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. 2014 Dec 16;3:e04543. doi: 10.7554/eLife.04543

An intracellular anion channel critical for pigmentation

Nicholas W Bellono 1, Iliana E Escobar 1, Ariel J Lefkovith 2,3, Michael S Marks 2,3, Elena Oancea 1,*
Editor: Christopher Miller4
PMCID: PMC4270065  PMID: 25513726

Abstract

Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

DOI: http://dx.doi.org/10.7554/eLife.04543.001

Research organism: human

eLife digest

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.

DOI: http://dx.doi.org/10.7554/eLife.04543.002

Introduction

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.

Results and discussion

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 (IOCA2)

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 (IOCA2) that was not present in mock-transfected cells (Figure 1A).

Figure 1. OCA2 contributes to an endolysosomal chloride current with ion channel properties.

(A) Heterologous GFP-OCA2 localized to LAMP1-mCherry-positive late endosomes and lysosomes (endolysosomes) individually dissected from AD293 cells for patch-clamp experiments (scale bar = 5 μm). Whole-endolysosomal currents elicited by voltage steps between −80 mV and +80 mV in a representative endolysosome expressing GFP-OCA2 or in an endolysosome from a mock-transfected cell. (B) Representative whole-endolysosome current–voltage (I–V) relationships in response to voltage ramps. Outwardly rectifying IOCA2 was nearly abolished and its reversal potential (Erev) shifted in the positive direction when luminal Cl− was substituted for gluconate (Gluc−), similar to currents measured from mock-transfected endolysosomes containing a Cl−-based luminal solution. (C) Average current densities (pA/pF) measured at 100 mV. Inset: Average Erev (±s.e.m., n = 6–9 endolysosomes for each condition). (D) In a representative OCA2-expressing endolysosome, Erev was dependent on cytoplasmic [Cl−] ([Cl−]cyto). NMDG-based solutions were used to inhibit endogenous cation permeabilities. (E) IOCA2 Erev varied linearly with [Cl−]cyto when [Cl−]luminal was kept at 48 mM. Each point represents one endolysosome at the [Cl−]cyto indicated on the x-axis.

DOI: http://dx.doi.org/10.7554/eLife.04543.003

Figure 1.

Figure 1—figure supplement 1. OCA2 is a highly conserved transmembrane protein expressed in pigment cells.

Figure 1—figure supplement 1.

(A) Predicted membrane topology and homology of OCA2 from human, mouse, zebrafish and cavefish. OCA2 sequences were aligned using MegAlign (DNASTAR). Accession numbers of OCA2 sequences used in alignments generated using Clustal W in MegAlign (DNASTAR): NP-000266.2 (human), NP-068679.1 (mouse), XP_695807.5 (zebrafish), ABB29299.1 (cavefish). Amino acid percent identity was determined using Protein BLAST (http://blast.ncbi.nlm.nih.gov) for the indicated regions. TM = transmembrane domain, AA = amino acid. Inset: OCA2 alignment showing conserved regions with albinism-associated mutations used in this study (K614E in dark blue, V443I in purple, 5mut in light blue). (B) OCA2 mRNA expression profile in human tissue. OCA2 expression levels were highest in retina, consistent with OCA2 expression in the densely pigmented retinal pigment epithelium (RPE). Human skin had the second highest levels of OCA2 mRNA, due to high expression in melanocytes and very low levels in keratinocytes and skin fibroblasts, consistent with OCA2 expression in pigment cells. (n = 4 experiments).
DOI: http://dx.doi.org/10.7554/eLife.04543.004
Figure 1—figure supplement 2. IOCA2 is not mediated by K+ flux or regulated by pH.

Figure 1—figure supplement 2.

(A) In the same endolysosome, IOCA2 was not significantly different when cytoplasmic K+ was substituted with Na+. (B) The IOCA2 amplitude at 100 mV or Erev was not significantly different when cytoplasmic K+ was substituted with Na+ (±s.e.m., n = 3 endolysosomes). (C) IOCA2 current density (pA/pF) measured in endolysosomes expressing OCA2 was similar with a luminal pH of 4.6 or 6.8. Inset: Average Erev (±s.e.m., n = 4 endolysosomes per condition).
DOI: http://dx.doi.org/10.7554/eLife.04543.005
Figure 1—figure supplement 3. IOCA2 is mediated by Cl− flux.

Figure 1—figure supplement 3.

