Adducin and the Membrane Skeleton

描述(申请人提供)：本研究的目的是验证以下假设：内收蛋白在红细胞、血小板和肾脏的膜骨架的组装、稳定性和功能中起关键作用。内收蛋白的遗传缺陷会导致贫血、血小板功能障碍和系统血压的异常控制。膜骨架是大多数脊椎动物细胞的重要组成部分。在哺乳动物细胞的体外研究和对其他生物如黑腹毛虫的研究表明，内收蛋白在红细胞(RBC)膜骨架的发育组装中起着关键作用，同时也有数据表明，内收蛋白是血小板膜骨架的一个组成部分，对血小板功能(如聚集、丝足和片脂的形成)具有重要作用。此外，内收蛋白多态与米兰高血压(MHS)大鼠的红细胞和肾脏阳离子转运活性改变以及高血压表型有关。值得注意的是，去除膜骨架后，MHS和正常血压(MNS)对照组大鼠之间的阳离子转运差异被消除。在人类中，内收蛋白基因的多态与某些人群的高血压有关，但不是所有人群。在这里，我们将利用基因定义的小鼠，在体内确定内收蛋白在红细胞膜骨架组装和稳定脂质双层、血小板功能、红细胞阳离子转运活性和调节全身血压中的作用。其具体目的是：(1)确定内收蛋白在红细胞膜骨架组装和稳定中的作用。我们将使用loxP系统和干扰素诱导的Cre表达启动子菌株(MXL-Cre)在小鼠中产生有条件的内收蛋白基因敲除，并对每个突变菌株的红细胞表型进行完整的分析(单独和组合)。(2)探讨内收蛋白在血小板生理和病理生理中的作用。我们将在内收蛋白缺失菌株中进行一整套血小板功能测试，以评估形状变化、粘连和扩散、凝块回缩、分泌、聚集、磷脂酰丝氨酸暴露以及血栓和梗塞的发生率。(3)确定内收蛋白在RBC阳离子转运和全身血压调节中的作用。我们将测定内收蛋白缺失品系的RBC阳离子转运率(反映肾脏转运活动)、收缩压和心率。后续研究将包括心电学和超声心动学。

DESCRIPTION (provided by applicant): The goal of this research is to test the following hypothesis: adducin plays a critical role in the assembly, stability, and function of the membrane skeleton in red blood cells (RBCs), platelets and the kidney. Genetic defects in adducin result in anemia, platelet dysfunction, and aberrant control of systemic blood pressure. The membrane skeleton is a critical component of most vertebrate cells. In vitro studies in mammalian cells and studies in other organisms such as D. melanogaster suggest that adducin plays a critical role in the developmental assembly of the membrane skeleton in the red blood cell (RBC), while data have also emerged showing that adducin is a component of the platelet membrane skeleton and is important in platelet function (e. g., aggregation, formation of filopodia and lamellipodia). Moreover, adducin polymorphisms have been associated with altered RBC and kidney cation transport activity and a hypertensive phenotype in the Milan hypertensive (MHS) rat strain. Notably, the cation transport differences between MHS and normotensive (MNS) control rats are abolished upon removal of the membrane skeleton. In humans, adducin polymorphisms have been associated with hypertension in some but not all populations. Here, we will determine the role of adducin in RBC membrane skeleton assembly and stabilization of the lipid bilayer, platelet function, and RBC cation transport activity and the regulation of systemic blood pressure in vivo using genetically defined mice. The specific aims are to: (1) Determine the role of adducin in RBC membrane skeleton assembly and stabilization. We will generate conditional knockouts of the adducin genes in mice using the loxP system and an interferon-inducible Cre-expressing promoter strain (Mxl-Cre) and perform a complete analysis of the RBC phenotype of each mutant strain (singly and in combination). (2) Determine the role of adducin in platelet physiology and pathophysiology. We will perform a complete battery of platelet function tests in adducin null strains to assess shape change, adhesion and spreading, clot retraction, secretion, aggregation, phosphatidylserine exposure and the incidence of thrombi and infarcts. (3) Determine the role of adducin in RBC cation transport and systemic blood pressure regulation. We will determine RBC cation transporter rates (which mirror transport activities in the kidney), systolic blood pressure and heart rates in adducin null strains. Follow up studies will include electrocardiology and echocardiology.