AGONIST BINDING-SITE OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR - TESTS WITH NOVEL SIDE-CHAINS AND WITH SEVERAL AGONISTS

Structure-function relations in the nicotinic acetylcholine receptor a re probed using a recently developed method based on chemical synthesi s of nonsense suppressor tRNAs with unnatural amino acid residues, sit e-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural ami no acids, as many as 14 at a given site, are incorporated at three sit es, alpha 93, alpha 190, and alpha 198, all of which are tyrosine in t he wild-type receptor and are thought to contribute to the agonist bin ding site. Confirming and expanding upon earlier studies using convent ional mutagenesis, the three tyrosines are shown to be in substantiall y different structural microenvironments. In particular, a crucial rol e is established for the hydroxyl group of alpha Tyr93, whereas a vari ety of substituents are functional at the analogous position of alpha Tyr198. Interestingly, consideration of three different agonists (acet ylcholine, nicotine, and tetramethylammonium) does not discriminate be tween these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutation s at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjus ting EC(50) values. These results establish the broad generality and g reat potential of the unnatural amino acid methodology for illuminatin g subtle structural distinctions in neuroreceptors and related integra l membrane proteins.