Proton transport through influenza A virus M2 protein reconstituted in vesicles.

Abstract:

:Influenza A virus M2 protein is known to form acid-activated, proton-selective, amantadine-sensitive channels. We directly measured proton uptake in vesicles containing reconstituted M2 by monitoring external pH after addition of valinomycin to vesicles with 100-fold-diluted external [K(+)]. External pH typically increased by a few tenths of a pH unit over a few minutes after valinomycin addition, but proton uptake was not significantly altered by acidification. Under neutral conditions, external addition of 1 mM amantadine produced a reduction in flux consistent with randomly ordered channels; however, experimental variation is high with this method and the block was not statistically significant. Amantadine block was reduced at pH 5.4. In accord with Lin and Schroeder's study of reconstituted M2 using a pH-sensitive dye to monitor intravesicular pH, we conclude that bath pH weakly affects or does not significantly affect proton flow in the pH range 5.4-7.0 for the reconstituted system, contrary to results from electrophysiological studies. Theoretical analysis of the relaxation to Donnan equilibrium utilized for such vesicle uptake assays illuminates the appropriate timescale of the initial slope and an important limitation that must be placed on inferences about channel ion selectivity. The rise in pH over 10 s after ionophore addition yielded time-averaged single-channel conductances of 0.35 +/- 0.20 aS and 0.72 +/- 0.42 aS at pH 5.4 and 7.0, respectively, an order of magnitude lower than previously reported in vesicles. Assuming complete membrane incorporation and tetramerization of the reconstituted protein, such a low time-averaged conductance in the face of previously observed single-channel conductance (6 pS at pH 3) implies an open channel probability of 10(-6)-10(-4). Based on leakage of potassium from M2-containing vesicles, compared to protein-free vesicles, we conclude that M2 exhibits approximately 10(7) selectivity for hydrogen over potassium.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Moffat JC,Vijayvergiya V,Gao PF,Cross TA,Woodbury DJ,Busath DD

doi

10.1529/biophysj.107.109082

subject

Has Abstract

pub_date

2008-01-15 00:00:00

pages

434-45

issue

2

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(08)70721-5

journal_volume

94

pub_type

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