THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 9, pp.
5486 –5495, February 29, 2008
SLC24A5 Encodes a trans-Golgi Network Protein with
Potassium-dependent Sodium-Calcium Exchange Activity
That Regulates Human Epidermal Melanogenesis*
Received for publication, September 7, 2007, and in revised form, December 5, 2007 Published, JBC Papers in Press, December 28, 2007, DOI 10.1074/jbc.M707521200
Rebecca S. Ginger‡1, Sarah E. Askew‡, Richard M. Ogborne‡, Stephen Wilson‡, Dudley Ferdinando‡, Tony Dadd‡,
Adrian M. Smith‡, Shubana Kazi‡, Robert T. Szerencsei§, Robert J. Winkfein§, Paul P. M. Schnetkamp§,
and Martin R. Green‡
From ‡Unilever Corporate Research, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, England, United Kingdom and
the §Department of Physiology and Biophysics, Hotchkiss Brain Institute, University of Calgary, Alberta, T2N 4N1 Canada
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 ⬃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 pre-
dicted 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-de-
pendent exchanger activity.
For many years the genetic determinants of human skin color
have attracted interest from both pigmentation biologists and
anthropologists (1–5). Using high density whole genome array
technology, we have defined the key genetic variants associated
with natural skin color variation in a population of South Asian
ancestry (6). We identified a non-synonymous single nucleo-
tide polymorphism (nsSNP)2 in the SLC24A5 gene (rs1426654)
that was previously unknown to skin pigmentation biologists.
Independently, a group of zebrafish researchers discovered that
a different mutation in the zebrafish orthologue of this gene was
responsible for the hypo-pigmented phenotype of the golden
* The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence should be addressed. Tel.: 44-1234-248092; Fax:
44-1234-248010; E-mail address: rebecca.ginger@unilever.com.
2
The abbreviations used are: nsSNP, non-synonymous single nucleotide
polymorphism; NHM, normal human epidermal melanocytes; NCKX,
potassium-dependent sodium calcium exchanger; siRNA, small interfering
RNA; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-
1,3-diol; PBS, phosphate-buffered saline; HEK cells, human embryonic kid-
ney cells; TGN, trans-Golgi network.
5486 JOURNAL OF BIOLOGICAL CHEMISTRY
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
zebrafish (7). Using data from the human HapMap project (8),
they observed that the alternate alleles of the nsSNP
(rs1426654) in human SLC24A5 were present at very different
frequencies in populations of African and European ancestry.
Furthermore, they demonstrated that the allele encoding thre-
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onine 111 of the protein encoded by SLC24A5 was associated
with lighter skin in an admixed African-American population.
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).
Amino acid sequence alignments predict that SLC24A5
encodes a member of the potassium-dependent sodium cal-
cium exchanger protein family, designated NCKX5. 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. NCKX proteins have been shown to operate
in retinal rod and cone photoreceptors as well as in several types
of neurons in the brain (for review, see Ref. 12). A sixth protein,
designated NCKX6, is now thought to belong to a distinct, but
related protein family (CCX) as it shows substantial divergence
in phylogenetic analyses (13) and lacks many of the residues
conserved between NCKX1–5 that are key for transport activ-
ity. Although SLC24A5 is predicted to encode an NCKX pro-
tein, the exchanger activity of NCKX5 has yet to be experimen-
tally demonstrated. The residue altered by the nsSNP in
SLC24A5 lies in a region of the protein that is highly conserved
across all NCKX family members. This region contains a num-
ber of residues that are critical for exchange function (14 –16),
and the experimentally determined topology for NCKX2 sug-
gests that the polymorphic residue of NCKX5 sits in a trans-
membrane domain (17). NCKX1– 4 are located in the plasma
membrane, yet surprisingly, overexpressed hemagglutinin-
tagged recombinant zebrafish NCKX5 could not be detected in
the plasma membrane of MNT1 cells (7), and NCKX5 has been
detected in fractionated melanocytes enriched for a subcellular
organelle, the melanosome, using sucrose gradient fraction-
ation and proteomic analysis (18).
Here we describe a full analysis of the transcript expression
profile of all known sodium-calcium exchangers in cultured
human and murine melanocytes together with an immunocy-
tochemical analysis of native NCKX5 expression. We show that
this protein has a novel trans-Golgi network intracellular local-
VOLUME 283 • NUMBER 9 • FEBRUARY 29, 2008
 SLC24A5 Regulates Human Epidermal Melanogenesis
ization in normal human epidermal melanocytes (NHM), and
using siRNA-mediated knockdown we demonstrate conclu-
sively that SLC24A5 expression is required for melanin synthe-
sis in both cultured mouse and human melanocytes. We also
demonstrate that, as predicted, SLC24A5 encodes a protein
with potassium-dependent sodium-calcium exchanger activity.
Through site-directed mutagenesis in a heterologous expres-
sion system, we demonstrate that the residue switch encoded
by the nsSNP in SLC24A5 markedly alters exchanger activity
when introduced into NCKX2 or NCKX5. Together, these data
support the hypothesis that a nsSNP in the SLC24A5 gene
directly alters human skin color through its effect on the sodi-
um-calcium exchanger activity of NCKX5.
EXPERIMENTAL PROCEDURES
Cell Culture—NHM from neonatal foreskin were cultured in
MGM (M254 medium supplemented with human melanocyte
growth supplement) (Cascade Biologics) at 37 °C, 9% CO2.
Melanocytes were dedifferentiated as previously described (19)
using M254 and 50 nM EDN3 for 7 days before knockdown
(cells denoted MC:MB). B16 F10 mouse melanoma cells
(ATCC) were cultured in minimum essential medium with
Earle’s salts supplemented with 10% fetal calf serum and 2 mM
L-glutamine at 37 °C, 5% CO2.
