Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides

Abstract

The neuronal alpha7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin alpha-bungarotoxin (alpha-Bgt). Fine mapping of the alpha-Bgt-binding site on the human alpha7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human alpha7 subunit (residues 1-206). Screening of these peptides for (125)I-alpha-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186-197, which exhibited the best (125)I-alpha-Bgt binding, and one at residues 159-165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the alpha7(186-197) peptide was almost completely inhibited by unlabelled alpha-Bgt or d -tubocurarine. Alanine substitutions within the sequence 186-198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the alpha-Bgt binding site of the alpha1 subunit (residues 188-200 in Torpedo AChR). In competition experiments, the soluble peptides alpha7(186-197) and Torpedo alpha1(184-200) inhibited the binding of (125)I-alpha-Bgt to the immobilized alpha7(186-197) peptide, to native Torpedo AChR, and to the extracellular domain of the human alpha1 subunit. These results suggest that the toxin-binding sites of the neuronal alpha7 and muscle-type AChRs bind to identical or overlapping sites on the alpha-Bgt molecule. In support of this, when synthetic alpha-Bgt peptides were tested for binding to the recombinant extracellular domains of the human alpha7 and alpha1 subunits, and to native Torpedo and alpha7 AChR, the results indicated that alpha-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail.

Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Increases Suppressive Capacity of Naturally Occurring CD4+CD25+Regulatory T Cells in Mice In Vitro

“…First, the expressions of ␣7 nAChR mRNA and protein were shown on CD4 ϩ CD25 ϩ Tregs using RT-PCR and Western blotting methods. We then confirmed the surface expression of ␣7 nAChR on CD4 ϩ CD25 ϩ Tregs using FITC-labeled ␣-bungarotoxin, which is a selective ␣7 nAChR antagonist (Marinou and Tzartos, 2003). Tregs play a critical role in the maintenance of self-tolerance by suppressing, in a dominant manner, immune activation of self-aggressive effector T cells.…”

Soluble, Oligomeric, and Ligand-binding Extracellular Domain of the Human α7 Acetylcholine Receptor Expressed in Yeast

“…This assumption is further reinforced by our previous observations on human ␣1 and ␣7-ECDs expressed in E. coli as a non-glycosylated form. E. coli ␣1-ECD was not able to bind 125 I-␣-Bgt (37) whereas E. coli expressed ␣7-ECD was able to bind 125 I-␣-Bgt, 2 although with low affinity (34). The observation that the deglycosylated form binds ligands to the same extent as the glycosylated form may be very useful for crystallization studies, as it means that either form could be used for structural studies on the ␣7 ligand-binding domain.…”

“…Hoping to obtain a more soluble protein, we therefore constructed a mutant in which, in addition to the C116S mutation, the ␣7 hydrophobic Cys loop (15 amino acids) was replaced by the corresponding hydrophilic AChBP loop (14 amino acids). These amino acid residues do not seem to contribute to the ␣-Bgt-binding site (34).…”

Creating an α7 Nicotinic Acetylcholine Recognition Domain from the Acetylcholine-binding Protein

“…This ␣-neurotoxin binds to the ␤9 -10 linker in the periphery of the ligand-binding region, and glycosylation of h␣7-nAChR has no effect upon its binding (47), yet the generation of the stable mutant AChBPs presented here has revealed the location and extent of glycosylation of the extracellular domain of h␣7-nAChR to be within the vicinity of ligand binding for those ligands that bind in the apical region of the pocket. In our case, the oligosaccharide did not interact with MLA but rather was pulled away from the binding site by symmetry mates (Fig.…”