Structure-function studies of the dimerization of the N-terminal domain of NBCe1-

描述（由申请人提供）：K01奖将使Harry Gill博士从目前的助理教授转变为独立的科学研究者，专注于理解肾NBCe1-A介导Na/HCO3共转运的机制。Gill博士的背景是分子生物学、生物化学和晶体学。然而，由于必要的功能分析是生理的，因此将Gill博士整合到他的导师Walter Boron教授的生理实验室是至关重要的。一个由来自三家机构的高级研究员组成的指导委员会将指导吉尔博士的研究进展。培训活动包括：(a)在细胞水平上发展新的实验（电生理）技术；(b)为全动物水平的新实验工作做准备；(c)参加教学生理学课程；(d)进行原创性研究；(e)参加实验室会议、期刊俱乐部和研讨会；(f)与导师一起参加生理学科学会议；(g)发起由导师建立的网络；(h)教学（有限），以加强培训；(1)在实验室指导本科生；(j)基金写作方面的培训。项目(d)将为Gill博士提供初步数据，并为他的第一次R01申请做准备。研究计划的具体目的是：(1)利用x射线晶体学阐明胞浆N端（Nt）和人R298S突变在NBCe1-A功能中的作用。(2)培养作为细胞生理学家的电生理技能，并利用由内到外的巨大斑块首次测量NBCe1- a对细胞质侧[Na]， [HCO3]和ph的依赖性。(3)探索似乎编织在NBCe1细胞质Nt内部的导管的功能意义，并为携带关键Nt突变的敲入小鼠的下一代制备靶向载体。这项从原子到细胞/分子水平的实验工作将为胞浆Nt在SLC4家族成员功能中的作用提供重要的新见解。K01培训计划将培养出一个全面发展的学术科学家，他将在提交自己的R01资助时具有很强的竞争力。

DESCRIPTION (provided by applicant): The K01 award will transition Dr. Harry Gill from a currently mentored Assistant Professor to an independent scientific investigator, focusing on understanding the mechanism of Na/HCO3 cotransport by renal NBCe1-A. Dr. Gill's background is in molecular biology, biochemistry, and crystallography. However, because the necessary functional assays are physiological, it is critical that Dr. Gill be integrated into the Physiology Lab of his mentor, Professor Walter Boron. A mentoring committee consisting of senior investigators from three institutions will guide Dr. Gill's progress. The training activities include: (a) developing new experimental (electrophysiological) techniques at the cellular level; (b) preparing for new experimental work at the whole-animal level; (c) taking didactic physiology coursework; (d) performing original research; (e) participating in lab meetings, journal clubs, and seminars; (f) attending mentor at physiologically oriented scientific meetings with mentor; (g) initiating mentor-generated networking; (h) teaching (limited) to enhance training; (i) mentoring undergraduate students in the Lab; and (j) training in grant writing. Item (d) will provide Dr. Gill with preliminary data and otherwise prepare him for his first R01 application. The specific aims of the research plan are: (1) To use X-ray crystallography to illuminate the role of the cytosolic N terminus (Nt) and the human R298S mutation in the function of NBCe1-A. (2) To develop electrophysiological skills as a cell physiologist, and exploit the inside-out giant patch to measure for the first time the dependence of NBCe1-A on cytosol-side [Na], [HCO3], and pH. (3) To explore the functional significance of conduits that appear to weave through the interior of the cytosolic Nt of NBCe1, and to prepare a targeting vector for the future generation of a knock-in mouse carrying a key Nt mutation. The proposed experimental work-from the atomic to the cellular/molecular levels-will provide important new insights into the role of the cytosolic Nt in the function of members of the broader SLC4 family. The K01 training program will produce a well rounded academic scientist who will be highly competitive in submitting his own R01 grant. PUBLIC HEALTH RELEVANCE: Bicarbonate (HCO3) transport across epithelial cells of the kidney is of great importance for maintaining the proper acid-base balance of the blood, which is in turn essential for nearly all body functions. NBCe1-a critically important protein in the membrane of kidney cells-transports HCO3 from the urine into the blood, and thereby prevents the blood acid-base balance from becoming too acidic. Patients with a defective NBCe1 gene are severely affected, having very acidic blood, problems with their eyes, and mental retardation.