MODES OF OPERATION OF THE SODIUM PUMP

描述：该项目的长期目标是提供一个完整的 钠泵的动力学和热力学描述(模型) Na，K-ATPase。钠泵是细胞质膜的一种不可或缺的蛋白质 几乎所有的动物细胞，并负责维持跨膜 Na和K梯度是产生静息和动作的基础 电位，细胞体积和pH调节，液体分泌和重吸收， 营养素和其他离子的二次转运等。洋地黄的作用 心脏上的药物几乎肯定是通过钠泵调节的。这个 人体内的钠泵消耗了人体所产生的全部三磷酸腺苷的相当一部分 尸体。 该建议包括四个具体目标：(1)继续分析 操作模式之间的稳态动力学和热力学关系 (正向Na/K；反向K/Na；Na/Na交换；K/K交换) 特别要注意细胞膜电位的影响。(2)继续 钠/钠系统稳态前电压跳跃感应电流暂态分析 交换(可能还有K/K交换)模式，目的是建立 的3个可分解组分之间的前体-产品关系 转瞬即逝。(3)产生和分析[ATP]跳跃感应泵电流 电压钳位下的瞬变。这三个目标将在内部实现 透析法观察到的巨型轴突能(I)控制离子和 生化条件和膜电位，以及(Ii)同位素的测定 通量和(微小)跨膜电流，从长达一小时的直流稳定性到 微秒分辨率。动力学分析将用严格的最小二乘法进行。 注意(I)某些参数之间的内在一致性的拟合 由各种模式共享以及(Ii)稳态和预稳态 描述给定反应的参数。人们非常感兴趣的是细胞的影响 膜电位对动力学参数的影响(即速率和平衡常数； 表观亲和力；希尔系数)，因为这些效应揭示了重要的 物质和结构上的限制，如通道的性质和深度 势垒或在给定步骤中穿过一个场的电荷量。这个 最终的具体目标是(4)利用非洲爪哇卵母细胞的表达(I)来鉴定 非洲爪哇α亚基克隆与内酯环相互作用的残基 强心类固醇和(Ii)进行强心类固醇的初步鉴定 克隆鱿鱼泵α亚基并检测少量可能的mRNA 编辑可能对宏观泵特性产生影响的站点。

DESCRIPTION: The long term objective of this project is to provide a complete kinetic and thermodynamic description ("model") of the sodium pump or Na,K-ATPase. The sodium pump is an integral protein of the plasma membrane of nearly all animal cells and is responsible for maintenance of the transmembrane Na and K gradients that underlie the generation of resting and action potentials, cell volume and pH regulation, fluid secretion and reabsorption, secondary transport of nutrients and other ions, etc. The action of digitalis drugs on the heart is almost certainly mediated through the sodium pump. The sodium pump in humans consumes an appreciable fraction of all ATP made in the body. The proposal comprises four specific aims: (1) To continue to analyze steady-state kinetic and thermodynamic relationships between modes of operation (forward Na/K; backward K/Na; Na/Na exchange; K/K exchange) of the pump with special attention to the effects of cell membrane potential. (2) To continue to analyze pre-steady-state voltage jump-induced current transients in the Na/Na exchange (and possibly K/K exchange) mode with the purpose of establishing precursor-product relationships between the 3 resolvable components of the transients. (3) To generate and analyze [ATP] jump-induced pump current transients under voltage clamp. These three aims will be pursued on internally dialyzed giant axons of Loligo pealei which allow (i) control of ionic and biochemical conditions and membrane potential, and (ii) measurement of isotopic fluxes and (minuscule) transmembrane currents, from hour-long DC stability to microsecond resolution. Kinetic analysis will be done by rigorous least-squares fitting with attention to internal consistency between (i) certain parameters shared by the various modes and (ii) steady-state and pre-steady-state parameters describing a given reaction. Of great interest is the effect of cell membrane potential on kinetic parameters (i.e. rate and equilibrium constants; apparent affinities; Hill coefficients) because these effects reveal important physical and structural constraints such as the nature and depth of access wells or the amount of charge tranversing a field during a given step. The final specific aim is (4) to use Xenopus oocyte expression (i) to identify on Xenopus alpha subunit clones the residues that interact with the lactone ring of cardiotonic steroids and (ii) to carry out initial characterization of the cloned squid pump alpha subunit and examine a small number of putative mRNA editing sites for possible effects on macroscopic pump characteristics.