The acetylcholine receptor is a well-characterized excitatory neurotransmitter receptor that upon ligand binding, shifts structural conformation such that the pore allows passive flux of ions, thus depolarizing the cell. This proposal outlines experiments intended to chemically probe the open-channel state using photoactivatable reagents. Briefly, acetylcholine receptor expressed in Xenopus oocytes will be incubated with benzophenone, a hydrophobic and photoactivated topological probe. Photoactivation will be coordinated with ligand- dependent channel activation using two-electrode voltage clamping. Mass spectrometry will be utilized to identify sites of probe incorporation. A similar approach will be used with substituted cysteine mutagenesis targeting the second and fourth transmembrane domains as well as the short extracellular loop connecting the second and third transmembrane domains. Channel-state dependent crosslinking using benzophenone coupled to thiol-reactive reagents will be utilized to identify structural elements that shift conformation upon channel activation. Thus, I will utilize electrophysiology to coordinate channel- state and photoirradiation as well as characterize changes in channel function after probe incorporation/crosslinking. These experiments should identify structural elements involved in gating mechanism and should yield a model for the gating mechanism.
