相互作用蛋白IRBIT调控碳酸氢根转运体NBCe1功能的分子机理

The electrogenic Na+/HCO3– cotransporter NBCe1 (SLC4A4) belongs to the HCO3– transporter superfamily of SLC4. NBCe1 plays an important role in acid-base balance regulation in intra- as well as extracellular fluid. It is of great significance in both physiology and pathophysiology. Mutations in SLC4A4 are associated with a series of human diseases, including severe metabolic acidosis, migraine, mental retardation, ocular abnormalities, short stature, bone development deficit, etc. Topologically, NBCe1 has a large intracellular amino-terminal domain, followed by a large transmembrane domain containing 14 transmembrane helices and then a short carboxyl-terminal domain. The unique portion of the Nt (Nt-VRB) of NBCe1-B, a specific variant of NBCe1, contains an auto-inhibitory domain (AID), removal of which greatly enhances the activity of NBCe1 by ~300%. Moreover, Nt-VRB contains an IRBIT-binding domain (IBD). IRBIT is a protein partner that can interact with and regulate the function of a broad range of proteins with highly diverse biological functions. IRBIT can stimulate the activity of NBCe1-B. However, it remains unknown what the molecular mechanism is underlying the regulation of NBCe1-B by IRBIT, and what the relationship is between IRBIT and AID. The present project is designed to address these questions. Here, we will investigate the structural mechanism underlying the specific binding of IRBIT to NBCe1-B by using structural approaches including circular dichroism and nuclear magnetic resonance in combination of electrophysiology. Moreover, we will examine whether the regulation of NBCe1 by IRBIT relies on the presence of AID, although it is not necessary for the binding of IRBIT, as demonstrated in our preliminary study. How would the AID affect the regulation of NBCe1 by IRBIT? Finally, we will examine the likely involvement of kinase in the regulation of NBCe1 by IRBIT. We will study the effect of IRBIT binding on the phosphorylation of NBCe1. We will identify the specific phosphorylation sites of NBCe1 by mass spectroscopy and examine the effect of mutations to the phosphorylation sites on the regulation of NBCe1 by IRBIT. The present study will shed light into understanding the structure and function, and molecular mechanism underlying the functional regulation of NBCe1.

钠离子碳酸氢根共转运体NBCe1（e1）是重要的pH调控蛋白，具有重要的生理及病理学作用，其基因突变导致一系列人类重大疾病。本项目拟研究e1的功能调控机理。e1氨基末端Nt含一个自抑制结构域AID，删除之可使e1活性增强约3倍。IRBIT参与调控多种功能各异的蛋白，具有广泛而重要的生物学作用。通过蛋白相互作用，IRBIT可增强e1活性约3倍。IRBIT调控e1的分子机理为何？其与AID是否及有何内在关联？本项目为研究这些问题而设计。我们将通过圆二色谱、核磁共振、电生理学等技术研究IRBIT与e1识别及结合的结构机理；通过基因突变结合电生理学、生化及免疫学等技术研究IRBIT对e1的调控与AID之间的关系；通过质谱分析鉴定e1的磷酸化靶点，突变靶点，通过电生理学研究分析IRBIT对e1的调控作用与e1磷酸化的关系。本研究对深入了解e1的功能调控机制及对基于e1的靶向应用开发将有重要意义。

Na+/HCO3–共转运体（NBC）属于SLC4家族，包括NBCe1、NBCe2、NBCn1、NBCn2、NDCBE 5个不同成员。NBC在体内具有十分广泛的表达与分布，具有极其重要的生理及病理学作用。在本项目的资助下，我们对NBC的结构功能、功能调控机制及NBC的生理学作用进行了深入研究，取得了多项重要成果。.在结构功能方面，我们深入研究了NBCe1离子跨膜转运的分子机制，阐明了NBCe1底物结合的动力学模型，发现在NBCe1中HCO3−结合先于Na+结合，Na+结合依赖于先结合的HCO3–。此外，我们鉴定了Na+和HCO3−在NBCe1中的底物结合位点，并阐明了NBCe1不同转运模式下的化学计量比。.在功能调控机制方面，我们研究了IRBIT激活NBC的分子机制。NBCe1、NBCn1、NBCn2等均存在自抑制现象，其氨基末端含有一个保守的自抑制结构域AID（auto-inhibitory domain）。我们以NBCe1为对象，发现AID通过静电相互作用力与跨膜区结合，阻碍跨膜区的构象转换，降低NBCe1的活性。IRBIT通过与跨膜区竞争结合AID，使AID从跨膜区解离，从而激活NBCe1。我们还发现，IRBIT可以显著激活NBCn1和NBCn2的活性。此外，我们还对磷酸化调控NBCe1活性的机制进行了研究。.在NBC的生理学作用方面，我们阐明了NBCn1是子宫内膜上皮细胞存在的一种HCO3–吸收途径，参与维持子宫腔微环境液体酸碱平衡稳态调控。其次，我们研究了NBC在肾脏酸碱平衡及水盐平衡调节中的作用。我们发现NBCn1和NBCn2在mTAL具有多重重要的生理学作用，在不同的生理学条件下，参与NH4+、HCO3–及NaCl重吸收调控。在NaCl重吸收中的作用，提示NBCn1、NBCn2可能参与机体血压调节。第三，我们在小肠上皮细胞中发现一种新的NBCn2异构体，NBCn2是小肠上皮细胞中一种新的NaCl吸收机制。.在本项目的支持下，本人以通讯作者在The Journal of Physiology（3篇）、Frontiers in Physiology（2篇）、BBA-Biomembranes（1篇）发表研究论文共6篇，以合作者在Cell Reports发表论文1篇。课题组共培养博士生毕业3人，硕士生毕业4人，在读博士生4人，在读硕士生3人。