Melanin is a pigment found in our skin, eyes and hair. Individuals who are unable to make or store melanin, a condition known as albinism, have unusually pale features and problems with vision. The pigment helps to protect us from harmful UV radiation, and so individuals with albinism also have an increased risk of developing skin and eye cancers.

In cells, melanin is made and stored in compartments called melanosomes. The most common type of albinism is caused by defects in a protein called OCA2, which is found in the membrane that surrounds melanosomes. However the role of OCA2 in melanin production is unclear.

It has been proposed that OCA2 may allow charged particles (or ions) to enter or leave melanosomes. Here, Bellono et al. used a technique called patch-clamp to study the movement of ions across the membrane of melanosomes from skin and eye cells. The experiments show that a flow of chloride ions out of the melanosome is required for melanin to be produced. OCA2 is involved in the ion movement, and it might alter the acidity of the melanosome when present.

Bellono et al. propose that OCA2 is part of an ion channel that allows chloride ions to pass through the membrane, to make the melanosome less acidic and enable melanin to be produced. The next challenge will be to identify other ion channels in the melanosome and understand their roles in producing melanin.

Ion channels are membrane proteins that regulate the concentration of key signaling ions to control a wide range of cellular functions. While plasma membrane ion channels have been extensively studied, much less is known about the identity and physiology of intracellular channels because they are less accessible to direct electrophysiological characterization.

Melanosomes are lysosome-related organelles that produce and store melanin, a natural pigment present in most organisms. Impaired melanin synthesis and storage affects visual system development and pigmentation of the skin, eyes, and hair, leading to reduced protection against ultraviolet radiation and predisposition for skin and eye cancers. A number of genes encoding putative melanosomal ion transport proteins are critical for melanosomal function, as mutations in these genes result in oculocutaneous albinism (OCA) (Montoliu et al., 2014). This suggests that ionic homeostasis plays an important role in melanin synthesis and storage, yet how ion channels might contribute to melanosome function and pigmentation remains poorly understood (Bellono and Oancea, 2014).

One of the most common forms of albinism is caused by mutations in a highly conserved protein encoded by the oculocutaneous albinism II gene (OCA2) (Gardner et al., 1992; Rinchik et al., 1993; Rosemblat et al., 1994; Lee et al., 1994a; Sitaram et al., 2009) (Figure 1—figure supplement 1). OCA2-deficient animals lack pigment (Gardner et al., 1992; Protas et al., 2006) and reduced OCA2 expression leads to decreased melanin underlying blue eye color in humans (Eiberg et al., 2008; Sturm et al., 2008). OCA2 has twelve predicted transmembrane domains (Gardner et al., 1992), is localized to melanosomal membranes (Rosemblat et al., 1994; Sitaram et al., 2009), and has been implicated in pH regulation of melanosomes and trafficking of the melanogenic enzyme tyrosinase (Puri et al., 2000; Manga and Orlow, 2001; Chen et al., 2002, 2004; Ni-Komatsu and Orlow, 2006). Despite its importance, the function of OCA2 and the molecular mechanism by which it regulates melanin are not known.

Here we identify and characterize a new intracellular ion channel that resides in the melanosomal membrane and requires OCA2 expression. Using whole-organelle and single-channel patch-clamp recordings we found that OCA2 contributes to an anion channel required for pigmentation. The OCA2-mediated Cl⁻ current was nearly abolished by a mutation identified in patients with oculocutaneous albinism type II. Interestingly, expression of OCA2 in endolysosomes increased organelle pH, providing a potential mechanism for how OCA2 regulates melanogenesis. Thus, a previously uncharacterized OCA2-dependent anion channel is critical for melanosomal function and pigmentation, revealing a novel function for intracellular ion channels.

Because intracellular ion channels are important regulators of organellar and cellular function and defects in melanosomes, lysosomal-related organelles present in melanocytes and retina, often result in severe pigmentation phenotypes, we wondered how ion channels contribute to melanosome function. We investigated OCA2, a melanosome-specific membrane protein of unknown function that, when mutated, results in albinism.

OCA2 expression in endolysosomes leads to a Cl⁻ conductance (I_OCA2)

In melanocytes and retinal pigment epithelium (RPE) OCA2 is restricted to melanosomes, but when expressed in heterologous systems it localizes to lysosomes and late endosomes (endolysosomes) (Sitaram et al., 2009). To investigate if OCA2 has ion transport activity, we expressed OCA2 tagged with GFP (GFP-OCA2) or mCherry (mCherry-OCA2) in AD293 cells, where it colocalized with the endolysosomal marker LAMP1 (Figure 1A). Endolysosomes are an ideal melanosome-related heterologous system because they can be enlarged by treating cells with 1 μM vacuolin-1 and mechanically released from the cytoplasm with a glass pipette (Cerny et al., 2004; Saito et al., 2007; Dong et al., 2008). We recorded whole-organelle currents from endolysosomes expressing GFP-OCA2 and found that voltage pulses evoked a large outwardly rectifying current (I_OCA2) that was not present in mock-transfected cells (Figure 1A).

