Localization and function of SLC24A5 in skin pigmentation

Children'S Hosp Of Philadelphia, Philadelphia PA

Investigators

Abstract

Melanins are pigments responsible for skin, hair, and eye color in mammals and function in visual acuity, ocular development, and protecting skin from harmful effects of ultraviolet light. Melanins are synthesized by skin melanocytes and eye pigment cells within membrane-bound organelles called melanosomes. Variation in the type or quantity of melanin synthesized underlies characteristic skin tone differences among humans, and impaired melanosome biogenesis and/or melanin synthesis result in oculocutaneous albinism (OCA) or ocular albinism (OA) with corresponding skin and visual defects. Among the nine genes that are mutated in non- syndromic OCA and OA patients, one of the least understood is SLC24A5. Inactivating mutations in SLC24A5 cause OCA type 6, and genetic variation at the SLC24A5 locus is a major cause of skin tone differences across human populations. SLC24A5 encodes NCKX5, a member of a family of potassium-dependent sodium/calcium exchangers that facilitate ion transport across membranes. While the ion antiporter activity of NCKX5 has been validated, neither the subcellular localization of NCKX5 nor its role in pigmentation are well understood. Competing published studies suggest that NCKX5 localizes to the Golgi, melanosomes, or mitochondria, and functional studies have suggested roles for NCKX5 in mitochondrial or Golgi function, control of melanosome protein levels, or regulation of melanosome pH – a key determinant of melanogenesis – with no mechanistic insights into how NCKX5 influences these parameters. Each of these studies lacks rigor and suffers from the use of non-validated and/or poorly designed reagents, overexpressed tagged NCKX5 transgenes, a lack of attention to protein or organelle maturation, and experiments that lack rigor and proper controls and fail to consider dynamic cellular processes. We propose to circumvent these concerns and to develop tools that will allow us to better define NCKX5 localization and function in melanocytes. We will generate a panel of congenic immortalized mouse melanocyte cell lines in which (i) epitope or fluorescent tags are inserted into the Slc24a5 gene locus at distinct sites so that tagged NCKX5 is expressed at endogenous levels, and (ii) the Slc24a5 gene is knocked out or mutagenized to variants corresponding to human OCA6 or light skin-associated alleles. These tools will be used to define NCKX5 localization, assess how SLC24A5 loss- of-function impacts melanosome and Golgi pH, protein content and trafficking, and ultimately to define how SLC24A5 function impacts melanogenesis. Given the importance to human health of SLC24A5 gene alterations, a clearer determination of NCKX5 localization and function in melanocytes will advance our basic understanding of pigmentation and inform strategies for novel OCA therapeutics and to mitigate the damaging effects of ultraviolet radiation in individuals with light skin-associated SLC24A5 alleles. The specific aims are: 1. Define the site of NCKX5 localization and function at endogenous levels of expression. 2. Assess the impact of impaired SLC24A5 function on organelle protein levels, trafficking, and pH.