Average IOCA2 current densities (pA/pF) were calculated at the indicated voltages. Currents recorded from mock-transfected AD293 endolysosomes (A) or endolysosomes expressing OCA2 in the presence of luminal Gluc− (B) were subtracted from currents measured from OCA2-expressing endolysosomes in the presence of luminal Cl−. Δ(OCA2 − mock) and Δ(luminal NaCl − NaGluc) currents have an Erev near ECl (ECl = Nernst potential for Cl−; ±s.e.m., n = 5 endolysosomes per condition).
DOI: http://dx.doi.org/10.7554/eLife.04543.006
Figure 1—figure supplement 4. Cl− channel blockers DIDS, NFA, and NPPB do not affect IOCA2.

Figure 1—figure supplement 4.

(A) In a representative endolysosome IOCA2 was not affected by Cl− channel inhibitors 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS), niflumic acid (NFA), or 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (each at 100 μM), compared to control (DMSO vehicle). (B) The average current amplitude of IOCA2 normalized to control conditions (<1% DMSO) was similar when DIDS, NFA, or NPPB was bath applied at 100 μM or 300 μM (n = 3–4 endolysosomes for each). DIDS blocks several members of the Chloride Channel (ClC) family, Ca2+-activated Cl− channels (CaCC), Maxi Cl− channels, and volume regulated Cl− channels (VRAC); NFA blocks several ClC members, VRAC, and CaCC; NPPB inhibits several ClC members, Maxi Cl−, VRAC, and cystic fibrosis transmembrane conductance regulator (CFTR). Pharmacological profile information was obtained from the International Union of Basic and Clinical Pharmacology (IUPHAR, http://www.iuphar-db.org).
DOI: http://dx.doi.org/10.7554/eLife.04543.007
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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 IOCA2 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 (Erev) (Figure 1—figure supplement 2A,B), suggesting that K+ is not required for IOCA2. We next tested if IOCA2 is mediated by luminal Cl− moving into the cytoplasm by replacing Cl− in the pipette solution with the impermeant anion gluconate (Gluc−). IOCA2 was nearly abolished with Gluc− in the pipette and Erev 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 Erev 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 IOCA2, the Erev for IOCA2 became similar to ECl predicted by the Nernst equation (Figure 1—figure supplement 3). The significant reduction in current amplitude in the presence of luminal Gluc− indicates that IOCA2 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 IOCA2 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 IOCA2 amplitude (Figure 1—figure supplement 4). Thus, IOCA2 has a different pharmacological profile from the channels and transporters that are inhibited by the tested compounds.

The Nernst equation predicts that if IOCA2 were due to selective transport of Cl−, Erev will be dependent on the Cl− concentration ([Cl−]) gradient across the endolysosomal membrane. To establish whether this is the case, we determined Erev for currents measured at variable cytoplasmic and constant luminal [Cl−], using NMDG-based solutions to prevent endogenous cationic permeability. Erev 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). Erev increased linearly as a function of cytosolic [Cl−] ([Cl−]cyto), with a 10-fold change in [Cl−]cyto corresponding to a shift in Erev of 58 mV, consistent with a highly Cl− selective current (Figure 1E).

Mutations associated with oculocutaneous albinism disorder affect IOCA2

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).

Figure 2. Effect of OCA2 disease-associated mutations on IOCA2.

(A) Predicted topology of OCA2 with albinism-associated mutations: K614E (dark blue) in a conserved cytoplasmic loop, V443I (purple) in a highly conserved luminal loop, and 5mut (light blue) consisting of 5 point mutations (V443I, M446V, I473S, N476D, N489D) in the same luminal loop. (B) mCherry-tagged WT, K614E, and V443I OCA2 localized to LAMP1-GFP-positive isolated endolysosomes (merged in yellow), while 5mut did not (scale bar = 5 μm). (C) Representative I–V relationships measured in response to voltage ramps from endolysosomes expressing WT, K614E, V443I, or from endolysosomes isolated from cells expressing 5mut OCA2. (D) Average IOCA2 current densities (pA/pF) measured at 100 mV (±s.e.m., n = 4–8 endolysosomes for each condition). (E) Melan-ink4a melanocytes expressing mCherry-tagged (red) WT, V443I, or K614E and stained with anti-tyrosinase-related protein one (TYRP1) antibodies (green). Insets, 3× magnification of boxed regions. Merged images (right) show that WT, V443I, and K614E OCA2 variants localize primarily to TYRP1-positive compartments (scale bar = 10 μm).

DOI: http://dx.doi.org/10.7554/eLife.04543.008

Figure 2.

