MECHANISM OF SODIUM DEPENDENT METABOLITE TRANSPORT

这项建议旨在评估 膜电位和膜电位的变化可以调节 钠离子偶联转运系统的功能。它强调了一种新的 从概念的角度来看，“Na+-Well”模型设想 运输蛋白上的离子结合部位可能是 “嵌入”在膜基质中相对较深的位置，并通过 表现出离子专一性的通道状访问路径，因此Na+ 离子将“感应”膜所代表的电场的一部分 达到或解离离子结合部位的潜力。这, 反过来，这表明在治理中潜力的主要作用 运输能力是电势依赖的Na+的结果 与传输周期相关联的绑定/解离事件。这 概念是通常被认为的“移位”的替代。 将潜力设想为改变转型的模式 不同载体构象状态之间的速率(向内与 面向外)，用于自由或装载的载体形式。 培养的LLC-PK1细胞是唯一采用的模型系统。二 描述了主要的实验方法。在一个完整的单元中 记录程序将用于监测Na+偶联的功能 两个主要热力学条件下的溶质运移 潜水力(钠离子梯度和膜电位)要小心 对整个运输周期的受控和由此产生的动力学影响 是可以测量的。描述了用于评估该机构的实验。 电位在两种不同的钠离子偶联转运系统中的作用 α-甲基葡萄糖苷和丙氨酸。这些体系在Na+偶联方面有所不同 化学计量学，因此在建模的复杂程度中 输运动力学与势能在调控中的作用 功能。 在第二种方法中，将使用同位素通量技术来测量 运输分段的潜在依赖性的速度和程度 全序列条件下Na+偶联体系的循环 不会发生周期事件。这两种方法提供了不同的 对传输机制的洞察，因为全细胞记录测量网络 整个运输周期催化的电荷传输，而同位素 通量实验为监测事件提供了高灵敏度，可以 由循环各段中的交换循环催化。14C-糖和 将使用22Na+交换来探索该比率和潜在的依赖性 为不同路段的全程运输提供有用的 全循环运行时与相关参数的比较。 交易所为感知潜在的依赖提供了特殊的价值 满载糖载体中的构象变化。非交易所 模式同位素通量实验和与化学探针的反应性将 用来确定潜在的依赖构象变化 可检测为无衬底的载体。

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.