Alternate CI-secretory pathways in cystic fibrosis

囊性纤维化（CF）表型的特征在于分泌性上皮细胞中顶端膜Cl-渗透性的调节缺陷，其与液体分泌减少相关。最近的研究Cl-分泌上皮细胞培养的特点是调节阴离子通道的细胞体积的变化，增加细胞内游离Ca 2+，并增加细胞内的环核苷酸。这是我们的假设，钙依赖性氯离子通道（I-Cl，Ca）将提供一个替代的调节氯离子传导途径，以纠正CF影响细胞的转运功能障碍。该途径在CF组织中没有缺陷，并且事实上可能在人气道组织中上调。分泌上皮中的Ca 2+依赖性Cl-传导可以分为两个不同的群体：直接受Ca 2+调节的群体和受CaMKII抑制剂抑制的群体。在初步研究中，我们已经从人结肠细胞系T84克隆了ClC-3氯离子通道的人同种型，并进行了电生理学研究，证明CaMKII参与hClC-3的激活。在本研究中，我们将通过研究其门控机制和药理学特性来进一步表征克隆的通道和I-Cl，CaMKII。(1)我们将通过反义和敲除小鼠来检测hClC-3和I-Cl、CaMK II的同一性以消除内源性hClC-3。我们将表征重组hClC-3对特异性CaMKII抑制剂的反应。(2)我们将使用已建立的细胞系和来自ClC-3（-/-）敲除小鼠的原代培养物，使用全细胞和单通道分析来确定CaMK II在通道门控和/或易位中的作用。(3)我们将通过定点诱变和磷酸化测定以及电生理学研究进一步研究CaMKII磷酸化在hClC-3活化中的作用。我们将评估的生理作用和可能的相互作用之间的磷酸化依赖性激活的通道CaMKII和抑制通道激活膜联蛋白IV和IP 4。(4)我们将研究通过组成型活性CaMKII或肽来增加CF细胞中的I-Cl、CaMKII的方法，所述肽阻断hClC-3与其特异性抑制剂（例如膜联蛋白IV和IP 4）之间的相互作用。本研究的最终目标是提供候选细胞内治疗靶点，这可能有助于改善与CF相关的慢性病理状态。

The cystic fibrosis (CF) phenotype is characterized by a regulatory defect in apical membrane Cl- permeability in secretory epithelia associated with a reduction in fluid secretion. Recent investigations on Cl- secretory epithelial cells in culture have characterized the regulation of anion channels by changes in cellular volume, increases in intracellular free Ca2+, and increases in intracellular cyclic nucleotides. It is our hypothesis that Ca2+-dependent Cl- channels (I-Cl, Ca) will provide for an alternative regulated Cl- conductance pathway to correct the transport dysfunction in CF affected cells. This pathway is not defective in CF tissue and may, in fact, be upregulated in human airway tissue. Ca2+-dependent Cl- conductances in secretory epithelium can be separated into two distinct populations: those that are directly regulated by Ca2+ and those which are inhibited by CaMKII inhibitors. In preliminary studies we have cloned the human isoform of the ClC-3 chloride channel from the human colonic cell line T84, and carried out electrophysiological studies which demonstrate that CaMKII is involved in the activation of hClC-3. In the proposed studies, we will further characterize the cloned channel and I-Cl,CaMKII by investigating its gating mechanism and pharmacological properties. (1) We will examine the identity of hClC-3 and I-Cl,CaMKII by antisense and knock-out mice to eliminate endogenous hClC-3. We will characterize the response of recombinant hClC-3 to specific CaMKII inhibitors. (2) We will determine the role of CaMKII in gating and/or translocation of the channel using established cell lines and primary culture from ClC-3 (-/-) knockout mice using whole cell and single channel analysis. (3) We will further investigate the role of CaMKII phosphorylation in the activation of hClC-3 by site-directed mutagenesis and phosphorylation assays as well as electrophysiological studies. We will evaluate the physiological role and possible interplay between phosphorylation-dependent activation of the channel by CaMKII and the inhibition of channel activation by annexin IV and IP4. (4) We will investigate an approach to augment I-Cl,CaMKII in CF cells via either a constitutively active CaMKII or peptide that blocks the interaction between hClC-3 and its specific inhibitors, such as annexin IV and IP4. The ultimate goal of this investigation is to provide candidate intracellular therapeutic targets which may be useful in ameliorating the chronic pathological condition associated with CF.