MECHANISM OF NA+-DEPENDENT METABOLITE TRANSPORT

NA依赖性代谢物转运机制

• 批准号: 2136908
• 负责人: GEORGE A KIMMICH
• 金额: $ 17.4万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-03-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2136908
• 关键词: alanine; aminoacid transport; carbohydrate transport; chemical kinetics; conformation; epithelium; membrane potentials; membrane transport proteins; molecular site; protein structure function; radiotracer; sodium; stoichiometry; tissue /cell culture

This proposal is aimed at evaluating the molecular mechanism by which membrane potentials and changes in membrane potential can regulate the function of Na+-coupled transport systems. It emphasizes a new conceptual perspective, the "Na+-well" model, which envisions the possibility that ion binding sites on the transport protein are "embedded" relatively deep in the membrane matrix and are reached via a channel-like access route that exhibits ion specificity, so that a Na+ ion will 'sense" a part of the electric field represented by the membrane potential in reaching or dissociating from the ion binding site. This, in turn, suggests that a primary role for the potential in governing transport capability is the result of potential-dependent Na+ binding/dissociation events associated with the transport cycle. This concept is an alternative to the usually considered "translocation" models in which the potential is envisioned as altering the transition rate between different carrier conformational states ("inward" vs. "outward" facing) for either the free or loaded carrier forms. Cultured LLC-PK1 cells are the only model system to be employed. Two primary experimental approaches are described. In one, whole-cell recording procedures will be used to monitor the function of Na+-coupled solute transport under conditions in which the two primary thermodynamic diving forces (Na+ gradient and membrane potential) are carefully controlled and the resulting kinetic effects on the full transport cycle can be measured. Experiments are described for evaluating the mechanistic role of potentials in two different Na+-coupled transport systems - for alpha-methylglucoside and alanine. These systems differ in Na+ coupling stoichiometry and therefore in the degree of complexity for modeling transport kinetics and the role that potentials play in governing function. In a second approach, isotope flux techniques will be used to measure the rate and degree of potential dependence for segments of the transport cycle for Na+-coupled systems under conditions where the full sequence of cycle events does not occur. The two approaches offer different insights to transport mechanism because whole cell recording measures net transport of charge catalyzed by the full transport cycle whereas isotope flux experiments offer high sensitivity for monitoring events that can be catalyzed by exchange loops in segments of the cycle. 14C-sugar and 22Na+ exchanges will be used to probe the rate and potential dependence for different segments in the full transport cycle and provide useful comparisons to related parameters when the full cycle is operative. Exchanges offer particular value for sensing potential dependent conformational changes in the full loaded sugar carrier. Non-exchanges mode isotope flux experiments and reactivity with chemical probes will be employed to determine if potential dependent conformational changes can be detected for the substrate-free carrier.

该提案旨在评估其分子机制 膜电位和膜电位的变化可以调节 Na+耦合运输系统的功能。 它强调了一种新的 从概念角度来看，“Na+-well”模型设想了 转运蛋白上的离子结合位点的可能性 “嵌入”在膜基质中相对较深的位置，并通过 具有离子特异性的通道状访问路径，因此 Na+ 离子将“感知”膜所代表的电场的一部分 到达离子结合位点或从离子结合位点解离的潜力。这， 反过来，表明潜力在治理中发挥着主要作用 传输能力是电位依赖性 Na+ 的结果 与运输循环相关的结合/解离事件。这 概念是通常认为的“易位”的替代方案 模型中潜力被设想为改变转变 不同载体构象状态之间的比率（“向内”与“向内”） “朝外”）适用于自由或装载的载体形式。 培养的 LLC-PK1 细胞是唯一使用的模型系统。二 描述了主要的实验方法。在一个全细胞中 记录程序将用于监测Na+耦合的功能 溶质在两个主要热力学条件下的迁移 潜水力（Na+梯度和膜电位）仔细 受控以及由此产生的对整个运输周期的动力学影响 可以测量。描述了用于评估机械性能的实验 电位在两种不同的 Na+ 耦合传输系统中的作用 - 对于 α-甲基葡萄糖苷和丙氨酸。这些系统的不同之处在于 Na+ 耦合 化学计量以及建模的复杂程度 传输动力学和电位在调控中的作用 功能。 在第二种方法中，同位素通量技术将用于测量 交通运输各部分的潜在依赖程度和比率 Na+耦合系统在完整序列的条件下的循环 循环事件不会发生。这两种方法提供了不同的 对运输机制的见解，因为全细胞记录测量网络 电荷传输由整个传输周期催化，而同位素 通量实验为监测事件提供了高灵敏度，可以 由循环片段中的交换循环催化。 14C-糖和 22Na+交换将用于探测速率和潜在依赖性 针对整个运输周期的不同环节，提供有用的 全循环运行时相关参数的比较。 交易所为感知潜在依赖提供了特殊的价值 满载糖载体的构象变化。非交换 模式同位素通量实验和与化学探针的反应将 用于确定是否存在潜在的依赖性构象变化 可以检测无底物载体。