Chimeric Analysis of a Neuronal Nicotinic Acetylcholine Receptor Reveals Amino Acids Conferring Sensitivity to α-Bungarotoxin

Abstract

We have investigated the molecular determinants responsible for ␣-bungarotoxin (␣Bgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous ␣ subunits, one highly sensitive to ␣Bgtx block (␣1) and the other, ␣Bgtx-insensitive (␣3). By replacing rat ␣3 residues 184 -191 with the corresponding region from the Torpedo ␣1 subunit, we introduced a cluster of five ␣1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the ␣3 subunit. Functional activity and ␣Bgtx sensitivity were assessed following co-expression in Nicotinic acetylcholine receptors (nAChRs)1 are multimeric ligand-gated ion channels expressed on skeletal muscle cells and on select groups of nerve cells in the peripheral and central nervous systems (1-3). Muscle nAChRs have pentameric structures made up of two ␣1 subunits and one each ␤1, ␥, and ␦(or ⑀) subunits; they are among the best characterized ion channels and serve as a model for understanding the structure and function of related ligand-gated channels responding to glycine, ␥-aminobutyric acid, and 5-hydroxytryptamine (4). Advances in the characterization of muscle nAChRs have been significantly aided by the discovery of a high affinity competitive antagonist, ␣-bungarotoxin (␣Bgtx). ␣Bgtx is used extensively in experiments on the molecular properties of nAChRs and for following expression, targeting, and clustering of these receptors on muscle during synapse formation (1-4). Much less is known about nAChRs on neurons, in part because comparable antagonists are in limited quantity or are nonexistent. The purpose of this paper is to define the amino acid residues that are essential for high affinity ␣Bgtx binding through a chimeric subunit approach, by conferring ␣Bgtx sensitivity to a neuronal ␣ subunit that is normally insensitive to ␣Bgtx. Previous work indicates that the main ␣Bgtx binding site is between residues 173 and 204 on the ␣1 subunit of the muscle nAChR. Specifically, studies of peptides derived from the Torpedo ␣1 sequence capable of binding ␣Bgtx with sub-micromolar affinity suggest that the major determinants of toxin binding are located in a region adjacent to the vicinal cysteines 192 and 193 (e.g. see Ref. 5). Recent studies of heterologously expressed muscle nAChRs have identified residues in this region of the native receptor that appear to interact with the short ␣-neurotoxin I from Naja mossambica mossambica (NmmI (6, 7)) and with ␣Bgtx (8, 9). Residues in this region are also involved in forming the binding sites for agonists and non-␣-neurotoxin antagonists (6, 7, 10 -12). In such studies, singlesite mutations in the muscle type ␣1 subunit have not been very helpful in fully defining the ␣-neurotoxin binding site in the native nAChR, as most mutations studied fail to produce large changes in ␣Bgtx affinity (6 -9). Therefore, in this study, rather than eliminate ␣Bgtx binding, we have used site-directed mutagenesis of a neuronal nAChR to introduce a toxin binding site. As a consequence, we identified the molecular determin...

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

“…Clearly the loop C that contains highly functional residues conserved in both receptors plays a predominant binding role. The evidence that supports that conclusion includes (i) mutational analyses with short chain (33,34) and long chain (35)(36)(37) toxins, (ii) the use of synthetic receptor peptides of the region 180-200 (38,39), (iii) studies on the resistance of various species to toxins (40,41), (iv) direct affinity labeling experiments (42), and (v) resolution of solution structures of the complexes formed between ␣-Bgtx and peptides (32,(43)(44)(45). Also, the additional binding function observed here for other ␣ and non-␣ loops was postulated for muscular receptors (46)(47)(48).…”

α4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors

“…For expression of neuronal nAChRs, typically 5 ng of each subunit cRNA was injected per oocyte. The oocytes were incubated at 15ºC in ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES at pH 7.5) with 1 mM glucose-6-phosphate, 50 mg/L gentamycin, and 5 mM pyruvic acid to increase their survival for up to 7 days after injection [20,36]. The oocyte recordings were obtained 1-6 days post-injection.…”

The cholinergic antagonist α-bungarotoxin also binds and blocks a subset of GABA receptors

“…From these results, we conclude that BTX binds to the amino-terminal extracellular domain of GABA A R ␤3. Knowing that BTX also binds to an amino-terminal site in nAChR ␣1 and ␣7 subunits (15)(16)(17), which are distantly related to GABA A Rs, we compared nAChR and GABA A R sequences. Surprisingly, GABA A R ␤3 did not share residues critical for BTX binding to nAChRs, and no distinctive differences between GABA A R ␤1 and ␤3 sequences occurred in this domain (Fig.…”