Real-time PCR—Total RNA was extracted using the RNeasy
mini kit (Qiagen) and quantified using Ribo Green (Invitrogen).
1 ␮g of total RNA was reverse-transcribed using the Roche
Applied Science first strand synthesis kit. Real-time PCR was
performed on a Bio-Rad iCycler using human or murine SLC24
or SLC8, human vSNARE, or murine TBP QuantiTect real-time
PCR primers (Qiagen) and SYBR Green PCR master mix (Bio-
Rad). All measurements from each cell type were made on a
single cDNA sample. Data analysis was performed using the
comparative cycle threshold method (⌬CT).
Anti-NCKX5 Antibodies—Peptides anti-NCKX5-(I) (DEG-
QPFIRRQSRTDSG) and anti-NCKX5-(C) (GNNKIRGCGG)
were coupled to keyhole limpet hemocyanin, and rabbits were
immunized in a 90-day protocol. Bleeds were affinity-purified
using individual peptides conjugated to bovine serum albumin
on a Sepharose support.
Immunoblotting—For membrane extracts NHM were lysed
in 0.25 M sucrose, PBS using a Dounce homogenizer (30
strokes). Homogenates were centrifuged at 20,000 ⫻ g, 20 min,
4 °C. Membrane-associated proteins were extracted using 1%
Triton X-100 in PBS plus protease inhibitor mixture and cen-
trifuged. Alternatively, whole cell lysates were prepared from
NHM using 1% Triton X-100 in PBS plus protease inhibitor
mixture, and cell debris was removed by centrifugation. Protein
content was determined by BCA assay. Proteins (15 ␮g/well
unless otherwise stated) were separated by SDS-PAGE on 10%
Novex bis-tris acrylamide gels, transferred to Invitrulon mem-
brane, blocked in PBS, 0.1% Tween 20 with 2% w/v skimmed
milk protein (PBS/T), and probed with primary antibody
diluted in PBS/T. Primary antibodies consisted of anti-
NCKX5-(C) at 0.5 ␮g/ml goat anti-Trp1 (G17), mouse anti-
tyrosinase clone T311, mouse anti-Lamp-1 clone H4A3, mouse
anti-Mart-1 clone A103 (all from Santa Cruz Biotechnology,
Inc.), mouse HMB45 (against pmel17) clone MCA2146 (Sero-
FEBRUARY 29, 2008 • VOLUME 283 • NUMBER 9
tec), and mouse anti-␤-actin (Abcam Plc). Secondary antibody
was horseradish peroxidase-conjugated anti-rabbit, anti-
mouse, or anti-goat IgG (Jackson ImmunoResearch) as appro-
priate diluted in PBS/T. Detection was via SuperSignal Western
picochemiluminescence substrate (Perbio) and visualized with
a Chemidoc XRS imaging system (Bio-Rad). The Mr of the
bands was determined using Quantity One 4.6.3 one-dimen-
sional analysis software from Bio-Rad. Myc-tagged hNCKX5
was expressed by transient transfection in HEK293 cells. Degly-
cosylation with peptide N-glycosidase F was carried out as
described before (17).
Immunofluorescence Microscopy—Cells grown on glass cov-
erslips were fixed with 2% paraformaldehyde for 20 min,
washed with PBS, and permeabilized with 0.5% saponin. Cells
were blocked with 0.2% bovine serum albumin, 0.1% saponin,
PBS for 1 h at room temperature, probed with primary and
secondary antibodies, and mounted with Vectashield威 (Vector
Laboratories). Primary antibodies were rabbit anti-human
NCKX5-(I) and NCKX5-(C) at 0.8 ␮g/ml, TGN46 and HMB45
(AbD Serotec), GM130 (BD Biosciences), anti-gp100 (AbCam),
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Mel-5 anti-TYRP1 (Signet), and T311 anti-tyrosinase
(AbCam). Other antibodies tested for co-localization were
Lamp 1, flotillin 2 and syntaxin 8 (BD Biosciences), EEA1, Sar1,
␤-COP, PEX-19 (AbCam), Lamp 2 (RDI), C423 (anti-clathrin
L) (Covance), and syntaxin 6 (Sigma-Aldrich). Secondary anti-
bodies were Alexa Fluor威 488 donkey anti-rabbit, 633 donkey
anti-sheep, or 633 goat anti-mouse (Invitrogen). Confocal
microscopy was performed using a Leica TCSSp1 confocal S laser
microscope with Leica LCS software. Images were acquired using
20⫻ (dry) or 60⫻ (oil immersion 1.4na) objectives. 488- and
633-nm excitation was via argon or HeNe lasers respectively. Opti-
cal series were collected at 0.5 ␮M z-steps. For co-localizations with
GM130, the topological separation of the signal from each channel
was determined by plotting the intensity profile of each signal in
three separate cross-sections of the Golgi stacks.
RNA-mediated Interference Transfections—B16 or NHM
were seeded at 2 ⫻ 104 cells/cm2 for melanogenesis experi-
ments or 1 ⫻ 104 cells/cm2 for immunofluorescence micros-
copy experiments; MC:MB cells were seeded at 1.5 ⫻ 104 cells/
cm2. Cells were adhered for 24 h and transfected with 2 ␮g/ml
Lipofectamine 2000 and 50 nM or 100 nM StealthTM RNA-me-
diated interference duplex. All dilutions were performed with
Opti-MEM. Murine siRNA targeted nucleotides 762–787, with
a corresponding scrambled control. Human siRNA targeted
nucleotides 185–210 and 492–517 with scrambled control cor-
responding to 260 –285. Cells were incubated with transfection
reagents for 6 – 8 h. NHM were returned to MGM and grown
for 5– 6 days before analysis. B16 cells were grown in phenol
red-free Dulbecco’s modified Eagle’s medium, 10% fetal calf
serum, 4 mM L-glutamine, after 72 h the media was analyzed for
melanin content, and the cells incubated for 1 h in WST-1 rea-
gent (Roche Applied Science) to determine viability.
