Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure.
The pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (alpha gamma alpha delta beta), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the alpha gamma and alpha delta interfaces. By interchanging residues on the gamma and delta subunits, and ascertaining how they interact with the alpha-subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins, which contain two disulfide loops and 12-14 amino acids, show a 10,000-fold preference for the alpha delta over the alpha gamma subunit interface with alpha epsilon falling between the two. The waglerins, as 22-24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for alpha epsilon over the alpha gamma and alpha delta interfaces. Finally, the 6700 Da short alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alpha gamma and alpha delta interfaces over alpha epsilon. Selective mutagenesis enables one to also distinguish alpha-neurotoxin binding at the alpha gamma and alpha delta subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.
Taylor
P
P
Department of Pharmacology, University of California, San Diego, La Jolla 92093, USA.
Osaka
H
H
Molles
B E
BE
Sugiyama
N
N
Marchot
P
P
Ackermann
E J
EJ
Malany
S
S
McArdle
J J
JJ
Sine
S M
SM
Tsigelny
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006031:Glycosylation; D008024:Ligands; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry* / Q000494:pharmacology; D009498:Neurotoxins / Q000737:chemistry / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; D046911:Macromolecular Substances; D008978:Mollusk Venoms; D009498:Neurotoxins; D010456:Peptides, Cyclic; D011978:Receptors, Nicotinic