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The measured outward current could result either from K⁺, the main cation in the bath, moving into the endolysosome, or Cl⁻, the main anion in the pipette solution, moving out of the endolysosome. We tested if I_OCA2 carries K⁺ into the lumen by replacing it with Na⁺ in the bath solution. We detected no significant change in the current amplitude or reversal potential (E_rev) (Figure 1—figure supplement 2A,B), suggesting that K⁺ is not required for I_OCA2. We next tested if I_OCA2 is mediated by luminal Cl⁻ moving into the cytoplasm by replacing Cl⁻ in the pipette solution with the impermeant anion gluconate (Gluc⁻). I_OCA2 was nearly abolished with Gluc⁻ in the pipette and E_rev was significantly shifted in the positive direction, consistent with the endogenous current recorded in endolysosomes from mock-transfected cells (Figure 1B,C). The similar current amplitude and positive E_rev measured in mock-transfected and OCA2-expressing endolysosomes with luminal Gluc⁻ suggests the presence of an endogenous cationic permeability. When we subtracted this endogenous current from I_OCA2, the E_rev for I_OCA2 became similar to E_Cl predicted by the Nernst equation (Figure 1—figure supplement 3). The significant reduction in current amplitude in the presence of luminal Gluc⁻ indicates that I_OCA2 is mediated by Cl⁻, consistent with OCA2 homology to bacterial anion transporters (Rinchik et al., 1993; Brilliant, 2001; Kobayashi et al., 2006). We tested whether a range of pharmacological inhibitors of different Cl⁻ channels and transporters affect I_OCA2 by bath-applying 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS), niflumic acid (NFA), or 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), but none significantly altered I_OCA2 amplitude (Figure 1—figure supplement 4). Thus, I_OCA2 has a different pharmacological profile from the channels and transporters that are inhibited by the tested compounds.

The Nernst equation predicts that if I_OCA2 were due to selective transport of Cl⁻, E_rev will be dependent on the Cl⁻ concentration ([Cl⁻]) gradient across the endolysosomal membrane. To establish whether this is the case, we determined E_rev for currents measured at variable cytoplasmic and constant luminal [Cl⁻], using NMDG-based solutions to prevent endogenous cationic permeability. E_rev for 48 mM luminal and 8 mM cytoplasmic [Cl⁻] was −42.7 ± 2.3 mV and shifted to 1.6 ± 2.5 mV under symmetrical 48 mM [Cl⁻] (Figure 1D). E_rev increased linearly as a function of cytosolic [Cl⁻] ([Cl⁻]_cyto), with a 10-fold change in [Cl⁻]_cyto corresponding to a shift in E_rev of 58 mV, consistent with a highly Cl⁻ selective current (Figure 1E).

Mutations associated with oculocutaneous albinism disorder affect I_OCA2

OCA2 shares little homology with known chloride channels or transporters; OCA2 might be an accessory subunit of a Cl⁻ transporter or form a Cl⁻ channel or carrier protein itself. To determine if specific OCA2 residues are required for Cl⁻ transport, we sought to identify mutations important for ion transport, but not melanosomal localization. We analyzed mCherry-OCA2 variants containing disease-related mutations within highly conserved regions of the protein (Figure 1—figure supplement 1A). We chose mutations identified in patients with OCA type II through human genetic studies: V443I, a common albinism-associated mutation in a predicted luminal loop and important for melanin content in vitro (Lee et al., 1994a, 1994b; Sviderskaya et al., 1997; King et al., 2003; Garrison et al., 2004; Hongyi et al., 2007; Preising et al., 2007; Hutton and Spritz, 2008; Rimoldi et al., 2014) and K614E, in a predicted cytoplasmic loop (Passmore et al., 1999). We also generated an OCA2 variant with five point mutations in the same predicted luminal loop as V443I (5mut: V443I, M446V, I473S, N476D, N489D) (Lee et al., 1994a, 1994b; Spritz et al., 1995; Hongyi et al., 2007; Preising et al., 2007) (Figure 2A).