Sucrose Density Gradient Fractionation—Light and Dark
NHM were extracted to obtain a total granule fraction and frac-
tionated essentially as described by Chen et al. (23). Briefly, cells
were washed, trypsinized, and resuspended in 0.25 M homoge-
nization buffer. After 60 strokes in a Dounce tissue-grinder,
they were centrifuged at 100 ⫻ g to remove debris, and the
JOURNAL OF BIOLOGICAL CHEMISTRY 5487
SLC24A5 Regulates Human Epidermal Melanogenesis
supernatant was centrifuged at 100,000 ⫻ g (4 °C) before loading
onto a stepped sucrose gradient (0.8–2 M in 0.2 M increments).
Gradients were centrifuged at 25,000 rpm (4 °C) in a Beckman
SW28 rotor. Interface fractions were collected, and 7.5 ␮l of each
fraction was loaded per well in SDS reducing buffer.
Melanin Assay—NHM were lysed in 1% Triton X-100, PBS
and centrifuged at 15,000 g for 10 min, and protein was quan-
tified by BCA assay. Melanin pellets washed in 1:1 ethanol/
diethyl ether were dissolved in 1 M NaOH, 10% Me2SO, 30 min,
60 °C. Melanin content of the supernatants or culture media
was measured at 450 nm.
Clustal Alignments—Protein sequences were aligned using
ClustalW program using the default settings. The resultant
alignment files were visualized using Jalview multiple align-
ment editor (20), and manual adjustments made as necessary.
Functional Analysis of Mutant NCKX2 and NCKX5—
Codons for the indicated residues were mutated using cDNA of
the Myc-tagged short splice variant of human NCKX2
(AAF25811) or human NCKX5 and cloned in to the pEIA vec-
tor as described before (14). The Myc tag (EQKLISEEDL) was
inserted between Ser-52 and Glu-53 of human NCKX5. The
mutant and wild-type NCKX2 and NCKX5 proteins were
expressed in insect High Five cells, and protein expression was
monitored by Western blotting with the Myc mAb (New Eng-
land Biolabs). NCKX function was assayed by measuring
45
Ca2⫹ uptake via reverse exchange in Na⫹-loaded cells as
described in detail previously (21, 14). External 45Ca2⫹ was
removed by a rapid filtration/washing procedure using borosili-
cate glass fiber filters (21). NaCl, KCl, LiCl, and choline chloride
were all Sigma Ultra grade. Protein content of cell samples was
determined with the Bio-Rad protein assay.
RESULTS
SLC24A5 mRNA Is the Predominant Sodium-Calcium
Exchanger Transcript in Human and Mouse Melanocytes—In
mammalian cells sodium-calcium exchange is mediated both
through NCKX proteins, encoded by the SLC24 gene family,
and potassium-independent sodium-calcium exchanger
(NCX) proteins encoded by the closely related, but distinct
SLC8 gene family. This gene family comprises three members,
NCX1–3, transcripts of which are found in many tissues, in
particular in the heart and brain (for review, see Ref. 22). The
transcript profile of all known sodium-calcium (potassium)
exchangers was determined in monolayer cultures of NHM and
mouse melanocytes using real-time PCR (Fig. 1). In both cul-
tures the predominant isoform was SLC24A5, which was ⬎100-
fold more highly expressed than any of the other SLC24A tran-
scripts, and transcripts of the SLC8 family were not detected in
either cell line. 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.
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 pro-
tein was assessed through siRNA-mediated knockdown in
NHM. Transfection with two different siRNA duplexes (siRNA
185 and siRNA 492) resulted in ⬎90% knockdown of SLC24A5
5488 JOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 1. SLC24A5 is the predominant sodium-calcium exchanger tran-
script in cultured human and mouse melanocytes. Real-time PCR was used to
detect mRNA expression of SLC24 and SLC8 in light and dark normal NHM and
B16 mouse melanocytes. Cycle threshold (Ct) values for each gene were com-
pared using a one-way analysis of variance, and the -fold differences between
each gene and SLC24A5 are shown. Bars indicate 95% lower and upper confi-
dence limits. Dunnet’s adjustment for multiple testing to a common control was
used, and all comparisons were significant at the p ⬍ 0.0001 level. -Fold values
were calculated by ⫺2⌬Ct. Data for SLC24A1 (A1), SLC24A3 (A3), and SLC24A4
(A4) are plotted. SLC8A1, SLC8A2, SLC8A3, and SLC24A2 were not detected in
any of the cell types, and SLC24A3 was not detected in dark NHM.
transcript levels compared with control cultures after 48 h.3
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 ⬃43 kDa were clearly detected in
extracts from control or untreated cells but were virtually
absent in extracts from SLC24A5 siRNA-treated cells (Fig. 2A).
The apparent size of these bands is surprisingly low com-
pared with the predicted molecular mass in the absence of
glycosylation of 54.9 kDa. However, the lower molecular
mass observed here is consistent with that observed for het-
erologously expressed recombinant hNCKX5 detected
through amino and carboxyl-terminal tags, which also runs
as a triplet of bands ⬃43 kDa (Fig. 2B). De-glycosylation of
the recombinant protein (Fig. 2B) removes the upper band,
suggesting that the lower bands reflect signal peptide cleav-
age of unglycosylated protein. 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.
