In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force

Abstract

Phagosomes employ lytic enzymes, cationic peptides, and reactive oxygen intermediates to eliminate invading microorganisms. The effectiveness of these microbicidal mechanisms is potentiated by the acidic pH created by H ؉ -pumping vacuolar-type ATPases (VATPases) on the phagosomal membrane. The degree of phagosomal acidification varies greatly among neutrophils, macrophages, and dendritic cells and can be affected by diseases like cystic fibrosis. The determinants of phagosomal pH 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. When counterion conductance is limiting, generation of a large membrane potential will dominate the proton-motive force (pmf), with a proportionally diminished pH gradient. Validation of this notion requires direct measurement of the electrical potential that develops across the phagosomal membrane (⌿ ⌽). We describe a noninvasive procedure to estimate ⌿ ⌽ in intact cells, based on fluorescence resonance energy transfer. This approach, in combination with measurements of phagosomal pH, enabled us to calculate the pmf across phagosomes of murine macrophages and to analyze the factors that limit acidification. At steady state, ⌿⌽ averaged 27 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A. The comparatively small contribution of the potential to the pmf suggests that proton pumping is not limited by the counterion permeability, a notion that was validated independently by using ionophores. Instead, phagosomal pH stabilizes when the rate of proton pumping, which decreases gradually as the lumen acidifies, is matched by the passive leak of proton equivalents.macrophage ͉ membrane potential ͉ pH ͉ phagosome ͉ ratiometric imaging

Direct recruitment of H+-ATPase from lysosomes for phagosomal acidification

“…7E). This time-course agrees with the acidification kinetics reported previously (Lukacs et al, 1990;Steinberg et al, 2007;Yates et al, 2007). The early recruitment of a3-GFP to phagosomes occurs via tubular structures extending from the perinuclear lysosomes along microtubules.…”

“…The phagosomal pH was measured by single-particle fluorescence ratiometry (Steinberg et al, 2007). Macrophages fed with FITC beads opsonized with human IgG were placed under a Leica ASMDW microscope.…”

VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification

“…pH measurements. Neutrophils were loaded with 2 µM BCECF/AM for 20 min, seeded on 25-mm glass coverslips that were inserted in a thermostatic chamber (Harvard Apparatus), and imaged on an Axiovert S100 TV through Ca 2+ is caused by the opening of SOCE channels on the membrane of phagosomes (Lundqvist-Gustafsson et al, 2000), small compartments which can depolarize significantly (Steinberg et al, 2007). Our observation that proton channel ablation depolarizes cells during oxidase activation suggests that phagosomes, where the oxidase normally assembles, are depolarized in cells lacking VSOP/Hv1.…”

Role of the phagosomal redox-sensitive TRP channel TRPM2 in regulating bactericidal activity of macrophages

“…Oxidant stress results in increased formation of ADPR, which activates TRPM2. TRPM2 activations gates the flux of cations (Na + and Ca 2+ ) from phagosomes, which are regulated by cation concentrations (Lundqvist-Gustafsson et al, 2000) and phagosomal membrane potential (Steinberg et al, 2007b). Cation efflux (cation counter flux) and H + influx decreases phagosomal pH and thus favors bacterial killing.…”