STRUCTURE/FUNCTION OF C1 CHANNEL GENES IN CILIARY BODIES

描述（改编自申请人的摘要）：眼睫状体 上皮是哺乳动物眼睛中的房水分泌部位。 的 睫状体上皮也是减少眼内压的主要靶组织。 青光眼的治疗方法有哪些 水溶液的离子组成 体液表明睫状体上皮转运的主要阴离子 是氯离子（Cl-）通过Cl-通道。 这些氯离子通道的激活 已经通过肿胀的非色素睫状上皮细胞观察到， 在体内和体外，在各向异性条件下， 在容量调节过程中决定房水分泌的速率。 两 候选基因已被确定参与体积调节， 无色素睫状上皮细胞的氯离子电导：a）人 pICln的同源物，一种Cl-通道调节剂，和B）人同源物 非突触Cl-通道ClC-3。 在这项研究资助中，PI建议 pICln是NPE细胞中ClC-3通道的Cl-通道调节剂。 的 该项目的主要目的是分析 pICln和ClC-3的结构和功能之间的联系。 为了实现这一目标，我们 目的如下：1）识别和表征独特的 pICln中一级序列的结构域（即核苷酸结合位点，Ca 2 + 结合位点、酸性结构域、磷酸化位点）和ClC-3（即， PKC和Ca 2 +/钙调蛋白依赖性蛋白激酶的磷酸化位点 II），以深入了解pICln和ClC-3的潜在机制 生物发生、亚基组装和与其他蛋白质的相互作用。 的 针对纯化的pIClin和ClC-3蛋白的多克隆抗体的产生 在细菌中表达并对抗合成肽，有助于阐明 它们的结构-功能特性。 2)应用分子 例如在独特结合位点中的位点特异性诱变的策略， pICln和ClC-3的磷酸化位点残基将允许研究这些 功能域的低渗肿胀刺激的反应。 这种类型的 方法，加上反义策略将有助于深入了解 睫状体上皮Cl-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也 应允许确定是否动力学行为和电流-电压 关系对应于特定的Cl-通道。 3)以确定是否 pICln和ClC-3沿着不同的基因表达， 人睫状上皮的局部区域或节段，即睫状体部 皱襞和睫状体平坦部的组织学特征。 的目标 这些研究将有助于获得pICln和ClC-3基因的基本信息 NPE和PE细胞的表达、分布以及是否为非等渗性 处理可以导致pICln和ClC-3在转录水平的调节， 和/或翻译后水平。 4)为了确定组织机构， 人pIClin和ClC-3基因的5'侧翼区。 在这些目标 研究将是从假定的基因组克隆中获得信息， 这些基因中存在的调控元件。

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.