Mechanisms of Hepatic Electrolyte Transport

描述(由申请人提供)：本提案中描述的研究涉及负责快速调节肝细胞离子转运以满足不断变化的生理需求的细胞机制。在肝细胞中，细胞代谢、细胞体积和离子转运之间的动态相互作用在调节对循环激素、底物可获得性和代谢刺激的反应中起着关键作用。先前的研究表明，这种反应在很大程度上是由通道介导的Na+内流和K+和Cl-外流之间的平衡所介导的，通道介导的Na+内流驱动细胞体积增加，K+和Cl-外流导致细胞体积减少。有趣的是，初步观察表明：i)离子通道蛋白的分布在质膜和细胞内膜(囊泡)地址之间受到仔细的调节，ii)不同的含有通道的囊泡可以被快速和选择性地动员起来，以形成质膜的导电特性。事实上，10,000个或更多的囊泡的胞吐对于细胞对激素的反应是必不可少的，并且足以在几分钟内替换20-40%的质膜。因此，特定的目标旨在解决以下工作假设：不同的含有通道的囊泡池的调节插入和恢复提供了一种快速调节肝细胞运输能力的机制，以响应不断变化的生理需求，并代表了激素和其他利用细胞体积作为调节细胞代谢和运输的中介信号的重要作用部位。具体目标集中在1)评估非选择性阳离子(NSC)通道作为容量调节性Na+内流的主导机制，2)表征将容量敏感信号与不同的含有通道的囊泡池的快速胞吐相结合的细胞信号，以及3)评估编码环核苷酸门控(CNG)阳离子通道作为肝细胞NSC电导的分子候选。细胞代谢、离子通道和细胞体积之间的界面代表着对肝脏运输、分泌和胆汁形成的生理调节的新的和强大的作用部位；也是开发药物方法来调节肝细胞和器官功能的重要靶点。

DESCRIPTION (provided by applicant): The studies described in this proposal address the cellular mechanisms responsible for rapid modulation of hepatocyte ion transport to meet changing physiologic demands. In liver cells, dynamic interactions between cell metabolism, cell volume and ion transport play a key role in regulation of the response to circulating hormones, substrate availability, and metabolic stimuli. Previous studies indicate that this response is mediated in large part by a balance between channel-mediated Na+ influx, which drives cell volume increases, versus reciprocal K+ and Cl- efflux, which are responsible for cell volume decreases. Interestingly, preliminary observations indicate that; i) the distribution of ion channel proteins is carefully regulated between plasma membrane and intracellular membrane (vesicular) addresses, and ii) distinct pools of channel-containing vesicles can be mobilized rapidly and selectively to shape the conductive properties of the plasma membrane. Indeed, exocytosis of 10,000 or more vesicles is essential for the cellular response to hormones, and is sufficient to replace 20-40% of the plasma membrane within minutes. Accordingly, the Specific Aims are designed to address the following working hypothesis: regulated insertion and retrieval of distinct pools of channel-containing vesicles provides a mechanism for rapid modulation of the transport capacity of hepatocytes in response to changing physiologic demands, and represents an important site of action for hormones and other factors that utilize cell volume as an intermediary signal regulating cell metabolism and transport. The Specific Aims focus on 1) evaluation of non-selective cation (NSC) channels as the dominant mechanism for volume-regulatory Na + influx, 2) characterization of the cellular signals that couple volume-sensitive signaling to rapid exocytosis of distinct pools of channel-containing vesicles, and 3) assessment of a cDNA encoding cyclic nucleotide-gated (CNG) cation channels as a molecular candidate for the NSC conductance in liver cells. The interface between cell metabolism, ion channels, and cell volume represents a novel and powerful site of action for physiologic modulation of hepatic transport, secretion, and bile formation; and an important target for development of pharmacological approaches to modulation of liver cell and organ function.