Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors.
Alpha-conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat alpha3beta2 receptors, expressed in Xenopus oocytes, are blocked with an IC(50) of 3.7 +/- 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of alpha3beta2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and approximately 2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the alpha3beta2 receptor.
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, P.O. Box 016189, Miami, Florida 33101, USA.
Drew
D
Everhart
G Edward
GE
Cartier
Arun
A
Malhotra
Aldrin V
AV
Gomes
J Michael
JM
McIntosh
Charles W
CW
Luetje
D000409:Alanine / Q000235:genetics; D000818:Animals; D002942:Circular Dichroism; D020916:Conotoxins / Q000737:chemistry* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000737:chemistry* / Q000494:pharmacology; D009865:Oocytes; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D040681:Structural Homology, Protein; D014982:Xenopus laevis
D020916:Conotoxins; D018733:Nicotinic Antagonists; D010455:Peptides; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C098872:alpha-conotoxin MII; C419362:nicotinic receptor alpha3beta2; D000409:Alanine