CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING |
Proton Transport Through Influenza A Virus M2 Protein Reconstituted in Vesicles
J Craig Moffat 1, Viksita Vijayvergiya 1, Fei Philip Gao 2, Timothy A. Cross 3, Dixon J. Woodbury 1 and David Busath 1*
1 Brigham Young University
2 National High magnetic Field Lab
3 Florida State Univ.
* To whom correspondence should be addressed. E-mail: david_busath{at}byu.edu.
Submitted on March 22, 2007
Revised on May 2, 2007
Accepted on 13 August 2007
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Abstract |
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Influenza A virus M2 protein is known to form acid-activated, proton-selective, amantadine-sensitive channels. We have 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+]. 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 a previous study of reconstituted M2 using a pH sensitive dye to monitor intravesicular pH (1), we conclude that bath pH weakly affects or does not significantly affect proton flow in the pH range of 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 time scale of the initial slope and an important limitation that must be placed on inferences about channel ion selectivity. The rise in pH over 10 seconds after ionophore addition yielded time-averaged single channel conductances 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 was previously reported in vesicles. Assuming complete membrane incorporation and tetramerization of the reconstituted protein, such a low time-average conductance in the face of the previously observed single channel conductance (6 pS at pH 3) implies a channel Po of 10-6 - 10-4. Based on leakage of potassium from M2 containing vesicles versus protein free vesicles, M2 exhibits ~107 selectivity for hydrogen over potassium.
Key Words:
Donnan equilibrium, amantadine, channel selectivity, lipid membranes, proton uptake, single channel conductance
