Function of calcium exchangers in vetebrate pigmentation

描述(申请人提供)：含有黑色素色素的细胞有助于视力，防止紫外线辐射，并可转化为肿瘤。这项建议是基于我们实验室的三项相关发现。1)斑马鱼色素减退突变体，金色，表现出与浅色皮肤人群相似的黑素体表型。2)该突变体的致病基因为SLC24A5，该基因编码一种钾依赖的钠/钙交换因子。3)人类同源基因SLC24A5的编码多态是非洲人和欧洲人肤色差异的重要决定因素。我们的长期目标是阐明SLC24A5蛋白在黑素合成中的作用和机制。我们提出了一种协作的、多学科的方法，包括超微结构分析、功能基因组学和运输研究。我们正在验证一种假设，即SLC24A5是一种位于黑素体或其前体细胞膜上的钙交换器，由其转运活性建立的细胞器离子环境在黑素体的形态发生和色素形成中具有重要作用。特定目的1利用双标记、共聚焦免疫荧光显微镜、亚细胞分级和免疫电子显微镜，确定SLC24A5蛋白在黑素小体及其前体中的亚细胞分布。特异靶2结合超微结构分析和反义吗啉基因敲除来确定SLC24A5的形态发生作用。具体目标3试图利用钙成像、电生理学和钙-45转运研究相结合的方法来确定SLC24A5蛋白的转运特性。特定目的4解决了SLC24A5中常见的人类多态的功能后果。这项拟议工作的发现将加强我们对包括人类在内的脊椎动物中黑素小体形态发生和黑色素沉积的理解。含有黑色素的细胞有助于视力，保护我们免受紫外线辐射，当转化时，会成为人类最致命的癌症之一，恶性黑色素瘤。这项工作将阐明一种新发现的钠钙交换蛋白是如何影响色素沉着的，并为治疗皮肤色素沉着问题的药物奠定基础，甚至可能是恶性黑色素瘤。

DESCRIPTION (provided by applicant): Cells containing melanin pigmentation aid vision, protect against UV radiation, and can transform into neoplasms. This proposal is based upon three related discoveries in our lab. 1) The zebrafish hypopigmentation mutant, golden, shows a melanosomal phenotype similar to that seen in light-skinned people. 2) The affected gene in this mutant is slc24a5, which encodes a putative K-dependent-Na/Ca exchanger. 3) A coding polymorphism in the human orthologue SLC24A5 is an important determinant of the difference in human skin color between Africans and Europeans. Our long-term goal is to elucidate the role and mechanism of the SLC24A5 protein in melanogenesis. We propose a collaborative, multidisciplinary approach that includes ultrastructural analysis, functional genomics, and transport studies. We are testing the hypothesis that slc24a5 is a calcium exchanger located in the membranes of melanosomes or their precursors, and that the organellar ionic milieu established by its transport activity is important in melanosome morphogenesis and pigment formation. Specific Aim 1 seeks to determine the subcellular distribution of the slc24a5 protein among melanosomes and their precursors using double-label, confocal immunofluorescence microscopy, subcellular fractionation, and immunoelectron microscopy. Specific Aim 2 combines ultrastructural analysis with antisense morpholino knockdown to define the morphogenetic roles of slc24a5. Specific Aim 3 seeks to establish the transport properties of the slc24a5 protein using a combination of calcium imaging, electrophysiology, and Ca-45 transport studies. Specific Aim 4 addresses the functional consequences of the common human polymorphism in SLC24A5. Findings from the proposed work will enhance our understanding of melanosome morphogenesis and melanin pigmentation in vertebrates, including humans. Melanin-containing cells aid vision, protect us from UV radiation, and when transformed, become one of the deadliest cancers in humans, malignant melanoma. This work will clarify how a newly-discovered sodium- calcium exchanger protein, first characterized in zebrafish, affects pigmentation, and lays a foundation for drugs to treat skin pigmentation problems, and perhaps malignant melanoma.