Figure 2—figure supplement 1. Localization of wild-type (WT), K614E, V443I, and 5mut OCA2.

Figure 2—figure supplement 1.

(A) HeLa cells expressing mCherry-tagged WT, K614E, V443I, or 5mut OCA2 (red) and stained with LAMP1 antibodies (green). All OCA2 variants, except 5mut, localized primarily to LAMP1-positive compartments (orange in merged images) (scale bar = 10 μm). (B) Melan-ink4a melanocytes expressing 5mut OCA2 tagged with mCherry (red) and stained with anti-TYRP1 antibodies show that 5mut OCA2-mCherry does not localize to TYRP1-positive compartments (green) in melanocytes (scale bar = 10 μm).
DOI: http://dx.doi.org/10.7554/eLife.04543.009
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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 IOCA2, 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 Erev as WT OCA2 (Figure 2C,D), but those expressing V443I had amplitudes reduced by ∼85% and Erev shifted to more positive values (Figure 2C,D). Endolysosomes of cells expressing 5mut had very small current amplitudes and a positive Erev, 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.

Figure 3. OCA2 expression regulates organellar pH.

(A) Ecliptic pHluorin-LAMP1 fluoresces when luminal pH > 6, but not when pH < 6. The V-ATPase inhibitor bafilomycin A1 (BafA1, 2 μM) was used to neutralize acidic endolysosomes and detect pHluorin-LAMP1 expression. (B) Endolysosomes from mock-transfected control (CTL) AD293 cells expressing pHluorin-LAMP1 were not fluorescent at baseline (Fo) but brightly fluoresced after BafA1 treatment (FBafA1). Endolysosomes coexpressing pHluorin-LAMP1 and mCherry-tagged WT or K614E OCA2 were fluorescent prior to BafA1 treatment (Fo), indicating that expression increased pH. Endolysosomes that coexpressed pHluorin-LAMP1 and V443I were dim at baseline (Fo), similar to CTL and mislocalized 5mut, representing an acidic lumen, and pHluorin-LAMP1 fluorescence increased upon BafA1 treatment (FBafA1) (scale bar = 10 µm). (C) Fluorescence intensity following BafA1 treatment was compared with baseline fluorescence to determine relative baseline acidity in enodolysosomes from CTL cells, expressing WT, K614E, V443I, OCA2 or from cells expressing mislocalized 5mut. Bars represent average normalized change in pHluorin-LAMP1 fluorescence (±s.e.m., n = 3 independent experiments, each experiment calculated as the average of n = 78–214 endolysosomes).

DOI: http://dx.doi.org/10.7554/eLife.04543.010

Figure 3.

Figure 3—figure supplement 1. LysoSensor pH measurements.

Figure 3—figure supplement 1.

(A) Lysosensor calibration curve determined from the emission ratio W1 (417–480 nm)/W2 (490–530 nm) at 405 nm excitation for pH values between 5 and 7. Representative curve from one experiment (±s.e.m., n = 231–350 endolysosomes). (B) Predicted pH values based on LysoSensor calibration in LAMP1-expressing control endolysosomes (CTL) compared with endolysosomes expressing WT, K614E, or V443I OCA2 (±s.e.m., n = 3 independent experiments, each experiment calculated as the average of n = 109–635 endolysosomes). (C) Right: Color scale used for Lysosensor. Acidic endolysosomes are shown in green and more neutral ones in blue. Left: AD293 cells loaded with Lysosensor and expressing LAMP1-mCherry (used as a control) or mCherry-tagged WT, K614E, V443I OCA2. Endolysosomes expressing LAMP1-mCherry are green, suggesting that they have acidic pH. Endolysosomes expressing WT or K614E, but not V443I, shift their pH to more neutral values as shown by the blue color (scale bar = 10 μm).
DOI: http://dx.doi.org/10.7554/eLife.04543.011
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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 (Imelano) 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 (Imelano) with a negative Erev 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 Imelano measured in the presence of Cl−, Erev was similar to ECl (Figure 4—figure supplement 1A). The Erev for Imelano 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.

Figure 4. Direct recording from melanosomes identifies an endogenous Cl− current (Imelano).

