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

Abstract

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.

Agmatine acts as an antagonist of neuronal nicotinic receptors

“…Specific binding was determined by subtracting the counts bound to quadruplicate samples incubated as above but including jM unlabelled NBT. Berg, 1986) and the chick retina (Loring et al, 1989 (Figure 5) a concentration at which agmatine significantly depressed nicotinic receptor function in the chick retina (Figure 4a). Agmatine did start blocking [1251]-NBT binding at 10mM, however the displacement was less than 50%.…”

Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture

“…Carbachol treatment decreases the number of internal sites to the same extent as surface sites on the neurons (Table 4). Chronic exposure to Bgt 3.1, an cu-neurotoxin that binds to AChRs on ciliary ganglion neurons in culture (Halvorsen and Berg, 1986a), has been shown to decrease the number of surface sites on the neurons (Smith et al, 1986). Incubation with Bgt 3.1 also decreases the number of internal sites, but to a smaller extent (Table 4).…”

“…Additional confirmation that mAb 35 distinguishes the neuronal AChR comes from studies with a second probe for neuronal AChRs, an a-neurotoxin that reversibly blocks the AChR function of chick autonomic neurons, The toxin has been called Bgt 3.1 (Ravdin and Berg, 1979;Ravdin et al, 198 l), K-bungarotoxin (Chiappinelli, 1983) and toxin F (Loring et al, 1984); by electrophoretic and amino acid sequence analyses the 3 toxins are indistinguishable (Loring et al, 1986). Bgt 3.1 binds to a class of sites on ciliary ganglion neurons in culture with the affinity, kinetics, pharmacology, and specificity expected for the neuronal AChR (Halvorsen and Berg, 1986a). Cross-linking studies with a photoaffinity derivative of Bgt 3.1 demonstrate that Bgt 3.1 and mAb 35 recognize the same neuronal component Berg, 1986b, 1987).…”

Rabies virus infection of cultured adult mouse dorsal root ganglion neurons

“…Glycolipids and nicotinic acetylcholine receptors are the most accepted candidates (King & Turner 1993). Both type of molecules are expressed by DRG cells (Halvorsen & Berg 1986) but the knowledge on nicotinic receptors on these cells is still fragmentary. An interesting finding was the observation that neurons are not susceptible to a "dilution effect" (at least in the range used).…”