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Upon expression in HeLa cells, the K614E and V443I mutants colocalized with the endolysosomal marker LAMP1, similar to WT mCherry-OCA2 (Figure 2—figure supplement 1A), indicating that both mutants traffic efficiently to endolysosomes when expressed in non-melanocytic cells. By contrast, 5mut OCA2 localized primarily to endoplasmic reticulum-like structures rather than endolysosomes (Figure 2—figure supplement 1A), therefore it could be used as a negative control. In pigment cells containing melanosomes (melan-ink4a), expression of OCA2 variants revealed that WT, as well as V443I and K614E mutants colocalized with the melanosomal tyrosinase-related protein 1 (TYRP1; overlap with TYRP1 = 60.1 ± 21.3% for WT; 50.7 ± 13.9% for V433I; and 53.6 ± 16.1% for K614E mCherry-OCA2) (Figure 2E), whereas overlap with the endolysosomal marker LAMP2 was minimal (11.8 ± 10.7% among all variants). A portion of K614E localized to vesicular structures without TYRP1 but that contained pigment when imaged by bright field microscopy.

To determine if albinism-associated mutations affect I_OCA2, we expressed mCherry-tagged WT, K614E, V443I or 5mut OCA2 in AD293 cells together with LAMP1-GFP (Figure 2B). Currents recorded from AD293 endolysosomes identified by LAMP1-GFP and expressing K614E had similar amplitudes and E_rev as WT OCA2 (Figure 2C,D), but those expressing V443I had amplitudes reduced by ∼85% and E_rev shifted to more positive values (Figure 2C,D). Endolysosomes of cells expressing 5mut had very small current amplitudes and a positive E_rev, similar to mock-transfected cells (Figure 2C,D).

Collectively, these data suggest that the V443I-containing luminal loop between transmembrane domains 5 and 6 is critical for the OCA2-mediated Cl⁻ current. The K614E mutation had little effect on localization or OCA2-mediated currents in our experiments. Because K614E was identified in albinism patients with an additional OCA2 mutation (Passmore et al., 1999), its associated phenotype might be too mild to detect in our assays or might be masked by overexpression.

OCA2 regulates organelle pH

How does the OCA2-mediated anion conductance affect pigmentation? Early stage melanosomes are highly acidic (Raposo et al., 2001), but because tyrosinase is inactive at pH < 6.0, melanosomal pH is thought to increase in order to allow for melanin synthesis (Ancans et al., 2001; Halaban et al., 2002). We tested the hypothesis that OCA2-mediated anion extrusion from melanosomes regulates luminal pH (Puri et al., 2000; Brilliant, 2001; Manga and Orlow, 2001; Chen et al., 2002, 2004; Ni-Komatsu and Orlow, 2006), similar to other anionic conductances (Stauber and Jentsch, 2013). We expressed in endolysosomes of AD293 cells ecliptic pHluorin-LAMP1, which becomes fluorescent at pH > 6 (Rak et al., 2011) (Figure 3A). Endolysosomes of control cells were acidic and lacked fluorescence emission at baseline (Figure 3B), but exhibited increased fluorescence upon neutralization (pH > 6) of organellar pH by cellular treatment with the vacuolar H⁺-ATPase inhibitor bafilomycin A1 (BafA1) (Figure 3A–C). In contrast, coexpression of WT OCA2 with pHluorin-LAMP1 resulted in endolysosomes that were fluorescent at baseline, with only a small increase in fluorescence in response to BafA1 (Figure 3B,C). This indicates that OCA2 expression increased the pH of endolysosomes to > 6.

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To determine if changes in pH required the OCA2-mediated anion conductance, we examined changes in pHluorin-LAMP1 fluorescence elicited by the expression of albinism-associated OCA2 mutants. Endolysosomes expressing K614E exhibited basal fluorescence and little change in fluorescence following treatment with BafA1 (Figure 3B,C), consistent with its intact conductance and localization to endolysosomes (Figure 2, Figure 3—figure supplement 1). However, endolysosomes expressing V443I, which have significantly reduced current amplitudes but intact localization (Figure 2, Figure 3—figure supplement 1), had dim fluorescence at baseline that increased dramatically following BafA1 treatment (Figure 3B,C). Endolysosomes from cells expressing mislocalized 5mut lacked basal fluorescence and increased their fluorescence with BafA1 treatment (Figure 3B,C), similar to untransfected cells. Together, these results support the notion that OCA2-mediated ion transport in organelles modulates luminal pH.

We confirmed the observed OCA2-associated changes in luminal pH using the ratiometric pH-sensitive dye LysoSensor DND-160. Based on LysoSensor calibration, the luminal pH of LAMP1-positive compartments in control cells had an approximate pH value of 5.12 ± 0.03, while the luminal pH of WT OCA2-expressing endolysosomes was significantly greater (6.67 ± 0.03, Figure 3—figure supplement 1A–C). Expression of the K614E mutant increased pH to 6.32 ± 0.03, while V443I only modestly raised the luminal pH of endolysosomes to 5.72 ± 0.04, (Figure 3—figure supplement 1B). Melanosomal pH measurements were not possible because fluorescent indicator uptake in melanosomes is impaired and melanin interferes with the emission of fluorescent proteins.

