SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis

Adrian M. Smith,Robert J. Winkfein,Paul P.M. Schnetkamp,Tony Dadd,Martin R. Green,Stephen Wilson,Richard M. Ogborne,Sarah E. Askew,Shubana Kazi,Robert T. Szerencsei,Rebecca S. Ginger,Dudley Ferdinando

doi:10.1074/jbc.m707521200

Adrian M. Smith, Robert J. Winkfein + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m707521200

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Abstract

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

Highlights

SLC24A5 Regulates Human Epidermal Melanogenesis ization in normal human epidermal melanocytes (NHM), and using siRNA-mediated knockdown we demonstrate conclusively that SLC24A5 expression is required for melanin synthesis in both cultured mouse and human melanocytes
Through site-directed mutagenesis in a heterologous expression system, we demonstrate that the residue switch encoded by the nsSNP in SLC24A5 markedly alters exchanger activity when introduced into NCKX2 or NCKX5
NCKX5 Protein Is Located in an Intracellular Membrane and Partially Co-localizes with the Trans-Golgi Network—The specificity of affinity-purified antisera for native NCKX5 protein was assessed through siRNA-mediated knockdown in NHM

Summary

Introduction

These data support the hypothesis that a nsSNP in the SLC24A5 gene directly alters human skin color through its effect on the sodium-calcium exchanger activity of NCKX5. Non-transfected HEK cells do not show any reactivity with the anti-Myc antibody.3 These results show that the affinity-purified polyclonal antibody preparation detects NCKX5 protein in human melanocyte extracts, with some nonspecific reactivity, suggested by the detection of other higher and lower molecular weight bands that are not affected by SLC24A5 knockdown.

Results

Conclusion