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Kinetics of proton transport into influenza virions by the viral M2 channel.

Ivanovic T, Rozendaal R, Floyd DL, Popovic M, van Oijen AM, Harrison SC - PLoS ONE (2012)

Bottom Line: Fusion-pore formation usually follows internal acidification but does not require it.The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second.The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m) of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

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A transporter model for M2-mediated proton transport into virions.Conformational changes in the channel histidines allow their protonation on the virion exterior and deprotonation on the virion interior facilitating transport of protons down the concentration gradient.
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pone-0031566-g005: A transporter model for M2-mediated proton transport into virions.Conformational changes in the channel histidines allow their protonation on the virion exterior and deprotonation on the virion interior facilitating transport of protons down the concentration gradient.

Mentions: Our single-virion assay permits measurement of M2 channel activity in the context of authentic influenza virions triggered to undergo membrane fusion. The experiments are consistent with two predictions of a transporter model (Figure 5) for passage of protons into the virion interior through M2 channels: 1) Michealis-Menten-like dependence of onset and dissipation rates with pKms coinciding with the pKa of the fourth channel histidine and 2) slow rate of proton transport. Our kinetic measurements of M2 channel activity on authentic virions complement recent findings from purified M2 protein reconstituted in liposomes and triggered to conduct protons in the pH range from 5 to 3.4 [13]. That work identified a second pseudosaturation step (pKm 6.25) corresponding to the measured pKa of the third protonation event in the channel histidine tetrad. Fluorescein is not a useful probe for detecting internal virion pH changes in this second regime, because of a combination of factors that include the very slow rate of proton transport in this regime, the photobleaching of fluorescein after prolonged laser-light exposure, and the almost identical pKas of fluorescein (∼6.4) and the third channel histidine (∼6.3). Nonetheless, the similar rates of proton transport and the similar dependence of these rates on proton concentration are strong evidence that the activity of M2 in authentic virions is insensitive to the presence of other viral proteins or the membrane remodeling induced by HA conformational changes during fusion.


Kinetics of proton transport into influenza virions by the viral M2 channel.

Ivanovic T, Rozendaal R, Floyd DL, Popovic M, van Oijen AM, Harrison SC - PLoS ONE (2012)

A transporter model for M2-mediated proton transport into virions.Conformational changes in the channel histidines allow their protonation on the virion exterior and deprotonation on the virion interior facilitating transport of protons down the concentration gradient.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295812&req=5

pone-0031566-g005: A transporter model for M2-mediated proton transport into virions.Conformational changes in the channel histidines allow their protonation on the virion exterior and deprotonation on the virion interior facilitating transport of protons down the concentration gradient.
Mentions: Our single-virion assay permits measurement of M2 channel activity in the context of authentic influenza virions triggered to undergo membrane fusion. The experiments are consistent with two predictions of a transporter model (Figure 5) for passage of protons into the virion interior through M2 channels: 1) Michealis-Menten-like dependence of onset and dissipation rates with pKms coinciding with the pKa of the fourth channel histidine and 2) slow rate of proton transport. Our kinetic measurements of M2 channel activity on authentic virions complement recent findings from purified M2 protein reconstituted in liposomes and triggered to conduct protons in the pH range from 5 to 3.4 [13]. That work identified a second pseudosaturation step (pKm 6.25) corresponding to the measured pKa of the third protonation event in the channel histidine tetrad. Fluorescein is not a useful probe for detecting internal virion pH changes in this second regime, because of a combination of factors that include the very slow rate of proton transport in this regime, the photobleaching of fluorescein after prolonged laser-light exposure, and the almost identical pKas of fluorescein (∼6.4) and the third channel histidine (∼6.3). Nonetheless, the similar rates of proton transport and the similar dependence of these rates on proton concentration are strong evidence that the activity of M2 in authentic virions is insensitive to the presence of other viral proteins or the membrane remodeling induced by HA conformational changes during fusion.

Bottom Line: Fusion-pore formation usually follows internal acidification but does not require it.The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second.The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m) of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

Show MeSH
Related in: MedlinePlus