Determinants of competitive antagonist sensitivity on neuronal nicotinic receptor beta subunits.
We constructed a series of chimeric and mutant neuronal nicotinic acetylcholine receptor beta subunits to map amino acid residues that determine sensitivity to competitive antagonists. The beta 2 and beta 4 subunits form pharmacologically distinct receptors when expressed in combination with the alpha 3 subunit in Xenopus oocytes. At equipotent acetylcholine concentrations, alpha 3 beta 2 is 56-fold more sensitive to blockage by dihydro-beta-erythroidine than is alpha 3 beta 4. The alpha 3 beta 2 combination is also sensitive to long-term blockade by neuronal bungarotoxin, whereas alpha 3 beta 4 is not. Pharmacological analysis of receptors formed by chimeric beta subunits reveals that amino acid residues that determine both dihydro-beta-erythroidine and neuronal bungarotoxin sensitivity are located within several sequence segments. The major determinant of sensitivity to both competitive antagonists is located between residues 54 and 63. A minor determinant of sensitivity to both antagonists lies between residues 1 and 54, whereas a minor determinant of NBT sensitivity lies between residues 74 and 80. Within region 54-63 of beta 2, mutant beta 2 subunits were used to identify threonine 59 as a residue critical in determining competitive antagonist sensitivity. Changing threonine 59 to lysine, as occurs in beta 4, causes a 9-fold decrease in dihydro-beta-erythroidine sensitivity and a 71-fold decrease in neuronal bungarotoxin sensitivity. Changing polar threonine 59 to negatively charged aspartate causes a 2.5-fold increase in neuronal bungarotoxin sensitivity and has no effect on dihydro-beta-erythroidine sensitivity.
Harvey
S C
SC
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101, USA.
Luetje
C W
CW
D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002038:Bungarotoxins / Q000494:pharmacology; D004079:Dihydro-beta-Erythroidine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005260:Female; D016297:Mutagenesis, Site-Directed; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009865:Oocytes / Q000502:physiology; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013912:Threonine / Q000032:analysis / Q000502:physiology; D014982:Xenopus laevis
D002038:Bungarotoxins; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D004079:Dihydro-beta-Erythroidine; D013912:Threonine