The Role of SLC24A5 (NCKX5) in Human Skin Pigmentation: The Importance of Cation Transport Activity

Human pigmentation is determined by melanosome development and melanin production through melanogenesis, a process regulated by melanogenic proteins. A single nucleotide polymorphism (A111T) in the SLC24A5 gene, which encodes the K⁺-dependent Na⁺/Ca²⁺ exchanger NCKX5, is a major determinant of normal variation in human skin and iris color. Despite the suggested role of NCKX5 in skin pigmentation, its ion transport function in melanogenesis remains unclear. To address this, we silenced SLC24A5 in pigmented human MNT1 melanoma cells using CRISPR/Cas9-mediated knockout. Loss of NCKX5 resulted in a marked reduction in eumelanin levels and the formation of abnormal, non-uniform melanosomes with defective fibrillar structure. To evaluate the functional role of NCKX5, we reconstituted depigmented cells with myc-tagged NCKX5 variants, including the transport-deficient mutant D383N. Reconstitution with the A111 variant of NCKX5 restored eumelanin synthesis more effectively than the T111 variant, whereas the D383N variant failed to rescue pigmentation. Moreover, the addition of the lysosomotropic weak base NH₄Cl to the growth medium, which neutralizes the acidic pH of intracellular organelles, markedly increased eumelanin content in depigmented cells lacking NCKX5, suggesting a link between NCKX5-mediated Ca²⁺ transport activity in the TGN and melanosomal pH regulation. We also discuss the many NCKX5 mutants that have been reported to affect pigmentation in both humans (Oculocutaneous albinism or OCA6) and animals in the context of their potential effects on NCKX5-mediated Ca²⁺ transport activity.