Phospholemman and Na-Pump Function in Cardiac Myocytes

描述（由申请方提供）：Na/K-ATP酶（NKA）是主要的Na排出途径，因此在[Na]i调节中至关重要。在心脏中，[Na]i通过Na/Ca交换（NCX）关键地调节[Ca]i和收缩性，这使得理解[Na]i调节极其重要。Phospholemman（PLM或FXYD 1）是已知与NKA相关并调节NKA的蛋白质的FXYD家族的跨膜蛋白。PLM是心脏中唯一丰富的FXYD蛋白，在心脏中它是蛋白激酶A（PKA）和C（PKC）磷酸化的主要底物。在最初的奖励期间，我们发现PLM调节NKA的方式类似于受磷蛋白（PLB）调节SR Ca-ATPase（SERCA）的方式。也就是说，PLM通过降低其[Na]i亲和力来抑制NKA，并且PLM磷酸化减轻这种抑制。我们还表明，PLM磷酸化和随后的NKA刺激是交感神经战斗或逃跑反应的组成部分（缓和[Na]i和细胞Ca负荷的上升，并限制Ca过载诱导的心律失常）。我们发现PLM与NKA 1 PLM磷酸化（通过免疫沉淀）物理缔合，但在荧光共振能量转移（FRET）测量中，PLM磷酸化改变了PLM-NKA相互作用，并且PLM形成同源寡聚体。所有这些都类似于SERCA-PLB。尽管其生理意义，PLM-NKA相互作用的机制知之甚少。因此，这个更新建议的总体目标是从机制上解释PLM和NKA如何在功能上相互作用。在此，我们将结合联合收割机FRET，[Na]i和[Ca]i测量，膜片钳和分子生物学技术在心肌细胞和HEK 293细胞。目的1关注NKA-PLM（和PLM-PLM复合物）的结构-功能。这一点很重要，原因有几个。首先，最近的NKA晶体结构与相关的FXYDs建议NKA和FXYDs的相互作用距离内的一些网站（至少在晶体中的特定稳定构象），但这需要在活细胞膜测试。这将在目标1中通过原位FRET测量、细胞中的Co-IP和NKA活性测量来测试。其次，PLM-PLM同源寡聚体如何相互作用（例如，它们是否形成具有类似PLB的结构基础的稳定多聚体）以及是否存在与PLM-NKA二聚体平衡的PLM多聚体池，其发挥功能调节（如PLB-SERCA）。这也将在目标1中进行评估。目的2更狭义地关注PLB（至少是与人类疾病相关的PLB人类突变体）可以被错误引导到肌膜并调节NKA的有趣的可能性。我们将测试WT PLB是否也可以与NKA活性相互作用并调节NKA活性，如果它是在肌膜中（反之，如果PLM在ER/SR中表达，则PLM是否可以与SERCA功能性相互作用）。目的3检查的问题，从我们的小说和令人惊讶的观察，哇巴因废除FRET之间的NKA和PLM或NKA的启发。这将提供关于NKA的二聚化（异或同）如何与其功能活性相关的重要信息，并且还可能提供NKA在激酶信号级联中的作用。

DESCRIPTION (provided by applicant): Na/K-ATPase (NKA) is the main Na extrusion pathway and therefore is essential in [Na]i regulation. In the heart, [Na]i critically modulates [Ca]i and contractility via Na/Ca exchange (NCX), which makes understanding [Na]i regulation extremely important. Phospholemman (PLM or FXYD1) is a transmembrane protein of the FXYD family of proteins that are known to associate with and modulate NKA. PLM is the only FXYD protein abundant in the heart, where it is a major substrate for phosphorylation by protein kinase A (PKA) and C (PKC). During the initial award period, we showed that PLM regulates NKA similar to the way phospholamban (PLB) regulates SR Ca-ATPase (SERCA). That is, PLM inhibits NKA by reducing its [Na]i affinity, and PLM phosphorylation relieves this inhibition. We also showed that PLM phosphorylation and the consequent NKA stimulation is integral to the sympathetic fight or flight response (tempering the rise in [Na]i and cellular Ca load, and limiting Ca overload-induced arrhythmias). We found that PLM physically associates with NKA 1PLM phosphorylation (via immunoprecipitation), but in fluorescence resonance energy transfer (FRET) measurements, that PLM phosphorylation alters the PLM-NKA interaction and that PLM forms homo- oligomers. All of this resembles SERCA-PLB. Despite its physiological significance, the mechanism of PLM- NKA interaction is poorly understood. Thus, the overall goal of this renewal proposal is to decipher mechanistically how PLM and NKA interact functionally. Here we will combine FRET, [Na]i and [Ca]i measurements, patch-clamp and molecular biology techniques in cardiac myocytes and HEK 293 cells. Aim 1 focuses on the structure-function of the NKA-PLM (and PLM-PLM complex). This is important for several reasons. First, the recent NKA crystal structures with associated FXYDs suggest some sites on NKA and FXYDs that are within interaction distance (at least in the particular stable conformation in the crystals), but this requires testing in live cell membranes. That will be tested in Aim 1 by in situ FRET measurements, Co-IP and NKA activity measurements in cells. Second, it is unknown how PLM-PLM homo-oligomers interact (e.g. whether they form stable multimers with a structural basis like PLB) and whether there is a pool of PLM multimers in equilibrium with PLM-NKA dimers that exerts functional regulation (as for PLB-SERCA). That will be assessed in Aim 1 as well. Aim 2 focuses more narrowly on the intriguing possibility that PLB (at least PLB human mutants that are relevant to human disease) can be misdirected to the sarcolemma and regulate NKA. We will test whether WT PLB can also interact with and modulate NKA activity if it is in the sarcolemma (and conversely if PLM can interact functionally with SERCA if it is expressed in the ER/SR). Aim 3 examines questions that are inspired from our novel and surprising observation that ouabain abolishes FRET between NKA and either PLM or NKA. This will provide important information about how dimerization (hetero or homo) of NKA relates to its functional activity, and possibly also the role of NKA in kinase signaling cascades.