Our pH measurements are consistent for the two indicators and show that OCA2-mediated Cl⁻ transport shifts the endolysosomal pH toward neutral values that in melanosomes would be optimal for tyrosinase activity and melanin synthesis. The mechanism by which OCA2 modulates pH is unclear. We hypothesize that OCA2-mediated Cl⁻ efflux from the lumen regulates the organelle membrane potential, reducing vacuolar H⁺-ATPase activity and resulting in less H⁺ being pumped in the lumen. Alternatively, the pH modulation by the OCA2-mediated Cl⁻ conductance could be a more complex mechanism involving the contribution of additional channels and transporters. Thus, our model for pH regulation by OCA2 remains tentative and awaits a better understanding of melanosomal membrane conductance and signaling.

OCA2 contributes to a melanosomal chloride conductance (I_melano) required for pigmentation

To investigate endogenous OCA2, we measured native currents by direct patch-clamp recordings from melanosomes. This is technically challenging due to the intracellular localization and small size (300–500 nm) of melanosomes and because treatment of melanocytes with vacuolin-1 did not significantly enlarge melanosomes. To circumvent these difficulties we exploited a dermal melanocyte cell line derived from mice deficient in ocular albinism 1 (Oa1) (Palmisano et al., 2008), in which melanosomes are enlarged to up to ∼1 μm diameter (Cortese et al., 2005). Recordings from individual melanosomes dissected from Oa1^−/− melanocytes under the same conditions used for endolysosome patch-clamp experiments (Figure 4A) revealed a large outwardly rectifying current (I_melano) with a negative E_rev that was dependent on luminal Cl⁻, similar to currents recorded from OCA2-expressing endolysosomes (Figure 4B,C). When currents recorded in the presence of luminal Gluc⁻ were subtracted from I_melano measured in the presence of Cl⁻, E_rev was similar to E_Cl (Figure 4—figure supplement 1A). The E_rev for I_melano measured with NMDG-based solutions was found to be −44.3 ± 2.5 mV for 48 mM luminal and 8 mM cytoplasmic [Cl⁻] and −2.3 ± 1.1 mV for symmetrical 48 mM [Cl⁻] (Figure 4—figure supplement 1B,C), consistent with the values measured for endolysosomes expressing OCA2 (Figure 1E) and with the Nernst potential for Cl⁻ selective currents.

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In addition to skin melanocytes, melanosomes are also present in the retinal pigment epithelium of the eye. We patch-clamped melanosomes dissected from freshly isolated RPE cells from American bullfrog (Lithobates catesbeianus) that are larger than those from mammalian RPE, thus allowing for patch-clamp experiments (Figure 4E, Figure 4—figure supplement 2). RPE whole-melanosome recordings identified an outwardly rectifying current with a negative E_rev that was nearly abolished (reduced by ∼90%) by substituting luminal Cl⁻ with Gluc⁻ (Figure 4E,F). Together, these data indicate that a current with similar properties as I_OCA2 is present in melanosomes from skin and RPE.

To determine if OCA2 contributes to I_melano, we used OCA2-targeted siRNA to reduce OCA2 expression (Figure 5—figure supplement 1A). Consistent with previous findings (Sviderskaya et al., 1997), reducing endogenous OCA2 expression markedly decreased melanin content in Oa1^−/− melanocytes (Figure 5A, Figure 5—figure supplement 1B). Melanosomes dissected from melanocytes expressing OCA2-targeted siRNA had dramatically reduced current amplitudes and melanin content compared with melanosomes from cells expressing scrambled siRNA (control), indicating that OCA2 is required for I_melano and pigmentation (Figure 5B,C). Expression of WT OCA2, but not of the V443I mutant, in siRNA-treated cells was sufficient to reconstitute I_melano and rescue melanization (Figure 5A–C), suggesting that the OCA2-mediated current is required for melanin production.

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Might melanin itself be critical for I_melano? To address this question, we treated Oa1^−/− melanocytes with phenylthiourea (PTU), a potent inhibitor of the key melanogenic enzyme tyrosinase, resulting in melanin depletion (Figure 5—figure supplement 2A). Recording from melanosomes dissected from PTU-treated melanocytes revealed a current with the same properties as in vehicle-treated cells and similar to I_melano, suggesting that melanin does not influence I_melano (Figure 5—figure supplement 2B,C). We thus concluded that OCA2 expression is required for I_melano and melanization, but melanin is not required for I_melano.

