Mechanisms of Ciliary Trafficking of the Olfactory CNG Channel

描述（由申请人提供）：本提案的总体目标是更好地理解蛋白质转运到嗅觉感觉神经元（OSN）纤毛的机制。纤毛是复杂的，微管丰富的，独特的细胞器适应不同的细胞功能。纤毛结构和功能的缺陷已被证明是许多人类疾病的基础。osn树突末端的感觉纤毛将信号分子区隔，包括环核苷酸门控（CNG）通道，这是气味诱导信号传导的主要目标。嗅觉CNG通道负责将来自气味受体的化学信号转化为在大脑中处理的动作电位。尽管对CNG通道的结构和功能有丰富的了解，但对其亚细胞靶向的机制知之甚少。蛋白质在纤毛内的运动是由鞭毛内运输（IFT）控制的，这是一个进化保守的过程，促进了货物沿着纤毛和鞭毛的运动。在哺乳动物系统中，异三聚体Kinesin II在IFT中起明显作用；然而，同源型KIF17在纤毛运输中的作用尚未确定。我在Martens博士的实验室进行的新发表的实验表明，KIF17是异源系统中嗅觉CNG通道的纤毛富集所必需的；然而，KIF17在天然嗅上皮中的作用尚不清楚。最近的研究表明，鼻呼吸道上皮细胞中另一种纤毛蛋白肾囊素1的正确靶向需要结合磷酸氟酸簇分类蛋白1 （PACS-1）。PACS-1是一种细胞内分选蛋白，与载货蛋白上酸性残基簇中磷酸化的丝氨酸残基结合。PACS-1已被证明与其他几种酸性含簇离子通道相互作用，但尚未确定PACS-1在嗅纤毛运输中的作用。我的假设是嗅觉CNG通道的纤毛靶向依赖于天然嗅觉上皮中的KIF17和PACS-1。具体目的1：研究KIF17在天然嗅觉感觉神经元嗅觉CNG通道纤毛运输中的作用。具体目标2：确定PACS-1对CNG通道纤毛靶向的影响。我将使用共聚焦成像、嗅觉功能测定和病毒表达系统来解决这些问题。我的建议为进一步阐明嗅觉CNG通道纤毛富集的机制提供了一个计划。表征通道靶向的一般机制将提供对纤毛转运的更好理解，并使我们进一步了解人类纤毛疾病的潜在病因。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a better understanding of the mechanisms underlying protein transport into olfactory sensory neuron (OSN) cilia. Cilia are complex, microtubule-rich, organelles uniquely adapted for diverse cellular functions. Defects in cilia structure and function have been shown to underlie a number of human diseases. Sensory cilia on dendritic endings of OSNs compartmentalize signaling molecules, including cyclic nucleotide-gated (CNG) channels that are the primary targets of odorant-induced signaling. Olfactory CNG channels are responsible for the translation of the chemical signal from the odorant receptor into action potentials that are processed in the brain. Despite a wealth of knowledge about CNG channel structure and function, little is known about the mechanisms of their subcellular targeting. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), an evolutionary conserved process that facilitates movement of cargo along cilia and flagella. In mammalian systems, the heterotrimeric Kinesin II plays a clear role in IFT; however, a role in ciliary transport has not yet been established for the homodirrieric KIF17. Newly published experiments I performed in Dr. Martens' laboratory have shown that KIF17 is required for ciliary enrichment of the olfactory CNG channel in a heterologous system; however, the role for KIF17 in native olfactory epithelium remains unknown. Recently it has been shown that the proper targeting of another ciliary protein, nephrocystin 1, in nasal respiratory epithelial cells requires the binding of phosphofurin acidic cluster sorting protein 1 (PACS-1). PACS-1 is an intracellular sorting protein that binds to phosphorylated serine residues contained in a cluster of acidic residues on the cargo protein. PACS-1 has been shown to interact with several other acidic cluster-containing ion channel, however no role has been defined for PACS-1 in olfactory cilia transport. It is my hypothesis that the ciliary targeting of olfactory CNG channels is dependent on both KIF17 and PACS-1 in native olfactory epithelium. The following specific aims are designed to test this hypothesis: Specific Aim 1: To investigate the role of KIF17 in the ciliary transport of the olfactory CNG channel in native olfactory sensory neurons. Specific Aim 2: To determine the effects of PACS-1 on CNG channel ciliary targeting. I will address these aims using confocal imaging, assays of olfactory function, and viral expression systems. My proposal provides a plan to further elucidate the mechanisms of ciliary enrichment of the olfactory CNG channel. Characterizing the general mechanisms of channel targeting will provide a better understanding of ciliary transport and give us further insight into the underlying etiologies of human ciliary disorders.