Phospholemman and Na-Pump Function in Cardiac Myocytes

描述（由申请人提供）：Na/ k - atp酶（NKA）是主要的Na挤压途径，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）对[Ca]i和收缩性进行关键调节，这使得了解[Na]i的调节非常重要。磷脂蛋白（PLM或FXYD1）是FXYD蛋白家族中的一种跨膜蛋白，已知与NKA相关并调节NKA。PLM是唯一在心脏中丰富的FXYD蛋白，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的方式与磷蛋白（PLB）调节SR ca - atp酶（SERCA）的方式相似。也就是说，PLM通过降低NKA的[Na]i亲和力来抑制NKA，而PLM磷酸化减轻了这种抑制作用。我们还发现，PLM磷酸化和随之而来的NKA刺激是交感搏击或逃跑反应的组成部分（缓和[Na]i和细胞钙负荷的上升，并限制钙负荷过载引起的心律失常）。我们发现PLM物理上与NKA 1PLM磷酸化相关（通过免疫沉淀），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，PLM形成同源低聚物。所有这些都类似于SERCA-PLB。尽管具有生理意义，但PLM- NKA相互作用的机制尚不清楚。因此，此更新建议的总体目标是从机制上解读PLM和NKA如何在功能上相互作用。在这里，我们将结合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。目标1侧重于NKA-PLM（以及PLM-PLM复合体）的结构-功能。这一点很重要，原因有几个。首先，最近与fxyd相关的NKA晶体结构表明NKA和fxyd上的一些位点在相互作用距离内（至少在晶体中特定的稳定构象中），但这需要在活细胞膜中进行测试。这将在Aim 1中通过原位FRET测量、Co-IP和细胞内NKA活性测量进行测试。其次，PLM-PLM同质寡聚物如何相互作用（例如，它们是否形成具有PLB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（如PLB- serca）发挥功能调节作用。这也将在Aim 1中进行评估。Aim 2更狭隘地关注PLB（至少与人类疾病相关的PLB人类突变体）可能被误导到肌膜并调节NKA的有趣可能性。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，如果它在肌膜中表达（反之，如果PLM在ER/SR中表达，是否可以与SERCA功能相互作用）。目标3考察了一些问题，这些问题来自于我们新颖而令人惊讶的观察，即ouabain废除了NKA与PLM或NKA之间的FRET。这将为NKA二聚化（异质或同源）与其功能活性的关系以及NKA在激酶信号级联中的作用提供重要信息。

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. PUBLIC HEALTH RELEVANCE: Altered cardiac myocyte sodium concentration [Na] is known to be a critical factor in modulating both mechanical action of the heart and induction of arrhythmias. Here we extend our novel work on the interaction and modulation of the Na/K-ATPase (responsible for regulating [Na]) by phospholemman. The proposed work will add important fundamental insight into the interaction, and how this regulatory complex regulates [Ni ain] the heart (using state of the art molecular, biophysical and cell biology approaches).