Discovery and function of ion channels in liver epithelia.

我的研究项目主要集中在离子通道的识别和在肝脏生理学中的作用。我们目前的重点是瞬时受体电位Melastatin（TRPM）通道，亚型7。TRPM 7是一种普遍表达的通道激酶，与许多增殖细胞的存活有关;敲除是胚胎致死的。最近的研究支持TRPM 7在器官发生中的重要作用。我们发现TRPM 7通道活性在分裂的肝细胞中比在终末分化的非分裂的成体肝细胞中显著更高。通道阻断导致分裂死亡率较高，与成年细胞相比，这意味着镁离子流入在支持肝细胞增殖的基础作用。除了在其他细胞中观察到的已建立的胞质、点状表达外，我们还将TRPM 7定位于所有肝细胞的核膜。TRPM 7在非分裂的成年肝细胞的核膜中的表达高于分裂的细胞。分化条件降低通道活性和增加核膜TRPM 7在分裂细胞。在有丝分裂期间，TRPM 7与有丝分裂纺锤体相关。因此，TRPM 7通道的活性似乎是重要的胚胎肝细胞扩增和肝癌细胞的生存，而核膜积累与终末分化。在接下来的五年里，我们将确定TRPM 7如何支持胚胎肝细胞扩增和去分化生长以及终末分化。将使用从分裂胚胎和肝癌细胞到非分裂成体细胞的大鼠肝细胞谱。这些细胞为研究TRPM 7在来自相同组织和物种的分裂和非分裂细胞中的表达和功能以及研究其在肝脏发育中的作用提供了独特的机会。基因破坏和过表达技术和通道突变体构建体将用于补充电生理学和分子方法，以鉴定将TRPM 7表达、定位和活性与胚胎扩增、不受调节的增殖和终末分化联系起来的分子事件。 描述了四个目标，为5-6篇研究生论文和众多本科项目提供基础。

My research program is focused on the identification and roles of ion channels in the physiology of the liver. Our current focus is on a Transient Receptor Potential Melastatin (TRPM) channel, isoform 7. TRPM7, a ubiquitously expressed channel-kinase, is associated with survival of many proliferating cells; knock-out is embryonic lethal. Recent studies support a significant role for TRPM7 in organogenesis. We showed that TRPM7 channel activity is significantly higher in dividing liver cells than in terminally differentiated, non-dividing adult hepatocytes. Channel block caused higher death rates in dividing as compared with adult cells, implying a fundamental role for Mg2+ influx in supporting liver cell proliferation. We also localized TRPM7 to the nuclear envelope in all liver cells in addition to the established cytosolic, punctate expression seen in other cells. TRPM7 expression was higher in the nuclear envelope of non-dividing adult hepatocytes as compared with dividing cells. Differentiating conditions reduced channel activity and increased nuclear envelope TRPM7 in dividing cells. During mitosis TRPM7 associates with the mitotic spindle. Thus TRPM7 channel activity seems to be important in embryonic hepatocyte expansion and hepatoma cell survival, whereas nuclear envelope accumulation is associated with terminal differentiation. Over the next five years we will define how TRPM7 supports embryonic hepatocyte expansion and de-differentiated growth, and terminal differentiation. A spectrum of rat hepatocytes from dividing embryonic and hepatoma cells to non-dividing adult cells will be used. These cells provide unique opportunities to study TRPM 7 expression and function in dividing and non-dividing cells from the same tissue and species and to study its role in liver development. Gene disruption and over-expression techniques and channel mutant constructs will be used to complement electrophysiological and molecular approaches to identify the molecular events linking TRPM7 expression, localization and activity to embryonic expansion, unregulated proliferation and terminal differentiation. Four aims are described providing the basis for 5-6 graduate theses and numerous undergraduate projects.