Cultured dark NHM probed by immunofluorescence with
either of our anti-NCKX5 antisera, raised against different pep-
3
R. S. Ginger, S. E. Askew, R. M. Ogborne, S. Wilson, D. Ferdinando, T. Dadd,
A. M. Smith, S. Kazi, R. T. Szerencsei, R. J. Winkfein, P. P. M. Schnetkamp, and
M. R. Green.
VOLUME 283 • NUMBER 9 • FEBRUARY 29, 2008
 SLC24A5 Regulates Human Epidermal Melanogenesis
on the punctate staining was
observed (Fig. 3C).3 This confirms
that the perinuclear staining we
have observed with both antibody
preparations is specific to NCKX5
and not due to the nonspecific reac-
tivity detected by Western blot.
However the punctuate staining is
either not specific to this protein, or
the protein localized to these struc-
tures is not removed by siRNA
knockdown even after 10 days.3 Co-
localization studies with multiple
markers of melanosomes, lyso-
somes, or endosomes were unable
to determine the identity of this
punctuate staining (Fig. 3D).3
NCKX5 Co-fractionates with
TGN46 in NHM Extracts—Subcel-
lular fractionation provides an alter-

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native approach to determine the
localization of proteins. Sucrose
density gradient centrifugation has
previously been used to enrich for
melanosomal proteins (23). Using
the same approach, fractionated
NHM extracts were probed with
anti-NCKX5 (C) and the TGN46
FIGURE 2. Detection of native NCKX5 protein in cultured NHM with affinity-purified polyclonal anti-sera. antibody (Fig. 4). Fractions 4 – 6
A, NHM were transfected with scrambled siRNA control (lane 2), siRNA 185 (lane 3), and siRNA 492 (lane 4). After
5 days proteins were extracted from these cultures and untransfected NHM (lane 1) and probed by Western (corresponding to the 1.2/1.4 M, 1.4/
blotting with anti-NCKX5-(C) and ␤-actin as a loading control. NCKX5 runs as ⬃43-kDa triplet, indicated by the 1.6 M, and 1.6/1.8 M sucrose inter-
arrow. The image shown is representative of three experiments. B, HEK293 cells transfected with Myc-tagged faces, respectively) contained pig-
hNCKX5 were extracted, and proteins were separated by SDS-PAGE after treatment with (⫹) or without (⫺)
peptide N-glycosidase. Western blots were probed with anti-Myc monoclonal antibody. C, NHM grown on ment and were similarly enriched
coverslips were fixed and probed with affinity-purified anti-NCKX5 (green) or TGN46 (red) and imaged at high for both NCKX5 and TGN46, sup-
magnification. The yellow color in merged images indicates co-localization. The same staining pattern is shown
with both of the anti-NCKX5 antisera raised against a carboxyl-terminal peptide (anti-NCKX5 (C)) and a peptide
porting the co-localization observed
from the large hydrophilic loop (anti-NCKX5 (I)). between these two antigens using
immunofluorescence and indicat-
tides and regions of NCKX5, showed the same strong perinu- ing that these fractions contain trans-Golgi membranes as well
clear staining and also punctuate staining distributed through- as melanosomes.
out the cytoplasm (Fig. 2C). We found no evidence of plasma SLC24A5 Knockdown Reduces Melanin Production in
membrane staining with these antisera using confocal micros- Mouse B16 Melanocytes—In melanocyte monolayer cul-
copy. A similar pattern of staining was observed in both light tures, where transfer is not possible, melanosome turnover is
and dark NHM, although the perinuclear staining was less limited to some degree by cellular turnover, making it diffi-
intense in lighter melanocytes.3 The perinuclear staining co- cult to measure the inhibition of melanin production over
localized with the trans-Golgi marker TGN46 (Fig. 2C), short time-courses. Mouse B16 cells are unusual in this
although merged images of serial optical sections revealed that respect because they secrete melanin directly into the sur-
NCKX5 only co-localized with part of the TGN (Fig. 3A). Co- rounding culture media, and melanogenesis is only triggered
staining of NHM with anti-NCKX5 and the cis-Golgi marker when cells reach the stationary phase of growth (24). This
GM130 (Fig. 3B) showed that the location of the two antigens affords the opportunity to assess the effect of SLC24A5
was close though not coincident with a separation of ⬃300 – knockdown against a low background of pre-existing mela-
500 nm, calculated from plots of signal intensity in each channel nin. Therefore, the effect of SLC24A5 siRNA knockdown on
across multiple sections the Golgi stacks.3 These data suggest melanin production was first assessed in mouse B16 melano-
that NCKX5 is partially located in a region of the TGN. cytes. Treatment with siRNA 762 elicited a marked and
Treatment of light or dark melanocytes with either of the reproducible reduction in melanin production 72 h post-
human siRNA duplexes for 5 days resulted in complete loss of knockdown (87.7% reduction p ⬍ 0.0001) compared with the
perinuclear NCKX5 in ⬎90% of cells. This is seen as a loss of control siRNA (Fig. 5, A and B) without any detectable tox-
yellow color in the merged images of cells double-stained for icity. This demonstrates that this gene and its products play
NCKX5 (green) and TGN46 (red). However no marked effect a controlling role in melanogenesis in this system.

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SLC24A5 Regulates Human Epidermal Melanogenesis
FIGURE 3. NCKX5 is partially localized to the trans-Golgi network in NHM.
