Functional Role of CaT1 and Calbindin D in Ca Transport

CaT1 和 Calbindin D 在 Ca 转运中的功能作用

• 批准号: 6741963
• 负责人: RICHARD James WOOD
• 金额: $ 21.2万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6741963
• 关键词: 1,25 dihydroxycholecalciferol; apical membrane; basolateral membrane; biological signal transduction; brush border membrane; calbindin; calcium binding protein; calcium channel; calcium flux; calcium ion; enzyme linked immunosorbent assay; gastrointestinal absorption /transport; gene expression; membrane transport proteins; phospholipase D; polymerase chain reaction; protein kinase C; protein structure function; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Absorbing too little calcium (Ca) in the intestine can be a risk factor for negative Ca balance and the development of osteoporosis. An important gap in our knowledge is that the molecular determinants of Ca influx into the enterocyte and transcellular Ca transport have not been unambiguously identified. This lack of knowledge limits the development of specifically targeted therapeutic agents to manipulate Ca absorption. The long-range goal of our research program is to identify the molecular factors responsible for age-related calcium malabsorption, and to target the development of safe and effective therapeutic agents that can modulate active calcium absorption. The recent discovery of the CaT1 (also known as ECaC2 and TRPV6) gene and the demonstration that its protein product functions in non-intestinal cells as a membrane Ca transporter, and our recent report that the expression of CaT1 in the Caco-2 human intestinal cell line is regulated by 1,25-dihydroxyvitamin D, offers an exciting new opportunity to further knowledge of the molecular mechanisms of vitamin D-mediated intestinal Ca absorption. We have two specific aims in this proposal (1) to establish the role of CaT1 and calbindin D in vitamin D-mediated calcium transport and (2) to identify to what extent CaT1 expression is a modulator of the rapid non-genomic vitamin D response in the enterocyte. We will determine the functional role of these proteins in the enterocyte by using Caco-2 cells transfected with sense and antisense CaT1 and calbindin D cDNA to alter the level of these proteins independently of vitamin D. The central hypothesis of our first research aim is that CaT1 acts as the primary gatekeeper of intestinal Ca absorption by controlling, under regulation by 1,25-dihydroxyvitamin D, the rate of Ca entry across the apical brush border membrane of the enterocyte. The cytosolic vitamin D-dependent calbindin D acts in concert with CaT1 as both intracellular buffer and transporter, delivering Ca that has crossed the brush border membrane to the basolateral membrane of the enterocyte for Ca exit into the blood. We hypothesize in our second research aim that the CaT1 molecule either is responsible for or modulates the rapid 'non-genomic' response to 1,25-dihydroxyvitamin D in Caco-2 cells by determining the rate of Ca entry into the enterocyte following 1,25-dihydroxyvitamin D treatment and thereby modulating the rise of free intracellular Ca and subsequent downstream activation of protein kinase C and phospholipase D.

描述（由申请人提供）：在肠中吸收太少的钙（CA）可能是负CA平衡和骨质疏松症的发展的危险因素。我们所知的一个重要差距是，尚未明确鉴定出CA涌入肠球菌和跨细胞Ca转运的分子决定因素。缺乏知识限制了特定靶向治疗剂来操纵CA吸收的发展。我们研究计划的远程目标是确定导致与年龄相关的钙吸收不良的分子因素，并针对可以调节活性钙吸收的安全有效治疗剂的开发。 The recent discovery of the CaT1 (also known as ECaC2 and TRPV6) gene and the demonstration that its protein product functions in non-intestinal cells as a membrane Ca transporter, and our recent report that the expression of CaT1 in the Caco-2 human intestinal cell line is regulated by 1,25-dihydroxyvitamin D, offers an exciting new opportunity to further knowledge of the molecular mechanisms of vitamin D-mediated肠吸收。在该提案（1）中，我们有两个具体的目的，以确定CAT1和Calbindin d在维生素D介导的钙转运中的作用，并且（2）确定CAT1表达在多大程度上是肠上皮细胞中快速非基因组维生素D反应的调节剂。我们将通过使用带有感官和反义CAT1和Calbindin d cDNA转染的CACO-2细胞来确定这些蛋白质在肠球细胞中的功能作用，以独立于维生素D。我们的第一个研究目标的中​​心假设是，Cat1的主要率是通过控制Informandy vyecruption capermation ocking ode capermation caperage captraption cat1的主要研究。 CA进入肠肠细胞的顶端刷边界膜。胞质维生素D依赖性的Calbindin D与CAT1一起起作用，作为细胞内缓冲液和转运蛋白，将Ca交付，将刷子边界膜越过了肠上皮细胞的基底外侧膜，以使CA出口进入血液。我们在第二项研究中假设，CAT1分子要么负责或调节CACO-2细胞中1,25-二羟基毒素D的快速“非基因组”反应，通过确定Ca进入肠ot肠地细胞的速率，并在1,25-二羟基蛋白D治疗后及时地进行了调制，并在其上进行了调制，并及时地进行了调制的均及时及时均可及时地进行了调节。激酶C和磷脂酶D.