SNARE REGULATION OF BCELL KCA--SUR POTENTIATES SECRETION

BCELL KCA--SUR 的圈套调节增强分泌

• 批准号: 2759688
• 负责人: HERBERT Young GAISANO
• 金额: $ 10万
• 依托单位: UNIVERSITY OF TORONTO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2759688
• 关键词: calcium channel; cell membrane; diabetes mellitus; exocytosis; laboratory mouse; laboratory rat; membrane fusion; membrane proteins; pancreatic islet function; potassium channel; protein structure function; receptor; receptor binding; site directed mutagenesis; sulfonylurea; syntaxin; tissue /cell culture; transfection; voltage /patch clamp

DESCRIPTION (taken from the application) It is now well established that the most distal stages of the beta cell insuli secretory machinery involves plasma membrane (PM) ion channels (L-type voltage-dependent Ca2+ channel, sulfonoylurea receptor- SUR/K/ATP, and K/Ca channels) and exocytotic proteins, collectively called SNAREs on the PM and insulin secretory granules (SG). Much of the recent attention has been directe towards the structure and function of the recently cloned SUR/K/ATP, and Ca2+ channels on the PM, and the role of SNARE proteins in insulin SG exocytosis. I contrast, little is known about beta cell K/Ca channel regulation, or the role of SURs on the SGs, which comprise 90% of beta cellular SUR. This proposal hypothesizes that SNARE proteins directly modulate beta cell K/Ca activity and insulin SG-SUR. Towards the first aim of elucidating the mechanisms by which SNARE proteins regulate the beta cell K/Ca channel function, preliminary patch clamp data hav been generated to demonstrate that overexpression of a mutant SNARE protein in insulinoma HIT cells sensitized the K/Ca channel to closure. This resulted in potentiation of glucose-evoked insulin secretion, as closure of K/Ca maintains the PM in a depolarized state after glucose stimulation. Using patch clamp methods, the studies proposed are threefold, including: 1) to identify the functional domains within this SNARE protein which interact with the K/Ca channel in a manner that amplifies insulin secretion, and 2) to explore for other SNARE proteins which might interact with this mutant SNARE to form complexes that further modulate the K/Ca channel, 3) to apply the insights gained from these studies in beta cell lines to a) normal islet beta cells, to amplify the insulin secretory response, and thereby identify potential novel therapeutic targets for drug development; and b) diabetic islet beta cells, to explore for possible distortions of SNARE-K/Ca interactions which might be a basis for the insulin secretory defects of diabetes. Towards the second aim of elucidating the functional interactions of SNAREs an insulin SG-SUR, the hypotheses are that these interactions may serve: 1) to effect SG-SG (homotypic) fusion as a mechanism which enhances insulin exocytosis, or 2) to recruit SURs from this reserve pool in the SGs to the PM where SURs can exert its action. For the proposed studies, the required antibody and cDNA probes (many already subcloned into expression vectors) have been obtained or generated. The combination of molecular biological (to manipulate the expression of these proteins), biochemical (to identify protein binding interactions), morphologic (to detect cellular translocation) and functional-patch clamp methods (to elucidate functional interactions) are in place.

描述（取自应用程序） 现在已经很好地确定了beta单元的最远端阶段 胰岛分泌机械涉及质膜（PM）离子通道 （L型电压依赖性Ca2+通道，磺酰脲受体/K/ATP， 和k/ca通道）和胞吐蛋白，统称在 PM和胰岛素分泌颗粒（SG）。 最近的许多关注 已直接朝着最近克隆的结构和功能 SUR/K/ATP和PM上的Ca2+通道，以及SNARE蛋白在 胰岛素SG胞吐作用。 我对比了，关于beta细胞K/CA知之甚少 渠道调节或SUR在SGS上的作用，其中占90％ Beta细胞SUR。 该建议假设直接吞噬蛋白质 调节β细胞K/CA活性和胰岛素SG-SUR。 要阐明弹药蛋白的机制的第一个目的 调节Beta单元/CA通道功能，初步贴片夹数据 生成的生成以证明突变圈的过表达 胰岛素瘤中的蛋白质击中细胞使K/CA通道敏感以闭合。 这导致了葡萄糖诱发的胰岛素分泌的增强， K/CA的闭合在葡萄糖后将PM保持在去极化状态 刺激。 使用斑块夹方法，提出的研究为三倍， 包括：1）识别此圈圈蛋白中的功能域 以放大胰岛素的方式与K/CA通道相互作用 分泌物，以及2）探索可能相互作用的其他军鼓蛋白 用这种突变的圈套形成复合物，以进一步调节K/CA 频道，3）应用这些研究中从β细胞中获得的见解 线至）正常胰岛β细胞，以扩增胰岛素分泌 反应，从而确定药物的潜在新型治疗靶标 发展; b）糖尿病胰岛β细胞，以探索可能 SNARE-K/CA相互作用的扭曲可能是 糖尿病的胰岛素分泌缺陷。 要阐明网罗的功能相互作用的第二个目的 胰岛素SG-SUR，假设是这些相互作用可能有用：1） 实现SG-SG（同型）融合作为增强胰岛素的机​​制 胞吞作用，或2）从SGS中的此储备库招募Surs到 surs可以采取行动的下午。 对于拟议的研究，所需的 抗体和cDNA探针（许多已经亚克隆到表达载体中） 已获得或生成。 分子生物学的组合 （要操纵这些蛋白质的表达），生化（确定 蛋白质结合相互作用），形态学（检测细胞 易位）和功能点夹夹方法（阐明功能 互动）就位。