Molecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel.
Molecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel.
复制标题
DOI：
10.1085/jgp.200709800
复制
发表时间：
2007-10
期刊：
影响因子：
--
通讯作者：
Stockand JD
中科院分区：
文献类型：
--
作者：
Pochynyuk O;Tong Q;Medina J;Vandewalle A;Staruschenko A;Bugaj V;Stockand JD
磷脂酰肌醇4，5-二磷酸（PI（4，5）P2）和磷脂酰肌醇3，4，5-三磷酸（PI（3，4，5）P3）是生理上重要的第二信使。这些分子结合效应蛋白以调节活性。几种类型的离子通道，包括上皮Na+通道（ENaC），是能够直接与这些信号分子相互作用的磷酸肌醇效应子。然而，很少有人知道ENaC和其他离子通道内的磷酸肌醇结合和调制的重要区域。此外，在许多情况下，这种调节的分子机制仍然不清楚。在这里，我们研究PI（3，4，5）P3和PI（4，5）P2对ENaC的调节，以开始鉴定这种调节的分子决定因素。我们鉴定了在β-和γ-ENaC而不是α-ENaC中的第二跨膜结构域之后的内膜界面附近的细胞内区域，其是PI（3，4，5）P2而不是PI（4，5）P2调节所必需的。这些区域内保守的碱性氨基酸的电荷中和表明，这些极性残基是磷酸肌醇调节的关键。单通道分析表明，PI（3，4，5）P3增加β-和γ-ENaC的第二个跨膜结构域后的区域是ENaC开放概率的关键，从而确定了ENaC开放的机制。出乎意料的是，β-和γ-ENaC的最N末端内的细胞内结构域被鉴定为响应于膜PI（4，5）P2的消耗而下调ENaC活性和Po的关键。通道的这些区域在PI（3，4，5）P3反应中没有可识别的作用。同样，这些结构域内保守的带正电荷的残基是特别重要的，对于外源PI（4，5）P2增加开放概率是必需的。我们得出结论，β和γ亚基赋予ENaC磷酸肌醇敏感性，通道的不同区域对PI（3，4，5）P3和PI（4，5）P2的调节至关重要。这表明这些磷酸肌醇占据ENaC内不同的配体结合位点以调节开放概率。
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na+ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P3 and PI(4,5)P2 to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P2 but not PI(4,5)P2 modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P3 augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and Po in response to depletion of membrane PI(4,5)P2. These regions of the channel played no identifiable role in a PI(3,4,5)P3 response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P2 to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P3 and PI(4,5)P2. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.
登录
查看更多内容
DOI：
10.1085/jgp.112.4.423
发表时间：
1998-10
期刊：
The Journal of general physiology
影响因子：
--
作者：
Fyfe GK;Canessa CM
通讯作者：
Canessa CM
DOI：
10.1152/ajpgi.1999.276.3.g567
发表时间：
1999-03-01
影响因子：
4.5
作者：
Hummler, E;Horisberger, JD
通讯作者：
Horisberger, JD
影响因子：
4.8
作者：
Du, XO;Zhang, HL;Logothetis, DE
通讯作者：
Logothetis, DE
影响因子：
5.3
作者：
Gamper, N;Reznikov, V;Shapiro, MS
通讯作者：
Shapiro, MS
影响因子：
3.8
作者：
Cukras, CA;Jeliazkova, I;Nichols, CG
通讯作者：
Nichols, CG