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。

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.