicate the presence of a 17-amino acid extension or signal peptide on this subunit. From these data, structural predictions for the y subunit are made such as potential membrane-spanning regions, possible asparagine-linked glycosylation sites, and the assignment of regions of the protein to the extracellular, internal, and cytoplasmic domains of the lipid bilayer.The nicotinic acetylcholine receptor (AcChoR) is a glycoprotein located in the postsynaptic membrane of the vertebrate neuromuscular junction. When the neurotransmitter acetylcholine binds to the receptor, a channel permeable to small cations opens. The resulting ion flow leads to depolarization and contraction of the muscle cell. AcChoR from the electric organ of Torpedo californica has been most extensively studied because it is a major component of the membrane in this tissue and can be purified in milligram amounts (for recent reviews, see refs. 1 and 2). The AcChoR from T. californica is an oligomeric complex with a molecular mass of --250,000 daltons. The complex is composed of four different polypeptide chains of approximately 40,000 (a), 50,000 (,B), 60,000 (y), and 65,000 (8) daltons (3, 4) with a stoichiometry of 2:1:1:1, respectively (5, 6). The subunits have a number of common features: each subunit is glycosylated (4, 7), the subunits have similar amino acid compositions (8) and show 35-50% sequence homology in the NH2-terminal regions (6), and each subunit spans the lipid bilayer (9). A number of experiments have demonstrated that a large part of the AcChoR extends into the synaptic cleft or extracellular side of the membrane (reviewed in ref. 10). The purified 250,000-dalton oligomer contains the binding sites for acetylcholine and the ligand-gated ion channel (11,12). Acetylcholine analogs are known to bind to the a-polypeptide chains (3); as yet, no specific functions have been assigned to the other polypeptide chains.This paper reports the complete nucleotide sequence of a cDNA clone (4D8) coding for the y subunit of AcChoR from the electric organ of T. californica. The deduced amino acid sequence of the y subunit and its putative signal peptide are also presented. Several structural predictions based on the amino acid sequence are discussed and incorporated into a model giving a possible partitioning of the y-polypeptide chain into the extracellular, ...

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The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 #M acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors,/3, the rate of dissociation of ACh from doubly liganded receptors, k_~, and the rate of channel closing, a. The rate of ACh dissociation from singly liganded receptors, k_l, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k l, k~, and ~ determined, the rates of ACh association, k+l and k+~, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca~+-free extracellular solution containing 1 mM free Mg 2+ at 22~ (b) Ca~+-free solution at 12~ and (c) extracellular Ca ~+ and Mg ~+, both at 0.5 mM, at 22~ For all three conditions the dissociation constant for the first agonist binding site is ~100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22~ ACh associates at diffusion-limited rates, whereas at 12~ association is 30-to 60-fold slower. Also slowed at 12~ are/3 (4-fold), k_~ (3-fold), k_ 1 (25-fold), and a (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12~ Changing from a Ca~+-free to a Ca2+-containing extracellular solution does not affect k+l and k+~, but increases/5 (twofold) and decreases k_2, k l, and a (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the openand closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.Address reprint requests to Dr.

Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.

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