Because of their extreme specificity and affinity, alpha-bungarotoxin and related snake neurotoxins have been of immense value for the assay, purification, and biochemical as well as histochemical characterization of nicotinic acetylcholine receptor in skeletal muscle and electric tissue. Specific macromolecular binding sites for alpha-bungarotoxin that resemble acetylcholine receptors with respect to biochemical and drug binding properties as well as synaptic location, have also been found in the celtral and autonomic nervous system of higher vertebrates. However, since the toxin is physiologically inert at these sites, the functional significance of the toxin receptors is unclear. It has recently become possible to incorporate purified acetylcholine receptor from Torpedo electric tissue into phospholipid vesicles and to analyze ion fluxes in response to cholinergic agonists in such preparations. It is the goal of the present proposal to use this approach to elucidate the physiological role of neuronal receptors for alpha-bungarotoxin. Such receptors will be purified from the optic lobe of the newly-hatched chick (a tissue of high receptor density) in the presence of phospholipid, and reconstituted into large unilamellar vesicles under conditions that permit incorporation of active Torpedo acetylcholine receptor. Measurement of 22 Na+ fluxes upon treatment with carbamylcholine will reveal, if alpha-bungarotoxin binding activity and acetylcholine receptors co-purify. Similar reconstitution experiments with acetylcholine receptors from chick skeletal muscle are also proposed. This research may settle the long-standing cotroversy regarding alpha-bungarotoxin binding sites and acetylcholine receptors on nerve cells, and contribute to our understanding of a neuronal target for nicotine, and of cholinergic transmission in the central nervous system and autonomic ganglia.
NIH ExPorter
NIH ExPorter