Regulation of volume in the lens

项目总结 这项拨款提案的总体目标是更好地理解缝隙连接、连接蛋白半通道的作用。 和钙激活的氯离子通道在维持晶状体内稳态和确定如何扰动 在这些系统中会导致白内障的发展和无法准确聚焦。这些渠道 似乎调节组成完整晶状体的单个晶状体纤维细胞的清晰度、体积和形状。 晶状体中缺乏适当的流动模式会导致白内障。这些透镜系统中的问题也可能影响 晶状体在调节过程中改变形状的能力。这个问题在个人中尤为严重。 患有老花眼。具体目标有三个： 目的1：研究与白内障相关的连接蛋白突变的影响。 实验和建模方法。我们将重点研究白内障相关区域的突变。 已知在未对接的半通道的门控中起重要作用。这些研究将有助于确定 突变导致疾病的机制。他们还可能确定潜在的目标 开发治疗药物来调节连接蛋白通道的开放概率。 目标2：使用我们实验室开发的一种新制剂，以更好地了解纤维如何 细胞对机械应力做出反应。将检验以下假设：(1)Cx46半通道是 被机械应力激活的。拟议的研究将检验这些通道的机械敏感性。 在缺乏CX50的外周纤维细胞中使用全细胞膜片钳技术。机械拉伸将是 使用两种不同的刺激诱导：(1)施加于贴片吸管的正压脉冲；(2)低张 压力。其他通道对晶状体机械敏感膜电导的可能贡献 将通过对双基因敲除(Cx46-/-)进行全细胞膜片钳实验来研究纤维细胞 ；CX50-/-)纤维。 目的3：了解钙激活氯离子通道在外周纤维容量调节中的作用 细胞。将检验以下两个假设：(3A)外周晶状体纤维细胞表达钙激活 可归因于阴离子通道蛋白TMEM16A的氯通道；(3b)这些通道可 膜拉伸通过嘌呤能信号间接激活或通过机械敏感性钙内流间接激活 频道。

PROJECT SUMMARY The overall goal of this grant proposal is to better understand the role of gap junctions, connexin hemichannels and calcium-activated chloride channels in maintaining lens homeostasis and to determine how perturbations in these systems lead to the development of cataracts and the inability to focus accurately. These channels appear to regulate the clarity, volume, and shape of the individual lens fiber cells that make up the intact lens. A lack of proper flow patterns in the lens leads to cataracts. Problems in these lens systems also likely affect the ability of the lens to alter its shape during accommodation. This problem is particularly acute in individuals suffering from presbyopia. There are three specific aims: Aim 1: To characterize the effects of cataract-associated connexin mutations using a combination of experimental and modeling approaches. We will focus on cataract associated mutations located in regions that are known to be important in the gating of undocked hemichannels. These studies will help to identify mechanisms by which the mutations lead to disease. They may also identify potential targets for the development of therapeutic agents to modulate the open probability of connexin channels. Aim 2: To use a new preparation developed by our laboratory to gain a better understanding about how fiber cells respond to mechanical stress. The following hypothesis will be tested: (1) Cx46 hemichannels are activated by mechanical stress. The proposed studies will examine the mechanosensitivity of these channels in peripheral fiber cells lacking Cx50 using the whole cell patch clamp technique. Mechanical stretch will be induced using two different stimuli: (1) positive pressure pulses applied to the patch pipette; (2) hypotonic stress. The possible contribution of other channels to the mechanosensitive membrane conductance in lens fiber cells will be investigated by performing whole cell patch clamp experiments on double knockout (Cx46-/- ;Cx50-/-) fibers. Aim 3: To understand the role of calcium-activated chloride channels in volume regulation of peripheral fiber cells. The following two hypotheses will be tested: (3a) peripheral lens fiber cells express calcium-activated chloride channels that can be attributed to the anion channel protein, TMEM16a; (3b) these channels can be indirectly activated by membrane stretch via purinergic signaling or calcium influx through mechanosensitive channels.