Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding.
The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.
Sugiyama
N
N
Department of Pharmacology, University of California, San Diego, La Jolla 92093, USA.
Marchot
P
P
Kawanishi
C
C
Osaka
H
H
Molles
B
B
Sine
S M
SM
Taylor
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D020916:Conotoxins; D017124:Conserved Sequence; D004622:Embryo, Mammalian; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007668:Kidney; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics
D020916:Conotoxins; D007457:Iodine Radioisotopes; D008978:Mollusk Venoms; D010456:Peptides, Cyclic; D011978:Receptors, Nicotinic; D014443:Tyrosine; C036155:conotoxin MI; D011392:Proline