Identification of sequence segments forming the alpha-bungarotoxin binding sites on two nicotinic acetylcholine receptor alpha subunits from the avian brain.
The relationship between neuronal alpha-bungarotoxin binding proteins (alpha BGTBPs) and nicotinic acetylcholine receptor function in the brain of higher vertebrates has remained controversial for over a decade. Recently, the cDNAs for two homologous putative ligand binding subunits, designated alpha BGTBP alpha 1 and alpha BGTBP alpha 2, have been isolated on the basis of their homology to the N terminus of an alpha BGTBP purified from chick brain. In the present study, a panel of overlapping synthetic peptides corresponding to the complete chick brain alpha BGTBP alpha 1 subunit and residues 166-215 of the alpha BGTBP alpha 2 subunits were tested for their ability to bind 125I-alpha BGT. The sequence segments corresponding to alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were found to consistently and specifically bind 125I-alpha BGT. The ability of these peptides to bind alpha BGT was significantly decreased by reduction and alkylation of the Cys residues at positions 190/191, whereas oxidation had little effect on alpha BGT binding activity. The relative affinities for alpha BGT of the peptide sequences alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were compared with those of peptides corresponding to the sequence segments Torpedo alpha 1-(181-200) and chick muscle alpha 1-(179-198). In competition assays, the IC50 for alpha BGTBP alpha 1-(181-200) was 20-fold higher than that obtained for the other peptides (approximately 2 versus 40 microM). These results indicate that alpha BGTBP alpha 1 and alpha BGTBP alpha 2 are ligand binding subunits able to bind alpha BGT at sites homologous with nAChR alpha subunits and that these subunits may confer differential ligand binding properties on the two alpha BGTBP subtypes of which they are components.
McLane
K E
KE
Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul 55108.
Wu
X D
XD
Schoepfer
R
R
Lindstrom
J M
JM
Conti-Tronconi
B M
BM
D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002038:Bungarotoxins / Q000378:metabolism*; D002645:Chickens; D004247:DNA / Q000235:genetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D012911:Snakes; D014101:Torpedo
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D004247:DNA; D000109:Acetylcholine