Molecular Characterization of the Sodium/Iodide Symporter (NIS)

描述（由申请人提供）：Na+/I-同向转运体（NIS）是介导甲状腺主动I-转运的关键质膜蛋白，是甲状腺激素生物合成的第一步。NIS也是有史以来设计的最成功的靶向内部放射癌症治疗的中心分子：甲状腺切除术后给予的甲状腺癌131 I治疗。该项目的最初启动导致了编码NIS的cDNA的克隆，这是数十年来一直寻求的甲状腺研究的重大突破。从那时起，该项目（在随后的资助周期中）取得了许多其他重大进展，包括大量关于NIS的结构/功能和机制信息，以及关于其调节，生物发生，翻译后修饰和甲状腺外组织表达的高度揭示性发现，包括原发性和转移性乳腺癌。我们还发现，NIS运输不同的阴离子底物与不同的化学计量，一个重要的机械性能，尚未被描述为任何其他转运。目前的建议集中在一个新发现的监管串扰相互作用，发生在甲状腺细胞之间的NIS和K+通道，KCNQ 1-KCNE 2，从而开始了新的篇章，在我们的研究NIS的调节。这种NIS/KCNQ 1-KCNE 2调节相互作用是第一个被确定的转运蛋白/通道相互作用。KCNQ 1是心室复极化所必需的电压门控钾（Kv）通道α亚基，而通道β亚基KCNE 2是可以调节KCNQ 1的基因家族的五个成员之一的产物。KCNQ 1和KCNE 2在人心室肌细胞中各自发挥重要作用，但我们发现它们也在甲状腺细胞的质膜以及其他上皮组织中共同形成异聚体通道。令人惊讶的是，敲除KCNQ 1或KCNE 2基因的小鼠，除了预期的心脏表现外，也会出现甲状腺功能减退，因为KCNQ 1-KCNE 2通过增加NIS活性在甲状腺功能中起着至关重要的作用。我们还表明，NIS反过来又刺激KCNQ 1-KCNE 2的活性。本研究的目的是阐明NIS和KCNQ 1-KCNE 2之间双向调控相互作用的潜在机制，并研究其他KCNE亚基在NIS调控和甲状腺生理中的作用。本研究的主要目的是：1.确定NIS/Q1-E2相互作用在生物合成途径中的发生时间和位置。2.验证了Q1-E2引起NIS活性增加是由于Q1-E2引起的局部膜电位（Δ E）变化所致的假说。3.确定NIS和Q1的哪些结构域参与这两种蛋白质之间的相互作用。4.阐明KCNE辅助亚基1、3、4和5在Q1调节NIS活性和甲状腺功能以及反过来NIS调节Q1活性中的作用。这项研究可能会激发人们对其他转运蛋白-通道相互作用的分析兴趣，这也可能被证明具有生理和临床意义。

DESCRIPTION (provided by applicant): The Na+/I- symporter (NIS) is the key plasma membrane protein that mediates active I- transport in the thyroid, the first step in the biosynthesis of the thyroid hormones. NIS is also the molecule at the center of the most successful targeted internal radiation cancer treatment ever devised: 131I- therapy of thyroid cancer administered post-thyroidectomy. The initial launching of this project resulted in the cloning of the cDNA that encodes NIS, a major breakthrough in thyroid research that had been sought for decades. Since then, this project has (in subsequent funding cycles) yielded many other major advances, including a wealth of structure/function and mechanistic information on NIS, and highly revealing findings about its regulation, biogenesis, posttranslational modifications, and expression in extrathyroidal tissues, including primary and metastatic breast cancer. We have also discovered that NIS transports different anion substrates with different stoichiometries, a significant mechanistic property that has not been described for any other transporters to date. The current proposal focuses on a newly discovered regulatory crosstalk interaction that takes place in thyroid cells between NIS and a K+ channel, KCNQ1-KCNE2, thereby beginning a new chapter in our study of NIS regulation. This NIS/KCNQ1-KCNE2 regulatory interaction was the first transporter/channel interaction ever to be identified. KCNQ1 is a voltage-gated potassium (Kv) channel α-subunit essential for the repolarization of the cardiac ventricles, and KCNE2, the channel β-subunit, is the product of one of five members of a gene family that can regulate KCNQ1. KCNQ1 and KCNE2 each perform essential roles in human ventricular myocytes, but we discovered that they also form a heteromeric channel together in the plasma membrane of thyrocytes, as well as in other epithelial tissues. Surprisingly, knockout mice devoid of either KCNQ1 or KCNE2, in addition to the expected cardiac manifestations, also develop hypothyroidism, because KCNQ1-KCNE2 plays a crucial role in thyroid function by increasing NIS activity. We have also shown that NIS, in turn, stimulates the activity of KCNQ1-KCNE2. The goal of this proposal is to elucidate the underlying mechanisms of the bi-directional regulatory interaction between NIS and KCNQ1-KCNE2 and to investigate the role of the other KCNE subunits in NIS regulation and thyroid physiology. We will pursue the following Aims: 1.To ascertain when and where in the biosynthetic pathway the NIS/Q1-E2 interaction occurs. 2.To test the hypothesis that the increase in NIS activity caused by Q1-E2 results from a local change in the membrane potential (Δψ) brought about by the activity of Q1-E2. 3. To determine which domains of NIS and Q1 participate in the interaction between these two proteins. 4. To elucidate the roles of KCNE ancillary subunits 1, 3, 4, and 5 in the regulation of NIS activity and thyroid function by Q1 and, conversely, of Q1 activity by NIS. This research is likely to spur interest in the analysis of other such transporter-channel interactions, which may also prove to be physiologically and clinically significant.