Schulze Dan H, Muqhal Muqeem, Lederer W Jon, Ruknudin Abdul M
Department of Microbiology and Immunology, School of Medicine, and Institute of Molecular Cardiology, Medical Biotechnology Center, University of Maryland Biotechnology Institute, University of Maryland, Baltimore, Maryland 21201, USA.
J Biol Chem. 2003 Aug 1;278(31):28849-55. doi: 10.1074/jbc.M300754200. Epub 2003 May 16.
The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings.
钠钙交换体NCX1是一种广泛表达的膜蛋白，对包括哺乳动物心脏和大脑细胞在内的许多细胞的钙稳态至关重要。NCX1的功能取决于亚细胞（“局部”）因素、NCX1的磷酸化状态以及NCX1在细胞内的亚细胞定位。在此，我们研究了心肌细胞内NCX1的分子组织。我们发现，在体外，NCX1可被蛋白激酶A（PKA）依赖性磷酸化动态磷酸化。我们还提供证据表明，这种磷酸化的调节归因于NCX1大分子复合物的存在。具体而言，我们发现该大分子复合物包括PKA的催化亚基和调节亚基。然而，在该大分子复合物中仅发现RI调节亚基，未发现RII。其他关键调节酶也与NCX1相关，包括蛋白激酶C（PKC）以及两种丝氨酸/苏氨酸蛋白磷酸酶PP1和PP2A。重要的是，发现了蛋白激酶A锚定蛋白mAKAP，其在大分子复合物中的存在表明，这些调节酶如在多种细胞中对许多通道蛋白的调节那样，被协调定位以调节NCX1。双重免疫细胞化学染色显示心肌细胞中NCX1蛋白与mAKAP和PKA-RI蛋白共定位。最后，亮氨酸/异亮氨酸拉链基序已被确定为可能的相互作用位点。我们在心脏中发现NCX1大分子复合物，提示了心脏和其他细胞中NCX1调节是如何实现 的。NCX1大分子复合物的存在也可能为最近有争议的研究结果提供一种解释。
