STRUCTURE/FUNCTION OF C1 CHANNEL GENES IN CILIARY BODIES

睫状体中 C1 通道基因的结构/功能

• 批准号: 2019753
• 负责人: MIGUEL COCA-PRADOS
• 金额: $ 26.44万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-06 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2019753
• 关键词: antisense nucleic acid; biological fluid transport; calmodulin dependent protein kinase; chloride channels; epithelium; gene expression; genetic regulatory element; genetic transcription; glaucoma; human genetic material tag; human tissue; ion transport; molecular pathology; nucleic acid sequence; phosphorylation; polymerase chain reaction; posttranslational modifications; protein kinase C; protein structure function; site directed mutagenesis; sodium potassium exchanging ATPase; tissue /cell culture; transcription factor; uvea ciliary body

DESCRIPTION (adapted from the applicant's abstract): The ocular ciliary epithelium is the site of aqueous humor secretion in the mammalian eye. The ciliary epithelium is also the main target tissue to reduce intraocular pressure in the treatment of glaucoma. The ionic composition of aqueous humor indicates that the major anion transported by the ciliary epithelium is chloride (Cl-) via Cl- channels. The activation of these Cl- channels have been observed by swelling nonpigmented ciliary epithelial cells, in vivo and in vitro, under anisosmotic conditions, and they are rate-determining the aqueous humor secretion during volume regulation. Two candidate genes had been identified to participate in volume regulation via chloride conductance in nonpigmented ciliary epithelial cells: a) the human homologue of pICln, a Cl- channel regulator, and b) the human homologous to the non-synaptic Cl-channel ClC-3. In this research grant the PI proposes that pICln, is the Cl-channel regulator of ClC-3 channels in NPE cells. The main objective of the proposed project is to analyze the relationship between structure and function of pICln, and ClC-3. To achieve this goal we intend the following: 1) The identification and characterization of unique domains of primary sequence in pICln (i.e. nucleotide binding site, Ca2+ binding sites, acidic domains, phosphorylation sites) and ClC-3 (i.e., phosphorylation sites for PKC, and Ca2+/calmodulin-dependent protein kinase II), to gain insight into the mechanisms underlying pICln and ClC-3 biogenesis, subunit assembly and interaction with other proteins. The generation of polyclonal antibodies to purified pICln and ClC-3 proteins expressed in bacteria and against synthetic peptides with help to elucidate their structural-functional properties. 2) Application of molecular strategies such as site specific mutagenesis in unique binding sites or phosphorylation sites residues of pICln and ClC-3 will permit to study these functional domains in response to hypotonic swelling stimuli. This type of approach, coupled with antisense strategy will help to gain insight into the mechanisms underlying Cl-transport in the ciliary epithelium. Overexpression of pICln or ClC-3 in cells deficient in these channels also should permit to determine whether the kinetic behavior and current-voltage relationship correspond to the specific Cl-channel. 3) To determine whether there is differential gene expression of pICln and ClC-3 along the distinct regional areas or segments of the human ciliary epithelium, namely the pars plicata and pars plana, by applying reverse transcript PCR. The goal of these studies will be to obtain basic information about pICln and ClC-3 gene expression, distribution of NPE and PE cells and whether anisosmotic treatments can lead to regulation of pICln and ClC-3 at the transcriptional and/or posttranslational level. 4) To determine the organization of the human pICln and ClC-3 genes at their 5' flanking region. The goal in these studies will be to obtain information from genomic clones of the putative regulatory elements present in these genes.

描述（改编自申请人的摘要）：眼部睫状 上皮是哺乳动物眼中水性幽默分泌的位置。 这 睫状上皮也是降低眼内的主要目标组织 青光眼治疗的压力。 水的离子成分 幽默表明由睫状上皮运输的主要阴离子 通过Cl-通道为氯化物（Cl-）。 这些Cl-通道的激活 已经通过肿胀的非色素睫状上皮细胞观察到 体内和体外，在动态性条件下，它们是 在体积调节过程中对水性幽默的分泌进行评分。 二 已确定候选基因通过 非色素睫状上皮细胞中的氯化物电导：a）人类 Cl-Channel调节器Picln的同源物，b）人类同源 非突触CL通道CLC-3。 在这项研究中，PI提出了 该PICLN是NPE细胞中CLC-3通道的CL通道调节剂。 这 拟议项目的主要目的是分析关系 PICLN的结构和功能和CLC-3之间。 为了实现这一目标，我们 打算以下内容：1）唯一的识别和表征 PICLN中原代序列的结构域（即核苷酸结合位点，Ca2+ 结合位点，酸性结构域，磷酸化位点）和CLC-3（即 PKC的磷酸化位点和Ca2+/钙调蛋白依赖性蛋白激酶 ii），以深入了解PICLN和CLC-3的机制 生物发生，亚基组装以及与其他蛋白质的相互作用。 这 纯化PICLN和CLC-3蛋白的多克隆抗体产生 在细菌中表达和反对合成肽，有助于阐明 它们的结构功能。 2）分子的应用 诸如独特结合位点中特定诱变的策略或 PICLN和CLC-3的磷酸化位点残基将允许研究这些 功能域响应低音肿胀刺激。 这类 方法，加上反义策略将有助于了解 睫状上皮中的Cl-Transport的机制。 在这些通道中缺乏细胞中PICLN或CLC-3的过表达也 应允许确定动力学行为和电流电压是否 关系对应于特定的CL通道。 3）确定是否 PICLN和CLC-3沿不同的基因表达差异 人类睫状上皮的区域区域或细分市场，即pars Plicata和Pars Plana，通过应用逆转录pcr。 目标 这些研究将获得有关PICLN和CLC-3基因的基本信息 表达，NPE和PE细胞的分布以及是否存在过度 治疗可以导致在转录上调节PICLN和CLC-3 和/或翻译后水平。 4）确定组织 人PICLN和CLC-3基因在其5'侧翼区域。 这些目标 研究将是从推定的基因组克隆中获取信息 这些基因中存在的调节元件。