Regulation of volume in the lens

镜片体积的调节

• 批准号: 9156474
• 负责人: Lisa Ebihara
• 金额: $ 39万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9156474
• 关键词: Acute; Affect; Anions; Applications Grants; Calcium; Cataract; Cells; Chloride Channels; Connexins; Development; Disease; Dyes; Experimental Models; Fiber; Gap Junctions; Goals; Homeostasis; Individual; Knock-out; Knockout Mice; Laboratories; Lead; Lens Fiber; Mechanical Stress; Mechanics; Membrane; Mutation; Patch-Clamp Techniques; Pattern; Peripheral; Play; Point Mutation; Preparation; Presbyopia; Probability; Proteins; Pulse Pressure; Regulation; Role; Shapes; Signal Transduction; Speed; Stimulus; Stress; Stretching; System; Testing; Therapeutic Agents; fiber cell; lens; patch clamp; pressure; research study; response; uptake; vision development

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.

项目摘要 该赠款提案的总体目标是更好地了解差距连接的作用，连接蛋白半通道 和钙激活的氯化物通道在维持晶状体稳态方面并确定扰动如何 在这些系统中，导致白内障的发展和无法准确集中。这些渠道 似乎可以调节组成完整晶状体的单个透镜纤维细胞的清晰度，体积和形状。 镜头中缺乏适当的流动模式会导致白内障。这些镜头系统中的问题也可能影响 镜头在适应期间改变其形状的能力。这个问题在个人中特别严重 患有长老会。有三个特定的目标： 目标1：表征与白内障相关的连接蛋白突变的影响 实验和建模方法。我们将专注于区域中的白内障相关突变 已知在不可锁定的半通道的门控中很重要。这些研究将有助于确定 突变导致疾病的机制。他们还可以确定 开发治疗剂以调节连接蛋白通道的开放概率。 目的2：使用我们实验室开发的新准备工作，以更好地了解如何纤维 细胞应对机械应力。将测试以下假设：（1）CX46半通道是 通过机械应力激活。拟议的研究将检查这些通道的机械敏感性 在使用整个细胞斑块夹技术缺乏CX50的外围纤维细胞中。机械拉伸将是 使用两种不同的刺激诱导：（1）施加在斑块移液器上的正压脉冲； （2）低音 压力。其他通道对镜头中机械敏感膜电导的可能贡献 将通过在双基因敲除（CX46 - / - 进行全细胞斑块夹实验）来研究纤维细胞 ; cx50 - / - ）纤维。 目标3：了解钙活化的氯化物通道在外周纤维体积调节中的作用 细胞。将测试以下两个假设：（3A）外围透镜纤维细胞表达钙激活 可以归因于阴离子通道蛋白TMEM16A的氯化物通道； （3B）这些频道可以是 通过嘌呤能信号传导或通过机械敏感的钙涌入间接激活的膜拉伸激活 频道。