Identifying and Characterizing Histamine Transporters

组胺转运蛋白的鉴定和表征

• 批准号: 6623259
• 负责人: ANN E STUART
• 金额: $ 14.44万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-23 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623259
• 关键词: Cirripedia; Drosophilidae; Xenopus oocyte; acyltransferase; complementary DNA; complementary RNA; electrophysiology; gene expression; genetic library; histamine; horseshoe crabs; hypothalamus; in situ hybridization; inflammation; membrane transport proteins; molecular biology information system; molecular cloning; neurons; neurotransmitter transport; nucleic acid sequence; polymerase chain reaction; vasoconstriction

DESCRIPTION (provided by applicant): Histamine is a vitally important messenger in the human body: In the periphery, it regulates vascular permeability, vasoconstriction, inflammation, and acid secretion by the stomach; in the brain it is a neurotransmitter employed by hypothalamic cells that project widely throughout the brain and spinal cord, and even to the retina. While histamine receptors are well known, a histamine transporter has been elusive. We propose to clone and study histamine transporters from invertebrate photoreceptors, neurons that clearly and specifically take up histamine. We will begin with Drosophila (fly). Candidate sequences will be identified by screening the Drosophila database, then will be further screened for their presence in photoreceptors of Drosophila compound eyes using in situ hybridization. Positive results will be followed by obtaining the full length clone using the PCR reaction, converting the cDNA to cRNA, injecting it into oocytes, and assaying for both Na-dependent (3H)histamine uptake and histamine-induced currents. Alternatively, we will pursue initial results using cDNA libraries from the eyes of Balanus (barnacle) and Limulus (horseshoe crab). Full-length candidate genes, identified using PCR amplification of sequences from the libraries, will be injected into oocytes for assay of histamine transport. Another alternative is injection of barnacle or fly mRNA into oocytes followed by expression cloning. Successful cloning of an invertebrate histamine transporter will be followed by attempt to clone a possible mammalian homologue. Cloned histamine transporters, expressed in Xenopus oocytes, will be characterized using electrophysiological techniques. The substrate selectivity of these carriers will be determined. The substrate and ionic dependence of the carriers will be defined which should permit understanding of how these transporters control the extracellular substrate concentration. This is of particular interest in the case of the photoreceptors, where the extracellular histamine concentration is thought to be continually maintained in a range that activates postsynaptic receptors. Determining the voltage dependence of the transporter should provide greater understanding of the observation (in barnacle photoreceptors) that histamine uptake is greater with depolarization. The pharmacology of each transporter will also be described, with special attention to drugs known to affect mental function. Successful cloning of a mammalian homologue would open the door to localizing this transporter within regions and cell types of the brain and determining whether it is present in other histamine-containing cells of the body.

描述（申请人提供）：组胺是一个非常重要的使者 在人体中：在外围，它调节血管渗透性， 胃的血管收缩，炎症和酸分泌；在大脑中 它是下丘脑细胞使用的神经递质，可广泛投射 在整个大脑和脊髓，甚至是视网膜。而组胺 受体是众所周知的，一种组胺转运蛋白是难以捉摸的。我们建议 克隆并研究来自无脊椎动物感光体的组胺转运蛋白， 明确，特别具体服用组胺的神经元。我们将从 果蝇（飞）。候选序列将通过筛选来确定 果蝇数据库，然后将进一步筛选其存在 果蝇复合眼的感光体使用原位杂交。 阳性结果将在使用该阳性的克隆中使用 PCR反应，将cDNA转化为CRNA，将其注入卵母细胞，然后 分析Na依赖性（3H）组胺摄取和组胺诱导的分析 电流。或者，我们将使用cDNA库取得初始结果 从Balanus（Barnacle）和Limulus（马蹄蟹）的眼睛。全长 候选基因，使用来自PCR的PCR扩增 图书馆将被注入卵母细胞，以进行组胺转运。 另一种选择是随后注射藤壶或将mRNA飞入卵母细胞 通过表达克隆。成功克隆无脊椎动物组胺 转运蛋白将尝试克隆可能的哺乳动物 同源物。克隆的组胺转运蛋白以爪蟾卵母细胞表达，将是 使用电生理技术进行表征。底物选择性 这些载体将被确定。底物和离子依赖性 将定义运营商，这应该允许了解这些 转运蛋白控制细胞外底物浓度。这是 在细胞外的光感受器的情况下特别感兴趣 组胺浓度被认为不断保持在一个范围内 激活突触后受体。确定电压依赖性 转运蛋白应提供对观察的更多了解（在 藤壶光感受器），随着去极化的变化，组胺摄取更大。 还将描述每个转运蛋白的药理学，并具有特殊 注意已知会影响心理功能的药物。成功克隆 哺乳动物同源物将为将该转运蛋白定位在内 大脑的区域和细胞类型，并确定它是否存在 人体的其他含组胺细胞。