Paul T. Wilson

Neurotoxin-binding site on the acetylcholine receptor.

Publisher Summary The nicotinic acetylcholine receptor (AChR) at the neuromuscular junction transduces a chemical signal, the neurotransmitter acetylcholine released in response to an action potential at the motor nerve terminal, into an electrical event in the muscle cell that eventually activates the contraction process. In response to acetylcholine, the AChR undergoes a conformational change in which a cation-selective channel is opened, allowing sodium ions to enter and depolarize the cell. Essential functions of the receptor during this process are the binding of acetylcholine to the AChR, the coupling of the binding event to elements of the receptor comprising the channel, the opening of the channel to allow inward passage of cations, and the mechanisms that modulate these steps. Identification of the acetylcholine-binding site on the AChR would increase understanding of the mechanism by which binding of the ligand leads to the changes in the receptor that result in opening of the channel. Localization of this site is greatly facilitated by the use of snake venom curaremimetic neurotoxins, which bind to the receptor with considerably higher affinity than acetylcholine. There is evidence that the toxin-binding site includes the acetylcholine-binding site so that use of the toxins as biological probes should permit characterization of the site on the AChR involved in the binding of acetylcholine. The use of toxins as probes along with techniques employing other ligands, peptide fragments, monoclonal antibodies, synthetic receptor peptides, and genetic engineering are yielding considerable information on the toxin-binding site. In this chapter, current information on the localization of the curaremimetic neurotoxin-binding site on the primary amino acid sequence of the nicotinic AChR is discussed.

Binding of rabies virus to purified Torpedo acetylcholine receptor

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

Monoclonal antibodies directed against a synthetic peptide corresponding to the α-bungarotoxin binding region of the acetylcholine receptor

Murine monoclonal antibodies have been produced against a 32 amino acid synthetic peptide corresponding to residues 173-204 on the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica. All of the monoclonal antibodies were of the IgM subtype and most cross-reacted with the purified native receptor. None of the antibodies were effective in blocking alpha-bungarotoxin binding to the receptor nor, conversely, did alpha-bungarotoxin interfere with antibody binding. However, two monoclonal antibodies, previously shown to bind near the ligand binding site on the native receptor, did compete partially (50%) with the binding of one of the IgM monoclonal antibodies.

Chapter 9 Synthetic Peptides in the Study of the Nicotinic Acetylcholine Receptor

Publisher Summary This chapter focuses on the information obtained through the use of synthetic peptides corresponding to the primary amino acid sequence of the four subunits in the nicotinic acetylcholine receptor (AChR). It discusses how such synthetic peptides have facilitated the identification of the bungarotoxin (BGTX)-binding site on the a-subunit, how peptides have been used to identify specificities of preexisting monoclonal antibodies (MAbs), and how some peptides have facilitated the production of MAbs with domain-directed specificity. Similar approaches involving synthetic peptides may prove useful in the study of other membrane proteins.