ION TRANSPORT MECHANISMS

工作总结：ATP依赖性离子泵产生跨膜阳离子梯度，这对电激发、溶质转运、细胞大小和形状调节以及细胞内阳离子稳态至关重要。哺乳动物细胞膜中的Na+/K+-ATP酶利用ATP水解产生的能量，在每个催化循环中将3个Na+转运出细胞，2个K+转运入细胞。Na+流出和K+流入的不相等化学计量导致可以用灵敏的电记录装置测量的向外流动的正电流。利用激光闪光光解/人工双层膜技术和猝灭流混合，我们测量了E1 P（Na 3）到E2 P（Na 3）构象转变的动力学。该反应被认为是产电的（电荷移位），因为它与Na+转运位点从细胞质到细胞外表面的重定向偶联。在两个不同的Na+/K+-ATP酶制剂（鳗鱼的电器官，猪肾）的速率常数的构象转变的猝灭流研究的评价一致的速率常数确定的电测量。使用电位敏感染料RH 421和停流混合来测量构象转变的动力学，得到了类似的结果。这表明，过渡本身是产电或它控制的动力学电荷易位在随后的快速步骤（Na+ deocclusion？）。由于K+激活的E2 P去磷酸化遵循构象转变，因此Na+可能在K+结合（激活E2 P水解）之前与酶解离（电荷移位步骤）。这种行为与Na+/K+-ATP酶的Albers-Post机制预测的阳离子结合和解离事件的顺序一致，并且有证据表明Na+和K+在α亚基中共享用于阳离子结合的相似氨基酸残基。- 泵，ATP，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