Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus
Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus
复制标题
DOI：
10.1128/jvi.75.8.3647-3656.2001
复制
发表时间：
2001-04-01
影响因子：
5.4
通讯作者：
Schroeder, C
中科院分区：
文献类型：
--
作者：
Lin, TI;Schroeder, C
病毒离子通道蛋白M2支持流感病毒及其糖蛋白通过细胞的酸性隔间转运，M2将内体质子传导到病毒粒子中以启动去涂层，并通过平衡跨高尔基膜上的pH保持对酸敏感的病毒血凝素的天然构象，M2通道的异常低电导阻碍了电生理技术对单通道的分解。脂质体重组M2的平均单一通道电流在中性pH时为-1.2aa(1.2×10(-18)A)，在pH 5.7时为2.7~4.1aa，激活了该通道。外推到生理温度分别为4.8和40 aA，单位电导为0.03对0.4 fS，这一微小的活性似乎足以使病毒繁殖，但低到足以避免流产的细胞毒性，M2的单位渗透性在其他质子通道的报道范围内，为了解决M2的离子选择性，我们利用密封脂质体中离子流入和流出的耦合，通过质子逆流监测金属离子的通量，使用比可用的Na(+)或KF探针灵敏1000倍的pH探针。即使低pH激活的M2不传导Na(+)和K(+)，M2的质子选择性估计至少为3×10(6)(超过钠或钾离子)，这与电生理研究一致。M2的严格质子选择性表明，流感病毒的细胞病理学不涉及细胞钠或钾梯度的直接扰动。
The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell, M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin, The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer-1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7 - which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS, This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity, The unitary permeability of M2 was within the range reported for other proton channels, To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes, Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na(+) or Kf probes. Even low-pH-activated M2 did not conduct Na(+) and K(+), The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.