(A) Dermal macromelanosomes were individually dissected from Oa1−/− melanocytes and patch-clamped. A NaCl-based luminal solution and KGluc-based cytoplasmic solution were used for recordings (scale bar = 10 μm). (B) Representative I–V relationships exhibit a Cl−-dependent outwardly rectifying current (Imelano) with a negative Erev. Substituting luminal Cl− for Gluc− reduced the current amplitude and shifted Erev of Imelano. (C) Average current densities (pA/pF) measured at 100 mV. Inset: Average Erev (±s.e.m., n = 4–5 dermal melanosomes for each condition). (D) Patch-clamp experiments using freshly isolated bullfrog RPE melanosomes (scale = 10 μm). (E) Representative whole-RPE melanosome current–voltage (I–V) relationships in response to voltage ramps. Outwardly rectifying Imelano was reduced and Erev shifted in the positive direction when luminal Cl− was substituted for Gluc−. (F) Average current densities (pA/pF) measured at 100 mV. Inset: Average Erev (±s.e.m., n = 5 RPE melanosomes for each condition).

DOI: http://dx.doi.org/10.7554/eLife.04543.012

Figure 4.

Figure 4—figure supplement 1. Imelano Erev is dependent on the Cl− concentration gradient.

Figure 4—figure supplement 1.

(A) Average current densities (pA/pF) at the indicated voltages were subtracted to calculate the Cl− component of Imelano. Δ(luminal NaCl − NaGluc) current has an Erev near ECl (ECl = Nernst potential for Cl−; ±s.e.m., n = 4 melanosomes per condition). (B) In a representative dermal melanosome, Erev was dependent on [Cl−]cyto. NMDG-based solutions were used to inhibit endogenous cation permeability. (C) Imelano Erev as a function of [Cl−]cyto, when [Cl−]luminal was kept at 48 mM. Each point represents a melanosome at the indicated [Cl−]cyto on the x-axis. Assuming linear Erev dependence on [Cl−]cyto, the slope of the fitted line is 56 mV/10-fold Δ[Cl−]cyto.
DOI: http://dx.doi.org/10.7554/eLife.04543.013
Figure 4—figure supplement 2. RPE melanosome dissection.

Figure 4—figure supplement 2.

Melanosomes were dissected from freshly isolated bullfrog RPE cells using two glass pipettes. The sequence of images shows one pipette used to hold the non-adherent cell and one to dissect the cell to isolate individual melanosomes (red circle) for patch-clamp experiments (scale = 10 μm).
DOI: http://dx.doi.org/10.7554/eLife.04543.014
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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 Erev 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 IOCA2 is present in melanosomes from skin and RPE.

To determine if OCA2 contributes to Imelano, 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 Imelano and pigmentation (Figure 5B,C). Expression of WT OCA2, but not of the V443I mutant, in siRNA-treated cells was sufficient to reconstitute Imelano and rescue melanization (Figure 5A–C), suggesting that the OCA2-mediated current is required for melanin production.

Figure 5. OCA2 is required for Imelano and pigmentation.

(A) Representative images of Oa1−/− melanocytes expressing scrambled or OCA2-targeted siRNA, the latter of which significantly decreased melanin content. Pigmentation was restored by transfection with mCherry-tagged WT OCA2, but not with the mCherry-tagged V443I mutant (scale bar = 10 μm). (B) Representative I–V relationships from melanosomes from cells expressing scrambled siRNA, OCA2-targeted siRNA, and rescued with WT or V443I OCA2. Insets: images of the individual melanosomes used for the shown recordings (scale bar = 3 μm). (C) Average Imelano current densities (pA/pF) measured at 100 mV in melanosomes from cells expressing scrambled siRNA or OCA2-targeted siRNA (±s.e.m., n = 4 melanosomes per condition). (D) Average current densities (pA/pF) measured at 100 mV in melanosomes expressing WT or V443I OCA2 from cells expressing OCA2-targeted siRNA (±s.e.m., n = 3–4 melanosomes per condition).

DOI: http://dx.doi.org/10.7554/eLife.04543.015

Figure 5.

Figure 5—figure supplement 1. OCA2-targeted siRNA reduces OCA2 mRNA and melanin content in Oa1−/− melanocytes.

Figure 5—figure supplement 1.

(A) OCA2 mRNA levels in melanocytes expressing OCA2-targeted siRNA was reduced by ∼90% compared with scrambled siRNA-expressing melanocytes (±s.e.m., n = 3 experiments). (B) Melanin content of melanocytes expressing OCA2-targeted siRNA was ∼73% lower than that of scrambled siRNA-expressing melanocytes (±s.e.m., n = 3 experiments).
DOI: http://dx.doi.org/10.7554/eLife.04543.016
Figure 5—figure supplement 2. Melanin is not required for Imelano.

Figure 5—figure supplement 2.

