Probing pore constriction in a ligand-gated ion channel by trapping a metal ion in the pore upon agonist dissociation.

ABSTRACT: Eukaryotic pentameric ligand-gated ion channels (pLGICs) are receptors activated by neurotransmitters to rapidly transport ions across cell membranes, down their electrochemical gradients. Recent crystal structures of two prokaryotic pLGICs were interpreted to imply that the extracellular side of the transmembrane pore constricts to close the channel (Hilf, R. J., and Dutzler, R. (2009) Nature 457, 115-118; Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J. P., Delarue, M., and Corringer, P. J. (2009) Nature 457, 111-114). Here, we utilized a eukaryotic acetylcholine (ACh)-serotonin chimeric pLGIC that was engineered with histidines to coordinate a metal ion within the channel pore, at its cytoplasmic side. In a previous study, the access of Zn(2+) ions to the engineered histidines had been explored when the channel was either at rest (closed) or active (open) (Paas, Y., Gibor, G., Grailhe, R., Savatier-Duclert, N., Dufresne, V., Sunesen, M., de Carvalho, L. P., Changeux, J. P., and Attali, B. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 15877-15882). In this study, the interactions of Zn(2+) with the pore were probed upon agonist (ACh) dissociation that triggers the transition of the receptor from the active conformation to the resting conformation (i.e. during deactivation). Application of Zn(2+) onto ACh-bound open receptors obstructed their pore and prevented ionic flow. Removing ACh from its extracellular binding sites to trigger deactivation while Zn(2+) is still bound led to tight trapping of Zn(2+) within the pore. Together with single-channel recordings, made to explore single pore-blocking events, we show that dissociation of ACh causes the gate to shut on a Zn(2+) ion that effectively acts as a "foot in the door." We infer that, upon deactivation, the cytoplasmic side of the pore of the ACh-serotonin receptor chimera constricts to close the channel.

SUBMITTER: Pittel I 

PROVIDER: S-EPMC2924088 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature