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高能量细胞活动和代谢过程中维持ATP水平起重要作用。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和肌酸转运蛋白的基因突变是许多肌酸缺乏症的基础，但肌酸缺乏症导致这种显著脑缺陷的分子和细胞机制仍然难以捉摸。在对秀丽隐杆线虫进行遗传筛选以鉴定与磷脂酶Cp有冗余功能的基因时，我们分离出snf-3的一个等位基因，该等位基因编码NaVCI依赖的神经递质转运蛋白，与哺乳动物肌酸转运蛋白序列高度相似。我们假设磷酸肌醇和肌酸转运蛋白信号通路汇聚调节[Ca2"，稳态（振荡）所需的线虫的超行为，其排便周期。我们将使用遗传方法结合钙和IP3成像以及电生理和生化技术来测试这一点。在目的1中，我们提出表征SNF-3的生化特性及其在体内的亚细胞定位和功能。在目的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[Ca2+]稳态中的作用。最近的发现强调了肌酸对大脑健康的重要性。肌酸缺乏综合征患者有严重的神经系统疾病，补充肌酸可以保护中风和神经系统综合征患者的大脑。我们认为秀丽隐杆线虫的排便周期为分析肌酸转运体的分子和细胞生物学提供了一个强有力的模型系统。

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.