Artigas Pablo, Gadsby David C
Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12613-8. doi: 10.1073/pnas.0602720103. Epub 2006 Aug 7.
The Na/K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na/K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na/K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na/K pump. We used outside-out patch recording of Xenopus alpha1beta3 Na/K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus alpha1beta3 Na/K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the alpha subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pump-channels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump's ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na/K pump.
钠钾泵在动物细胞膜上建立了重要的离子浓度梯度。强心甾类化合物，如哇巴因，是钠钾泵的特异性抑制剂。我们利用海洋毒素——刺尾鱼毒素，来探究钠钾泵的离子转运途径及其与哇巴因相互作用的位点。刺尾鱼毒素使泵的门控解偶联，正常情况下泵的门控严格交替打开，而现在两个门控有时会同时打开，从而将泵转变为离子通道。因此，刺尾鱼毒素甚至允许对单个钠钾泵进行电生理分析。我们使用非洲爪蟾α1β3钠钾泵的外向膜片钳记录，这些泵在非洲爪蟾卵母细胞中表达后通过点突变使其对哇巴因产生抗性。内源性的、对哇巴因敏感的非洲爪蟾α1β3钠钾泵通过持续暴露于哇巴因而失活。我们发现，α亚基第一个细胞外环两端的两个残基的侧链电荷，已知其对哇巴因亲和力有重要贡献，通过静电机制强烈影响单个与刺尾鱼毒素结合的泵通道的电导。用带不同电荷的甲硫基磺酸试剂修饰在这两个位置引入的半胱氨酸可模拟这些效应。这些修饰的结果表明，这两个残基都位于泵离子通道口附近的一个宽阔前庭中。结合的哇巴因保护对电导影响最强的位点免受甲硫基磺酸修饰，而对影响较弱的位点则不保护。这些结果提示了一种绘制结合的强心甾在钠钾泵细胞外表面足迹的方法。
