Synthetic peptides used to locate the .alpha.-bungarotoxin binding site and immunogenic regions on .alpha. subunits of the nicotinic acetylcholine receptor

Biochemistry ◽  
1987 ◽  
Vol 26 (12) ◽  
pp. 3261-3266 ◽  
Author(s):  
Scott Ralston ◽  
Virender Sarin ◽  
Lam Thanh Hung ◽  
Jean Rivier ◽  
J. Lawrence Fox ◽  
...  
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Synthetic Peptides ◽  
Nicotinic Acetylcholine ◽  
Alpha Subunits ◽  
Alpha Bungarotoxin

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

2003 ◽  
Vol 372 (2) ◽  
pp. 543-554 ◽  
Author(s):  
Martha MARINOU ◽  
Socrates J. TZARTOS
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Binding Sites ◽  
Synthetic Peptides ◽  
Extracellular Domain ◽  
Muscle Type ◽  
Nicotinic Acetylcholine ◽  
Toxin Binding ◽  
Α1 Subunit

The neuronal α7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin α-bungarotoxin (α-Bgt). Fine mapping of the α-Bgt-binding site on the human α7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human α7 subunit (residues 1–206). Screening of these peptides for 125I-α-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186–197, which exhibited the best 125I-α-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 α7(186–197) peptide was almost completely inhibited by unlabelled α-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 α-Bgt binding site of the α1 subunit (residues 188–200 in Torpedo AChR). In competition experiments, the soluble peptides α7(186–197) and Torpedo α1(184–200) inhibited the binding of 125I-α-Bgt to the immobilized α7(186–197) peptide, to native Torpedo AChR, and to the extracellular domain of the human α1 subunit. These results suggest that the toxin-binding sites of the neuronal α7 and muscle-type AChRs bind to identical or overlapping sites on the α-Bgt molecule. In support of this, when synthetic α-Bgt peptides were tested for binding to the recombinant extracellular domains of the human α7 and α1 subunits, and to native Torpedo and α7 AChR, the results indicated that α-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail.

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