We next sought to compare the biophysical properties of the endogenous OCA2-mediated melanosome current (I_melano) in dermal and RPE melanosomes with those of the OCA2-expressing endolysosome current (I_OCA2). Excised cytoplasmic-side-out patches from partially dissected endolysosomes of OCA2-expressing AD293 cells (Figure 6A) exhibited single-channel currents (i_OCA2) with greatest activity at positive membrane potentials (Figure 6B), consistent with the outward rectification measured for whole-endolysosome I_OCA2 (Figure 1B). Patches from mock-transfected endolysosomes did not exhibit similar single-channel activity (Figure 6—figure supplement 1A,B). The E_rev of i_OCA2 was −66 mV, close to E_Cl predicted by the Nernst equation (−68 mV), and the unitary slope conductance was 58 ± 2 pS (Figure 6C).

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Ion channels are pore-proteins that allow rapid diffusion of ions (>10⁶ ions/s) down their concentration gradients, while carriers and pumps typically move ions at slower rates (<10⁵ ions/s). We thus calculated the OCA2-mediated transport rate at 80 mV, where we measured the largest single-channel amplitude, and found a transport rate of ∼5.5 × 10⁷ ions/s. The large conductance recorded in isolated patches from OCA2-expressing endolysosomes indicates that OCA2 functions as a critical component of an electrodiffusive anion channel. Similar properties were measured for single-channel currents in excised cytoplasmic-side-out patches of dermal melanosomes from Oa1^−/− melanocytes (i_melano) (E_rev = −67 mV and unitary slope conductance = 60 ± 1 pS) (Figure 6D,E). Thus, the single-channel properties of this endogenous melanosomal current are nearly identical to those recorded from OCA2-expressing endolysosomes.

To determine the anion selectivity of the recorded currents, we estimated the permeability to different anions by determining E_rev under symmetrical concentrations (48 mM) of luminal Cl⁻ and cytoplasmic Cl⁻, Br⁻, I⁻, F⁻ or Gluc⁻. E_rev for I_OCA2 was close to zero for cytoplasmic Cl⁻ and Br⁻ and shifted to negative voltages for I⁻, F⁻, and Gluc⁻ (Figure 6F), suggesting that in endolysosomes OCA2 mediates a current selective for Cl⁻ and Br⁻, but poorly permeable to I⁻, F⁻, and Gluc⁻. A similar shift in E_rev was measured for I_melano in dermal or RPE melanosomes (Figure 6F), indicating that the endogenous currents have the same selectivity profile. Moreover, the calculated permeability ratios (P_x/P_Cl) for the heterologously expressed currents were nearly the same as the endogenous ones (Figure 6G). Collectively, these results suggest that I_OCA2 and I_melano are mediated by the same anion channel.

1. 1. Ancans J
   2. Tobin DJ
   3. Hoogduijn MJ
   4. Smit NP
   5. Wakamatsu K
   6. Thody AJ

   (2001) Melanosomal pH controls rate of melanogenesis, eumelanin/phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells

   Experimental Cell Research 268:26–35.

   https://doi.org/10.1006/excr.2001.5251

   • Google Scholar
2. 1. Bellono NW
   2. Kammel LG
   3. Zimmerman AL
   4. Oancea E

   (2013) UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes

   Proceedings of the National Academy of Sciences of USA 110:2383–2388.

   https://doi.org/10.1073/pnas.1215555110

   • Google Scholar
3. 1. Bellono NW
   2. Oancea EV

   (2014) Ion transport in pigmentation

   Archives of Biochemistry and Biophysics 563C:35–41.

   https://doi.org/10.1016/j.abb.2014.06.020

   • Google Scholar
4. 1. Brilliant MH

   (2001) The mouse p (pink-eyed dilution) and human P genes, oculocutaneous albinism type 2 (OCA2), and melanosomal pH

   Pigment Cell Research 14:86–93.

   https://doi.org/10.1034/j.1600-0749.2001.140203.x

   • Google Scholar
5. 1. Calvo PA
   2. Frank DW
   3. Bieler BM
   4. Berson JF
   5. Marks MS

   (1999) A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery

   Journal of Biological Chemistry 274:12780–12789.

   https://doi.org/10.1074/jbc.274.18.12780

   • Google Scholar
6. 1. Cerny J
   2. Feng Y
   3. Yu A
   4. Miyake K
   5. Borgonovo B
   6. Klumperman J
   7. Meldolesi J
   8. Mcneil PL
   9. Kirchhausen T

   (2004) The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing

   EMBO Reports 5:883–888.

