title: Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry.
abstract: Site-specific fluorescence recordings have shown great promise in understanding conformational changes in signaling proteins. The reported applications on ion channels have been limited to extracellular sites in whole oocyte preparations. We are now able to directly monitor gating movements of the intracellular domains of cyclic nucleotide-gated channels using simultaneous site-specific fluorescence recording and patchclamp current recording from inside-out patches. Fluorescence signals were reliably observed when fluorophore was covalently attached to a site between the cyclic nucleotide-binding domain and the pore. While iodide, an anionic quencher, has a higher quenching efficiency in the channel's closed state, thallium ion, a cationic quencher, has a higher quenching efficiency in the open state. The state and charge dependence of quenching suggests movements of charged or dipolar residues near the fluorophore during CNG channel activation.
authors.affiliation: Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle 98195, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zheng
authors.forename: W N
authors.initials: WN
authors.lastname: Zagotta
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D007454:Iodides / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000379:methods*; D011487:Protein Conformation / Q000187:drug effects; D013793:Thallium / Q000494:pharmacology
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D005456:Fluorescent Dyes; D007454:Iodides; D007473:Ion Channels; D013793:Thallium; D006152:Cyclic GMP; D003545:Cysteine