Characterization of the Na+/Cl--dependent Neurotransmitter Transporter (SNF-3)

描述（由申请人提供）：肌酸及其磷酸肌酸形式在维持高能细胞活动和代谢期间的ATP水平方面发挥重要作用。在人类中，肌酸缺乏综合征的特征是中度至重度智力迟钝、言语和语言迟缓、癫痫和自闭症行为。编码肌酸生物合成酶的基因以及肌酸转运蛋白的突变是许多肌酸缺乏症的基础，但肌酸缺乏症导致如此突出的脑缺陷的分子和细胞机制仍然难以捉摸。同时对C.为了鉴定与磷脂酶Cp具有冗余功能的基因，我们分离了snf-3的等位基因，其编码与哺乳动物肌酸转运蛋白具有高度序列相似性的NaVCI依赖性神经递质转运蛋白。我们假设磷酸肌醇和肌酸转运蛋白信号通路会聚以调节线虫超日行为所需的[Ca 2 +]、稳态（振荡）及其排便周期。我们将使用遗传方法结合钙和IP 3成像以及电生理和生化技术来测试这一点。在目标1中，我们提出了表征SNF-3的生化特性及其在体内的亚细胞定位和功能。在目标2中，我们将确定SNF-3在磷脂酶信号传导中的作用。最后，我们将描述SNF-3在[Ca 2 +]稳态中的作用。最近的发现强调了肌酸在大脑健康中的重要性。肌酸缺乏综合征患者有严重的神经系统疾病和肌酸补充剂保护中风和神经系统综合征患者的大脑。我们建议C. elegans排便循环代表了分析肌酸转运蛋白的分子和细胞生物学的强大模型系统。

DESCRIPTION (provided by applicant): Creatine and its phospho-creatine form play an important role maintaining ATP levels during high energy cellular activity and metabolism. In humans, creatine deficiency syndromes are characterized by moderate to severe mental retardation, speech and language delays, epilepsy and autistic behavior. Mutations in genes encoding the creatine biosynthetic enzymes as well as in the creatine transporter underlie many creatine deficiency disorders but the molecular and cellular mechanisms by which creatine deficiency leads to such prominent brain defects remain elusive. While conducting a genetic screen in C. elegans to identify genes that function redundantly with phosopholipase Cp, we isolated an allele of snf-3, which encodes a NaVCI" dependent neurotransmitter transporter with high sequence similarity to the mammalian creatine transporter. We hypothesize that phosphoinositol and creatine transporter signaling pathways converge to regulate [Ca2"], homeostasis (oscillation) required for a nematode ultradian behavior, its defecation cycle. We will test this using genetic methods in combination with calcium and IP3 imaging as well as electrophysiological and biochemical techniques. In aim 1, we propose to characterize the biochemical properties of SNF-3 and its subcellular localization and function in vivo. In aim 2, we will determine a role for SNF-3 in phospholipase signaling. Lastly, we will characterize the role of SNF-3 in [Ca2+], homeostasis. Recent discoveries highlight the importance of creatine in brain health. Patients with creatine deficiency syndromes have severe neurological disorders and creatine supplementation protects the brain of patients with stroke and neurological syndromes. We propose that C. elegans defecation cycle represents a powerful model system to analyze the molecular and cellular biology of the creatine transporter.