Mapping the Structural Requirements for Nicotinic Acetylcholine Receptor Activation by Using Tethered Alkyltrimethylammonium Agonists and Antagonists

• Published in: Biochemistry (Easton) 2006-09, Vol.45 (35), p.10641-10653
• Main Authors: Stewart, Deirdre S, Chiara, David C, Cohen, Jonathan B
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Binding Sites; Dose-Response Relationship, Drug; In Vitro Techniques; Kinetics; Models, Chemical; Nicotinic Agonists - chemistry; Nicotinic Antagonists - chemistry; Protein Subunits - chemistry; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - genetics; Structure-Activity Relationship; Torpedo; Xenopus - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi060686t

A molecule as simple in structure as tetramethylammonium gates the nicotinic acetylcholine receptor (nAChR) with high efficacy. To compare the structure of the nAChR transmitter binding site in the open channel state with that of the ACh binding protein, we determined the efficacy of nAChR gating by −S(CH2) n N(CH3)3 + (n = 1−4) tethered to substituted cysteines at positions in the α subunits or γ and δ subunits predicted to contribute to the ACh binding sites in mutant Torpedo nAChRs expressed in Xenopus oocytes. For tethered thiocholine [−S(CH2)2N(CH3)3 +], we previously reported that within α195−201 gating was observed only at αY198C while at αY93C it acted as an antagonist. We now show that within α191−194, thiocholine activates when tethered at αCys192 or αCys193. Thiocholine also activates when tethered at αY190C or αW149C in nAChRs containing a β subunit mutation (βL257S) that destabilizes the closed channel, but not from γW55C/δW57C, where longer adducts can activate. When tethered at positions in binding site segment E, thiocholine activates only from γL119C/δL121C, where the shorter −S(CH2)1N(CH3)3 + acts as an antagonist. Longer adducts tethered at γL109C/δL111C or γL119C/δL121C also activate, but less efficiently. The length requirements for efficient gating by tethered agonists agree closely with predictions based upon the structure of the agonist site in a nAChR homology model derived from the ACh binding protein structure, which suggests that this structure is an excellent model of the nAChR agonist binding site in the open channel conformation. The inability of thiocholine to activate from αY93C, which is not predicted by the model, is discussed in terms of the structure of the nAChR in the closed state.