ENAC CHANNEL CONTROLS EPIDERMAL DIFFERENTIATION

ENAC 通道控制表皮分化

• 批准号: 6197174
• 负责人: Theodora M Mauro
• 金额: $ 19.83万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6197174
• 关键词: Xenopus; Xenopus oocyte; amiloride; antisense nucleic acid; calcium binding protein; calcium flux; cell differentiation; cyclic AMP; gene targeting; genetically modified animals; histology; keratinocyte; laboratory mouse; protein structure function; skin; sodium channel; tissue /cell culture; voltage /patch clamp

Ionic fluxes control both early and late stages of keratinocytes differentiation, directing process such as synthesis of differentiation-specific proteins and lipid secretion. Pharmacologic experiments, which demonstrated that an amiloride-sensitive ionic conductance controlled Ca2+ stimulated keratinocyte differentiation, led up to probe keratinocytes for the amiloride-blockable epithelial sodium channel (EnaC). EnaC alpha, beta and gamma subunits were identified in cultured keratinocytes and epidermis. Two aspects of EnaC subunit expression were dependent on the differentiation state of the keratinocytes. First, the beta subunit was expressed only in more differentiated keratinocytes, while the alpha and gamma subunits were expressed throughout differentiation. Second, alpha and beta subunits were expressed in adult but not fetal epidermis. Studies demonstrating hyperplastic, vacuolated epidermis in alpha-subunit knockout mice validated our hypothesis that this channel controls early aspects of differentiation. Further, electron micrography of this skin demonstrated an additional defect, i.e. premature lipid secretion. We will define EnaC channel properties and functions by comparing keratinocytes and epidermis of EnaC alpha-subunit knockout mice versus normal controls. We will examine how expression of different subunits during differentiation changes channel properties by comparing preconfluent versus post-confluent normal keratinocytes and by expressing these different subunit combinations in xenopus oocytes. Finally, we will define the keratinocyte EnaC channel regulation, testing three possible regulators: plasma membrane Ca2+ receptor, cAMP and stretch. This study will delineate the mechanism(s) by which this EnaC channel mediates keratinocytes differentiation.

离子通量控制角质形成细胞分化的早期和晚期，指导过程，例如分化特异性蛋白质和脂质分泌的合成。药理学实验表明，Amiloride敏感的离子电导控制CA2+刺激的角质形成细胞分化，导致了可探测可依thimoride可嵌合的可胺可抑制可氨氧化钠的上皮钠通道（ENAC）。在培养的角质形成细胞和表皮中鉴定出ENAC Alpha，beta和Gamma亚基。 ENAC亚基表达的两个方面取决于角质形成细胞的分化状态。首先，仅在更分化的角质形成细胞中表达了β亚基，而α和伽马亚基在整个分化过程中均表达。其次，α和β亚基在成人表皮中表达，但没有表达。研究表明，α-亚基敲除小鼠中增生的，空泡的表皮验证了我们的假设，即该通道控制分化的早期方面。此外，该皮肤的电子显微照片表现出额外的缺陷，即早产脂质分泌。我们将通过比较ENAC Alpha-Subunit敲除小鼠的角质形成细胞和表皮来定义ENAC通道的性质和功能。我们将通过比较Xenopus卵母细胞中的这些不同的亚基组合来研究在分化变化过程中不同亚基的表达如何通过比较偶联的正常角质形成细胞和共体会后的正常角质形成细胞和这些不同的亚基组合来进行表达。最后，我们将定义角质形成ENAC通道调节，测试三个可能的调节剂：质膜Ca2+受体，cAMP和拉伸。这项研究将描述该ENAC通道介导角质形成细胞分化的机制。