title: In situ measurement of the electrical potential across the lysosomal membrane using FRET.
abstract: The progressive acidification of the endocytic pathway is generated by H(+) pumping of electrogenic vacuolar-type ATPases (V-ATPases) on the endosomal/lysosomal membrane. The determinants of pH during endosome maturation are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. If counter-ion conductance becomes limiting, the generation of a large membrane potential would dominate the proton-motive force (pmf), diminishing the pH gradient proportionally. Validation of this notion requires direct measurement of the electrical potential that develops across the endosomal/lysosomal membrane. To date, the measurement of lysosomal membrane potential (ψ(φ) ) in situ has been hampered by the inability to access endosomes by electrophysiological means and the fact that individual organelles cannot be discerned when using potentiometric fluorescent dyes. Here, we describe a noninvasive procedure to estimate ψ(φ) in intact cells, based on fluorescence resonance energy transfer (FRET). At steady state, ψ(φ) averaged 19 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A (CcA). ψ(φ) was considerably increased by alkalinization of the lysosome lumen by NH(4) Cl, implying that at steady state the V-ATPase operates at submaximal rates and that the contribution of ψ(φ) to pmf is relatively small. Our method should enable systematic studies of endosomal/lysosomal potential.
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8.
authors.forename: Mirkka
authors.initials: M
authors.lastname: Koivusalo
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Steinberg
authors.forename: David
authors.initials: D
authors.lastname: Mason
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology*; D008247:Lysosomes / Q000378:metabolism / Q000502:physiology*; D018942:Macrolides / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology*; D015388:Organelles / Q000378:metabolism / Q000502:physiology; D010539:Permeability; D018892:Proton-Motive Force / Q000502:physiology; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D018942:Macrolides; C036978:concanamycin A; D025262:Vacuolar Proton-Translocating ATPases