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Cited by 158melanin and skin pigmentationCalcium signaling and nucleotide metabolismIon Transport and Channel Regulation

SLC24A5 encodes a trans-Golgi network protein with potassium-dependent sodium-calcium exchange activity that regulates human epidermal melanogenesis

Open access
Ginger, Rebecca S., Askew, Sarah E., Ogborne, Richard M., Wilson, Stephen, Ferdinando, Dudley, Dadd, Tony, Smith, Adrian M., Kazi, Shubana +4 more·2008·Journal of Biological Chemistry
DOI 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.

AI snapshot

Written from the paper’s full text.

TL;DR

SLC24A5 encodes NCKX5, a potassium-dependent sodium-calcium exchanger that is partially localized to the trans-Golgi network in human epidermal melanocytes and is required for melanin synthesis. The skin-color-associated nsSNP rs1426654 changes a conserved residue and reduces NCKX5/NCKX2 exchange activity.

Key findings

  • SLC24A5 was the predominant sodium-calcium exchanger transcript in cultured human and mouse melanocytes, >100-fold more highly expressed than any other SLC24A transcript; SLC8 family transcripts were not detected.
  • Native NCKX5 ran as a ~43-kDa triplet on SDS-PAGE, partially co-localized with the trans-Golgi marker TGN46, showed no plasma membrane staining, and co-fractionated with TGN46 and pigment/TYRP1-enriched fractions.
  • SLC24A5 knockdown reduced melanin production: 87.7% in mouse B16 melanocytes (p < 0.0001), 22% and 30.5% in NHM with siRNA 185 and 492 (p < 0.0001), and 80–90% in re-differentiating melanoblasts (p < 0.0001); melanosomal markers pmel17, MART1, TYR, and TYRP1 were reduced, while LAMP-1 increased.
  • NCKX5 showed K+-dependent Na+/Ca2+ exchange activity, and the threonine-encoding allele at NCKX5 residue 111 or the equivalent NCKX2 residue 177 caused substantially reduced exchanger activity.

Methods

  • Cultured normal human melanocytes (NHM), B16 F10 mouse melanoma cells, HEK293 cells, and High Five insect cells were used with real-time PCR, immunoblotting, immunofluorescence confocal microscopy, sucrose density gradient fractionation, and melanin assays.
  • SLC24A5 was knocked down with siRNA duplexes in B16 and NHM, including de-differentiated melanoblasts; melanin was normalized to protein and analyzed by ANOVA with Dunnett’s adjustment.
  • NCKX2 and NCKX5 mutants were expressed in High Five cells, and reverse exchange was measured as K+-dependent 45Ca2+ uptake in Na+-loaded cells.

Limitations

  • The paper states that the unique intracellular localization of NCKX5 necessitated new assays because current methodologies were not suitable for measuring intracellular sodium-calcium exchange activity, and NCKX5 activity in the heterologous system was low.
  • The paper states that it cannot rule out a cryptic melanosomal localization for NCKX5, that the identity of the punctate staining was not determined, and that no co-localization between NCKX5 and melanosomal markers was detected by immunofluorescence.
  • Evident from the text: the NHM total-melanin knockdown effect was modest (22–30.5%), and the anti-NCKX5 antisera showed some nonspecific reactivity on Western blots.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1074/jbc.m707521200

Cites

49 of 49 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 41 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Native human NCKX5 in cultured human epidermal melanocytes runs as a triplet of approximately 43 kDa on SDS-PAGE.

    “In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of ϳ43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network.”
    • Also stated in: § NCKX5 Protein Is Located in an Intracellular · boosted
      “Western blots of cell extracts harvested 5 days after transfection and probed with affinity-purified anti-NCKX5-(C) sera showed that a triplet of bands of ϳ43 kDa were clearly detected in extracts from control or untreated cells but were virtually absent in extracts from SLC24A5 siRNA-treated cells (Fig. 2A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Native human NCKX5 is partially localized to the trans-Golgi network in cultured human epidermal melanocytes.

    “In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of ϳ43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network.”
    • Also stated in: § NCKX5 Protein Is Located in an Intracellular · plain
      “The perinuclear staining colocalized with the trans-Golgi marker TGN46 (Fig. 2C), although merged images of serial optical sections revealed that NCKX5 only co-localized with part of the TGN (Fig. 3A).”
    Quote in the paperSubstantial claim
  • Findingown data

    The non-synonymous SNP in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 exchange 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.”
    • Also stated in: § SLC24A5 Knockdown Reduces Melanin Production in NHM- · plain
      “Constructs carrying the threonine variant had a greatly reduced exchange activity compared with those carrying the alanine variant (Fig. 8C).”
    Quote in the paperSubstantial claim
  • Findingown data

    NCKX5 possesses potassium-dependent sodium-calcium exchanger activity.

