Characterization of the ubiquitous choline transporter

性状（由申请人提供）：胆碱是一种膳食必需营养素，是合成膜磷脂、甜菜碱、渗透压调节剂和神经递质乙酰胆碱所必需的。为了实现这些代谢作用，胆碱通过至少两种转运机制被递送到细胞中。一种机制是钠依赖性高亲和力机制，仅在含乙酰胆碱的神经元中发现。第二种机制是钠非依赖性低亲和力转运（LACT），存在于所有细胞中。目前对LACT蛋白知之甚少，甚至不知道是否存在多种LACT蛋白。其他人的生化研究和本实验室的证实表明，LACT是一种有机阳离子转运蛋白（OCT）。然而，LACT的底物特异性与已知OCT蛋白的底物特异性不匹配。通过对OCT基因家族的分析，我们鉴定了几个与已知OCT蛋白密切相关的未知基因。我们假设LACT基因是这些孤儿基因之一。本提案中的实验将测试这一假设，并包括2个具体目标，一个遗传和一个生化。1. NG108细胞系中孤儿OCT基因的表达将通过用siRNA寡核苷酸转染来消除。将在mRNA沉默的细胞中测量胆碱的转运，以确定LACT活性是否也降低。2.孤儿OCT基因将在酵母酿酒酵母中表达，并测试胆碱转运活性。这两个具体目标是互补的方法，将决定性地检验这一假设。如果孤儿基因不显示LACT活性，则将筛选表达新蛋白质的酵母对候选底物的摄取，以鉴定孤儿基因的生理底物。然后进行RNA沉默，以确认孤儿转运蛋白在已鉴定底物转运中的作用。这些实验将提供对这个重要基因家族的更好理解，并深入了解胆碱向细胞的传递。这些见解将提供途径，以扭转损失的膜完整性和受损的细胞功能的疾病，如中风。OCT蛋白功能的分析也将为阳离子药物的摄取、排泄和分布提供新的信息。

DESCRIPTION (provided by applicant): Choline is a dietary essential nutrient that is required for the synthesis of membrane phospholipids, betaine, osmolytes and the neurotransmitter acetylcholine. To carry out these metabolic roles, choline is delivered into cells by at least two transport mechanisms. One mechanism is a sodium-dependent high-affinity mechanism found only in acetylcholine-containing neurons. The second mechanism is a sodium-independent, low-affinity transport (LACT) that is present in all cells. Little is known about the LACT protein or even whether there might be multiple LACT proteins. Biochemical studies by others and confirmed by the present laboratory have shown that LACT is a type of organic cation transporter (OCT). However, the substrate specificity of LACT does not match that of known OCT proteins. By analysis of the OCT gene family, we identified several uncharacterized genes closely related to known OCT proteins. We hypothesize that the LACT gene is one of these orphan genes. The experiments in this proposal will test this hypothesis and consist of 2 specific aims, one genetic and one biochemical. 1. Expression of orphan OCT genes in the NG108 cell line, will be eliminated by transfection with siRNA oligonucleotides. Transport of choline will be measured in mRNA-silenced cells to determine if LACT activity is also reduced. 2. Orphan OCT genes will be expressed in yeast Saccharomyces cerevisiae and tested for choline transport activity. These two specific aims are complementary approaches that will test, decisively, the hypothesis. Should the orphan genes not display LACT activity, yeast expressing the novel proteins will be screened for uptake of candidate substrates in order to identify the physiological substrate(s) for the orphan genes. RNA silencing will then be done to confirm the role of the orphan transporter in transport of identified substrates. These experiments will provide a better understanding of this important gene family and insight into delivery of choline to cells. Such insights would provide avenues to reverse the loss of membrane integrity and impaired cellular function in disorders such as stroke. Analysis of OCT protein function will also provide new information on the uptake, excretion and distribution of cationic drugs.