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Ca ++的调节， 质子和磷酸化。 目标是看看Cx46和Cx50（或Cx56）是否 和Cx45.6，鸡的对应物）可以形成异聚通道， 新的功能特性。 PI将突变连接蛋白以定位Ca ++ 负责通道关闭的结合位点，从而定位氨基酸 在通道的孔中或附近。 她将决定 C-末端在连接通道pH调节中的作用 缺乏部分细胞质C-末端的透镜纤维连接蛋白。 的 申请人在她的实验系统中具有独特的优势， 从而能够确定解离 Ca ++与半通道相互作用的常数。 她将研究 细胞外和细胞内Ca ++对连接通道的影响。 通过异聚体实验，PI将共表达两种连接蛋白， 对于具有与仅从父预测的那些特性不同的特性的通道。 这是一种可行的方法，因为其他连接蛋白已经混合， 导致了它们细胞间通道的独特生理学。 的pi 还将研究Cx46和Cx56的Ca ++敏感性的差异 半通道 她将构建嵌合体，看看哪一个会导致开关， 的敏感性。 PI将观察Cx56 pH敏感性的变化 截短突变体 具体目标#2：PI将表征透镜的功能特性 通道转染到N2A或HeLa细胞中。 需要解决的问题是： 半通道的特性在多大程度上与 细胞间通道？ 46和50有什么区别？ 46和50是不是 异聚连接体 刺激磷酸化的作用是什么 频道财产的事吗 申请人已安排与博士合作。 埃里克·拜尔。 拜尔已经有了Cx50 N2A转染子。 这些研究将 用双全细胞膜片钳技术，聚焦于一些突变体， 连接蛋白在卵母细胞系统中发育。 PI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.