Primary Structure of Nicotinic Acetylcholine Receptor
Annual rept. 15 Jul 1985-14 Jul 1986
SALK INST FOR BIOLOGICAL STUDIES LA JOLLA CA
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Cholinergic transmission occurs between nerve and muscle and between nerve and nerve. In both cases, acetylcholine is released by the nerve, bound by a nicotinic acetylcholine receptor, and destroyed by the enzyme acetylcholinesterase. Our goal has been to isolate cDNA clones encoding the nicotinic acetylcholine receptor at the neuromuscular junction and to use these clones to determine the structure of the receptor. In this report we present the amino acid sequence of the receptor and show that the clones we have isolated will direct the synthesis of the receptor. We have isolated cDNA clones encoding the alpha-, Beta-, Gamma- and delta-subunits of the mouse muscle nicotinic acetylcholine receptor and have determined the sequences for the alpha-beta-, and gamma-subunits. The clones were engineered to make them suitable for expression studies and inserted into plasmids downstream of a promotor. These plasmids have been used to synthesize RNA which will direct the synthesis of functional acetylcholine receptors in the Xenopus oocyte. We screened a cDNA library prepared from a neuronal cell line. We isolated three clones out of one million examined and determined the sequence of the one with the longest insert. This sequence revealed that the clone encoded a protein with considerable structural homology to the muscle receptor alpha-subunits. We demonstrated that transcripts hybridizing to the clone are expressed in regions of the brain known to have cholinergic transmission. We have proposed that the clone encodes the alpha-subunit of a neuronal nicotinic receptor.
- Genetic Engineering and Molecular Biology
- Anatomy and Physiology