Cholesterol Depletion of the Plasma Membrane Prevents Activation of the Epithelial Sodium Channel (ENaC) by SGK1
Cholesterol Depletion of the Plasma Membrane Prevents Activation of the Epithelial Sodium Channel (ENaC) by SGK1
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DOI：
10.1159/000257516
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发表时间：
2009-01-01
影响因子：
--
通讯作者：
Diakov, Alexei
中科院分区：
文献类型：
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作者：
Krueger, Bettina;Haerteis, Silke;Diakov, Alexei
上皮钠通道（ENaC）的脂质环境及其与所谓的脂筏的可能联系可能与其功能有关。本研究的目的是证实ENaC与脂筏的关联，并分析甲基-β-环糊精（M β CD）对细胞膜胆固醇消耗对通道功能和调节的影响。使用蔗糖密度梯度离心，我们证明了ENaC蛋白的显著部分分布到被认为是典型的脂筏组分的低密度组分。重要的是，通过M β CD对细胞裂解物的胆固醇消耗将ENaC转移到更高密度的非筏级分。活细胞成像表明，随着时间的推移，M β CD治疗大大减少了菲律宾染色，证实了质膜的胆固醇耗竭。对于电生理学研究，将完整的卵母细胞暴露于20 mM M β CD 3小时。M β CD治疗对基线全细胞ENaC电流没有一致的影响。除了典型的约5 pS的单通道电导，亚电导状态的ENaC偶尔观察到的补丁从M β CD治疗，但不是从控制卵母细胞。重要的是，在外向膜片钳记录中，在用M β CD预处理的卵母细胞中，移液器溶液中重组SGK 1的刺激作用基本上被消除。这些结果表明，通过细胞溶质SGK 1的ENaC活化通过从质膜去除胆固醇而受到损害。因此，通过SGK 1激活ENaC可能需要存在完整的脂质环境和/或脂筏作为信号平台。版权所有（C）2009 S. Karger AG，巴塞尔
The lipid environment of the epithelial sodium channel (ENaC) and its possible association with so-called lipid rafts may be relevant to its function. The aim of our study was to confirm the association of ENaC with lipid rafts and to analyze the effect of cholesterol depletion of the plasma membrane by methyl-beta-cyclodextrin (M beta CD) on channel function and regulation. Using sucrose density gradient centrifugation we demonstrated that a significant portion of ENaC protein distributes to low density fractions thought to be typical lipid raft fractions. Importantly, cholesterol depletion of cell lysate by M beta CD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with M beta CD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM M beta CD for three hours. M beta CD treatment had no consistent effect on baseline whole-cell ENaC currents. In addition to the typical single channel conductance of about 5 pS, subconductance states of ENaC were occasionally observed in patches from M beta CD treated but not from control oocytes. Importantly, in outside-out patch clamp recordings the stimulatory effect of recombinant SGK1 in the pipette solution was essentially abolished in oocytes pretreated with M beta CD. These results indicate that ENaC activation by cytosolic SGK1 is compromised by removing cholesterol from the plasma membrane. Thus, ENaC activation by SGK1 may require the presence of an intact lipid environment and/or of lipid rafts as signalling platform. Copyright (C) 2009 S. Karger AG, Basel