Purification and characterization of the α-bungarotoxin binding protein from rat brain

doi:10.1016/0006-8993(85)90187-8

Brain Research

Volume 347, Issue 2, 18 November 1985, Pages 274-283

https://doi.org/10.1016/0006-8993(85)90187-8 Get rights and content

Abstract

The α-bungarotoxin (BGT) binding protein from rat brain has been purified and its polypeptide chain composition has been examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polypeptide chains with M_rs of 55,000, 53,500 and 49,000 have been identified as constituents of the protein. The affinity ligand [³H]maleimidobenzyl trimethylammonium bromide ([³H]MBTA), used to identify the ligand binding site on neuromuscular junction acetylcholine receptors (NMJ AChRs), binds to the 55,000 dalton polypeptide chain. Using a technique where ligands are bound to the protein while the protein is immobilized on α-cobratoxin-Sepharose 4B, it was established that the brain BGT binding protein, like NMJ AChRs, possesses two binding sites for BGT. These experiments reinforce previous evidence that the brain BGT binding protein is closely related but not identical to NMJ AChRs.

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Citation Excerpt :
The cobratoxin resin was rinsed four times with solubilization buffer, and toxin receptors were eluted with SDS-polyacrylamide gel electrophoresis sample buffer (300 mm Tris-Cl, pH 6.8, 30% glycerol, 8% SDS, 1 murea, 1 m dithiothreitol, 1% β-mercaptoethanol). Samples were resolved with SDS-polyacrylamide gel electrophoresis (4% stacking, 8% separating gels) transferred to Immobilon membrane (Millipore, Bedford, MA), and immunodetected with antibodies specific for the extracellular domain of the α7 nAChR subunit (31, 32). All antibodies were diluted in PBS, 0.1% Tween 20, and 5% nonfat dry milk.
Transient transfection has not been a successful method to express the α7 nicotinic acetylcholine receptor such that these receptors are detected on the cell surface. This is not the case for all ligand-gated ion channels. Transient transfection with the 5-hydroxytryptamine type 3 subunit cDNA results in detectable surface receptor expression. Cell lines stably expressing the α7 nicotinic acetylcholine receptor produce detectable, albeit variable, levels of surface receptor expression. α7 nicotinic acetylcholine receptor surface expression is dependent, at least in part, on cell-specific factors. In addition to factors provided by the cells used for receptor expression, we hypothesize that the surface expression level in transfected cells is an intrinsic property of the receptor protein under study. Employing a set of α7–5-hydroxytryptamine type 3 chimeric receptor subunit cDNAs, we expressed these constructs in a transient transfection system and quantified surface receptor expression. We have identified amino acids that control receptor distribution between surface and intracellular pools; surface receptor expression can be manipulated without affecting the total number of receptors. These determinants function independently of the cell line used for expression and the transfection method employed. How these surface expression determinants in the α7 nicotinic acetylcholine receptor might influence synaptic efficacy is discussed.
The α-bungarotoxin-binding nicotinic acetylcholine receptor from rat brain contains only the α7 subunit
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When expressed in Xenopus oocytes, the rat α7 subunit forms homo-oligomeric nicotinic acetylcholine receptors, which are blocked by α-bungarotoxin. Since the pharmacological and physiological properties of the α7 receptor expressed in oocytes are similar to those of the α-bungarotoxin-sensitive nicotinic currents recorded from neuronal preparations and the distribution patterns of α7 mRNA and α-bungarotoxin-binding sites in the rat brain are very similar, α7 is thought to be the main component of the α-bungarotoxin-binding nicotinic receptor in the mammalian brain. However, while α7 is found in purified α-bungarotoxin-binding complexes from rat brain or PC12 cells, other proteins copurify with it. Therefore, the question whether α7 forms a homo-oligomeric α-bungarotoxin-binding nicotinic receptor in the mammalian brain remains. We have developed and characterized affinity-purified polyclonal antibodies and used these antibodies in Western blot analyses of α-bungarotoxin-binding proteins purified from rat brains. We report here that our experimental data support the current working hypothesis that the α-bungarotoxin-binding nicotinic receptor is a homo-oligomer of α7 subunits in the rat brain.
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Neuronal/nicotinic α-bungarotoxin binding sites (nBgtS) found in the nervous system are not well characterized. In this study, photolabile toxin derivatives have been used in affinity labeling protocols to investigate the subunit composition of nBgtS expressed by different neuron-like cell lines. Data obtained was compared to the known subunit composition of toxin-binding muscle-type nicotinic acetylcholine receptors (nAChR). Muscle-type nAChR-rich membranes prepared from Torpedo electroplax contain components with corrected apparent molecular sizes of 41, 46, 50, 62 and 66 kDa that are reactive with toxin. The photoaffinity labeling patterns for preparations derived from cells of the TE671 clone, which express muscle-type nAChR, are very similar to that of cells of the IMR-32 or SH-SY5Y clonal lines, which express nBgtS. There is consistent labeling of four polypeptides with corrected apparent molecular weights of 40, 43, 47 and 56 kDa. These results suggest that both mammalian muscle-type nAChR and mammalian nBgtS are similarly composed of at least four kinds of subunits.
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It has recently been demonstrated that α-bungarotoxin receptors, which behave as functional nicotinic receptors, are present in chick CNS.
In this paper, we report the purification and characterization of a functional α-bungarotoxin receptor from chick cerebellum, a nervous tissue in which a clear inhibition of induced nicotine effects has been reported in vivo. This receptor contains at least three subunits of apparent mol. wt 52,000, 57,000 and 67,000. The use of monoclonal antibodies specific for the α7 subunit demonstrated that 75% of the molecules present in our purified preparation belong to the α7 subtype and that this antibody labels the 57,000 band in western blot, thus indicating that this is the toxin binding subunit.
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Although neuronal [¹²⁵I]-α-bungarotoxin binding proteins are similar in many respects to muscle nicotinic acetylcholine receptors, their functional significance has eluded researchers for the past fifteen years. Over this period, their status became increasingly doubtful, as almost all attempts failed to demonstrate that α-bungarotoxin could block neuronal nicotinic responses. Recently, these enigmatic proteins have been cloned and expressed in oocytes, and have been examined afresh in their native state. As Paul Clarke explains, it is time to recognize neuronal α-bungarotoxin binding proteins as distinct members of the nicotinic acetylcholine receptor gene family, even if perhaps they do not function quite like other members.

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Purification and characterization of the α-bungarotoxin binding protein from rat brain

Abstract

Brain Research

Biochem. Pharmacol.

Neuroscience

J. Mol. Biol.

Brain Research

Neuroscience

J. Biol. Chem.

Brain Research

Studies on the mechanism of action of acetylcholine antagonists on rat parasympathetic ganglion cells

J. Physiol. (London)

Characterization of the α-bungarotoxin receptor in chick-embryo retina

Eur. J. Biochem.

Non-equivalence of α-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons

Affinity labeling of one of two α-neurotoxin binding sites in acetylcholine receptor fromTorpedo californica

Biochemistry

Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle

Mammalian muscle acetylcholine receptor purification and characterization

Biochemistry

Shared antigenic determinant betweenElectrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons

The affinity-labeling of partially purified acetylcholine receptor from electric tissue ofElectrophorus

Purification and characterization of nicotinic acetylcholine receptors from muscle

Membr. Biochem.

Cleavage of the structural proteins during the assembly of the head of bacteriophage T4

Nature (London)