    “Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity.”
    • Also stated in: § SLC24A5 Knockdown Reduces Melanin Production in NHM- · boosted
      “Reverse exchange of NCKX5 was only observed when extracellular K ϩ was present and no reverse exchange was observed in the presence of either extracellular Na ϩ or Li ϩ , confirming K ϩ -dependent Na ϩ /Ca 2ϩ exchange function.”
    Quote in the paperSubstantial claim
  • Findingown data

    Removal of the NCKX5 protein through siRNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production.

    “Removal of the NCKX5 protein through small interfering RNAmediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production.”
    Quote in the paperSubstantial claim
  • May combine two claims. 12
  • Classified as probably not a substantial claim. 12

  • May combine two claims.

    Findingown data

    In mouse melanocytes, low levels of SLC24A4 and very low levels of SLC24A1 and SLC24A3 were found.

    “In NMH low levels of SLC24A1 and very low levels of SLC24A3 and SLC24A4 were detected, and in the mouse melanocytes, low levels of SLC24A4 and very low levels of SLC24A1 and SLC24A3 were found.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Constructs carrying the threonine variant at residue 111 of NCKX5 or 177 of NCKX2 had greatly reduced exchange activity compared with those carrying the alanine variant.

    “Constructs carrying the threonine variant had a greatly reduced exchange activity compared with those carrying the alanine variant (Fig. 8C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A similar pattern of NCKX5 staining was observed in both light and dark NHM, although the perinuclear staining was less intense in lighter melanocytes.

    “A similar pattern of staining was observed in both light and dark NHM, although the perinuclear staining was less intense in lighter melanocytes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The apparent size of the NCKX5 bands (approximately 43 kDa) is surprisingly low compared with the predicted molecular mass in the absence of glycosylation of 54.9 kDa.

    “The apparent size of these bands is surprisingly low compared with the predicted molecular mass in the absence of glycosylation of 54.9 kDa.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Reverse exchange of NCKX5 was only observed when extracellular K+ was present and no reverse exchange was observed in the presence of either extracellular Na+ or Li+, confirming K+-dependent Na+/Ca2+ exchange function.

    “Reverse exchange of NCKX5 was only observed when extracellular K ϩ was present and no reverse exchange was observed in the presence of either extracellular Na ϩ or Li ϩ , confirming K ϩ -dependent Na ϩ /Ca 2ϩ exchange function.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    When SLC24A5 is disrupted through siRNA knockdown, melanosomal maturation is blocked, leading to a down-regulation of pmel17 and late melanosomal markers TYR and TYRP1.

    “Thus, when SLC24A5 is disrupted through siRNA knockdown, melanosomal maturation is blocked, leading to a down-regulation of pmel17 and late melanosomal markers TYR and TYRP1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    This SNP and another in SLC45A2 participated in the evolution of light skin in Europeans but not East Asians.

    “Subsequent analyses have pinpointed this SNP and another in the SLC45A2 gene as participants in the evolution of light skin in Europeans but not East Asians (9 -11).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [9] Norton, 2006, Mol. Biol. Evol. source

      Not checked in the free run.

    • cites [10] Myles, 2007, Hum. Genet. source

      Not checked in the free run.

    • cites [11] Soejima, 2007, Int. J. Med.

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    De-glycosylation of the recombinant NCKX5 protein removes the upper band, suggesting that the lower bands reflect signal peptide cleavage of unglycosylated protein.

    “De-glycosylation of the recombinant protein (Fig. 2B) removes the upper band, suggesting that the lower bands reflect signal peptide cleavage of unglycosylated protein.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    SLC24A5 plays a role very early in melanosome biogenesis and this process is intimately linked with lysosome biogenesis.

    “Conversely, expression of the lysosomal marker LAMP-1 was up-regulated after SLC24A5 knockdown, suggesting that SLC24A5 plays a role very early in melanosome biogenesis and that this process is intimately linked with lysosome biogenesis.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Reduced tyrosinase activity is measured in Caucasian compared with Negroid-derived melanocytes.

