INTERCELLULAR COMMUNICATION IN THE LENS

描述：具体目标#1：PI将比较单通道 Cx46和Cx56半通道的性质及其受钙离子的调节， 质子和磷酸化。目标将是查看Cx46和CX50(或Cx56 和Cx45.6，鸡的对应物)可以与 新的功能特性。PI将突变连接蛋白以定位Ca++ 负责通道关闭从而定位氨基酸的结合位点 在水道的毛孔里或附近的。她将决定 突变体在连接通道pH调节中的C末端 缺少部分细胞质C-末端的晶状体纤维连接蛋白。这个 申请者在她的视觉实验系统中有独特的优势 随着时间的推移在单通道上，因此能够确定解离 钙离子与半沟槽相互作用的常数。她将研究 细胞外和细胞内钙离子对连接通道的影响 在异构体实验中，Pi将共表达两个连接蛋白并查看 用于具有与仅从父级预测的属性不同的属性的频道。 这是一种可行的方法，因为其他连接蛋白已经混合在一起 导致它们的细胞间通道具有独特的生理特性。《少年派》 还将研究Cx46和Cx56对Ca++敏感性的差异 半条腿。她将构建嵌合体，看看哪些会导致转换 敏感度。PI将观察Cx56的pH敏感性的变化 截断突变体。 具体目标2：PI将描述镜片的功能特性 转染N2A或HeLa细胞的通道。需要解决的问题包括： 半通道的特性可以在多大程度上与 细胞间通道？46和50有什么不同？做46和50做 异构体连接子？刺激磷酸化的效果是什么 关于频道属性的？申请人已经安排了与Dr。 埃里克·拜尔。拜尔已经有了CX50 N2A转染体。这些研究将 用双全细胞膜片钳完成，专注于一些突变型 连接蛋白在卵母细胞系统中发育。公安部已经提出了一些 初步数据显示，她可以获得DWCPC数据。

DESCRIPTION: Specific Aim #1: The PI will compare single channel properties of Cx46 and Cx56 hemichannels and their regulation by Ca++, protons and phosphorylation. Goals will be to see if Cx46 and Cx50 (or Cx56 and Cx45.6, the chicken counterparts) can form heteromeric channels with novel functional properties. The PI will mutate connexins to locate Ca++ binding sites responsible for channel closure and thus localize amino acids in or near the pore of the channel. She will determine the role of the C-terminus in pH regulation of junctional channels by engineering mutants of lens fiber connexins which lack portions of the cytoplasmic C-terminus. The applicant has the unique advantage in her experimental system of looking over time at single channels and thus being able to determine dissociation constants for Ca++ interaction with the hemichannels. She will study the effects of extracellular and intracellular Ca++ on the junctional channels. With the heteromeric experiments, PI will co-express two connexins and look for channels with properties unique from those predicted from parents alone. This is a feasible approach since other connexins have been mixed and have resulted in unique physiologies in their intercellular channels. The PI will also study differences in Ca++ sensitivity of Cx46 and Cx56 hemichannels. She will construct chimeras to see which result in a switch of sensitivity. The PI will look at changes in pH sensitivity of Cx56 truncation mutants. Specific Aim #2: The PI will characterize the functional properties of lens channels transfected into N2A or HeLa cells. Questions to be addressed are: to what extent can the properties of hemichannels be correlated with intercellular channels? How do 46 and 50 differ? Do 46 and 50 make heteromeric connexons? What are the effects of stimulating phosphorylation on channel properties? The applicant has arranged a collaboration with Dr. Eric Beyer. Beyer already has Cx50 N2A transfectants. These studies will be done with double whole-cell patch clamp, focusing on some of the mutant connexins developed in the oocyte system. The PI has presented some preliminary data showing that she can obtain DWCPC data.