Nicotinic acetylcholine receptor from chick optic lobe.

125

105

Monoclonal antibodies raised against purified acetylcholine receptor from muscle and electric organ were tested for cross-reaction with surface components on chicken ciliary ganglion neurons. Indirect immunofluorescence indicated that antibodies to a determinant in the "main immunogenic region" of the receptor bind to the neurons in culture. Ultrastructural studies on 16-day embryonic ganglia, using horseradish peroxidase-conjugated monoclonal antibody, revealed that most of the conjugate labeling was associated with synaptic membrane on the neurons. A lesser amount of labeling was associated with the short processes extending from the neuronal somata in the region of preganglionic innervation. The labeling was blocked by coincubation with unlabeled antibodies of appropriate specificity and not by nonimmune serum. The pattern of labeling was clearly different from that previously found for a horseradish peroxidase conjugate of a-bungarotoxin: the toxin conjugate bound extensively to the short processes but not to synaptic membrane on the neurons. The synaptic antigen identified here by the crossreacting antibodies is a candidate for the synaptic acetylcholine receptor on chicken ciliary ganglion neurons.Chicken ciliary ganglion neurons have nicotinic acetylcholine (AcCho) receptors that mediate chemical synaptic transmission through the ganglion (1, 2). Efforts to study the regulation and distribution of such receptors on neurons have been limited by the absence of suitable probes. a-Bungarotoxin has been a very useful probe for studying nicotinic AcCho receptors in vertebrate skeletal muscle and electric organ, where the toxin binds tightly and specifically to the receptor and blocks its function (3). Ciliary ganglion neurons also have high-affinity binding sites for a-bungarotoxin, but in this case the binding sites appear to be distinct from synaptic AcCho receptors. Ultrastructural studies demonstrate that toxin binding sites on the neurons are located in the immediate vicinity of presynaptic terminals but are not present in the postsynaptic membrane (4), as would be expected for synaptic receptors. Moreover, a-bungarotoxin does not block AcCho receptor function on the neurons (5, 6), and, when the levels of AcCho sensitivity and toxin binding are compared for the neurons grown under various conditions in cell culture, no simple correlation is found between the two properties (7), as would be expected if they represented the same membrane component.An alternative approach is to use antibodies against the AcCho receptor as probes. Monoclonal antibodies (mAbs) against AcCho receptors from skeletal muscle and electric organ have been useful in studying the structure and synthesis of these receptors (8)(9)(10)(11)(12). Recent studies have shown that mAbs specific for each of the four subunits of AcCho receptors from muscle and electric organ bind to neurons in the lateral spiriform nucleus of chicken brain (13). These neurons do not bind a-bungarotoxin. Best cross-reaction was obtained with mAbs to the "...

Section: Discussionmentioning

confidence: 87%

Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons.

Jacob¹,

Berg²,

Lindstrom³

1984

125

105

“…We conclude that, within our detection limits, the a-subunit gene is encoded by a unique locus in chicken, as was found for T. marmorata (26) and mouse (31). Gene families could be suggested to account for the occurrence of a-bungarotoxin-binding sites and nicotinic AcChoR in the central nervous system of vertebrates (34), particularly in the optic lobe of the chicken (35), for developmental changes in several properties of the AcChoR (36), and for differences between the two agonist binding sites, carried mainly by the two a subunits present per pentameric AcChoR (1). All of the results obtained up to now indicate that, if such a gene family were to exist for the a subunit, then its members would not be very homologous to each other.…”

Section: Discussionmentioning

confidence: 91%

Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.

Klarsfeld¹,

Changeux²

1985

136

In vitro blocking the spontaneous activity of primary cultures of chicken embryo myotubes with tetrodotoxin increases~2-fold their content in surface acetylcholine receptor. To investigate this effect at the level of gene expression, chicken genomic DNA sequences coding for the acetylcholine receptor a subunit were isolated and characterized. They were shown to belong to a single-copy, polymorphic gene with at least two alleles in the chicken strain utilized. Probes derived from these genomic clones were used to quantitate levels of a-subunit mRNA. In culture, a 2-day exposure to tetrodotoxin increased these mRNA levels up to 13-fold, a value similar to that observed after denervation of chick leg muscle (-17-fold). Actin mRNA levels varied little in any of these experiments. These results support the notion that membrane electrical activity affects acetylcholine receptor expression by regulating the accumulation ofthe corresponding mRNAs.The acetylcholine receptor (AcChoR) is an oligomeric protein composed of five transmembrane subunits with an a213y8 structure (reviewed in ref.

“…In conclusion, we note that there is sound immunological and biochemical evidence for the presence in chicken brain of a nicotinic AcChoR with properties similar to but distinguishable from those of the AcChoR at the neuromuscular junction (31,32). It will be interesting to see if our set of chicken AcChoR probes detects related sequences in brain cDNA libraries.…”

Section: Ggccrtcr209rccgccctgcrcggrrgrtc-tc-rttc-cgcttc-cccc-rtg-c-tcmentioning

confidence: 99%

Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor.

Nef¹,

Mauron²,

Stalder³

et al. 1984

125

We have cloned and sequenced a fragment of the chicken genome approximately 9 kilobases in length that comprises the genes encoding the 6 and 'y subunits of the nicotinic acetyicholine receptor. The two genes are homologous and have identical structures: both consist of 12 exons, some of which precisely correspond to predicted structural domains of the receptor subunits. The 8 and y subunit gehes are encoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of 8 and the initiator codon of y. Blot analysis demonstrates that the genes we describe are unique in the genome. Comparison of the predicted protein sequence for the correspodding subunits of chicken and of the elasmobranch Torpedo reveals a high degree of conservation in Aome but not all of the protein domains.The nicotinic acetylcholine receptor (AcChok) mediates synaptic transmission at the vertebrate neuromuscular junction. Located in the folds of the postsynaptic membrane, the AcChoR is a cation channel whose opening is triggered by acetylcholine. AcChoR activation normally results in an influx of Na' that depolarizes the postsynaptic membrane and leads to muscle contraction. The AcChoR is the best understood of all ligand-gated ion channels due to its relative abundance in the electrocytes of Torpedo or Electrophorus, from which workable quantities of receptor can be purified. The receptor is a pentamer of four different subunits in the stoichiometty a2f8W. All subunits are glycosylated and span the membrane. They assemble into a barrel-shaped structure whose central cavity is thought to be the gatpd ion channel (for recent reviews see refs. 1-3). That the four subunits participate in the assembly of a single functional AcChoR molecule was elegantly verified by showing that all four mRNA species must be injected in Xenopus oocytes to allow detection of physiologically active receptor (4). Microsequencing of the amino ends of the four purified Torpedo subunits demonstrated that they were related and suggested that they had evolved from an ancestral gene by a series of duplications (5).Several laboratories recently succeeded in cloning and sequencing some (6-9) or all (10-12) of the cDNAs encoding the four subunits of Torpedo AcChoR. It was found that there is significantly more homology between the deduced protein sequences of the a-,8 and y-8 pairs than between any other combinations of subunits, suggesting that an initial duplication of the ancestral gene had given rise to two protogenes, one of which later yielded the y and 8 genes while the other gave rise to the a and p genes (12). Moreover, all subunits were shown to have four very hydrophobic stretches three of them closely grouped-of sufficient length to span the membrane. This finding suggested a model for the transmembrane insertion of the receptor whereby each subunit weaves four times through the membrane to yield an assembly of twenty transmembrane a helices, a subset of which constitutes the channel per se (8,9,12). A relate...