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

In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force
In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force
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 (V-ATPases) 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 (Psi(Phi)). We describe a noninvasive procedure to estimate Psi(Phi) 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, Psi(Phi) 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
0027-8424
9523-9528
Steinberg, Benjamin E.
b0613ad4-c50e-49d9-ba09-2975a44cde36
Touret, Nicolas
abeaf2ae-6d96-414c-903d-f0535b1850f6
Vargas-Caballero, Mariana
de2178ac-77fd-4748-9fe5-109ab8ad93e1
Grinstein, Sergio
1ecff327-d9e3-43f9-9df5-1877d2ec2a62
Steinberg, Benjamin E.
b0613ad4-c50e-49d9-ba09-2975a44cde36
Touret, Nicolas
abeaf2ae-6d96-414c-903d-f0535b1850f6
Vargas-Caballero, Mariana
de2178ac-77fd-4748-9fe5-109ab8ad93e1
Grinstein, Sergio
1ecff327-d9e3-43f9-9df5-1877d2ec2a62

Steinberg, Benjamin E., Touret, Nicolas, Vargas-Caballero, Mariana and Grinstein, Sergio (2007) In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force. Proceedings of the National Academy of Sciences, 104 (22), 9523-9528. (doi:10.1073/pnas.0700783104). (PMID:17517624)

Record type: Article

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 (V-ATPases) 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 (Psi(Phi)). We describe a noninvasive procedure to estimate Psi(Phi) 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, Psi(Phi) 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.

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More information

Published date: 29 May 2007
Keywords: macrophage, membrane potential, pH, phagosome, ratiometric imaging
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 348673
URI: http://eprints.soton.ac.uk/id/eprint/348673
ISSN: 0027-8424
PURE UUID: 6606a2a6-85c5-4ad2-9b0c-79d60d2488aa
ORCID for Mariana Vargas-Caballero: ORCID iD orcid.org/0000-0003-2326-4001

Catalogue record

Date deposited: 13 May 2013 13:23
Last modified: 03 Dec 2019 01:38

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