Rougier Jean-Sébastien, van Bemmelen Miguel X, Bruce M Christine, Jespersen Thomas, Gavillet Bruno, Apothéloz Florine, Cordonier Sophie, Staub Olivier, Rotin Daniela, Abriel Hugues
Department of Pharmacology and Toxicology, Service of Cardiology, University of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland.
Am J Physiol Cell Physiol. 2005 Mar;288(3):C692-701. doi: 10.1152/ajpcell.00460.2004. Epub 2004 Nov 17.
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.
电压门控钠通道（Na(v)）由10个基因组成一个家族。Na(v)的COOH末端包含一簇氨基酸，在10个成员中的7个成员中几乎相同。这个COOH末端序列PPSYDSV是一个PY基序，已知可与Nedd4家族的E3蛋白-泛素连接酶的WW结构域结合。我们最近报道心脏Na(v)1.5受Nedd4-2调节。在本研究中，我们进一步研究了Na(v)蛋白调节的分子决定因素。当在HEK-293细胞中表达并使用全细胞电压钳进行研究时，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的下拉实验表明，小鼠脑Nedd4-2与Na(v) PY基序结合。使用WW结构域的内在色氨酸荧光成像，我们发现Na(v)1.5 PY基序优先与Nedd4-2的第四个WW结构域结合，解离常数（K(d)）约为55μM。我们测试了三种Nedd4样E3（Nedd4-1、Nedd4-2和WWP2）与Na(v)1.5结合的特性以及泛素化和下调Na(v)1.5的能力。尽管Nedd4-1和WWP2与Nedd4-2一样与Na(v)1.5 PY基序结合，但只有Nedd4-2能强烈地泛素化并下调Na(v)1.5。有趣的是，WWP2的共表达与Nedd4-2的作用相互竞争。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的内化。这项研究表明，Na(v)通道的Nedd4依赖性泛素化可能是一种通过调节质膜上通道密度来调节神经元和心肌细胞兴奋性的普遍机制。