    “This might account for the reduced tyrosinase activity measured in Caucasian compared with Negroid-derived melanocytes (30).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Iozumi, 1993, J. Investig. Dermatol. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The nsSNP associated with skin color variation is causative, directly mediating phenotypic variation in skin color by altering NCKX5 exchanger activity.

    “These data also support the hypothesis that the nsSNP associated with skin color variation is causative, directly mediating phenotypic variation in skin color by altering NCKX5 exchanger activity.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The Golgi apparatus is an established calcium store.

    “The Golgi apparatus is an established calcium store (31), and its role in sorting and processing secretory and membrane proteins is highly sensitive to changes in calcium concentration within the lumen.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Pinton, 1998, EMBO J. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    All other NCKX proteins traffic to the plasma membrane where they remove Ca2+ from the cytoplasm to the extracellular space.

    “These data, supporting an intracellular, partial TGN localization, are surprising since all other NCKX proteins have been shown to traffic to the plasma membrane where they operate to remove Ca 2ϩ from the cytoplasm to the extracellular space (26 -28).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [26] Prinsen, 2000, J. Neurosci. source

      Not checked in the free run.

    • cites [27] Sheng, 2000, J. Biophys. source

      Not checked in the free run.

    • cites [28] Visser, 2007, J. Biol. Chem. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The NCKX family consists of five members (NCKX1-5) that use both the inward sodium gradient and the outward potassium gradient to extrude calcium across the plasma membrane.

    “The NCKX family currently consists of five members (NCKX 1-5) that are thought to use both the inward sodium gradient and the outward potassium gradient to extrude calcium across the plasma membrane.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Altimimi, 2007, Channels source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lamp1 is enriched in lysosomes.

    “The 85-kDa form of pmel17 that is down-regulated by SLC24A5 knockdown is a marker of early melanosomes (25), whereas Lamp1 is enriched in lysosomes (47).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Raposo, 2001, J. Cell Biol. source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    NHE7 is located in the TGN and post-Golgi vesicles and is differentially expressed in light and dark NHM.

    “In addition, the Na ϩ /H ϩ exchanger NHE7 is located in the TGN and post-Golgi vesicles (35,36) and is differentially expressed in light and dark NHM (37) melanosomal pH could also be applied to the TGN.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [35] Numata, 2001, J. Biol. Chem. source

      Not checked in the free run.

    • cites [36] Nakamura, 2005, J. Biol. Chem. source

      Not checked in the free run.

    • cites [37] Smith, 2004, Exp. Cell Res. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ca2+ is a co-factor for phenylalanine hydroxylase.

    “Ca 2ϩ is also a co-factor for phenylalanine hydroxylase (45); the latter has been suggested to protect the redox balance and prevent DNA damage (46).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Schallreuter, 1999, Biochem. Biophys. Res. Commun. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Endosomal membrane fusion events are Ca2+-mediated and likely modulated by changing local Ca2+ gradients.

    “Endosomal membrane fusion events, which are essential processes in melanosome and lysosome-related organelle biogenesis, are Ca 2ϩ -mediated (49, 50) and are likely modulated by changing local Ca 2ϩ gradients.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [49] Mayorga, 1994, J. Cell Biol.

      Not checked in the free run.

    • cites [50] Pryor, 2000, J. Cell Biol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the model proposed by Raposo et al., the coated endosome serves as a critical sorting point between endocytic and pre-melanosomal pathways.

    “In the model proposed by Raposo et al. (47,48), the coated endosome (also referred to as a stage 1 melanosome) serves as a critical sorting point between endocytic and pre-melanosomal pathways, from which macromolecules are specifically directed into melanosomal or lysosomal pathways.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [47] Raposo, 2001, J. Cell Biol. source

      Not checked in the free run.

    • cites [48] Raposo, 2007, Curr. Opin. Cell Biol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    NCKX5 is required for the correct delivery of melanosomal components such as pmel17 into the melanosomal pathway.

    “In this model NCKX5 is required for the correct delivery of melanosomal components such as pmel17 into the melanosomal pathway.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    De-pigmenting effects of certain tricyclic compounds elicit mis-trafficking of tyrosinase leading to co-localization with Lamp1.

    “The importance of correctly regulating such intracellular trafficking mechanisms to melanogenesis is highlighted by the de-pigmenting effects of certain tricyclic compounds that elicit the mis-trafficking of tyrosinase leading to its co-localization with Lamp1 (51).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Ni-Komatsu, 2007, J. Investig. Dermatol.

