Abstract
We identify residues in the epsilon and delta subunits of the adult nicotinic acetylcholine receptor that give the alphaepsilon and alphadelta binding sites different affinities for the curariform antagonist dimethyl d-tubocurarine (DMT). By constructing epsilon-delta subunit chimeras, coexpressing them with complementary subunits, and measuring DMT binding, we identify two pairs of residues, Ileepsilon58/Hisdelta60 and Aspepsilon59/Aladelta61, responsible for DMT site selectivity in the adult receptor. The two determinants contribute approximately equally to the binding site and interact in contributing to the site. Exchange of these residues from one subunit to the other exchanges the affinities of the resulting binding sites. These determinants in the adult receptor are far from those that confer site selectivity in the fetal receptor; determinants in the fetal receptor are Ilegamma116/Valdelta118, Tyrgamma117/Thrdelta119, and Sergamma161/Lysdelta163. Thus, alternative residues confer DMT selectivity in fetal and adult acetylcholine receptors.
Highlights
Acetylcholine receptors (AChRs)1 from vertebrate skeletal muscle are pentamers of homologous subunits with the compositions ␣2␥␦ in fetal muscle and ␣2⑀␦ in adult muscle (2)
These results suggest that two pairs of adjacent residues confer DMT selectivity in the adult receptor, Ile⑀58/His␦60 and Asp⑀59/Ala␦61
Exchange of Selectivity Determinants between the ⑀ and ␦ Subunits—To further confirm that the pairs of equivalent residues Ile⑀58/His␦60 and Asp⑀59/Ala␦61 are solely responsible for DMT selectivity in the adult receptor, we expressed the double mutants ⑀I58H/⑀D59A and ␦H60I/␦A61D, alone or together, and measured DMT binding
Summary
Materials—Dimethyl d-tubocurarine was generously provided by Lilly. 125I-Labeled ␣-bungarotoxin was purchased from NEN Life Science Products, d-tubocurarine chloride from ICN Pharmaceuticals, Inc., and the 293 human embryonic kidney cell line from the American Type Culture Collection. Chimeric subunit cDNAs were constructed by bridging naturally occurring or mutagenically installed restriction sites with synthetic double-stranded oligonucleotides (1). The chimera ⑀63␦ was constructed by bridging a 35-base pair (bp) synthetic double-stranded oligonucleotide from a PflMI restriction site in the ⑀ subunit to a mutagenically installed SalI site in the ␦ subunit. Expression of Mutant Receptors and Ligand Binding Measurements—Human embryonic kidney cells were transfected with mutant or wild-type AChR subunit cDNAs using calcium phosphate precipitation as described (1). The cells were briefly centrifuged, resuspended in potassium Ringer high solution, and divided into aliquots for DMT binding measurements. Binding was terminated by the addition of 2 ml of potassium Ringer solution containing 300 M d-tubocurarine chloride.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
