Abstract
Neuronal bungarotoxin (NBT) is a highly selective, slowly reversible, competitive antagonist of the α3β2 neuronal nicotinic receptor. Contributions to NBT sensitivity are made by both the α3 and β2 subunits. We used a chimeric α subunit to demonstrate that the entire α3 contribution lies within sequence segment 84–215. Construction and analysis of a series of mutant α3 subunits identified seven amino acid residues (Thr143, Tyr184, Lys185, His186, Ile188, Gln198, Ser203) within this region that contribute to NBT sensitivity. Changing Thr143 to lysine, as in α2, resulted in a ∼1000-fold loss of NBT sensitivity. The effect on NBT sensitivity of changing each of the other six residues ranged from 1.8- to 40.5-fold. More extensive mutagenesis demonstrated that Thr143 serves as part of the consensus sequence for glycosylation at N141, and it is this glycosylation that is the determinant of NBT sensitivity. Only serine could substitute for threonine to maintain full NBT sensitivity, and changing Asn141 to alanine resulted in a ∼300-fold loss of NBT sensitivity. The chimera α2–181-α3, containing all identified determinants except the glycosylation site, formed receptors insensitive to 300 nmNBT. Installation of threonine to complete the glycosylation consensus site in this chimera conferred NBT sensitivity only 10-fold less than that of wild-type α3β2. These seven determinants of NBT sensitivity are located in close proximity to a series of conserved residues that are common features of all nicotinic receptor binding sites.
Footnotes
-
Send reprint requests to: Dr. Charles W. Luetje, Department of Molecular and Cellular Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189, Miami, FL 33101. E-mail:cluetje{at}chroma.med.miami.edu
-
↵1 Current affiliation: Eli Lilly and Co., Lilly Corporate Center, Indianapolis, IN 46285.
-
This work was supported by grants to C.W.L. from the National Institute on Drug Abuse (DA08102), the American Heart Association, Florida Affiliate, and the Pharmaceutical Research and Manufacturers of America Foundation. C.W.L. was an Initial Investigator of the American Heart Association Florida Affiliate. S.C.H. was supported in part by T32-HL07188.
- Abbreviations:
- ACh
- acetylcholine
- DHβE
- dihydro-β-erythroidine
- nAChR
- nicotinic acetylcholine receptor
- NBT
- neuronal bungarotoxin
- PCR
- polymerase chain reaction
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- Received November 19, 1997.
- Accepted February 24, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|