Ubiquitylation as a regulator of potassium channel function

描述(由申请人提供)：电压门控离子通道是膜兴奋性的主要决定因素。Kv1.2钾通道在大脑中广泛表达，但对Kv1.2在大脑中的调控知之甚少。在模型细胞系统中，Kv1.2被发现受酪氨酸和丝氨酸/苏氨酸磷酸化的调节。最近，我们发现泛素化能够调节Kv1.2的功能。在初步研究中，我们表明泛素化影响Kv1.2的内吞和循环，并且它的作用独立于Kv1.2的降解。有趣的是，Kv1.2内泛素化位点的突变揭示了Kv1.2内不同的泛素化模式以截然不同的方式影响通道运输，有些降低，有些增加细胞表面的Kv1.2水平。这些初步研究利用模型细胞系统和异位表达的野生型或突变形式的Kv1.2来揭示泛素化在Kv1.2调控中的微妙作用。然而，尽管它提供了丰富的信息，但这种方法只表明泛素化如何影响Kv1.2在大脑中的内源性表达。鉴于Kv1.2在大脑中的关键作用，完全缺乏关于Kv1.2泛素化的发生和影响的信息存在着显著的差距。目前的挑战和这项拨款提案的目标是确定我们的初步研究预测的Kv1.2异位表达的泛素化类型是否发生在大脑内源性表达的Kv1.2中。为此，我们将使用Aqua和SILAC质谱学方法来定量确定刺激诱导的Kv1.2在脑内表达的泛素化的数量和分子内模式。

DESCRIPTION (provided by applicant): Voltage gated ion channels are major determinants of membrane excitability. The Kv1.2 potassium channel is expressed widely throughout the brain, but little is known about Kv1.2 regulation in the brain. Using model cell systems, Kv1.2 was found to be regulated by tyrosine and serine/threonine phosphorylation. More recently, we have identified ubiquitylation as being capable of modulating Kv1.2 function. In preliminary studies we show that ubiquitylation affects Kv1.2 endocytosis and recycling and that it does so independently of Kv1.2 degradation. Intriguingly, mutagenesis of ubiquitylation sites within Kv1.2 revealed that distinct patterns of ubiquitylation within Kv1.2 affect channel trafficking in dramatically different ways, some decreasing and some increasing Kv1.2 levels at the cell surface. These preliminary studies employed model cell systems and ectopically expressed wild type or mutant forms of Kv1.2 to reveal a nuanced role for ubiquitylation in Kv1.2 regulation. As informative as it is, however, this approach only suggests how ubiquitylation affects Kv1.2 endogenously expressed in the brain. Given the key role of Kv1.2 in the brain, the complete lack of information on the occurrence and effects of Kv1.2 ubiquitylation there is a striking gap. The current challenge, and goal of this grant proposal, is to determine whether the types of ubiquitylation predicted by our preliminary studies with ectopically expressed Kv1.2 occur in Kv1.2 expressed endogenously within the brain. To do so, we will use AQUA and SILAC mass spectrometry methods to quantitatively determine the amount and intra-molecular patterns of stimulus-induced ubiquitylation of Kv1.2 expressed endogenously within the brain.