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CA 1锥体神经元的兴奋性非常重要。由于定位支配h通道在神经元兴奋性的稳态中的功能，并且h通道功能障碍与颞叶癫痫有关，因此我们的长期目标是了解海马中h通道的未知运输和靶向机制，CA 1锥体神经元以及新皮层V层神经元的h通道表现出显著的远端树突富集（DDE），对控制细胞兴奋性和同步性至关重要。参与h通道运输的一种分子候选物是含有（TPR）Rab 8b相互作用蛋白（TRIPSb）的四方三肽重复序列。TRIPSb是唯一已知的h通道的相互作用，以证明海马和皮质中的锥体神经元DDE模仿HCN 1和2。此外，TRIPSb已被证明可以调节卵母细胞中的Ih密度。TRIPSb通过其保守的C-末端TPR结构域以与过氧化物酶体输入受体蛋白同源的方式与h通道相互作用。然而，TRIPSb的N-末端区域与任何已知的蛋白质没有同源性，并且其功能未知。我们的初步数据表明，N-末端是高度选择性剪接的发育调节的方式，从而特定的亚型上调与DDE发作的同时。值得注意的是，选择性剪接改变了细胞分选信号的存在，这些信号被认为在贩运中很重要。由于这个相当大的，但间接的证据与TRIPSb的h通道贩运，我们的理由，TRIPSb起着重要的作用，在建立和/或维持h通道DDE在海马。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TRIPSb在一般情况下，特别是TRIPSb的N-末端，是否需要在CA 1锥体神经元h通道DDE。我们将在切片培养物和活体动物中使用慢病毒递送shRNA和显性阴性TRIPSb构建体来确定对体内海马中h通道DDE的影响。2)确定TRIPSb N端选择性剪接在h通道运输和DDE中的作用。我们将使用单细胞RT-PCR，生物素化和TRIPSb剪接异构体的病毒递送来确定TRIPSb剪接如何影响h通道表面运输和DDE。

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.