ENAC CHANNEL CONTROLS EPIDERMAL DIFFERENTIATION

离子通量控制角化细胞分化的早期和晚期，指导分化特异性蛋白质的合成和脂质分泌等过程。药理学实验表明，阿米洛利敏感的离子电导控制Ca2+刺激角质细胞分化，导致角质细胞探测阿米洛利可阻断上皮钠通道（EnaC）。在培养的角质形成细胞和表皮中鉴定出EnaC α、β和γ亚基。EnaC亚基表达的两个方面依赖于角质形成细胞的分化状态。首先，β亚基仅在分化程度较高的角质形成细胞中表达，而α和γ亚基则在整个分化过程中表达。其次，α和β亚基在成人表皮中表达，而在胎儿表皮中不表达。研究表明，α亚基敲除小鼠表皮增生，空泡化，证实了我们的假设，即该通道控制分化的早期方面。此外，该皮肤的电子显微摄影显示了一个额外的缺陷，即过早的脂质分泌。我们将通过比较EnaC α亚单位敲除小鼠与正常对照组的角质形成细胞和表皮来定义EnaC通道的特性和功能。我们将通过比较融合前和融合后正常角质形成细胞以及在非洲爪蟾卵母细胞中表达这些不同亚基组合来研究分化过程中不同亚基的表达如何改变通道特性。最后，我们将定义角化细胞EnaC通道调节，测试三种可能的调节因子：质膜Ca2+受体，cAMP和拉伸。本研究将描述EnaC通道介导角质形成细胞分化的机制。

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.