Agonist-driven conformational changes in the inner beta-sheet of alpha7 nicotinic receptors.

Cys-loop ligand-gated ion channels assemble as pentameric proteins, and each monomer contributes two structural elements: an extracellular ligand-binding domain (LBD) and a transmembrane ion channel domain. Models of receptor activation include rotational movements of subunits leading to opening of the ion channel. We tested this idea using substituted cysteine accessibility to track conformational changes in the inner beta sheet of the LBD. Using a nondesensitizing chick alpha7 background (L(247)T), we constructed 18 consecutive cysteine replacement mutants (Leu(36) to Ile(53)) and tested each for expression of acetylcholine (ACh)-evoked currents and functional sensitivity to thiol modification. We measured rates of modification in the presence and absence of ACh to identify conformational changes associated with receptor activation. Resting modification rates of eight substituted cysteines in the beta1 and beta2 strands and the sequence between them (loop 2) varied over several orders of magnitude, suggesting substantial differences in the accessibility or electrostatic environment of individual side chains. These differences were in general agreement with structural models of the LBD. Eight of 18 cysteine replacements displayed ACh-dependent changes in modification rates, indicating a change in the accessibility or electrostatic environment of the introduced cysteine during activation. We were surprised that the effects of agonist exposure were difficult to reconcile with rotational models of activation. Acetylcholine reduced the modification rate of M(40)C but increased it at N(52)C despite the close physical proximity of these residues. Our results suggest that models that depend strictly on rigid-body rotation of the LBD may provide an incomplete description of receptor activation.