Na/K Pump Current in Isolated Heart Cells

描述（由申请人提供）：Na/K泵产生Na和K离子的跨膜电化学梯度，这是电信号和二次偶联运输的基础，并且是地高辛的受体，地高辛是一种广泛使用的心脏强直类固醇，可以特异性抑制Na/K泵。遗传的Na/K泵突变现在与快速发作的肌张力障碍-帕金森病和家族性偏瘫偏头痛有关。我们的长期目标仍然是详细了解Na/K泵是如何工作的，即离子转运途径和相关门是什么样子的，以及它们的协调相互作用是如何在细胞膜上以相反的方向先运输3个Na离子，然后运送2个K离子的。我们将Na/K泵视为具有胞质门和细胞外门的特殊离子通道，其运动紧密耦合，因此两个门通常不会同时打开（与离子通道的门不同）。因此，我们通过应用强大的电生理方法来研究Na/K泵，这些方法已被证明在了解离子通道如何工作方面是成功的。因此，由Na和K离子的不均匀传输产生的固定电流可以灵敏地测定稳定循环期间的周转率，电压跳跃后的电荷弛豫可以监测速率限制某些部分反应的构象转变。综上所述，这些信号揭示了Na/K泵离子传输的分子机制。现在，为了进一步研究离子输运机制，我们将这些记录方法与三种新工具结合起来：(a)基于相关SR - Ca泵关键构象的最新高分辨率晶体结构的Na/K泵α亚基同源性模型；(b)基于这些结构模型的位点特异性突变，以及在爪蟾卵母细胞中的表达，抗性突变爪蟾Na/K泵，其中一些含有新的半胱氨酸残基，以测试它们对小巯基特异性试剂的可及性；(c)海洋毒素，palytoxin，它破坏Na/K泵的两个门之间的耦合，将其转化为由泵的生理配体Na和K离子和核苷酸门控的离子通道。具体目标是使用这些工具：(1)确定运输的Na和K离子穿过的离子转运途径（或多个途径）的位置、结构和物理化学特征；(2)确定Na/K泵的两个主要闸门的位置和结构；(3)检查控制闸门开启和关闭的机制。

DESCRIPTION (provided by applicant): The Na/K pump generates the transmembrane electrochemical gradients of Na and K ions that underlie electrical signaling and secondary coupled transport, and is the receptor for digoxin, the widely prescribed cardiotonic steroid that specifically inhibits the Na/K pump. Inherited Na/K pump mutations are now linked to rapid-onset dystonia-parkinsonism and familial hemiplegic migraines. Our long term goal remains to understand in detail how the Na/K pump works, i.e. what the ion translocation pathways and associated gates look like, and how their orchestrated interaction transports first 3 Na and then 2 K ions in opposite directions across the cell membrane. We view the Na/K pump as a specialized ion channel with cytoplasmic and extracellular gates whose movements are tightly coupled so that both gates are never normally open at the same time (unlike the gates of ion channels). Accordingly, we investigate the Na/K pump by applying powerful electrophysiological methods that have proven successful in learning how ion channels work. Thus, the stationary current that results from the unequal transport of Na and K ions sensitively assays turnover rate during steady cycling, and charge relaxations following voltage jumps monitor conformational transitions that rate limit certain partial reactions. Together, these signals have shed light on the molecular mechanism of ion transport by the Na/K pump. Now, to further investigate the ion transport mechanism, we combine these recording methods with three new tools: (a) homology models of the Na/K pump alpha subunit based on recent high-resolution crystal structures of key conformations of the related SR Ca pump; (b) site-specific mutagenesis based on those structural models, and expression in Xenopus oocytes, of ouabain-resistant mutant Xenopus Na/K pumps, some bearing novel cysteine residues introduced to test their accessibility to small sulfhydryl-specific reagents; (c) the marine toxin, palytoxin, which disrupts the coupling between the Na/K pump's two gates, transforming it into an ion channel gated by the pump's physiological ligands, Na and K ions and nucleotides. The specific aims are to use these tools: (1) to determine the location, structure, and physico-chemical characteristics of the ion-translocation pathway (or pathways) traversed by the transported Na and K ions, (2) to determine the location and structure of the Na/K pump's two principal gates, and (3) to examine the mechanisms controlling opening and closing of the gates.