title: Dynamics of internal pore opening in K(V) channels probed by a fluorescent unnatural amino acid.
abstract: Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions.
authors: Groupe d'étude des protéines membranaires (GÉPROM) and Department of Physics, Université de Montréal, Montreal, QC, Canada H3C 3J7.
authors: Tanja
authors: T
authors: Kalstrup
authors: Rikard
authors: R
authors: Blunck
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D018360:Crystallography, X-Ray; D004331:Drosophila melanogaster / Q000502:physiology; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000494:pharmacology; D005470:Fluorometry; D015640:Ion Channel Gating / Q000502:physiology*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010957:Plasmids; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry; D051637:Shaker Superfamily of Potassium Channels / Q000737:chemistry*; D014982:Xenopus laevis
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D051637:Shaker Superfamily of Potassium Channels; D012343:RNA, Transfer
keywords: Anap; two-color VCF
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