Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating
| Autor: | Nikolai M. Soldatov, Evgeny Kobrinsky, Louisa Stevens, Yasir Kazmi, Dennis Wray |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Yellow fluorescent protein
Patch-Clamp Techniques Time Factors Gating Transfection Biochemistry Shab Potassium Channels Tetramer Chlorocebus aethiops Fluorescence Resonance Energy Transfer Animals Patch clamp Molecular Biology Models Statistical biology Chemistry Cell Membrane Depolarization Blood Proteins Cell Biology Phosphoproteins Potassium channel Protein Structure Tertiary Electrophysiology Pleckstrin homology domain Crystallography Förster resonance energy transfer COS Cells biology.protein |
| Zdroj: | Journal of Biological Chemistry. 281:19233-19240 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m601231200 |
| Popis: | The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFP(N)-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization. |
| Databáze: | OpenAIRE |
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