氨基端结构域在NBCe1功能调制中的作用及机制研究

The Na-coupled HCO3– transporters (NCBTs) of SLC4 family include two electrogenic members NBCe1 (SLC4A4) and NBCe2 (SLC4A5) as well as three electroneutral members NBCn1 (SLC4A7), NBCn2 (SLC4A10), and NDCBE (SLC4A8). These NCBTs are widely expressed in diverse tissues and play critical physiological and pathological roles in the body. Dysfunctions of the NCBTs are associated with a series of diseases in human, such as metabolic acidosis (the hallmark phenotype), mental retardation, epilepsy, migraine, hypertension, breast cancer, etc. The NCBTs contain a large (~60kDa) intracellular amino-terminal (Nt) domain accounting for ~45% of the full-length polypeptide of the transporters. However, the 3-dimensional structure of this Nt domain and its role in the ion-transport machinery of NCBTs remains mystic. Our preliminary study showed that the isolated Nt domain of NBCn2, as an independent “subunit”, was able to interact with the transporter and modulate its function, e.g., the surface expression in cells. Taking together the previous studies from our group as well as colleagues, we hypothesize that the Nt domain is a regulatory domain modulating the activity and the biophysical properties of the NCBTs and that it is not a component essential for the ion transport machinery. To test this hypothesis, we developed the present project using the electrogenic NBCe1 as a model molecule..Firstly, we will examine, by truncation mutation, the role of different portion of the Nt domain in the ion-transport activity as well as the surface expression of NBCe1. Moreover, we will examine whether coexpressing the isolated Nt domain and the transmembrane domain (TMD) is able to reconstitute the full activity of wild type NBCe1. These studies will address whether the Nt domain is necessary for the activity of NBCe1. And if it is, what is the minimal structural requirement?.Secondly, we will investigate the role of the Nt domain in the modulation of the biophysical properties of NBCe1, such as the single-molecule activity, sensitivity to intracellular as well as extracellular pH alteration, and substrate affinity of NBCe1. By point mutations based on the structure, we will further investigate the mechanism underlying such modulations by the Nt domain..Finally, we will employ structural biology approaches, including structural modeling as well as X-ray diffraction, to investigate the fine 3-D structure of the Nt domain of NBCe1..The study will provide critical insight for understanding the molecular mechanism underlying NCBT-mediated ion transport. It will also be important for NCBT-targeted drug design and development.

碳酸氢根转运体NCBT包括NBCe1等5个成员，具有重要的生理及病理学意义，其功能失常可导致代谢性酸中毒、智障、癫痫、偏头疼、高血压、肿瘤等一系列重大疾病。NCBT的胞内氨基端结构域Nt分子量约60kD，占全长多肽45%。Nt的三维结构及其在NCBT离子转运机制中的作用尚不清楚。综合我们的前期结果及文献分析，在此我们提出新假设：Nt结构域不是NCBT活性必需的结构，而是一个功能调控结构域，作为pH或底物浓度感受器对NCBT的活性及生物物理学特性进行调制。我们将以NBCe1为对象，通过结构生物学手段研究Nt的三维结构，通过突变结合电生理学、生化及细胞生物学技术研究Nt对NBCe1活性与表达定位的必要性，及其对NBCe1单分子活性、pH敏感性及底物亲和性等生物物理学特性的调制作用及机制。本研究对深入了解NCBT的工作机制将有重要的理论意义，对以NCBT为靶向的药物开发与设计亦有重要实用价值。

Na+/HCO3–共转运体（NBC）在pH调控及离子跨膜运输中具有重要的生理学作用。其中NBCe1-B，NBCn1，NBCn2等均含自抑制结构域（AID），存在自抑制现象，可被调控因子IRBIT激活。在本项目的资助下，我们在NBC的结构功能与功能调控机制，及NBC的生理学作用两方面取得了良好的进展。.在NBC的结构功能及功能调控机制方面，研究了自抑制结构域AID抑制NBCe1-B的机理，发现AID通过静电相互作用与跨膜区结合抑制转运体活性；IRBIT与AID竞争结合，使其从跨膜区解离，从而激活NBCe1-B。从爪蟾卵母细胞中克隆了两种IRBIT同源基因，过表达爪蟾IRBIT可激活小鼠NBCe1-B；发现IRBIT的Nt是一个重要的调节结构域，影响IRBIT与NBCe1-B互作。.在NBC的生理学作用方面，发现在大鼠肾脏中，NBCn2表达在近段肾小管顶膜，是一种新的完全不同于传统的依赖于NHE3的HCO3–重吸收途径。发现NBCn1和NBCn2在肾小管髓袢升支粗段可促进NH4+重吸收，抑制HCO3–及NaCl重吸收的作用。发现NBCn1是子宫内膜上皮细胞介导Na+和HCO3–吸收，参与维持子宫腔微环境液体酸碱平衡稳态调控。在大鼠小肠上皮细胞中克隆了一种新的NBCn2剪接异构体，发现NBCn2表达在小肠上皮细胞顶膜，参与NaCl吸收。.以上研究成果，部分已经在Frontiers in Physiology （2018、2019），Journal of the American Society of Nephrology（2017），BBA-Biomembranes（2020）等杂志上。部分结果论文正在审稿中。通过项目的研究，培养博士生2人，硕士生3人。在读博士5人，在读硕士4人。课题组成员参加国际国内会议24人次。