Voltage sensing by fluorescence resonance energy transfer in single cells.
A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nernst equation. A voltage-sensitive fluorescent readout is created by labeling the extracellular surface of the cell with a second fluorophore, here a fluorescently labeled lectin, that can undergo energy transfer with the membrane-bound sensor. Fluorescence resonance energy transfer between the two fluorophores is disrupted when the membrane potential is depolarized, because the anion is pulled to the intracellular surface of the plasma membrane far from the lectin. Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl is n-hexyl and n-decyl (DiSBA-C6-(3) and DiSBA-C10-(3), respectively) can function as donors to Texas Red labeled wheat germ agglutinin (TR-WGA) and acceptors from fluorescein-labeled lectin (FI-WGA). In voltage-clamped fibroblasts, the translocation of these oxonols is measured as a displacement current with a time constant of approximately 2 ms for 100 mV depolarization at 20 degrees C, which equals the speed of the fluorescence changes. Fluorescence ratio changes of between 4% and 34% were observed for a 100-mV depolarization in fibroblasts, astrocytoma cells, beating cardiac myocytes, and B104 neuroblastoma cells. The large fluorescence changes allow high-speed confocal imaging.
Howard Hughes Medical Institute, University of California, San Diego, La Jolla 92093-0647, USA.
J E
JE
González
R Y
RY
Tsien
D000818:Animals; D001254:Astrocytoma; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D007739:L Cells / Q000502:physiology; D008564:Membrane Potentials; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes