Fluorescent probe based on intramolecular proton transfer for fast ratiometric measurement of cellular transmembrane potential.
Development of fast-response potentiometric probes for measuring the transmembrane potential Vm in cell plasma membranes remains a challenge. To overcome the limitations of the classical charge-shift potentiometric probes, we selected a 3-hydroxychromone fluorophore undergoing an excited-state intramolecular proton transfer (ESIPT) reaction that generates a dual emission highly sensitive to electric fields. To achieve the highest sensitivity to the electric field associated to Vm, we modified the fluorophore by adding two rigid legs containing terminal polar sulfonate groups to allow a deep vertical insertion of the fluorophore into the membrane. Fluorescence spectra of the new dye in lipid vesicles and cell membranes confirm the fluorophore location in the hydrophobic region of the membranes. Variation of Vm in lipid vesicles and cell plasma membranes results in a change of the intensity ratio of the two emission bands of the probe. The ratiometric response of the dye in cells is approximately 15% per 100 mV, and is thus quite large in comparison with most single-fluorophore, fast-response probes reported to date. Combined patch-clamp/fluorescence data further show that the ratiometric response of the dye in cells is faster than 1 ms. Analysis of the excitation and emission shifts further suggests that the probe responds to changes in Vm by a mechanism based on electrochromic modulation of its ESIPT reaction. Thus, for the first time, the ESIPT reaction has been successfully applied as a sensing principle for detection of transmembrane potential, allowing to couple classical electrochromic band shifts with changes in the relative intensities of the two well-separated emission bands. The fast two-band ratiometric response as well as the relatively high sensitivity of the new probe are the key features that make it useful for rapid detection of Vm changes in cell suspensions and single cells. Moreover, the new design principles proposed in the present work should allow further improvement of the probe sensitivity.
Klymchenko
Andrey S
AS
UMR 7175 CNRS, Département Pharmacologie et Physicochimie, Université Louis Pasteur (Strasbourg 1), Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France. aklymchenko@pharma.u-strasbg.fr
Stoeckel
Herrade
H
Takeda
Kenneth
K
Mély
Yves
Y
D005456:Fluorescent Dyes; D009682:Magnetic Resonance Spectroscopy; D008564:Membrane Potentials; D011522:Protons; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D011522:Protons