Agonist-Induced Conformational Changes in the Extracellular Domain of α7 Nicotinic Acetylcholine Receptors

Abstract

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 ␤9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E 172 in chick ␣7 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 L 247 T ␣7 nAChRs. In the absence of agonist, ACh-evoked responses of E 172 C/L 247 T ␣7 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 N 170 C and did not change inhibition of G 171 C receptors. The antagonist dihydro-␤-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 N 170 to a more accessible position and E 172 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.

Spontaneous conformational change and toxin binding in α7 acetylcholine receptor: Insight into channel activation and inhibition

“…We propose that this subunit has undergone a transition from the closed to the open conformation, whereas the other four subunits remain in the closed conformation. The details of our open conformation correlate well with the SCAM results of Rosenberg and coworkers (10,19). In particular, relative to the closed conformation, both our MD simulations and SCAM find that residues M37 and M40 in the ␤1 strand have decreased solvent accessibility, whereas N52 in ␤2 has increased solvent accessibility.…”

“…The agonist-induced conformational change of the LBD in the chicken ␣7 receptor has been probed by SCAM (10,19). After channel activation, residues M37 and M40 on the ␤1 strand become more buried, but N52 on the neighboring ␤2 strand becomes more exposed (10).…”

“…S1 A); the inner ␤ sheet is assumed to be coupled to the TMD, and hence when the former rotates, the latter is dragged along, leading to channel opening. However, the proposed relative rotation between the two ␤ sheets is not seen in MD simulations (9, 13) and does not appear to be consistent with acetylcholine-induced changes in residue solvent accessibilities measured by the substituted cysteine accessibility method (SCAM) (10,19).…”

“…S1 A); the inner ␤ sheet is assumed to be coupled to the TMD, and hence when the former rotates, the latter is dragged along, leading to channel opening. However, the proposed relative rotation between the two ␤ sheets is not seen in MD simulations (9, 13) and does not appear to be consistent with acetylcholine-induced changes in residue solvent accessibilities measured by the substituted cysteine accessibility method (SCAM) (10,19).From normal mode analysis, Changeux, Karplus, and coworkers (14, 18) proposed the lowest-frequency mode as the model for channel activation. In this ''quaternary twist'' model, the LBD rotates counterclockwise around the central axis, and the TMD rotates in the opposite direction (top view).…”

“…allostery ͉ spontaneous opening ͉ ligand-gated ion channel ͉ ligand binding N icotinic AChRs (nAChRs) are a well studied prototype for ligand-gated ion channels (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). nAChRs are pentamers consisting of either homo or hetero subunits.…”