   https://doi.org/10.1038/sj.embor.7400243

   • Google Scholar
7. 1. Chen K
   2. Manga P
   3. Orlow SJ

   (2002) Pink-eyed dilution protein controls the processing of tyrosinase

   Molecular Biology of the Cell 13:1953–1964.

   https://doi.org/10.1091/mbc.02-02-0022

   • Google Scholar
8. 1. Chen K
   2. Minwalla L
   3. Ni L
   4. Orlow SJ

   (2004) Correction of defective early tyrosinase processing by bafilomycin A1 and monensin in pink-eyed dilution melanocytes

   Pigment Cell Research 17:36–42.

   https://doi.org/10.1046/j.1600-0749.2003.00106.x

   • Google Scholar
9. 1. Cortese K
   2. Giordano F
   3. Surace EM
   4. Venturi C
   5. Ballabio A
   6. Tacchetti C
   7. Marigo V

   (2005) The ocular albinism type 1 (OA1) gene controls melanosome maturation and size

   Investigative Ophthalmology & Visual Science 46:4358–4364.

   https://doi.org/10.1167/iovs.05-0834

   • Google Scholar
10. 1. Dong XP
    2. Cheng X
    3. Mills E
    4. Delling M
    5. Wang F
    6. Kurz T
    7. Xu H

    (2008) The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

    Nature 455:992–996.

    https://doi.org/10.1038/nature07311

    • Google Scholar
11. 1. Eiberg H
    2. Troelsen J
    3. Nielsen M
    4. Mikkelsen A
    5. Mengel-From J
    6. Kjaer KW
    7. Hansen L

    (2008) Blue eye color in humans may be caused by a perfectly associated founder mutation in a regulatory element located within the HERC2 gene inhibiting OCA2 expression

    Human Genetics 123:177–187.

    https://doi.org/10.1007/s00439-007-0460-x

    • Google Scholar
12. 1. Gardner JM
    2. Nakatsu Y
    3. Gondo Y
    4. Lee S
    5. Lyon MF
    6. King RA
    7. Brilliant MH

    (1992) The mouse pink-eyed dilution gene: association with human Prader-Willi and Angelman syndromes

    Science 257:1121–1124.

    https://doi.org/10.1126/science.257.5073.1121

    • Google Scholar
13. 1. Garrison NA
    2. Yi Z
    3. Cohen-Barak O
    4. Huizing M
    5. Hartnell LM
    6. Gahl WA
    7. Brilliant MH

    (2004) P gene mutations in patients with oculocutaneous albinism and findings suggestive of Hermansky-Pudlak syndrome

    Journal of Medical Genetics 41:e86.

    https://doi.org/10.1136/jmg.2003.014902

    • Google Scholar
14. 1. Halaban R
    2. Patton RS
    3. Cheng E
    4. Svedine S
    5. Trombetta ES
    6. Wahl ML
    7. Ariyan S
    8. Hebert DN

    (2002) Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway

    The Journal of Biological Chemistry 277:14821–14828.

    https://doi.org/10.1074/jbc.M111497200

    • Google Scholar
15. 1. Hongyi L
    2. Haiyun W
    3. Hui Z
    4. Qing W
    5. Honglei D
    6. Shu M
    7. Weiying J

    (2007) Prenatal diagnosis of oculocutaneous albinism type II and novel mutations in two Chinese families

    Prenatal Diagnosis 27:502–506.

    https://doi.org/10.1002/pd.1713

    • Google Scholar
16. 1. Hutton SM
    2. Spritz RA

    (2008) A comprehensive genetic study of autosomal recessive ocular albinism in Caucasian patients

    Investigative Ophthalmology & Visual Science 49:868–872.

    https://doi.org/10.1167/iovs.07-0791

    • Google Scholar
17. 1. King RA
    2. Willaert RK
    3. Schmidt RM
    4. Pietsch J
    5. Savage S
    6. Brott MJ
    7. Fryer JP
    8. Summers CG
    9. Oetting WS

    (2003) MC1R mutations modify the classic phenotype of oculocutaneous albinism type 2 (OCA2)

    American Journal of Human Genetics 73:638–645.

    https://doi.org/10.1086/377569

    • Google Scholar
18. 1. Kobayashi M
    2. Katoh H
    3. Ikeuchi M

    (2006) Mutations in a putative chloride efflux transporter gene suppress the chloride requirement of photosystem II in the cytochrome c550-deficient mutant

    Plant & Cell Physiology 47:799–804.

    https://doi.org/10.1093/pcp/pcj052

    • Google Scholar
19. 1. Lee ST
    2. Nicholls RD
    3. Bundey S
    4. Laxova R
    5. Musarella M
    6. Spritz RA

    (1994a) Mutations of the P gene in oculocutaneous albinism, ocular albinism, and Prader-Willi syndrome plus albinism

    The New England Journal of Medicine 330:529–534.

    https://doi.org/10.1056/NEJM199402243300803

    • Google Scholar
20. 1. Lee ST
    2. Nicholls RD
    3. Schnur RE
    4. Guida LC
    5. Lukuo J
    6. Spinner NB
    7. Zackai EH
    8. Spritz RA

    (1994b)

    Diverse mutations of the P gene among African-Americans with type II (tyrosinase-positive) oculocutaneous albinism (OCA2)

    Human Molecular Genetics 3:2047–2051.