A, NHM grown on coverslips were fixed and probed with anti-NCKX5-(I)
(green) and anti-TGN46 (red) antisera. Staining with each antibody was visu-
alized by high magnification immunofluorescence confocal microscopy, and
co-localization is seen as yellow in the merged images. Two merged images of
different optical sections from the same cell are shown (500 nm apart). B, NHM
grown on coverslips were fixed and probed with anti-NCKX5-(I) (green) and
anti-GM130 (red) antisera and imaged by high magnification immunofluores-
cence microscopy. The red and green and merged images of part of a cell are
shown. C, NHM were grown on coverslips and transfected with Lipofectamine
alone (vehicle control), siRNA 492, or scrambled siRNA control. After 5 days
cells were fixed and probed by low magnification immunofluorescence con-
focal microscopy with anti-NCKX5-(I) (green) and anti-TGN46 (red) antisera.
Merged low power images from each treatment are shown and are represent-
ative of three experiments. The yellow color indicates co-localization of
NCKX5 and TGN46, whereas the red color indicates the loss of NCKX5 staining
but retention of the TGN46 antigen. D, NHM grown on coverslips were fixed
and probed with anti-NCKX5-(I) (green) and anti-Tyr, Mel-5, anti-gp100, or
HMB45 (red) antisera and imaged by high magnification immunofluores-
cence microscopy. Merged images are shown.
SLC24A5 Knockdown Reduces Melanin Production in NHM—
The effect of SLC24A5 knockdown on melanogenesis in
cultured NHM was investigated by comparing the melanin
content of cell pellets 5 days after siRNA transfection. NHM
transfected with either of the SLC24A5 knockdown siRNAs
(Fig. 6A) resulted in significant percentage reductions of 22 and
30.5% (siRNA 185 and 492, respectively; p ⬍ 0.0001) of total
cellular melanin compared with the control cells, demonstrat-
ing that SLC24A5 expression plays an important role in mela-
nin synthesis in these cells. The effect of SLC24A5 knockdown
on melanogenesis was even clearer when assessed in depig-
5490 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 4. NCKX5 co-fractionates with melanosomal and trans-Golgi
markers. Dark NHM cultures were extracted and fractionated by sucrose
density centrifugation as described under “Experimental Procedures.” Frac-
tions 1–7 were collected from different points of the sucrose gradient, corre-
sponding to steps of 0.8, 1, 1.2, 1.4, 1.6, 1.8, and 2 M sucrose, respectively.
Fractions were probed by Western blotting with anti-TGN46, anti-NCKX5 (C),
and anti-TYRP1 antisera. Arrows indicate reactive bands.
mented melanocytes (Fig. 6B). In these cultures the difficulty of
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measuring a reduction in de novo melanin synthesis against a
large background of pre-existing melanin was substantially
lessened. The pigment content of NHM cultured in melano-
blast media for 1 week was greatly reduced (Fig. 6B, MB), and
switching these cells back into normal melanocyte media
restored pigment production after 5 days (Fig. 6B, MGM).
Transfection of de-differentiated cells with SLC24A5 siRNA
knockdown duplexes (185 and 492) almost completely blocked
pigment synthesis after switching back into melanocyte media
compared with the control transfection (Fig. 6B, SC) with a
normalized percentage difference in melanin content of 80–90%
(p ⬍ 0.0001). These results demonstrate for the first time that
SLC24A5 expression is necessary for melanin production in
human epidermal melanoblasts stimulated to differentiate.
Melanosomal Markers Are Down-regulated after SLC24A5
Knockdown—Protein extracts prepared from re-pigmented
melanoblasts treated with control or SLC24A5 knockdown
siRNAs were probed by Western blotting for known melanoso-
mal markers (Fig. 7). The expression of early melanosome
markers pmel17 (85-kDa form) and MART1 and late melano-
somal markers TYR and TYRP1 was substantially reduced after
SLC245A5 knockdown. Conversely, expression of the lysoso-
mal marker LAMP-1 was up-regulated after SLC24A5 knock-
down, suggesting that SLC24A5 plays a role very early in mela-
nosome biogenesis and that this process is intimately linked
with lysosome biogenesis.
Switching Threonine for Alanine at Residue 111 of NCKX5 or
Residue 177 of NCKX2 Reduces Exchange Activity—The NCKX
proteins have a high degree of amino acid sequence similarity in
two regions that have been identified as essential for exchange
activity (denoted ␣1 and ␣2). The non-synonymous SNP
(rs1426654) that is associated with natural skin color variation
lies within the first of these highly conserved regions. All NCKX
proteins and all cross-species orthologues of NCKX5 have an
alanine at this position, except the NCKX5 variant identified in
light-skinned humans, which has a threonine (Fig. 8, A and B).
Consistent with the notion that native NCKX5 is localized to an
intracellular membrane, no NCKX activity could be measured
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 SLC24A5 Regulates Human Epidermal Melanogenesis
across the plasma membranes of
B16 or NHM cells using established
methods (12) and neither was traf-
ficking to the plasma membrane
observed when Myc-tagged NCKX5
was expressed in HEK293 cells.
Therefore, we were also unable to
use current methodology (12, 15) to
measure the NCKX activity of
NCKX5 in the heterologous system
used successfully for the NCKX2
isoform. In contrast, some traffick-
ing of Myc-tagged NCKX5 to the
plasma membrane was observed in
FIGURE 5. SLC24A5 knockdown disrupts melanogenesis in cultured B16 melanocytes. A, B16 melanocytes a High Five insect cell expression
growing in quadruplicate in 48-well plates were transfected with Lipofectamine alone (vehicle control), scram-
bled siRNA control, siRNA 762 cultured for 72 h and photographed alongside untransfected cells. B, the ratio of
system, although the protein
total melanin (␮g) in the media to total protein (␮g) in the cells recovered 72 h after B16 cells were transfected expression level of Myc-tagged
with the scrambled siRNA control or siRNA 762. Individual data points are plotted for four replicates of three NCKX5 was considerably lower
experiments. A two-way analysis of variance taking account of variability between experiments and siRNA
duplexes indicated that there is a difference in normalized melanin content between cells transfected with than that observed for Myc-tagged
siRNA 762 and scrambled siRNA control (p ⬍ 0.0001). NCKX2. We, therefore, used this

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FIGURE 6. SLC24A5 knockdown disrupts melanogenesis in cultured ants of NCKX2 when expressed in HEK293 cells assayed with
NHM. A, comparison of the ratio of total melanin (␮g) to total protein (␮g)
extracted from light NHM treated with the scrambled siRNA control (SC), fluorescent Ca -indicating dyes.
