Molecular determinants conferring alpha-toxin resistance in recombinant DNA-derived acetylcholine receptors.
Sequences of the alpha-subunits of the nicotinic acetylcholine receptor from the snake and mongoose contain several differences in the region between amino acids 183 and 200. Receptors from both of these species reveal resistance to the snake alpha-toxins presumably arising as a protective evolutionary mechanism. Sequence differences include the added glycosylation signals at residue 187 in the mongoose and at residues 189 and 111 in snake. Although previous observations with peptides and fusion proteins either synthesized chemically or in a bacterial expression system indicate that certain amino acid residues may contribute to the resistance, our findings with the intact receptor in an eukaryotic expression system indicate the major role for glycosylation. In this study, we show that addition of glycosylation signals gives rise to virtually complete glycosylation at the added sites, although heterogeneity of oligosaccharide processing is evident. By analysis of combinations of mutants, we document that glycosylation exerts the predominant influence on alpha-toxin binding. Substitutions at other residues are largely without influence as single mutations but appear to decrease affinity further in multiple mutants, particularly where the receptor is glycosylated at the 187 and 189 positions. Glycosylation exerts a major influence on the dissociation as well as the association rates of the alpha-toxin-receptor complex, suggesting that the decrease for alpha-toxin affinity is not simply a consequence of restricted diffusional access, rather glycosylation affects the conformation and stability of the bound complex.
Keller
S H
SH
Department of Pharmacology, University of California, La Jolla 92093.
Kreienkamp
H J
HJ
Kawanishi
C
C
Taylor
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002478:Cells, Cultured; D004274:DNA, Recombinant; D004351:Drug Resistance; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000494:pharmacology*
D002038:Bungarotoxins; D004274:DNA, Recombinant; D011978:Receptors, Nicotinic; D010738:Type C Phospholipases