    • Google Scholar
21. 1. Manga P
    2. Orlow SJ

    (2001) Inverse correlation between pink-eyed dilution protein expression and induction of melanogenesis by bafilomycin A1

    Pigment Cell Research 14:362–367.

    https://doi.org/10.1034/j.1600-0749.2001.140508.x

    • Google Scholar
22. 1. Montoliu L
    2. Grønskov K
    3. Wei AH
    4. Martínez-García M
    5. Fernández A
    6. Arveiler B
    7. Morice-Picard F
    8. Riazuddin S
    9. Suzuki T
    10. Ahmed ZM
    11. Rosenberg T
    12. Li W

    (2014) Increasing the complexity: new genes and new types of albinism

    Pigment Cell & Melanoma Research 27:11–18.

    https://doi.org/10.1111/pcmr.12167

    • Google Scholar
23. 1. Ni-Komatsu L
    2. Orlow SJ

    (2006) Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in oculocutaneous albinism type 2: effects of altering intracellular pH and pink-eyed dilution gene expression

    Experimental Eye Research 82:519–528.

    https://doi.org/10.1016/j.exer.2005.08.013

    • Google Scholar
24. 1. Oancea E
    2. Vriens J
    3. Brauchi S
    4. Jun J
    5. Splawski I
    6. Clapham DE

    (2009) TRPM1 forms ion channels associated with melanin content in melanocytes

    Science Signaling 2:ra21.

    https://doi.org/10.1126/scisignal.2000146

    • Google Scholar
25. 1. Palmisano I
    2. Bagnato P
    3. Palmigiano A
    4. Innamorati G
    5. Rotondo G
    6. Altimare D
    7. Venturi C
    8. Sviderskaya EV
    9. Piccirillo R
    10. Coppola M
    11. Marigo V
    12. Incerti B
    13. Ballabio A
    14. Surace EM
    15. Tacchetti C
    16. Bennett DC
    17. Schiaffino MV

    (2008) The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells

    Human Molecular Genetics 17:3487–3501.

    https://doi.org/10.1093/hmg/ddn241

    • Google Scholar
26. 1. Passmore LA
    2. Kaesmann-Kellner B
    3. Weber BH

    (1999) Novel and recurrent mutations in the tyrosinase gene and the P gene in the German albino population

    Human Genetics 105:200–210.

    https://doi.org/10.1007/s004390051090

    • Google Scholar
27. 1. Preising MN
    2. Forster H
    3. Tan H
    4. Lorenz B
    5. de Jong PT
    6. Plomp AS

    (2007)

    Mutation analysis in a family with oculocutaneous albinism manifesting in the same generation of three branches

    Molecular Vision 13:1851–1855.

    • Google Scholar
28. 1. Protas ME
    2. Hersey C
    3. Kochanek D
    4. Zhou Y
    5. Wilkens H
    6. Jeffery WR
    7. Zon LI
    8. Borowsky R
    9. Tabin CJ

    (2006) Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism

    Nature Genetics 38:107–111.

    https://doi.org/10.1038/ng1700

    • Google Scholar
29. 1. Puri N
    2. Gardner JM
    3. Brilliant MH

    (2000) Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes

    The Journal of Investigative Dermatology 115:607–613.

    https://doi.org/10.1046/j.1523-1747.2000.00108.x

    • Google Scholar
30. 1. Rak GD
    2. Mace EM
    3. Banerjee PP
    4. Svitkina T
    5. Orange JS

    (2011) Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse

    PLOS Biology 9:e1001151.

    https://doi.org/10.1371/journal.pbio.1001151

    • Google Scholar
31. 1. Raposo G
    2. Tenza D
    3. Murphy DM
    4. Berson JF
    5. Marks MS

    (2001) Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells

    The Journal of Cell Biology 152:809–824.

    https://doi.org/10.1083/jcb.152.4.809

    • Google Scholar
32. 1. Rimoldi V
    2. Straniero L
    3. Asselta R
    4. Mauri L
    5. Manfredini E
    6. Penco S
    7. Gesu GP
    8. Del Longo A
    9. Piozzi E
    10. Soldà G
    11. Primignani P

