MECHANISM OF NA+-DEPENDENT METABOLITE TRANSPORT

该建议旨在评估分子机制， 膜电位和膜电位的变化可以调节 Na+耦合运输系统的功能。 它强调了一个新的 概念上的观点，“Na+井”模型，它设想了 转运蛋白上的离子结合位点可能 “嵌入”在膜基质中相对较深，并通过 具有离子特异性的通道样进入途径，因此Na+ 离子将“感应”由膜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电位。这个， 反过来，这表明，一个主要的作用， 转运能力是电位依赖性Na+的结果 结合/解离事件与运输周期。这 概念是通常认为的“易位”的替代方案。 模型中的潜力被设想为改变过渡 不同载体构象状态之间的速率（“向内”对 “面向外”）用于自由或负载载体形式。 培养的LLC-PK 1细胞是唯一采用的模型系统。两 描述了主要的实验方法。在一个，全细胞 记录程序将用于监测Na+-偶联的功能， 在两种主要热力学 潜水力（Na+梯度和膜电位）仔细 以及由此产生的对整个运输周期的动力学影响 是可以测量的。描述了用于评估该机制的实验 电位在两种不同Na+耦合转运系统中的作用--对 α-甲基葡糖苷和丙氨酸。这些系统在Na+耦合方面有所不同 化学计量，因此在建模的复杂程度 运输动力学和电位在控制 功能 在第二种方法中，将使用同位素通量技术来测量 运输环节的潜在依赖率和程度 循环的Na+耦合系统的条件下，全序列 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净 电荷的传输由整个传输循环催化，而同位素 通量实验为监测事件提供了高灵敏度， 被循环中的交换环所催化。14 C-糖和 22 Na+交换将用于探测速率和潜在依赖性 在整个运输周期的不同部分，并提供有用的 当全周期运行时，与相关参数进行比较。 交换机提供特殊的价值感测电位依赖 在满载的糖载体中的构象变化。非交换 模式同位素通量实验和与化学探针的反应性将 用于确定潜在的依赖性构象变化 可以检测到无基片载体。

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.