siRNA 185, and siRNA 492. Individual data points are plotted for three
replicates of three experiments. A two-way analysis of variance taking
account of variability between experiments and siRNA duplexes indicated
that there is a difference in normalized melanin content between each of
the siRNA duplexes and the scrambled siRNA control (p ⬍ 0.0001 after a
Dunnett’s adjustment for multiple comparisons to the common control).
B, light NHM were cultured for 1 week in melanoblast medium, plated into
6-well plates, and transfected with siRNA 185, siRNA 492, or scrambled
siRNA control (SC) or untransfected (MGM). These cells were then grown in
MGM media for 5 days or left in melanoblast media (MB) and trypsinized,
and 450,000 cells from each treatment were pelleted in Eppendorf tubes
and photographed.
insect cell expression system to test
for NCKX5 activity and compare
the effect of the nsSNP on NCKX activity when introduced into
either the NCKX5 or NCKX2 background. The High Five cell
system has been shown previously to generate very consistent
NCKX2 protein expression and processing for functional anal-
ysis of NCKX2 mutants (14). Reverse sodium-calcium-potas-
sium exchange was measured as potassium-dependent 45Ca2⫹
uptake in Na⫹-loaded cells. Different forms of SLC24A2 and
SLC24A5 encoding either alanine or threonine at residues 111
or 177 of NCKX5 and NCKX2, respectively (177 is the equiva-
lent of residue 111 in NCKX5), were transfected into High Five
cells, and the NCKX activity conferred by each construct was
compared. Constructs carrying the threonine variant had a
greatly reduced exchange activity compared with those carry-
ing the alanine variant (Fig. 8C). However, three other substi-
tutions at residues that differ between NCKX1– 4 and NCKX5
in regions of high sequence conservation (A150S, L193F, and
I554L) had little impact on NCKX2 activity. Reverse exchange
of NCKX5 was only observed when extracellular K⫹ was pres-
ent and no reverse exchange was observed in the presence of
either extracellular Na⫹ or Li⫹, confirming K⫹-dependent
Na⫹/Ca2⫹ exchange function. Consistent with K⫹-dependent
45
Ca uptake via reverse Na⫹/Ca2⫹ exchange, this uptake was
abolished by the addition of the ionophore monesin which will
dissipate intracellular Na⫹.3 Furthermore, the cation depen-
dences did not vary significantly between the two allelic vari-
2⫹ 3
DISCUSSION
Recent publications have focused on the importance of
SLC24A5 in the variation of human skin color across global
populations (9 –11). However, little is understood about the
biological mechanisms that mediate the association between
SLC24A5 polymorphisms and vertebrate pigmentation.
SLC24A5 Plays a Key Role in Melanogenesis—In vivo epider-
mal melanocytes synthesize melanin within specialized

FEBRUARY 29, 2008 • VOLUME 283 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5491
SLC24A5 Regulates Human Epidermal Melanogenesis
FIGURE 7. Effect of SLC24A5 knockdown on expression of melanosomal
protein expression. Whole cell protein extracts from melanoblast SLC24A5
knockdown experiments were probed with anti-Tyr, anti-TYRP1, anti-
MART-1, anti-PMEL17, anti-LAMP-1, and anti-␤-actin antisera. Lanes were
loaded as follows: MB, melanoblast; MGM, re-pigmented melanoblast/mela-
nocyte; VC, knockdown vehicle control; SC, knockdown with scrambled siRNA
control; 185, knockdown with siRNA 185; 492, knockdown with siRNA 492. For
pmel17, the arrow indicates the 85 kDa for of the protein found in stage 1
melanosomes. For MART-1 and HMB45 blots, 5 ␮g of protein were loaded per
well.
organelles, termed melanosomes, which are transported to the
cell periphery and transferred to neighboring keratinocytes
through dendritic processes. Direct quantitation of melanin
secreted from B16 melanocytes post knockdown shows a
marked inhibition of melanogenesis with no effect on cell via-
bility (Fig. 5). SLC24A5 knockdown with two different siRNA
duplexes in cultured NHM reproducibly results in greater than
a 20% reduction in the total normalized melanin content of
these cells (Fig. 6A). Because NHM grown in monolayer culture
do not secrete melanin into the culture media, this apparently
small reduction is good evidence of an appreciable effect on
melanogenesis and is equivalent to the degree of inhibition
measured after treatment with known tyrosinase inhibitors.3
This is the first demonstration that SLC24A5 is directly
involved in human melanogenesis. In addition, the same
SLC24A5 siRNA duplexes block de novo pigment production in
partially de-differentiated melanocytes grown in melanoblast
media and returned to full melanocyte growth media, suggest-
ing that SLC24A5 may operate at an early stage of melanosome
biogenesis (Fig. 6B). A role for NCKX5 in early melanosome
biogenesis is also supported by the reduction in partially pro-
cessed 85-kDa pmel17 protein observed after SLC24A5 knock-
down (Fig. 7), as this is a marker of early melanosomes (25). Our
knockdown data support a central function for SLC24A5 in
mammalian pigmentation, conserved from fish to man.
