Alternate CI-secretory pathways in cystic fibrosis

囊性纤维化（CF）表型的特征是分泌上皮的顶端膜Cl-通透性的调节缺陷与液体分泌减少有关。最近对培养的Cl-分泌上皮细胞的研究表明，阴离子通道的调节通过细胞体积的变化、细胞内游离Ca2+的增加和细胞内环核苷酸的增加来实现。我们的假设是，Ca2+依赖的Cl-通道（I-Cl, Ca）将提供另一种调节的Cl-传导途径，以纠正CF影响细胞的运输功能障碍。这一途径在CF组织中没有缺陷，实际上可能在人气道组织中被上调。分泌上皮中Ca2+依赖性Cl-电导可分为两种不同的人群：直接受Ca2+调节的人群和受CaMKII抑制剂抑制的人群。在初步研究中，我们从人结肠细胞系T84中克隆出了hcc -3氯通道的人异构体，并进行了电生理研究，证明CaMKII参与了hcc -3的激活。在未来的研究中，我们将通过研究I-Cl，CaMKII的门控机制和药理特性来进一步表征克隆通道和I-Cl，CaMKII。(1)我们将通过反义和敲除小鼠来检测hClC-3和I-Cl、CaMKII的身份，以消除内源性hClC-3。我们将描述重组hClC-3对特定CaMKII抑制剂的反应。(2)我们将利用全细胞和单通道分析，利用已建立的细胞系和原代培养的ClC-3（-/-）敲除小鼠，确定CaMKII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实验以及电生理研究进一步研究CaMKII磷酸化在hClC-3激活中的作用。我们将评估CaMKII磷酸化依赖性通道激活与膜联蛋白IV和IP4抑制通道激活之间的生理作用和可能的相互作用。(4)我们将研究一种增加CF细胞中I-Cl、CaMKII的方法，通过组成型活性CaMKII或阻断hClC-3与其特异性抑制剂（如膜联蛋白IV和IP4）之间相互作用的肽。这项研究的最终目的是提供候选的细胞内治疗靶点，这可能有助于改善与CF相关的慢性病理状况。

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.