NOVEL INTERACTION OF AMP-KINASE WITH CFTR C1 CHANNEL

CFTR是囊性纤维化患者中存在缺陷的基因产物，它不仅作为一个C1通道发挥作用，而且在上皮细胞中也是其他膜转运蛋白的协调者和调节者。鉴定和鉴定与cftr相互作用的细胞蛋白对于了解cftr的调控及其对其他膜转运蛋白的调控作用是必要的。这一信息可能会为治疗CF提供新的治疗方法。这个项目将检验我们发现的CFTR和AMP激活的蛋白激酶(AMPK)之间的一种新的相互作用的后果。AMPK是一种已知的对细胞代谢状态做出反应的激酶，在营养应激条件下磷酸化并抑制几种关键的生物合成酶，从而保护细胞的ATP。它与cftr的相互作用可能是重要的，因为cftr受ATP水解酶的调控，可能介导细胞释放ATP。对于相互作用重要的两种蛋白质的区域将使用截断突变体和酵母双杂交系统来确定。AMPK-CFTR在体外和体内的结合将通过与表位标记的构建物的免疫共沉淀进行探索，并将定义调节相互作用强度的条件。AMPK-CFTR相互作用的功能后果将通过以下方面进行研究：检测AMPK与CFTR相关的细胞表达和定位，确定CFTR是否是体外和体内AMPK磷酸化的底物，使用膜片钳技术研究这种相互作用对CFTR单通道特性和细胞膜表达的影响，以及确定AMPK-CFTR相互作用对上皮Na+通道(ENaC)功能的影响。

CFTR, the gene product which is defective in patients with cystic fibrosis (CF), functions not only as a C1- channel, but also as a coordinator and regulator of other membrane transporters in epithelia. Identifying and characterizing cellular proteins that interact with CFTR is necessary to understand the regulation of CFTR and its regulatory effects on other membrane transporters. This information could suggest new therapies for CF. This project will examine the consequences of a novel interaction we have discovered between CFTR and AMP-activated protein kinase (AMPK). AMPK is a kinase which is known to respond to the metabolic status of cells, phosphorylating and inhibiting several key biosynthetic enzymes under conditions of nutritional stress, thereby conserving cellular ATP. Its interaction with CFTR may be significant because CFTR is gated by ATP hydrolysis and may mediate ATP release from cells. The regions of both proteins that are important for the interaction will be determined using truncation mutants and the yeast two-hybrid system. AMPK-CFTR binding in vitro and in vivo will be explored through co-immunoprecipitation with epitope-tagged constructs, and conditions that modulate the strength of the interaction will be defined. The functional consequences of the AMPK-CFTR interaction will be studied by: examining the cellular expression and localization of AMPK in relation to CFTR, determining whether CFTR is a substrate for AMPK phosphorylation in vitro and in vivo, investigating the effects of this interaction on CFTR single channel properties and expression in cell membranes using patch-clamp techniques, and determining the effects of the AMPK-CFTR interaction on epithelial Na+ channel (ENaC) function.