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    The calcium concentration of the Golgi compartment is highly regulated in keratinocytes.

    “Recent work has shown that the calcium concentration of the Golgi compartment is highly regulated in keratinocytes (32,33), and the calcium gradients across the Golgi play a fundamental part in intracellular calcium signaling and homeostasis (for review, see Ref. 34).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [32] Hu, 2000, Nat. Genet. source

      Not checked in the free run.

    • cites [33] Sudbrak, 2000, Hum. Mol. Genet. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S1 (asserted).

    Interpretationreasoning

    NCKX5 modulates melanogenesis through a role in the trafficking/sorting decisions that occur within the coated endosome.

    “We, therefore, propose a second model in which NCKX5 modulates melanogenesis through a role in the trafficking/sorting decisions, which occur within the coated endosome.”
    Quote in the paperNot a substantial claim
23 of 23

  • The paper relies on this without support.

    The exchanger activity of NCKX5 has not yet been experimentally demonstrated.

    “Although SLC24A5 is predicted to encode an NCKX protein, the exchanger activity of NCKX5 has yet to be experimentally demonstrated.”

  • The paper relies on this without support.

    NCKX5's credentials as a sodium-calcium-potassium exchanger had not previously been established experimentally.

    “However, despite a high degree of similarity at the amino acid level (Fig. 8B), the credentials of NCKX5 as a sodium-calcium-potassium exchanger have not previously been established experimentally.”

  • The paper relies on this without support.

    Currently available methodologies are not suitable for measuring intracellular sodium-calcium exchange activity.

    “The unique intracellular localization of NCKX5 necessitates the development of new assays as the currently available methodologies are not suitable for measuring intracellular sodium-calcium exchange activity.”

  • The paper relies on this without support.

    The more alkaline environment of Negroid melanosomes favors the activity of tyrosinase.

    “The more alkaline environment of Negroid melanosomes is believed to favor the activity of tyrosinase, a rate-limiting catalyst of melanin synthesis that operates optimally at neutral pH.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Involvement of NCKX5 in this process is highly likely given the role of this family of proteins in transmembrane Ca2+ transport and the higher ion exchange activity of NCKX5-111A compared to NCKX5-111T.

    “The involvement of NCKX5 in this process is highly likely given the role of this family of proteins in transmembrane Ca 2 þ transport (Schnetkamp, 2004), and the higher ion exchange activity of NCKX5-111A compared to NCKX5-111T (Ginger et al., 2008).”

    From Analysis of Cultured Human Melanocytes Based on Polymorphisms within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P Loci (2009) · cites this paper as Ginger et al., 2008

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. Soejima, 2007, Int. J. Med., 121, 36
  2. Kang, 1998, Pfluegers Arch. Eur. J. Physiol., 435, 350
  3. Mayorga, 1994, J. Cell Biol., 269, 30927
  4. Ni-Komatsu, 2007, J. Investig. Dermatol.

Cited by

Sorted by citations

Related

Authors
Ginger, R. S., Askew, S. E., Ogborne, R. M., Wilson, S., Ferdinando, D., Dadd, T., Smith, A. M., Kazi, S., Szerencsei, R. T., Winkfein, R. J., Schnetkamp, P. P. M., & Green, M. R.
Affiliations
  • Unilever (United Kingdom) (GB)
  • University of Calgary (CA)
Corresponding author
Ginger, Rebecca S.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2008-02
Volume
283
Issue
9
Pages
5486-5495
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m707521200
OpenAlex
W2109627824
Cited by
158
References
49
Citations by year
2026: 3 · 2025: 10 · 2024: 4 · 2023: 3 · 2022: 9 · 2021: 8 · 2020: 14 · 2019: 7 · 2018: 10 · 2017: 8 · 2016: 8 · 2015: 7 · 2014: 10 · 2013: 11 · 2012: 9
FWCI
3.58
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › melanin and skin pigmentation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
Keywords
SLC24A5, NCKX2, Na+/Ca2+ exchanger, melanogenesis, melanocytes, trans-Golgi network, skin color variation, single nucleotide polymorphisms
MeSH terms
Animals, Calcium, Golgi Apparatus, Humans, Male, Melanins, Melanocytes, Pigmentation, Potassium, Sodium, Ion Transport, Antiporters, Sodium-Calcium Exchanger, Polymorphism, Single Nucleotide, Cell Line, Tumor, Mice

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