Molecular Mechanisms of Ion Transport by the SMG

描述（由申请人提供）：液体和电解质运输是唾液腺（SG）的核心功能，对口腔健康至关重要。SG分泌的一个关键方面是CFTR起核心作用的导管分泌近140 mM的HCO3-。除了作为Cl通道外，CFTR还调节几种Cl和HCO3-转运蛋白在管腔膜上的活性，包括新发现的CI/HCO3和I-SLC26转运蛋白（SLCTs）。另一个从未被研究过的生理和临床重要功能是肠管的i转运和体内平衡。在甲状腺癌的1311治疗过程中，SGs被破坏。我们在本课题中的贡献是a) slc26t的电致性，b) slc26t在导管HCO3-分泌中的作用以及CFTR对其的调节，c) slc26t对CFTR的调节，d) i转运体slc26a4在SG细胞中的表达。最近我们还发现了一种新的adp激活的H？腮腺腺泡和导管细胞的7HCO3-可渗透通路。这些发现导致了一种关于导管液体和HCO3-分泌的新假设，其中大部分HCO3-分泌是由CFTR调节的导管上不同的slc26t介导的。反过来，slc26t在静息状态下抑制CFTR活性，在受刺激状态下增强CFTR活性，这决定了唾液中最终的HCO3-含量。我们还提出了SG细胞转运i的新假设。我们将在WT、slc26a4-/-和slc26a6-/-小鼠的模型系统和天然SG细胞中验证这一假设，具体目的有三个：确定ADP活化的H？/HCO3-途径及其在导管HCO3分泌中的作用2. 用KO小鼠研究slc26a4和slc26a6在SG液和HCO3-分泌中的作用。我们还在原代培养中开发了密封管，其中转运蛋白可以被dicer siRNA敲除，并且我们可以直接测量液体和HCO3-分泌。SG SLC26Ts、pNBC和其他HCO3-转运体的系统KD将用于研究它们在导管分泌中的作用。3. 表征slc26a4作为i和HCO3-转运体的功能，并检查基底侧2Na/l转运体NIS和腔体slc26a4在SG i稳态和积累中的作用及其在SG i毒性中的作用。这项工作将促进我们对健康和疾病中SG液体和电解质分泌的理解，并首次揭示甲状腺癌放射治疗中SG i毒性的分子基础。

DESCRIPTION (provided by applicant): Fluid and electrolyte transport is the central function of salivary glands (SG) that is critical for oral health. A critical aspect of SG secretion is the secretion of nearly 140 mM HCO3- by the duct in which CFTR plays a central role. In addition to functioning as a Cl channel CFTR regulates the activity of several Cl and HCO3- transporters at the luminal membrane, including the newly discovered CI/HCO3 and I-SLC26 transporters (SLCTs). Another physiologically and clinically important function of the duct that has never been studied is I-transport and homeostasis. SGs are destroyed during 1311 therapy of thyroid cancers. Our contributions to this topic are a) the electrogenicity of the SLC26Ts, b) the role of the SLC26Ts in ductal HCO3- secretion and their regulation by CFTR, c) the regulation of CFTR by the SLC26Ts, d) expression of the I-transporter slc26a4 in SG cells. Recently we also found a novel ADP-activated H?7HCO3- permeable pathway in parotid acinar and duct cells. These findings led to a new hypothesis of ductal fluid and HCO3- secretion in which the bulk of HCO3- secretion is mediated by different SLC26Ts along the duct that are regulated by CFTR. In turn, the SLC26Ts suppress CFTR activity in the resting state and enhance CFTR activity at the stimulated state, which determines the final HCO3- content in the saliva. We also develop a new hypothesis of I-transport by SG cells. We will test this hypothesis in model systems AND NATIVE SG CELLS of WT, slc26a4-/- and slc26a6-/- mice in three Specific Aims: 1. Determine the transport mechanisms of the ADP- activated H?/HCO3- pathway and its role in ductal HCO3 secretion. 2. Study the role of slc26a4 and slc26a6 in SG fluid and HCO3- secretion using the KO mice. We also developed sealed ducts in primary culture in which transporters can be knocked down by dicer siRNA and in which we can measure directly fluid and HCO3- secretion. Systematic KD of SG SLC26Ts, pNBC and other HCO3- transporters will be used to study their role in ductal secretion. 3. Characterize the function of slc26a4 as an I-and HCO3- transporter and examine the role of the basolateral 2Na/l transporter NIS and the luminal slc26a4 in SG I-homeostasis and accumulation and their role in SG I-toxicity. The proposed work should advance our understanding of SG fluid and electrolyte secretion in health and disease and reveal for the first time the molecular bases for I-toxicity of SG during radiation therapy of thyroid cancers.