Physiologically relevant energetic interactions within the Na/K Pump

描述(申请人提供)：所有动物细胞都需要通过Na/K泵，一种质膜P型ATPase来维持Na+和K+离子的跨膜梯度。在病理生理学上，钠/钾泵是强心类固醇的靶点，强心类固醇传统上用于治疗心力衰竭。此外，基因的突变 编码两种钠/钾泵亚型与偏头痛和帕金森病有关。我们实验室的长期目标是了解Na/K泵的结构、功能及其在心血管和神经疾病中的多种作用之间的关系。所有的P型ATPase都在两个主要构象E1和E2之间交替。在循环中稳定中间态的分子作用力必须是一系列特定于状态的残基-残基和残基-配体相互作用的结果。这一领域项目的主要研究目标是确定和表征Na/K泵中残基对之间以及残基与离子之间的能量和力学相关的相互作用。为了解决这个问题，我们在前期工作的基础上，利用诱变、Na/K泵在非洲爪哇卵母细胞中的异源表达、电压钳(双电极电压钳、切开卵母细胞和膜片钳)、工程半胱氨酸残基的化学修饰和计算化学来解决两个相互独立和相互关联的目标：1)鉴定和表征稳定Na/K泵构象的关键构象特异性相互作用；2)阐明离子诱导构象变化的机制。考虑到压倒性的证据表明，E1-E2转化率是这一关键转运蛋白的分子机制的基础，这里提出的工作将对理解和解决临床相关问题的病因至关重要。具体地说，功能障碍 大多数Na，K-ATPase被归因于高血压、充血性心力衰竭、家族性偏瘫、偏头痛和多囊肾病。

DESCRIPTION (provided by applicant): All animal cells require maintenance of transmembrane gradients for Na+ and K+ ions by the Na/K pump, a plasma membrane P-type ATPase. Pathophysiologically, the Na/K pump is the target specifically inhibited by cardiotonic steroids, drugs traditionally used for the treatment of heart failure. Also, mutations in the genes encoding for two Na/K pump isoforms have been linked to migraine and Parkinsonism. The long-term goal of our laboratory is to understand the relationship between the Na/K pump structure, its function, and its multiple roles in cardiovascular and neurological diseases. All P-type ATPases alternate between two major conformers E1 and E2. The molecular forces that stabilize intermediate states in the cycle must arise as a consequence of a set of state-specific residue-residue and residue-ligand interactions. The main research goal of this AREA project is to identify and characterize energetically and mechanistically relevant interactions between pairs of residues and between residues and ions within the Na/K pump. To address this problem we use mutagenesis, heterologous expression of Na/K pumps in Xenopus oocytes, voltage clamp (two electrode voltage clamp, cut-open oocyte and patch clamp), chemical modification of engineered cysteine residues and computational chemistry to address two independent and interrelated aims, which we propose on the basis of extensive preliminary work: 1) To identify and characterize critical conformation-specific interactions that stabilize Na/K pump conformations, 2) to elucidate the mechanisms of ion-induced conformational changes. Considering the overwhelming evidence that the rate of E1 - E2 conversion underlies the molecular mechanism of this critical transporter, the work proposed here will be crucial to understanding and resolving the etiology of clinically relevant problems. Specifically, dysfunction of the Na,K-ATPase has been attributed to hypertension, congestive heart failure, familial hemiplegic migraine, and polycystic kidney disease.