STRUCTURE/FUNCTION OF C1 CHANNEL GENES IN CILIARY BODIES

描述(改编自申请人的摘要)：眼部纤毛 上皮细胞是哺乳动物眼内房水分泌的场所。这个 睫状体上皮也是减少眼内压力的主要靶组织 青光眼治疗中的压力。水的离子组成 体液显示睫状体上皮运输的主要阴离子 是氯(Cl-)，通过氯离子通道。这些氯离子通道的激活 通过肿胀无色素的睫状体上皮细胞观察到 在体内和体外，在非等渗条件下，它们是 在容量调节过程中决定房水分泌的速率。二 已确定的候选基因通过以下途径参与体积调节 非色素睫状体上皮细胞的氯离子电导：a)人 氯离子通道调节剂pICln的同源物和b)人的同源物 非突触氯通道ClC-3。在这项研究拨款中，国际和平基金会建议 PICln是NPE细胞ClC-3通道的氯离子通道调节剂。这个 拟议项目的主要目标是分析 PICln和ClC-3的结构和功能之间的关系。为了实现这一目标，我们 意在：1)独一无二的鉴定和表征 PICln中主要序列的结构域(即核苷酸结合位点，钙 结合部位、酸性结构域、磷酸化部位)和ClC-3(即， 蛋白激酶C的磷酸化位点和钙/钙调素依赖的蛋白激酶 二)，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。这个 纯化pICln和ClC-3蛋白多克隆抗体的制备 在细菌中的表达和针对合成肽的表达有助于阐明 它们的结构-功能特性。2)分子的应用 策略，如在唯一结合位点上的定点突变或 PICln和ClC-3的磷酸化位点残基可以用来研究这些 功能域对低张肿胀刺激的反应。这种类型的 方法，再加上反义策略，将有助于深入了解 睫状体上皮氯离子转运的潜在机制。 PICln或ClC-3在缺乏这些通道的细胞中的过表达 应允许确定动力学行为和电流-电压 关系对应于特定的氯离子通道。3)确定是否 PICln和clc-3基因在不同的肿瘤细胞中表达差异。 人睫状体上皮的区域或节段，即纤毛部分 皱褶和扁平部，应用逆转录-聚合酶链式反应。的目标是 这些研究将获得有关pICln和clc-3基因的基本信息。 NPE和PE细胞的表达、分布及是否不等渗 处理可导致pICln和ClC-3在转录水平上的调节 和/或翻译后水平。4)确定组织机构 人类pICln和clc-3基因位于其5‘侧翼区。其中的目标是 研究将从推定的基因组克隆中获得信息 这些基因中存在调控元件。

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.