The N terminus of α-ENaC mediates ENaC cleavage and activation by furin
The N terminus of α-ENaC mediates ENaC cleavage and activation by furin
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DOI：
10.1085/jgp.201711860
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发表时间：
2018-08-01
影响因子：
3.8
通讯作者：
Stutts, M. Jackson
中科院分区：
文献类型：
--
作者：
Kota, Pradeep;Gentzsch, Martina;Stutts, M. Jackson
上皮Na+通道包含三个同源亚基（α、β和γ），它们受可变剪接和蛋白水解切割的调节。在这里，我们确定了由先前鉴定的天然存在的α亚基剪接变体（α-ENaC）的表达引起的降低的Na+电流（I-Na）的基础，其中残基34-82缺失（α（Delta 34）（-）（82））。在非洲爪蟾卵母细胞中用WT p和y亚基表达α（Delta 34）（-）（82）-ENaC产生降低的基础I Na，其可在很大程度上被外源胰蛋白酶回收。对于这种含有α（δ）（34）（-）（82）的ENaC，α和γ亚基都显示减少的切割片段，这与弗林蛋白酶或弗林蛋白酶样转化酶的加工减少一致。使用半胱氨酸的MTSET修饰（引入β-ENaC中的简并蛋白基因座）的数据表明，α（Delta 34）（-）（82）-ENaC的I-Na减少是由于未切割、接近沉默的ENaC群体增加，而不是由于所有通道均匀分布的开放概率降低。在用布雷菲德菌素A处理以破坏通道亚基的顺行运输后，表达α（δ）（34）（-）（82）-ENaC的卵母细胞中的I-Na比表达WT ENaC的卵母细胞中的I-Na重建得更慢。表达WT ENaC的卵母细胞在孔阻断剂阿米洛利中过夜或急性孵育增加基础ENaC蛋白水解刺激，与Na+反馈抑制的缓解一致。在表达α（δ）（34）（-）（82）-ENaC的卵母细胞中，这些反应降低。我们得出结论，α-ENaC N末端介导了控制切割和未切割的ENaC群体向卵母细胞膜递送的相互作用。
Epithelial Na+ channels comprise three homologous subunits (a, (3, and y) that are regulated by alternative splicing and proteolytic cleavage. Here, we determine the basis of the reduced Na+ current (I-Na) that results from expression of a previously identified, naturally occurring splice variant of the a subunit (alpha-ENaC), in which residues 34-82 are deleted (alpha(Delta 34)(-)(82)). alpha(Delta 34)(-)(82)-ENaC expression with WT p and y subunits in Xenopus oocytes produces reduced basal I Na , which can largely be recovered by exogenous trypsin. With this alpha(Delta)(34)(-)(82)-containing ENaC, both a and y subunits display decreased cleavage fragments, consistent with reduced processing by furin or furin-like convertases. Data using MTSET modification of a cysteine, introduced into the degenerin locus in beta-ENaC, suggest that the reduced I-Na of alpha(Delta 34)(-)(82)-ENaC arises from an increased population of uncleaved, near-silent ENaC, rather than from a reduced open probability spread uniformly across all channels. After treatment with brefeldin A to disrupt anterograde trafficking of channel subunits, I-Na in oocytes expressing alpha(Delta)(34)(-)(82)-ENaC is reestablished more slowly than that in oocytes expressing WT ENaC. Overnight or acute incubation of oocytes expressing WT ENaC in the pore blocker amiloride increases basal ENaC proteolytic stimulation, consistent with relief of Na+ feedback inhibition. These responses are reduced in oocytes expressing alpha(Delta)(34)(-)(82)-ENaC We conclude that the alpha-ENaC N terminus mediates interactions that govern the delivery of cleaved and uncleaved ENaC populations to the oocyte membrane.