The Alphavirus 6K protein activates endogenous ionic conductances when expressed in Xenopus oocytes
The Alphavirus 6K protein activates endogenous ionic conductances when expressed in Xenopus oocytes
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DOI：
10.1007/s00232-007-9003-6
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发表时间：
2007-01-01
影响因子：
2.4
通讯作者：
Dubuisson, Jean
中科院分区：
文献类型：
--
作者：
Antoine, Anne-Frederique;Montpellier, Claire;Dubuisson, Jean
甲病毒 Sindbis 6K 蛋白参与多种功能。它有助于 E1 和 PE2 病毒包膜糖蛋白的加工和膜插入以及病毒出芽。它还可以透化大肠杆菌和哺乳动物细胞。这些类似于病毒孔蛋白的特性被认为可以通过改变膜的通透性来帮助病毒出芽。我们在爪蟾卵母细胞中表达辛德比斯病毒 6K cRNA，以进一步表征 6K 对膜电导和透化的影响。尽管没有看到内在的通道特性，但在 24 小时内观察到细胞收缩。电压钳实验表明，6K 上调内源电流：超极化激活的内向电流 (I (in)) 和钙依赖性氯电流 (I (Cl))。 6K 位于质膜和内质网膜上。质膜电流上调可能是由于细胞内质网水平的钙稳态被破坏所致。事实上，6K cRNA 表达诱导网状钙储存耗尽和电容性钙进入激活。通过对培养介质的实验修改，我们发现这些事件的下游细胞收缩是由 6K 诱导的 KCl 外流引起的（I (Cl) 上调导致氯离子外流，其本身电驱动钾外流），这负责渗透水外流。我们的数据证实，6K 特异性触发一系列连续级联事件，导致细胞质钙升高和细胞透化，这可能在辛德比斯病毒生命周期中发挥作用。
The Alphavirus Sindbis 6K protein is involved in several functions. It contributes to the processing and membrane insertion of E1 and PE2 viral envelope glycoproteins and to virus budding. It also permeabilizes Escherichia coli and mammalian cells. These viroporin-like properties have been proposed to help virus budding by modifying membrane permeabilities. We expressed Sindbis virus 6K cRNA in Xenopus oocytes to further characterize the effect of 6K on membrane conductances and permeabilization. Although no intrinsic channel properties were seen, cell shrinkage was observed within 24 h. Voltage-clamp experiments showed that 6K upregulated endogenous currents: a hyperpolarization-activated inward current (I (in)) and a calcium-dependent chloride current (I (Cl)). 6K was located at both the plasma and the endoplasmic reticulum membranes. The plasma membrane current upregulation likely results from disruption of the calcium homeostasis of the cell at the endoplasmic reticulum level. Indeed, 6K cRNA expression induced reticular calcium store depletion and capacitative calcium entry activation. By experimental modifications of the incubation medium, we showed that downstream of these events cell shrinkage resulted from a 6K -induced KCl efflux (I (Cl) upregulation leads to chloride efflux, which itself electrically drives potassium efflux), which was responsible for an osmotic water efflux. Our data confirm that 6K specifically triggers a sequential cascade of events that leads to cytoplasmic calcium elevation and cell permeabilization, which likely play a role in the Sindbis virus life cycle.