A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the 8 subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector XgtlO from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the 8 clone was selected by hybridization with cDNA encoding the y subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR 8 and y subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo y subunit cDNA, but not with 8. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences.

The nicotinic acetylcholine (AcCho) receptor (AcChoR) Is a multisubunit protein complex of stoichiometry a2PVY6. The several subunits show homology with each other within a given species; in addition, homology Is found between analogous subunits between species. We have used the phage SP6 RNA polymerase ascription system to produce singlespeiesI RNA in vitro for various AcChoR subunits from cDNAs.Injection of an equimolar mixture of RNA for the a, 3, y, and The recent cloning of cDNAs for the subunits of the Torpedo electric organ AcChoR (6-11) makes it possible to apply the powerful techniques of molecular biology to the study of the structure, evolution, biosynthesis, and mechanisms that underlie the operation of the complex. Our interest lies in the mechanism of ligand activation and ion permeation through the channel. Through the use of site-directed mutagenesis (12), we hope to identify the structural features and, thus, the mechanisms involved in the functioning of the receptor. Since the mutagenesis involves manipulations at the DNA level, a suitable expression system must be developed to study the properties of these "mutant" receptors.Xenopus oocytes have proved to be an attractive system for the expression ofproteins coded for by exogenous nucleic acids. Nuclear injection ofDNA (13) or cytoplasmic injection of mRNA (14) In this report, we describe another approach to the expression of Torpedo AcChoRs in Xenopus oocytes. We utilize the highly efficient phage SP6 RNA polymerase in vitro transcription system developed by Melton and colleagues (18,19). This system allows the synthesis of microgram quantities of pure RNA from cDNA. When used with cDNAs for the individual subunits of the AcChoR, injection of the in vitro transcripts into oocytes gives rise to functional Torpedo AcChoRs in large quantities that can be studied readily by both biochemical and electrophysical techniques.While this manuscript was in preparation, Mishina et al. (20) described an expression system essentially similar to that described here. They have used this system to study the effects of segment deletion or single amino acid changes introduced into the Torpedo AcChoR a subunit on receptor function. For our first study, we have chosen to test the effect of a much larger "mutation" in that we have asked whether a hybrid receptor containing the Torpedo a, (3, and y subunits and the mouse 8 subunit is functional. This study necessarily required examination of the effects of one-by-one deletion of each individual Torpedo subunit on receptor assembly and function. MATERIALS AND METHODSPlasmids. Full-length Torpedo AcChoR cDNA clones were provided by D. Noonan of Scripps Research Institute (a subunit), T. Claudio of Yale University (J3 and 8 subunits) and S. Heinemann of Salk Institute (y subunit; ref. 10). The mouse BC3H-1 cell line AcChoR 8 subunit cDNA clone was isolated in this laboratory (21). The cDNA inserts were excised from the vector and inserted into plasmid pSP62-PL (provided by D. Melton of Harvard University), ...

A B S T R A C T This study used messenger RNA encoding each subunit (a, fl, % and 6) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of a, fl, % and ~ were injected into oocytes. After allowing 2-8 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [12sI]a-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (>l,000-fold range at 1 #M). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of a-bungarotoxin-binding sites. Five combinations were tested for dtubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 #M. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the fl subunit on voltage sensitivity of the response: gACh(--90 mV)/gAch(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if fl~a replaces fiT. Also, combinations including "YT or 6M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 #S/fmol at -60 mV; EACh is consistently lower for am. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.

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