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首页> 外文期刊>American Journal of Physiology >Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins.
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Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins.

机译：Nedd4 / Nedd4样蛋白对电压门控性钠通道调节的分子决定因素。

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The voltage-gated Na(+) channels (Na(v)) form a family composed of 10 genes. The COOH termini of Na(v) contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na(v)1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na(v) proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na(v)1.2 and Na(v)1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na(v)1.2, Na(v)1.3, and Na(v)1.5 indicated that mouse brain Nedd4-2 binds to the Na(v) PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na(v)1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K(d) of approximately 55 muM. We tested the binding properties and the ability to ubiquitinate and downregulate Na(v)1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na(v)1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na(v)1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na(v)1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na(v) channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.
机译：电压门控的Na（+）通道（Na（v））形成由10个基因组成的家族。 Na（v）的COOH末端含有10个成员中的7个几乎相同的氨基酸簇。该COOH末端序列PPSYDSV是一个PY基序，已知可与Nedd4家族E3蛋白泛素连接酶的WW域结合。我们最近报道心脏Na（v）1.5受Nedd4-2调控。在这项研究中，我们进一步研究了Na（v）蛋白质调控的分子决定因素。当在HEK-293细胞中表达并使用全细胞电压钳制研究时，神经元Na（v）1.2和Na（v）1.3也被Nedd4-2下调。使用带有Na（v）1.2，Na（v）1.3和Na（v）1.5的PY基序的融合蛋白进行下拉实验表明，小鼠脑Nedd4-2与Na（v）PY基序结合。使用WW域的固有色氨酸荧光成像，我们发现Na（v）1.5 PY基序优先结合Nedd4-2的第四个WW域，其K（d）约为55μM。我们测试了三种Nedd4样E3的结合特性以及泛素化和下调Na（v）1.5的能力：Nedd4-1，Nedd4-2和WWP2。尽管事实与Nedd4-2，Nedd4-1和WWP2绑定到Na（v）1.5 PY基序，只有Nedd4-2鲁棒地泛素化并下调了Na（v）1.5。有趣的是，WWP2的共表达与Nedd4-2的作用竞争。最后，使用布雷菲德菌素A，我们发现Nedd4-2加速了在HEK-293细胞中稳定表达的Na（v）1.5的内在化。这项研究表明，Na（v）通道的Nedd4依赖性泛素化可能代表通过调节质膜通道密度来调节神经元和肌细胞兴奋性的一般机制。

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