cDNA and genomic clones encoding alpha 7, a novel neuronal nicotinic acetylcholine receptor (nAChR) alpha subunit, were isolated and sequenced. The mature alpha 7 protein (479 residues) has moderate homology with all other alpha and non-alpha nAChR subunits and probably assumes the same transmembrane topology. alpha 7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the alpha 7 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The alpha 7 channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by alpha-bungarotoxin. A bacterial fusion protein encompassing residues 124-239 of alpha 7 binds labeled alpha-bungarotoxin. We conclude that alpha-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.

Neuronal nicotinic acetylcholine receptors are members of a gene family of ligand-gated transmitter receptors that includes muscle nicotinic receptors, GABAA receptors and glycine receptors. Several lines of evidence indicate that neuronal nicotinic receptors can be made up of only two subunits, an alpha (alpha) subunit which binds ligand, and a non-alpha (n alpha) or beta (beta) subunit. The stoichiometry of each subunit in the functional receptor has been difficult to assess, however. Estimates of the molecular weight of neuronal nicotonic receptor macromolecules suggest that these receptors contain at least four subunits but probably not more than five. We have examined the subunit stoichiometry of the chick neuronal alpha 4/n alpha 1 receptor by first using site-directed mutagenesis to create subunits that confer different single channel properties on the receptor. Co-injection with wild-type and mutant subunits led to the appearance of receptors with wild-type, mutant and hybrid conductances. From the number of hybrid conductances, we could deduce the number of each subunit in the functional receptor.

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, ...

Three cDNAs (ALS, D alpha 2 and ARD) isolated from the nervous system of Drosophila and encoding putative nicotinic acetylcholine receptor subunits were expressed in Xenopus oocytes in order to study their functional properties. Functional receptors could not be reconstituted from any of these subunits taken singly or in twos and threes. In contrast, large evoked currents (in the microA range) were consistently observed upon agonist application on oocytes co-injected with ALS or D alpha 2 in combination with the chick beta 2 structural subunit. The ALS/beta 2 and D alpha 2/beta 2 receptors are highly sensitive to acetylcholine and nicotine, and their physiological properties resemble those of native or reconstituted receptors from vertebrates. Although the physiological properties of ALS/beta 2 and D alpha 2/beta 2 receptors are quite similar, clear differences appear in their pharmacological profiles. The ALS/beta 2 receptor is highly sensitive to alpha-bungarotoxin while the D alpha 2/beta 2 receptor is totally insensitive to this agent. These results demonstrate that the Drosophila ALS and D alpha 2 cDNAs encode neuronal nicotinic subunits responding to physiological concentrations of the agonists acetylcholine and nicotine.