Chick sympathetic neurons develop receptors for α-bungarotoxin in vitro, but the toxin does not block nicotinic receptors

105

Section: Discussionmentioning

confidence: 99%

Unmasking the functions of the chromaffin cell α ₇ nicotinic receptor by using short pulses of acetylcholine and selective blockers

López

Montiel

Herrero

et al. 1998

105

“…If they bind specifically to the AcCho receptor, they will be useful agents for studying the distribution and regulation of this membrane component. a-Bungarotoxin [Bgt 2.2 (1)1, a small protein toxin isolated from the venom of the elapid snake Bungarus multicinctus, binds tightly and specifically to nicotinic acetylcholine (AcCho) re (2,3), rat superior cervical ganglia (4), and the pheochromocytoma cell line PC12 (5). The (6).…”

mentioning

confidence: 99%

Inhibition of neuronal acetylcholine sensitivity by α-toxins from Bungarus multicinctus venom

Ravdin

Berg

1979

146

Bungarus multicinctus venom contains several a-toxins in addition to the widelv used a-bungarotoxin (Bgt 2.2). We have found that two of the a-toxins (Bgt 3.1 and 3.3) inhibit neuronal acetylcholine (AcCho) sensitivity when tested on ciliary ganglion neurons in cell culture. Over 90% of the AcCho sensitivity recorded in response to iontophoretic application of AcCho was blocked when the neurons were incubated with either of the toxins at 10-7 M for I hr at 370C. component in the cell homogenate different from the component that bound Bgt 2.2 (6). It has been reported that high concentrations of Bgt 2.2 (10-6 M) can reversibly block nicotinic transmission through the ciliary ganglion (7). An earlier study found no effect of Bgt 2.2 on transmission through the ganglion (8).B. multicinctus venom contains several a-toxins that block AcCho receptors in skeletal muscle (1, 9) in addition to the widely used Bgt 2.2. We examined the effects of these toxins on ciliary ganglion neurons in cell culture to determine if they could also block neuronal AcCho receptors. Two of the a-toxins (Bgt 3.1 and ,3.3) were very effective in blocking sensitivity of the neurons to iontophoretically applied AcCho. Studies with rhodamine-labeled Bgt 2.2 demonstrated that the neurons also had a high-affinity binding site for Bgt 2.2, but concentrations of Bgt 2.2 adequate to saturate the site did not inhibit AcCho sensitivity. MATERIALS AND METHODSPreparation of Cell Cultures. Ciliary ganglion neuronmyotube cultures were prepared and maintained as described (10). Briefly, ciliary ganglia from 8-day-old chicken embryos were dissociated and plated at a density of about 104 neurons on 5-day-old myotube cultures (35-mm dishes) prepared from embryonic chicken pectoral muscle. The cultures were grown for 1-2 weeks in culture medium containing Eagle's minimal essential medium supplemented with 10% (vol/vol) horse serum, 5% (vol/vol) chicken embryo extract, 50 units of penicillin per ml, and 50 ,ug of streptomycin per ml.For experiments with fluorescence microscopy, glass-bottomed culture dishes were prepared by drilling an 18-mm hole in a 35-mm plastic culture dish and attaching a 25-mm Vanlab glass coverslip with Sylgard. The surface was coated with collagen and sterilized by UV irradiation. Cells were then plated and maintained in the cultures as described above.Purification of a-Toxins. a-Toxins from B. multicinctus venom were purified by ion-exchange chromatography as described (1), with the following modifications. The venom (20O mg in 6 ml) was applied to a column (40 X 2.5 cm) of carboxymethyl-Sephadex C-50 equilibrated with 50 mM ammonium acetate (pH 5.0). The column was eluted at 22 ml/hr with a 500-ml linear gradient of 50 mM ammonium acetate (pH 5.0) to 1.0 M ammonium acetate (pH 6.9). Fractions for peaks II and III (1) were pooled separately, lyophilized, and redissolved in 0.1 M ammonium acetate (pH 6.9). Further fractionation was achieved by chromatography on Whatman CM32 carboxymethyl-cellulose. Peak II was applied to a colu...

“…In fact, it has been much questioned whether these a-BTX-binding sites in the central nervous system or in neurons in culture are functional AcChoR because of the inability to demonstrate a-BTX blockade of cholinergic neurotransmission at a number ofsuch loci (reviewed in refs. 7, 9, and 10), because ofthe molecular separation made in some neuronal cell cultures between the AcChoR-mediated ion flux and the binding of 'Ilabeled monoiodo a-BTX (l25I-a-BTX) (11)(12)(13), and because of the frequent lack ofrelationship between cholinergic neural input and the '"I-a-BTX binding (7,9,14).…”

mentioning

confidence: 99%

Nicotinic acetylcholine receptor from chick optic lobe.

Norman¹,

Mehraban²,

Barnard³

et al. 1982

An a-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest ofthe chick brain which can also bind a-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the a-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors.