Identification of a nicotinic acetylcholine receptor on neurons using an alpha-neurotoxin that blocks receptor function.

An alpha-neurotoxin, Bgt 3.1, that reversibly blocks the ACh response of chick ciliary ganglion neurons has been used to identify 2 classes of high-affinity binding sites on the cells in culture. The first class appears to be the alpha-bungarotoxin binding site on the neurons. The second class of Bgt 3.1 sites is distinct from the alpha-bungarotoxin binding sites and has the properties expected for the functional nicotinic ACh receptor on the cells. Equilibrium binding and kinetic studies indicate a Kd value of 5-6 nM for Bgt 3.1 at the second class of sites. The kinetics and affinity of binding are consistent with those inferred from previous physiological studies for Bgt 3.1 inhibition of receptor function. Bgt 3.1 binding to the sites is completely inhibited by each of the cholinergic ligands ACh, carbachol, nicotine, d-tubocurarine, and trimethaphan, but not by alpha-bungarotoxin. Highest site densities are found in cultures of ciliary and sympathetic ganglion neurons, cell types known to have ganglionic nicotinic ACh receptors. Low levels of sites may be present in cultures of spinal cord and dorsal root ganglion neurons; no binding is found in cultures of skeletal myotubes or cardiac cells when alpha-bungarotoxin is used to block Bgt 3.1 binding to alpha-bungarotoxin sites. These results demonstrate that Bgt 3.1 can be used as a specific probe for the nicotinic ACh receptor on chick autonomic neurons.