D Goldman

Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system

Nicotinic acetylcholine receptors found in the peripheral and central nervous system differ from those found at the neuromuscular junction. Recently we isolated a cDNA clone encoding the alpha subunit of a neuronal acetylcholine receptor expressed in both the peripheral and central nervous system. In this paper we report the isolation of a cDNA encoding the alpha subunit of a second acetylcholine receptor expressed in the central nervous system. Thus it is clear that there is a family of genes coding for proteins with sequence and structural homology to the alpha subunit of the muscle nicotinic acetylcholine receptor. Members of this gene family are expressed in different regions of the central nervous system and, presumably, code for subtypes of the nicotinic acetylcholine receptor.

Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor

The mouse cell line BC3H-I synthesizes an acetylcholine receptor (AChR) with the pharmacological properties of a muscle nicotinic cholinergic receptor. We have purified mRNA from this cell line and used the size- fractionated poly(A)+RNA to produce a cDNA library of approximately 50,000 clones. The library was screened with a subclone containing genomic sequences coding for the putative acetylcholine-binding site of the alpha-subunit of chicken AChR. We obtained a plasmid, pMAR alpha 15, with a 1,717-base pair insert. The insert cDNA has 26 nucleotides at the 5′-end which code for a portion of the signal peptide followed by a single open reading frame of 1,311 nucleotides which code for a protein of 49,896 daltons. The insert has 377 bases of 3′-untranslated sequence with 3 polyadenylation sites. Radiolabeled plasmid DNA has been used to identify homologous RNA species of about 2 kilobases in Northern blot analyses of poly(A)+ selected RNA from BC3H-I cells. A similar size mRNA is seen in innervated mouse diaphragm and leg muscle, and both mouse and rat brain. Comparisons of the deduced amino acid sequence of the mouse AChR alpha-subunit with Torpedo marmorata, T. californica, chicken, human, and calf sequences show overall homologies of 80%, 80%, 86%, 96%, and 95%, respectively. More detailed analyses reveal a non-random distribution of amino acid substitutions in several structural domains. Based on the absolute conservation of cysteine residues, a new model for the arrangement of the disulfide bonds in the extracellular portion of the alpha-subunit is proposed.

Molecular Biology of Nicotinic Acetylcholine Receptors

Acetlylcholine is the neurotransmitter at the vertebrate neuromuscular junction, sympathetic ganglia, and in the central nervous system. Although in each location the presynaptic neuron releases acetylcholine, the receptor on the postsynaptic cell may differ. The nicotinic acetylcholine receptors at the neuromuscular junction are pharmacologically distinguishable from those in the sympathetic ganglia, and the nicotinic receptors in the central nervous system may further be distinguished from both the muscle and ganglionic type receptors. We know much more about the nicotinic acetylcholine receptors on muscle than about their counterparts on neurons. The reasons for this are twofold: (1) the identification of sources rich in nicotinic receptors, such as the electric organs of various rays and fishes; and (2) the identification of aneurotoxins, such as a-bungarotoxin, which bind with very high affinity to the muscle nicotinic acetylcholine receptor. The availability of a specific ligand and a rich source of receptor made possible biochemical studies of the receptor and led to our current understanding of this molecule. However, unlike its muscle counterpart, a rich source of neuronal nicotinic acetylcholine receptors has not been found. Furthermore, abungarotoxin has not provided the access to the neuronal nicotinic acetylcholine receptor that it did to the muscle-type nicotinic receptor. Consequently, our appreciation of its structure and regulation is less well developed. However, molecular biological approaches, which have been so valuable for studies of the muscle-type nicotinic acetylcholine receptors, now also provide access to the neuronal nicotinic acetylcholine receptor. In the following paragraphs we discuss our studies of the mouse muscle nicotinic acetylcholine receptor and describe a clone that, we propose, encodes a neuronal nicotinic acetylcholine receptor a-subunit.

Molecular Biology of the Neural and Muscle Nicotinic Acetylcholine Receptors

Most theories of nervous system function depend heavily on the existence and properties of the synapse. For this reason, this structure has been a focal point for neuroscience research for many decades. The best understood synapse is the neuromuscular junction because of its accessibility to biochemical and electrophysiological techniques and because of its elegant, well-defined structure. The nicotinic acetylcholine (ACh) receptor found in the postsynaptic membrane binds ACh released from the nerve. The binding of ACh results in a conformational change in the receptor that opens a channel permeable to cations. The resulting ion flux depolarizes the muscle and ultimately leads to muscle contraction. Thus the ACh receptor contains both a ligand-binding domain as well as a channel domain. Biological and structural studies have shown that the muscle nicotinic ACh receptor is a glycoprotein made up of five subunits with the stoichiometry α2βγδ; each of these subunits has a molecular weight between 40,000 and 60,000, and is encoded by a separate gene. This complex has been shown in reconstitution experiments to be a functional receptor containing both a ligand-binding site and a ligand-gated channel (for recent reviews, see Conti-Tronconi and Raftery, 1982; Popot and Changeux, 1984; Stroud and Finer-Moore, 1985; Karlin et al., 1986; McCarthy et al.,1986).

Neural Regulation of Acetylcholine Receptor Gene Expression

The muscle nicotinic acetylcholine receptor is the best characterized neuroreceptor to date.’ It is a pentameric integral membrane protein consisting of four different subunits with a stoichiometry of a&& The acetylcholine receptor is a ligand-gated ion channel that mediates transmission between nerve and muscle. Acetylcholine binds to the a-subunits of the receptor, which results in ion translocation across the membrane. In adult innervated skeletal muscle this receptor is localized to the neuromuscular junction. During development of the neuromuscular junction and upon denervation of adult muscle the levels and properties of the acetylcholine receptor change dramatically. During muscle fiber development myoblasts fuse to form multinucleated myotubes. Few, if any, acetylcholine receptors are found on myoblasts. However, once these myoblasts have fused to form myotubes, a large amount of acetylcholine receptor can be detected throughout the muscle fiber’s surface. After innervation of the muscle fiber the acetylcholine receptor is concentrated at the neuromuscular junction with a density over 1000-fold higher than that found in extrajunctional regions. Denervation of adult skeletal muscle results in a large increase in the number of acetylcholine receptors found in extrajunctional regions of the fiber.’” The receptors found in extrajunctional regions of either denervated muscle or muscle prior to innervation have properties that distinguish them from those receptors found at the neuromuscular The differences between these two receptor types result in a different channel open time, half life, isoelectric point, and immunologic reactivity. These data imply an influence of nerve on the levels and properties of the acetylcholine receptor. We tested the idea that muscle innervation inhibits acetylcholine receptor gene expression by comparing the levels of acetylcholine receptor-specific-RNA in innervated and denervated muscle. S 1 nuclease protection experiments using heteroduplexes formed between muscle a-subunit encoding cDNA and RNA isolated from either innervated or denervated muscle were performed in order to determine whether the junctional and extrajunctional acetylcholine receptors are coded for by a single gene product or by multiple ones. The fusing mouse muscle cell line, C2, is also being used as a model system to study the regulation of acetylcholine receptor expression. The levels of RNA coding