    (2014) Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II

    Gene 537:79–84.

    https://doi.org/10.1016/j.gene.2013.11.102

    • Google Scholar
33. 1. Rinchik EM
    2. Bultman SJ
    3. Horsthemke B
    4. Lee ST
    5. Strunk KM
    6. Spritz RA
    7. Avidano KM
    8. Jong MT
    9. Nicholls RD

    (1993) A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism

    Nature 361:72–76.

    https://doi.org/10.1038/361072a0

    • Google Scholar
34. 1. Rosemblat S
    2. Durham-Pierre D
    3. Gardner JM
    4. Nakatsu Y
    5. Brilliant MH
    6. Orlow SJ

    (1994) Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene

    Proceedings of the National Academy of Sciences of USA 91:12071–12075.

    https://doi.org/10.1073/pnas.91.25.12071

    • Google Scholar
35. 1. Saito M
    2. Hanson PI
    3. Schlesinger P

    (2007) Luminal chloride-dependent activation of endosome calcium channels: patch clamp study of enlarged endosomes

    The Journal of Biological Chemistry 282:27327–27333.

    https://doi.org/10.1074/jbc.M702557200

    • Google Scholar
36. 1. Setty SR
    2. Tenza D
    3. Truschel ST
    4. Chou E
    5. Sviderskaya EV
    6. Theos AC
    7. Lamoreux ML
    8. Di Pietro SM
    9. Starcevic M
    10. Bennett DC
    11. Dell'angelica EC
    12. Raposo G
    13. Marks MS

    (2007) BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles

    Molecular Biology of the Cell 18:768–780.

    https://doi.org/10.1091/mbc.E06-12-1066

    • Google Scholar
37. 1. Sitaram A
    2. Piccirillo R
    3. Palmisano I
    4. Harper DC
    5. Dell'Angelica EC
    6. Schiaffino MV
    7. Marks MS

    (2009) Localization to mature melanosomes by virtue of cytoplasmic dileucine motifs is required for human OCA2 function

    Molecular Biology of the Cell 20:1464–1477.

    https://doi.org/10.1091/mbc.E08-07-0710

    • Google Scholar
38. 1. Spritz RA
    2. Fukai K
    3. Holmes SA
    4. Luande J

    (1995)

    Frequent intragenic deletion of the P gene in Tanzanian patients with type II oculocutaneous albinism (OCA2)

    American Journal of Human Genetics 56:1320–1323.

    • Google Scholar
39. 1. Stauber T
    2. Jentsch TJ

    (2013) Chloride in vesicular trafficking and function

    Annual Review of Physiology 75:453–477.

    https://doi.org/10.1146/annurev-physiol-030212-183702

    • Google Scholar
40. 1. Sturm RA
    2. Duffy DL
    3. Zhao ZZ
    4. Leite FP
    5. Stark MS
    6. Hayward NK
    7. Martin NG
    8. Montgomery GW

    (2008) A single SNP in an evolutionary conserved region within intron 86 of the HERC2 gene determines human blue-brown eye color

    American Journal of Human Genetics 82:424–431.

    https://doi.org/10.1016/j.ajhg.2007.11.005

    • Google Scholar
41. 1. Sviderskaya EV
    2. Bennett DC
    3. HO L
    4. Bailin T
    5. Lee ST
    6. Spritz RA

    (1997) Complementation of hypopigmentation in p-mutant (pink-eyed dilution) mouse melanocytes by normal human P cDNA, and defective complementation by OCA2 mutant sequences

    The Journal of Investigative Dermatology 108:30–34.

    https://doi.org/10.1111/1523-1747.ep12285621

    • Google Scholar
42. 1. Sviderskaya EV
    2. Hill SP
    3. Evans-Whipp TJ
    4. Chin L
    5. Orlow SJ
    6. Easty DJ
    7. Cheong SC
    8. Beach D
    9. DePinho RA
    10. Bennett DC

    (2002) p16(Ink4a) in melanocyte senescence and differentiation

    Journal of the National Cancer Institute 94:446–454.

    https://doi.org/10.1093/jnci/94.6.446

    • Google Scholar

Author details

1. Nicholas W Bellono

   Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, United States

   Contribution

   NWB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Iliana E Escobar

   Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, United States

   Contribution

   IEE, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Ariel J Lefkovith

   1. Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, United States
   2. Department of Physiology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, United States

   Contribution

   AJL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Michael S Marks

   1. Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, United States
   2. Department of Physiology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, United States

   Contribution

   MSM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Elena Oancea

   Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, United States

   Contribution

   EO, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   elena_oancea@brown.edu

   Competing interests

   The authors declare that no competing interests exist.

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