钠钾ATP酶作为细胞膜"感知分子"对胆固醇代谢调节机制研究

The Na+/K+-ATPase, also known as "Sodium Pump", is a ubiquitous transmembrane enzyme that transports Na+ and K+ arcoss the plasma membrane by hydrolyzing ATP. Recently it has been demonstrated that cardiotonic steroids, such as ouabain binds to Na+/K+-ATPase generating a large number of secondary messengers. Binding of ouabain to the Na+/K+-ATPase/Src complex changes their interaction, results in the activation of Src/ERK cascades.Caveolae are flask-shaped invaginations of plasma membrane and are enriched in cholesterol, glycosphingolipids and sphingomyelin.The non-pumping pool of Na+/K+-ATPase and more than 50% of total cellular cholesterol mainly resides in caveolae. We have reported that Na+/K+-ATPase regulates cholesterol synthesis, distribution and trafficking. This project is to further clarify the molecular mechanism and link it to cholesterol-associated syndromes. Due to a large number of Na+/K+-ATPase colocalizes with cholesterol in caveolae and its activity is affected by cholesterol, it is possible that the two interact with each other. When we search Na+/K+-ATPaseα1 subunit for a potential cholesterol binding consensus (-L/V-(X) 1-5-Y-(X) 1-5-R/K-), we find two potential binding sites, one around the first transmembrane domain，the other at the last transmembrane domain. Interestingly, they are highly conserved within mammals and even toad and fish. Thus, we will test whether these consensus domains are responsible for directly interaction with cholesterol.We further hypothesize that this interaction is responsible for maintaining a normal plasma membrane distribution of cholesterol and the Na+/K+-ATPase could act as a cholesterol sensor in the plasma membrane. Our previous data clearly demonstrate that reduction of cellular Na+/K+-ATPase is sufficient to decrease ER cholesterol and subsequently alter cellular cholesterol metabolism via activation of a SREBP2-dependent pathway. Since SREBP2 is known to be regulated by ERK, we reason that Na+/K+-ATPase may regulate SREBP2 by stimulating Src/ERK pathway. Overall, we propose that first, Na+/K+-ATPase and cholesterol can directly bind; second, Na+/K+-ATPase stimulates Src/ERK signaling pathway in response to changes in cholesterol level; third, the signaling pathway of SCAP/SREBP2 was stimulated by Na+/K+-ATPase-Src/ERK activation. Cholesterol is an important substance for human tissues, whose metabolic disorder can cause atherosclerosis,hypertension,diabetes etc.Na+/K+-ATPase is significantly reduced in those cholesterol metabolic diseases. Thus, our new findings warrant further investigation of the role of the Na+/K+-ATPase in regulation of cholesterol metabolism and open doors for development of new therapeutic interventions.

钠钾ATP酶是细胞维持跨膜离子梯度的关键结构，除经典的离子泵功能，它还具有信号传导等非离子泵功能；约50%的钠钾ATP酶及胆固醇分布在膜穴内。基于我们前期研究成果：钠钾ATP酶可调控胆固醇合成、转运及分布；本项目进一步推测，钠钾ATP酶是细胞膜胆固醇感知分子，通过"胆固醇-钠钾ATP酶-Src/ERK-SREBP2-胆固醇"信号通路调节胆固醇代谢。本课题拟通过FRET技术，研究钠钾ATP酶与胆固醇作用方式，其次分析两者相互作用是否激活Src/ERK信号通路，最后证明通过激活Src/ERK从而激活SREBP2信号通路调节胆固醇合成及分布。胆固醇是人体组织不可缺少的重要物质，研究发现许多胆固醇代谢障碍性疾病，如动脉粥样硬化、高血压等都伴随钠钾ATP酶表达改变。本研究结果将阐明钠钾ATP酶调节胆固醇代谢的作用机制，为以钠钾ATP酶做为靶蛋白研制出针对动脉粥样硬化等疾病的治疗提供新方案和理论依据。

钠/钾ATP酶是细胞维持跨膜离子梯度的关键结构，除经典的离子泵功能，它还具有信号传导等非离子泵功能；约50%的钠/钾ATP酶及胆固醇分布在膜穴内。基于我们前期研究成果：钠/钾ATP酶可调控胆固醇合成、转运及分布；本项目进一步阐明Na/K-ATPase与胆固醇的相互作用方式及钠/钾ATP酶调节胆固醇代谢的分子作用机制。首先在空间结构上，通过FRET技术证明了钠/钾ATP酶和胆固醇可以直接结合，这也进一步证实钠/钾ATP酶和胆固醇的确存在相互作用关系。然后，通过降脂药物“瑞舒伐他汀”作用于细胞后，抑制了胆固醇代谢关键酶SREBP2及HMG-CoAR的基因表达，但胆固醇含量减少时，Na+/K+-ATPase/Src/ERK信号传导通路被激活，我们推测Na+/K+-ATPase/Src/ERK信号通路对维持胆固醇的稳定，调节胆固醇合成及分布密切相关。接着，在动物水平，通过高脂饲料喂养改变了细胞内胆固醇含量及分布。进一步实验发现：高脂喂养后，细胞内Na/K-ATPase/Src信号通路相关基因表达改变，与此同时脂代谢相关基因HMG-CoAR、SREBP-2的表达受到明显抑制。综合目前实验数据可以证实：钠/钾ATP酶和胆固醇通过直接结合的方式，感知细胞膜胆固醇水平； 钠/钾ATP酶和胆固醇结合后，通过激活Src/ERK信号传导通路，进而调控SREBP2表达，最终达到调节胆固醇合成的目的，同时也改变了胆固醇的分布。