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Proton transport through influenza a virus m2 protein reconstituted in vesicles.
| Content Provider | CiteSeerX |
|---|---|
| Author | Moffat, J. Craig Vijayvergiya, Viksita Gao, Philip F. Cross, Y. Timothy A. Woodbury, Y. Dixon J. Busath, David D. |
| Abstract | 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 [K1]. 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 6 0.20 aS and 0.72 6 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 106–104. Based on leakage of potassium from M2-containing vesicles, compared to protein-free vesicles, we conclude that M2 exhibits;107 selectivity for hydrogen over potassium. |
| File Format | |
| Access Restriction | Open |
| Subject Keyword | Influenza Virus M2 Protein Reconstituted Proton Transport External Ph Low Time-averaged Conductance Valinomycin Addition Bath Ph Weakly Affect Intravesicular Ph External Addition Affect Proton Flow Time-averaged Single-channel Conductance Experimental Variation Theoretical Analysis Appropriate Timescale Abstract Influenza Virus M2 Protein Amantadine Block 100-fold-diluted External K1 Electrophysiological Study M2-containing Vesicle Amantadine-sensitive Channel Neutral Condition Ph-sensitive Dye Protein-free Vesicle Important Limitation Ph Unit Open Channel Probability Ph Range Mm Amantadine Donnan Equilibrium Initial Slope Complete Membrane Incorporation Channel Ion Selectivity Single-channel Conductance Reconstituted Protein Reconstituted System Ionophore Addition |
| Content Type | Text |
