REGULATION OF NA TRANSPORT FUNCTION BY CFTR IN THYROID EPITHELIAL CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 甲状腺激素的生成需要摄取和释放碘进入甲状腺滤泡腔。碘的顶端退出被认为是由SLC26A4基因编码的阴离子交换蛋白Pendrin介导的。然而，有几条证据表明，顶端碘外流的替代途径存在于甲状腺中。 通过研究现有文献，以及挖掘微阵列数据库，我们确定了潜在的介导碘外流的转运体。它们包括囊性纤维化跨膜电导调节器(CFTR)和ClC-5，这两个基因都是先前在甲状腺组织中发现的Cl-/H+交换器。从野生型和CFTR-/-猪甲状腺培养极化原代培养物，以便于使用短路电流(ISC)测量和125I通量来评估阴离子传输。ISC研究表明，cAMP激活的阴离子(氯化物和碘化物)的分泌在CFTR-/-猪的甲状腺培养中被取消。野生型甲状腺表达另一种候选阴离子转运蛋白SLC26A7，分别使用qRT-PCR和免疫印迹方法测量其mRNA和蛋白质水平。有趣的是，在CFTR-/-甲状腺培养中，SLC26A7蛋白的表达被取消，尽管mRNA的表达没有显著差异。SLC26A7电流的生物物理和药理学特征将在表达系统和最终信息中进一步研究，以探索其在甲状腺培养物中的极化表达，以及其对碘分泌的功能影响。此外，未来使用极化原代培养的研究将测试ClC-5和/或SLC26A7 shRNA敲除的效果，以评估它们对载体碘运输的相对贡献。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Thyroid hormonogenesis requires iodide uptake and release into the thyroid follicular lumen. The apical exit of iodide is thought to be mediated by Pendrin, an anion exchanger encoded by the SLC26A4 gene. However, several lines of evidence suggest that alternative pathways for apical iodide efflux exist in the thyroid gland. By researching the existing literature, as well as by mining microarray databases, we identified transporters that potentially mediate iodide efflux. These include the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and ClC-5, a Cl-/H+ exchanger, both of which previously have been identified in thyroid tissue. Polarized primary cultures were grown from wild type and CFTR -/- pig thyroids to facilitate evaluations of anion transport using short-circuit current (Isc) measurements as well as 125I fluxes. Isc studies indicate the presence of cAMP-activated anion (chloride and iodide) secretion that is abolished in CFTR-/- pig thyroid cultures. Wild type thyroid expresses SLC26A7, another candidate anion transport protein, based on measurements of both the mRNA and protein levels, using qRT-PCR and immunoblotting, respectively. Interestingly, SLC26A7 protein expression is abolished in CFTR -/- thyroid cultures, although mRNA expression did not differ significantly. The biophysical and pharmacological profile of SLC26A7 currents will be further examined in expression systems and resultant information exploited to probe not only its polarized expression in thyroid cultures, but also its functional impact on iodide secretion. In addition, future studies using polarized primary cultures will test the effects of ClC-5 and/or SLC26A7 shRNA knockdown, to evaluate their relative contributions to vectorial iodide transport.