NCKX5 Is Localized to Subcellular Membranes—Multiple
sequence alignments predict that the protein encoded by
SLC24A5 is a member of the NCKX protein family (⬃42% sim-
ilarity across the whole protein and 70% similarity in the con-
served ␣1 and ␣2 regions). 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. Data generated
5492 JOURNAL OF BIOLOGICAL CHEMISTRY
using our affinity-purified polyclonal antisera suggest that,
unlike other previously characterized members of the sodium-
calcium exchanger and NCKX families, native NCKX5 is
located in a subcellular compartment that partially overlaps
with a region of the TGN (Figs. 2 and 3). We have found no
evidence to suggest that NCKX5 is located at the plasma mem-
brane of cultured melanocytes, consistent with previous find-
ings (7), which reported an intracellular localization for tagged
recombinant zebrafish NCKX5. In a proteomic analysis of frac-
tionated melanocytes, NCKX5 was detected in melanosome-
enriched fractions (18), although we have not detected any co-
localization between NCKX5 and melanosomal markers by
immunofluorescence microscopy with our antisera (Fig. 3D).
Fractionation of our NHM extracts using a sucrose density gra-
dient similar to that used in the proteomic study (18) revealed
that NCKX5, dark pigment, and TGN46 were all enriched in
the fractions enriched for melanosomal marker TYRP1. 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
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Ca2⫹ from the cytoplasm to the extracellular space (26 –28).
NCKX5 Possesses NCKX Activity That Is Altered by the nsSNP
in SLC24A5—The unique intracellular localization of NCKX5
necessitates the development of new assays as the currently
available methodologies are not suitable for measuring intra-
cellular sodium-calcium exchange activity. The cell surface-lo-
calized recombinant NCKX5 generated by heterologous
expression of SLC24A5 cDNA in insect High Five cells enabled
us to demonstrate for the first time that NCKX5 possesses
authentic NCKX activity (Fig. 8C). However, the level of activity
conferred by SLC24A5 was much lower than that generated by
SLC24A2 in the same system, possibly reflecting differences in
subcellular distribution or the considerably lower expression
level observed for NCKX5 compared with NCKX2. Because the
activity of NCKX5 in this heterologous expression system was
low, we used both NCKX2 and NCKX5 assays to test the effect
of the amino acid change encoded by the SLC24A5 nsSNP on
NCKX activity. In both proteins, switching alanine to threonine
at the critical residue (NCKX5 residue 111 and NCKX2 residue
177) caused a substantial reduction in exchanger activity. This
suggests that this residue is important for the exchanger activity
of both NCKX2 and NCKX5, and a high level of cross-species
and cross-family sequence conservation around this residue
implies is likely to be important for all NCKX proteins. 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.
SLC24A5, a pH Regulator?—Intracellular sodium-calcium
exchange raises potential questions as to how the necessary ion
gradients are maintained across an intracellular membrane and
in which direction the exchanger operates under physiological
conditions. Lamason et al. (7) propose a model based upon a
melanosomal localization for NCKX5. They suggest that
NCKX5 moves Ca2⫹ from the cytoplasm into the melanosome
using a Na⫹ gradient that is generated through coupling to a
Na⫹/H⫹ exchanger and a V-ATPase. They further propose that
calcium accumulation in the melanosome activates a calcium-
dependent furin-like protease that participates in the process-
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 SLC24A5 Regulates Human Epidermal Melanogenesis

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FIGURE 8. The threonine-encoding allele of the SLC24A5 nsSNP confers reduced exchanger activity in NCKX2 and NCKX5. A, cross-species comparison
of NCKX5 amino acid sequence within the highly conserved ␣1 region. Residues are numbered for human NCKX5. The degree of shading indicates the level of
residue conservation. The arrow indicates the position of the residue altered by the nsSNP in SLC24A5. Species included are: Homo sapiens (human), Mus
musculus (mouse), Xenopus tropicalis (frog), Danio rerio (zebrafish), and Gallus gallus (chicken). B, amino acid alignment of NCKX family proteins within the
highly conserved ␣1 region. Residues are numbered for human NCKX1. The degree of shading indicates the level of residue conservation. The arrow indicates
the position of the residue altered by the nsSNP in SLC24A5. C, insect High Five cells were transfected with expression constructs containing cDNA encoding
wild-type NCKX2 (WT), NCKX2 with threonine at residue 177 (A177T), NCKX2 with serine 150, phenylalanine 193 and leucine 554 (A150S/L193F/I554L), NCKX2
with threonine at residue 177, serine 150, phenylalanine 193, and leucine 554 (A150S/A177T/L193F/I554L), wild-type NCKX5 (WT), NCKX5 with threonine at
residue 111 (A111T), or vector alone, and reverse exchange activity was measured as described under “Experimental Procedures.” Data are the mean of 5– 8
(hNCKX2) or 8 –15 (hNCKX5) experiments ⫾S.E. (indicated by error bars).

ing of the melanosomal scaffold protein pmel17. If this were the
case, a reduction in NCKX5 exchange activity, which we predict
would result from the threonine 111 substitution encoded by
the nsSNP, would cause a net acidification of the melanosome
as the activity of the Na⫹/H⫹ exchanger is impaired. This is
consistent with the observation that melanosomal pH is higher
in cultured melanocytes derived from Negroid donors com-
pared with those derived from Caucasian donors (29). The
more alkaline environment of Negroid melanosomes is
believed to favor the activity of tyrosinase, a rate-limiting cata-
lyst of melanin synthesis that operates optimally at neutral pH.
