ION TRANSPORT MECHANISMS

工作总结：依赖于ATP的离子泵产生跨膜阳离子梯度，这对于电激发、溶质运输、细胞大小和形状调节以及细胞内阳离子平衡是必不可少的。哺乳动物细胞膜上的Na+/K+-ATPase在每个催化循环中利用ATP水解的能量将3个Na+送出细胞，2个K+送入细胞内。Na+外流和K+内流的不同化学计量比导致了流出的正电流，可以用灵敏的电记录设备来测量。利用激光闪光光解/人工双层膜技术和猝灭流动混合技术，测量了E1P(Na3)到E2P(Na3)构象转变的动力学。这个反应被认为是产生电的(电荷转移)，因为它耦合到Na+从细胞质到细胞外表面的转运位置的重新定位。在两种不同的Na+/K+-ATPase制剂(鳗鱼电器官和猪肾)中，由猝灭流动研究估算的构象转变速率常数与由电测得的速率常数一致。使用电位敏感染料RH421和停流混合测量构象转变的动力学也得到了类似的结果。这表明这种转变本身是电生的，或者它控制了随后的快速步骤(Na+脱遮挡？)中电荷转移的动力学。由于K+激活的E2P去磷酸化遵循构象转换，因此很可能在K+结合(激活E2P水解酶)之前，Na+从酶上解离(电荷转移步骤)。这一行为与Albers-Post机制预测的Na+/K+-ATPase阳离子结合和解离事件的序列一致，并证明了Na+和K+在α亚基上具有相似的阳离子结合氨基酸残基。-泵、三磷酸腺苷、钠、钾

SUMMARY OF WORK:The ATP-dependent ion pumps generate transmembrane cation gradients that are essential for electrical excitation, solute transport, cell size and shape regulation and intracellular cation homeostasis. The Na+/K+-ATPase in mammalian cell membranes utilizes the energy available from ATP hydrolysis to transport 3 Na+ out of and 2 K+ into the cell during each catalytic cycle. The unequal stoichiometry of Na+ efflux and K+ influx results in an outward-flowing positive current that can be measured with sensitive electrical recording devices. Using the laser flash photolysis/artificial bilayer technique and quenched- flow mixing, we measured the kinetics of the E1P(Na3)to E2P(Na3) conformational transition. This reaction is presumed to be electrogenic (charge-translocating) because it is coupled to reorientation of the Na+ transport sites from the cytoplasmic to the extracellular surface. In two different Na+/K+-ATPase preparations (eel electric organ,pig kidney)the rate constant for the conformational transition evaluated from the quenched-flow studies coincided with the rate constant determined from the electrical measurements. Similar results were obtained using the potential-sensitive dye RH421 and stopped-flow mixing to measure the kinetics of the conformational transition. This suggests that the transition itself is electrogenic or that it controls the kinetics of charge translocation in a subsequent fast step (Na+ deocclusion?). Because K+-activated dephosphorylation of E2P follows the conformational transition, it is likely that Na+ dissociates from the enzyme (charge translocating step) before K+ binds (activating E2P hydrolysis). This behavior is consistent with the sequence of the cation binding and dissociation events predicted by the Albers-Post mechanism for the Na+/K+-ATPase and with evidence that Na+ and K+ share similar amino acid residues for cation binding in the alpha subunit. - Pumps, ATP, Sodium, Potassium