Physiologically relevant energetic interactions within the Na/K Pump

描述（由申请方提供）：所有动物细胞都需要通过Na/K泵（质膜P型ATP酶）维持Na+和K+离子的跨膜梯度。在病理生理学上，Na/K泵是强心类固醇（传统上用于治疗心力衰竭的药物）特异性抑制的靶点。同时，基因突变 两种Na/K泵同种型的编码与偏头痛和帕金森综合征有关。我们实验室的长期目标是了解Na/K泵结构，功能及其在心血管和神经系统疾病中的多重作用之间的关系。所有的P型ATP酶在两个主要构象E1和E2之间交替。在循环中稳定中间状态的分子力必须作为一组特定状态的残基-残基和残基-配体相互作用的结果而产生。该区域项目的主要研究目标是确定和表征能量和机械相关的相互作用对残基之间和残基和离子之间的Na/K泵。为了解决这个问题，我们使用诱变，在非洲爪蟾卵母细胞中异源表达Na/K泵，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，以解决两个独立和相互关联的目标，我们在广泛的前期工作的基础上提出：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）阐明离子诱导的构象变化的机制。考虑到压倒性的证据表明，E1 - E2转换率的基础上的分子机制，这个关键的转运蛋白，这里提出的工作将是至关重要的理解和解决临床相关问题的病因。特别是功能障碍 高血压、充血性心力衰竭、家族性偏瘫性偏头痛和多囊肾病都与Na，K-ATP酶活性降低有关。

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.