Molecular Characterization of the Sodium/Iodide Symporter (NIS)

描述（由申请人提供）：Na+/I-同调体（NIS）是介导甲状腺中I-转运的关键质膜蛋白，这是甲状腺激素生物合成的第一步。NIS也是迄今为止设计的最成功的靶向内放疗癌症治疗的核心分子：甲状腺癌切除术后的131I治疗。这个项目的最初启动导致了编码NIS的cDNA的克隆，这是几十年来甲状腺研究的一个重大突破。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，以及关于其调控、生物发生、翻译后修饰和甲状腺外组织（包括原发性和转移性乳腺癌）表达的高度揭示性发现。我们还发现NIS运输具有不同化学计量的不同阴离子底物，这是迄今为止尚未描述的任何其他转运蛋白的重要机制性质。目前的建议侧重于新发现的发生在甲状腺细胞中NIS和K+通道KCNQ1-KCNE2之间的调控串扰相互作用，从而开启了我们研究NIS调控的新篇章。这种NIS/KCNQ1-KCNE2调控相互作用是迄今为止发现的第一个转运体/通道相互作用。KCNQ1是电压门控钾（Kv）通道α-亚基，对心室复极至关重要，而KCNE2是通道β-亚基，是可以调节KCNQ1的基因家族中五个成员之一的产物。KCNQ1和KCNE2各自在人心室肌细胞中发挥重要作用，但我们发现它们也在甲状腺细胞质膜以及其他上皮组织中共同形成异质通道。令人惊讶的是，缺乏KCNQ1或KCNE2的敲除小鼠，除了预期的心脏表现外，还会发生甲状腺功能减退，因为KCNQ1-KCNE2通过增加NIS活性在甲状腺功能中起关键作用。我们还表明，NIS反过来刺激KCNQ1-KCNE2的活性。本研究的目的是阐明NIS与KCNQ1-KCNE2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。我们将努力实现以下目标：为了确定在生物合成途径中NIS/Q1-E2相互作用发生的时间和位置。2.为了验证Q1-E2引起的NIS活性增加是由于Q1-E2活性引起的局部膜电位变化（Δψ）的假设。3. 确定NIS和Q1的哪些结构域参与了这两个蛋白之间的相互作用。4. 阐明KCNE辅助亚基1、3、4和5在通过Q1调节NIS活性和甲状腺功能以及反过来通过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体-通道相互作用分析的兴趣，这也可能被证明具有生理学和临床意义。

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.