Unlike the well characterized nicotinic receptors of muscle, little is known about the structure and distribution of neuronal nicotinic acetylcholine receptors even though these receptors participate in synaptic transmission in autonomic ganglia and at several sites in the central nervous system. Muscle receptors are well characterized in part due to the use of curarimimetic snake toxins, such as Alpha-bungarotoxin. However, the use of Alpha-bungarotoxin for the characterization of neuronal nicotinic receptors is controversial since the toxin does not block nicotinic transmission between neurons in most cases. The objective of this proposal is to use another snake neurotoxin (named toxin F which has been shown to block nicotinic transmission at a variety of neuronal nicotinic synapses) as a probe to characterize and purify neuronal receptors.
One aim i s to complete the amino acid sequence of toxin F in order to compare this toxin with other snake toxins. Bovine chromaffin cells will be tested to demonstrate whether or not toxin F blocks nicotinic function in these cells. Radiolabeled toxin F will then be used in binding assays to determine whether these cells can service as a rich source of receptors. Immunological techniques are a powerful adjunct in the characterization of muscle receptors. Antibodies to receptor of electric fish will be tested for cross-reactivity to neuronal receptors by a) determining at the electron microscope level whether the antibodies co-localize with toxin F at a neuronal synapse and b) by immunoprecipitation of toxin-receptor complexes. Depending on the results of these experiments, solubilized neuronal receptor will be purified by affinity columns containing lectins, antibodies or toxin F followed by isoelectric focusing of radiolabeled toxin-receptor complexes. The subunit structure of the receptor will be examined using SDS-polyacrylamide electrophoresis after the receptor has been directly radiolabeled.
| Lee, H K; Gwalani, L; Mishra, V et al. (2009) Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera. Brain Res 1259:7-16 |
