Proton transport through influenza a virus M2 protein reconstituted in vesicles
Proton transport through influenza a virus M2 protein reconstituted in vesicles
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DOI：
10.1529/biophysj.107.109082
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发表时间：
2008-01-15
影响因子：
3.4
通讯作者：
Busath, David D.
中科院分区：
文献类型：
--
作者：
Moffat, J. Craig;Vijayvergiya, Viksita;Busath, David D.
已知甲型流感病毒M2蛋白可形成酸激活、质子选择性、金刚烷胺敏感的通道。我们将valinomycin加入100倍稀释的外部[K+]后，通过监测外部pH，直接测量了含有重组M2的囊泡中的质子摄取。在加入万霉素后的几分钟内，外部pH值通常增加了十分之一的pH单位，但质子摄取并未因酸化而显着改变。在中性条件下，外加1 mM金刚烷胺可产生符合随机有序通道的还原内流；然而，该方法的实验变异较大，阻滞不具有统计学意义。pH为5.4时，金刚烷胺阻滞减少。根据Lin和Schroeder使用pH敏感染料监测囊泡内pH值对重组的M2进行的研究，我们得出结论，在pH值5.4-7.0范围内，对重组体系的质子流动有微弱影响或不显著影响，这与电生理研究的结果相反。对用于此类囊泡摄取试验的唐南平衡弛豫的理论分析阐明了初始斜率的适当时间尺度和必须放置在通道离子选择性推断上的重要限制。加入离子载体后，pH值在10秒内上升，在pH 5.4和7.0时，平均单通道电导分别为0.35 +/- 0.20 aS和0.72 +/- 0.42 aS，比先前报道的囊泡低一个数量级。假设完全的膜结合和重组蛋白的四聚化，面对先前观察到的单通道电导率（pH 3时6 pS），如此低的时间平均电导率意味着开放通道的概率为10(-6)-10（-4）。与不含蛋白质的囊泡相比，基于含M2囊泡中钾的泄漏，我们得出结论，M2对氢的选择性与钾的选择性相似，为10(7)。
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 influx 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 similar to 10(7) selectivity for hydrogen over potassium.