The Role of TRIP8b in Neuronal HCN Channel Trafficking

描述(由申请人提供)： 超极化激活的环核苷酸门控(HCN)通道(h通道)介导超极化激活电流(Ih)，对调节海马CA1区锥体神经元的兴奋性具有重要作用。由于h通道在神经元兴奋性的动态平衡中起支配作用，h通道功能障碍与颞叶癫痫有关，我们的长期目标是了解h通道在海马区未知的转运和靶向机制，CA1锥体神经元和新皮质V层神经元的h通道表现出显著的远端树突状丰富(DDE)，这被证明是控制细胞兴奋性和同步性的关键。一个与h通道转运有关的候选分子是含有四肽重复序列(TPR)的Rab8b相互作用蛋白(TRIPSb)。TRIPSb是唯一已知的h通道的相互作用者，在海马区和皮质显示锥体神经元DDE类似于HCN1和2。此外，TRIPSb还被证明调节卵母细胞中的iH密度。TRIPSb通过其保守的C末端TPR结构域与h通道相互作用，其方式与过氧化体输入受体蛋白同源。然而，TRIPSb的N-末端区域与任何已知蛋白质没有同源性，其功能尚不清楚。我们的初步数据表明，N-末端以发育调节的方式高度选择性地剪接，从而特定的异构体在DDE发病的同时上调。值得注意的是，选择性剪接改变了被认为在贩运中重要的细胞分选信号的存在。由于TRIPSb与h通道转运有关的大量间接证据，我们推测TRIPSb在海马区h通道DDE的建立和/或维持中起重要作用。为了确定TRIPSb在海马DDE中的作用，我们提出了以下具体目标：1)确定CA1锥体神经元的h通道DDE是否需要TRIPSb，特别是TRIPSb的N端。我们将在切片培养和活体动物中使用慢病毒传递shRNA和显性负的TRIPSb结构来确定其对活体海马区h通道DDE的影响。2)确定TRIPSb N端选择性剪接在h通道转运和DDE中的作用。我们将使用单细胞RT-PCR、生物素化和病毒传递TRIPSb剪接异构体来确定TRIPSb剪接如何影响h通道表面运输和DDE。公共卫生相关性：癫痫仍然是终生的主要负担。通过了解大脑如何调节兴奋性，我们将深入了解癫痫的原因和靶向治疗的机制。

DESCRIPTION (provided by applicant): Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) mediate the hyperpolarization-activated current (Ih) and are important for regulating excitability in CA1 pyramidal neurons of the hippocampus. Because localization governs h channel function in the homeostasis of neuronal excitability, and h channel dysfunction has been implicated in temporal lobe epilepsy, our longterm goal is to understand the unknown trafficking and targeting mechanism of h channels in the hippocampus, h channels of CA1 pyramidal neurons as well as neocortical layer V neurons exhibit a striking distal dendritic enrichment (DDE), shown to be critical for control of cellular excitability and synchrony. One molecular candidate implicated in h channel trafficking is the tetratricopeptide repeat-containing (TPR) Rab8b interacting protein (TRIPSb). TRIPSb is the only known interactor of h channels to demonstrate pyramidal neuron DDE in both the hippocampus and cortex mimicking that of HCN1 and 2. Additionally, TRIPSb has been shown to regulate Ih density in oocytes. TRIPSb interacts with h channels via its conserved C-terminal TPR domains in a manner homologous to the peroxisomal import receptor proteins. However, the N-terminal region of TRIPSb shares no homology with any known protein and its function is unknown. Our preliminary data indicate the N-terminus is highly alternatively spliced in a developmentally regulated manner, whereby specific isoforms are upregulated concurrent with DDE onset. Notably, alternative splicing alters the presence of cellular sorting signals thought to be important in trafficking. Because of this considerable but indirect evidence associating TRIPSb with h channel trafficking, we reason that TRIPSb plays an important role in the establishment and/or maintenance of h channel DDE in the hippocampus. To determine TRIPSb's role in hippocampal DDE, we propose the following specific aims: 1) Determine whether TRIPSb in general, and the N-terminus of TRIPSb in particular, are required for h channel DDE in CA1 pyramidal neurons. We will use lentiviral delivery of shRNA and dominant negative TRIPSb constructs in both slice culture and the living animal to determine the effect on h channel DDE in the hippocampus in vivo. 2) Determine the role of TRIPSb N-terminal alternative splicing in h channel trafficking and DDE. We will use single-cell RT-PCR, biotinylation, and viral delivery of TRIPSb splice isoforms to determine how TRIPSb splicing impacts h channel surface trafficking and DDE. PUBLIC HEALTH RELEVANCE: epilepsy remains a major lifelong burden. By understanding how the brain regulates excitability, we will gain insight into the causes of epilepsy and mechanisms for targeted treatment.