This might account for the reduced tyrosinase activity meas-
ured in Caucasian compared with Negroid-derived melano-
cytes (30). Although our studies cannot rule out a cryptic mela-
nosomal localization for NCKX5, our work has definitively
demonstrated that NCKX5 is partially localized to the TGN,
which leads us to propose two alternative models for the role of
NCKX5 in pigmentation.
A Role for SLC24A5 in the TGN?—The Golgi apparatus is an
established calcium store (31), and its role in sorting and proc-
essing secretory and membrane proteins is highly sensitive to
changes in calcium concentration within the lumen. 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). In addition, the Na⫹/H⫹ exchanger NHE7 is
located in the TGN and post-Golgi vesicles (35, 36) and is dif-
ferentially expressed in light and dark NHM (37). The model
proposed by Lamason et al. (7) in which SLC24A5 modulates

FEBRUARY 29, 2008 • VOLUME 283 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5493
SLC24A5 Regulates Human Epidermal Melanogenesis
melanosomal pH could also be applied to the TGN. Data on the
role of yeast Nhx1 in protein trafficking (38) and the effects of
alkalinization of tyrosinase on its maturation in melanoma cells
and mouse melanocytes (39, 40) led Smith et al. (37) to suggest
that NHE-7 might regulate tyrosinase maturation by raising the
pH of the TGN to allow correct folding and maturation of tyro-
sinase. This complex process is tightly regulated, and there is
evidence that it is essential that it is navigated correctly for
active tyrosinase to be localized appropriately (41, 42). In our
first alternative model we propose that the nsSNP in SLC24A5
reduces the Na⫹/Ca2⫹ exchange activity of NCKX5 located in
the TGN, which alters Na⫹/H⫹ exchange activity through
NHE-7 coupled to a V-ATPase, acidifying the TGN, potentially
impairing the maturation of tyrosinase. In addition, the accom-
panying changes to calcium gradients in the Golgi lumen could
impactanumberofprocesses,includingtheactivationofcalcium-
dependent furin-like protease activity in a manner analogous to
the melanosomal model proposed by Lamason et al (7). The
Golgi Ca2⫹ may also act as a store for Ca2⫹-mediated signaling
such as the proposed melanogenic response to agouti signaling
protein (43) or the UV-induced response to endothelin-1 (44).
Ca2⫹ is also a co-factor for phenylalanine hydroxylase (45); the
latter has been suggested to protect the redox balance and pre-
vent DNA damage (46).
SLC24A5 in Melanosome Biogenesis?—Our alternative
model takes into account the dramatic effect of SLC24A5
knockdown on pigmentation in re-differentiating melanocytes,
and the effect that this has on the expression of pmel17 and
Lamp1. 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). 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 melanoso-
mal or lysosomal pathways.
We, therefore, propose a second model in which NCKX5
modulates melanogenesis through a role in the trafficking/sort-
ing decisions, which occur within the coated endosome. In
this model NCKX5 is required for the correct delivery of
melanosomal components such as pmel17 into the melano-
somal pathway. Thus, when SLC24A5 is disrupted through
siRNA knockdown, melanosomal maturation is blocked, lead-
ing to a down-regulation of pmel17 and late melanosomal
markers TYR and TYRP1. Consequently, protein and mem-
brane resources from within the coated endosome are diverted
into the endosome-lysosome pathway, leading to an up-regula-
tion of Lamp1. Endosomal membrane fusion events, which
are essential processes in melanosome and lysosome-related
organelle biogenesis, are Ca2⫹-mediated (49, 50) and are likely
modulated by changing local Ca2⫹ gradients. We propose that
NCKX5 could play a role in maintaining local calcium concen-
trations in or near to the coated endosomes, which are closely
associated with the TGN (47), to facilitate appropriate mem-
brane fusion “kiss and run” events and ensure the delivery of
important proteins such as pmel17 into the melanosomal sys-
tem. The importance of correctly regulating such intracellular
trafficking mechanisms to melanogenesis is highlighted by the
5494 JOURNAL OF BIOLOGICAL CHEMISTRY
de-pigmenting effects of certain tricyclic compounds that elicit
the mis-trafficking of tyrosinase leading to its co-localization
with Lamp1 (51). A more precise examination of NCKX5 local-
ization by immunoelectron microscopy and of the effect of
SLC24A5 knockdown on different components of the endoso-
mal, lysosomal, and melanosomal pathways would help to fur-
ther our understanding of the mechanisms by which NCKX5
regulates melanogenesis and human skin color variation.
We have shown that NCKX5 is localized to the TGN of epi-
dermal melanocytes and plays a direct and important role in
human melanogenesis. Furthermore, the nsSNP in SLC24A5
associated with natural human skin color variation encodes an
amino acid change that alters both NCKX2 and NCKX5 activ-
ity. This strongly suggests that NCKX5 exchange function
modulates melanogenesis in epidermal melanocytes and
thereby in human skin.
Acknowledgments—We thank Jenny Pople, Carl Jarman, Wendy
Filsell, Amelia Fereday, Frans van der Ouderaa, and C. V. Natraj for
support and helpful discussions.
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JOURNAL OF BIOLOGICAL CHEMISTRY 5495
 SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent
Sodium-Calcium Exchange Activity That Regulates Human Epidermal
Melanogenesis
Rebecca S. Ginger, Sarah E. Askew, Richard M. Ogborne, Stephen Wilson, Dudley
Ferdinando, Tony Dadd, Adrian M. Smith, Shubana Kazi, Robert T. Szerencsei, Robert
J. Winkfein, Paul P. M. Schnetkamp and Martin R. Green
J. Biol. Chem. 2008, 283:5486-5495.
doi: 10.1074/jbc.M707521200 originally published online December 28, 2007

Access the most updated version of this article at doi: 10.1074/jbc.M707521200

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