Function of calcium exchangers in vetebrate pigmentation

描述（由申请人提供）：含有黑色素沉着的细胞有助于视力，保护免受紫外线辐射，并可转化为肿瘤。这个建议是基于我们实验室的三个相关发现。1)斑马鱼色素减退突变体，金色，显示出与浅色皮肤的人相似的黑素体表型。2)在这个突变体中受影响的基因是slc 24 a5，它编码一个假定的K-依赖性Na/Ca交换。3)人类直系同源基因SLC 24 A5的编码多态性是非洲人和欧洲人之间人类肤色差异的重要决定因素。我们的长期目标是阐明SLC 24 A5蛋白在黑素生成中的作用和机制。我们提出了一个合作，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。我们正在验证这样一个假设：slc 24 a5是一种位于黑素体或其前体细胞膜上的钙离子交换剂，其转运活性所建立的细胞器离子环境在黑素体形态发生和色素形成中很重要。具体目标1旨在确定使用双标记，共聚焦免疫荧光显微镜，亚细胞分级分离，和免疫电子显微镜的slc 24 a5蛋白的黑素体和它们的前体之间的亚细胞分布。特异性目标2结合超微结构分析与反义吗啉代敲低来定义slc 24 a5的形态发生作用。具体目标3旨在使用钙成像、电生理学和Ca-45转运研究的组合来建立slc 24 a5蛋白的转运特性。具体目标4解决了SLC 24 A5中常见人类多态性的功能后果。这项工作的发现将增强我们对脊椎动物（包括人类）黑素体形态发生和黑色素沉着的理解。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，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（首先在斑马鱼中发现）是如何影响色素沉着的，并为治疗皮肤色素沉着问题（可能是恶性黑色素瘤）的药物奠定基础。

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.