A fluorometric approach to local electric field measurements in a voltage-gated ion channel.
Site-specific electrostatic measurements have been limited to soluble proteins purified for in vitro spectroscopic characterization or proteins of known structure; however, comparable measurements have not been made for functional membrane bound proteins. Here, using an electrochromic fluorophore, we describe a method to monitor localized electric field changes in a voltage-gated potassium channel. By coupling the novel probe Di-1-ANEPIA to cysteines in Shaker and tracking field-induced optical changes, in vivo electrostatic measurements were recorded with submillisecond resolution. This technique reports dynamic changes in the electric field during the gating process and elucidates the electric field profile within Shaker. The extension of this method to other membrane bound proteins, including transporters, will yield insight into the role of electrical forces on protein function.
Asamoah
Osei Kwame
OK
Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Wuskell
Joseph P
JP
Loew
Leslie M
LM
Bezanilla
Francisco
F
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology*; D004560:Electricity; D001330:Electronic Data Processing; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods*; D005057:Eukaryotic Cells / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology*; D015394:Molecular Structure; D009865:Oocytes; D015221:Potassium Channels / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated / Q000502:physiology*; D051637:Shaker Superfamily of Potassium Channels; D015398:Signal Transduction / Q000502:physiology; D055672:Static Electricity
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051637:Shaker Superfamily of Potassium Channels; D002118:Calcium