Agonist-induced conformational changes in the extracellular domain of alpha 7 nicotinic acetylcholine receptors

• Published in: Molecular pharmacology 2003-09, Vol.64 (3), p.650
• Main Authors: Lyford, Lisa K, Sproul, Adrian D, Eddins, Donnie, McLaughlin, James T, Rosenberg, Robert L
• Format: Article
• Language: English
• Subjects: Acetylcholine - metabolism; Acetylcholine - pharmacology; alpha7 Nicotinic Acetylcholine Receptor; Animals; Chickens; Dose-Response Relationship, Drug; Extracellular Space - chemistry; Extracellular Space - drug effects; Extracellular Space - metabolism; Female; Nicotinic Agonists - metabolism; Nicotinic Agonists - pharmacology; Oocytes; Protein Binding - drug effects; Protein Binding - physiology; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - metabolism; Xenopus laevis
• ISSN: 0026-895X
• DOI: 10.1124/mol.64.3.650

The molecular mechanisms that couple agonist binding to the gating of Cys-loop ionotropic receptors are not well understood. The crystal structure of the acetylcholine (ACh) binding protein has provided insights into the structure of the extracellular domain of nicotinic receptors and a framework for testing mechanisms of activation. Key ligand binding residues are located at the C-terminal end of the beta9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E172 in chick alpha7 nicotinic acetylcholine receptors (nAChRs)] that is important for modulating activation. We hypothesize that agonist binding induces the movement of loop 9. To test this, we used the substituted-cysteine accessibility method to examine agonist-dependent changes in the modification of cysteines introduced in loop 9 of L247T alpha7 nAChRs. In the absence of agonist, ACh-evoked responses of E172C/L247T alpha7 nAChRs were inhibited by 2-trimethylammonioethylmethane thiosulfonate (MTSET). Agonist coapplication with MTSET reduced the extent and rate of modification. The dose-dependence of ACh activation was nearly identical with that of ACh-dependent protection from modification. ACh increased the inhibition by methanethiosulfonate reagents of N170C and did not change inhibition of G171C receptors. The antagonist dihydro-beta-erythroidine did not mimic the effects of ACh. Combined with a structural model, the data suggest that receptor activation includes subunit rotation and/or intrasubunit conformational changes that move N170 to a more accessible position and E172 to a more protected position away from the vestibule. Thus, loop 9, located near the junction between the extracellular and transmembrane domains, participates in conformational changes triggered by ligand binding.