(A) Representative images of melanosomes from Oa1−/− melanocytes treated with the tyrosinase inhibitor phenylthiourea (PTU, 300 μM) have significantly reduced pigment (scale bar = 3 μm). (B) A similar Imelano was measured from melanosomes of PTU-treated and control Oa1−/− melanocytes. (C) Average current densities (pA/pF) measured at 100 mV. Inset: Average Erev (±s.e.m., n = 5 melanosomes per condition).
DOI: http://dx.doi.org/10.7554/eLife.04543.017
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Might melanin itself be critical for Imelano? 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 Imelano, suggesting that melanin does not influence Imelano (Figure 5—figure supplement 2B,C). We thus concluded that OCA2 expression is required for Imelano and melanization, but melanin is not required for Imelano.

We next sought to compare the biophysical properties of the endogenous OCA2-mediated melanosome current (Imelano) in dermal and RPE melanosomes with those of the OCA2-expressing endolysosome current (IOCA2). Excised cytoplasmic-side-out patches from partially dissected endolysosomes of OCA2-expressing AD293 cells (Figure 6A) exhibited single-channel currents (iOCA2) with greatest activity at positive membrane potentials (Figure 6B), consistent with the outward rectification measured for whole-endolysosome IOCA2 (Figure 1B). Patches from mock-transfected endolysosomes did not exhibit similar single-channel activity (Figure 6—figure supplement 1A,B). The Erev of iOCA2 was −66 mV, close to ECl predicted by the Nernst equation (−68 mV), and the unitary slope conductance was 58 ± 2 pS (Figure 6C).

Figure 6. Endolyosomal IOCA2 has the same properties as melanosomal Imelano.

(A) Cytoplasmic-side-out patches were excised from partially dissected organelles to carry out single-channel recordings. (B) Representative single-channel currents recorded in response to voltage steps between −80 mV and +80 mV from a patch excised from an OCA2-expressing endolysosome (O indicates the open state and C the closed state of the channel). (C) Average single-channel current amplitudes from patches excised from OCA2-expressing endolysosomes (±s.e.m., n = 4 patches) (ECl = Nernst potential for Cl−). (D) Single-channel currents from a representative patch excised from a dermal melanosome that contains at least two channels (O1 indicates one open channel, O2 indicates two channels open simultaneously, and C both channels closed). (E) Average single-channel current amplitudes from patches excised from dermal melanosomes (±s.e.m., n = 3 patches). (F) In a representative OCA2-expressing endolysosome, dermal melanosome, and RPE melanosome, currents were selective for Cl− and Br−, but not I−, F−, or Gluc−. In the same organelle, currents were recorded with 48 mM luminal NMDG-Cl while substituting symmetrical concentrations of cytoplasmic anions in an NMDG-based solution. (G) IOCA2 permeability ratios based on Erev measurements (±s.e.m., n = 3–7 organelles for each condition).

DOI: http://dx.doi.org/10.7554/eLife.04543.018

Figure 6.

Figure 6—figure supplement 1. OCA2-mediated single-channel current characteristics.

Figure 6—figure supplement 1.

(A) Top: Representative single-channel currents recorded at 80 mV from cytoplasmic-side-out patches excised from an endolysosome isolated from a mock-transfected AD293 cell, OCA2-expressing endolysosome, and dermal melanosome. Bottom: Associated amplitude histograms. Inset: None of the endolysosome patches from mock-transfected cells exhibited significant single-channel activity (scale = 10 pA, 250 ms). (B) Average maximal current amplitudes at 80 mV calculated from the Gaussian fit of amplitude histograms of individual membrane patches excised from mock-transfected, OCA2-expressing endolysosomes, or dermal melanosomes (±s.e.m., n = 4–6 patches for each condition, i = 0.18 ± 0.18 pA for mock-transfected AD293 endolysosomes, 8.59 ± 0.46 pA for OCA2-expressing endolysosomes, 9.00 ± 0.51 pA for dermal melanosomes).
DOI: http://dx.doi.org/10.7554/eLife.04543.019
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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 (imelano) (Erev = −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 Erev under symmetrical concentrations (48 mM) of luminal Cl− and cytoplasmic Cl−, Br−, I−, F− or Gluc−. Erev for IOCA2 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 Erev was measured for Imelano in dermal or RPE melanosomes (Figure 6F), indicating that the endogenous currents have the same selectivity profile. Moreover, the calculated permeability ratios (Px/PCl) for the heterologously expressed currents were nearly the same as the endogenous ones (Figure 6G). Collectively, these results suggest that IOCA2 and Imelano are mediated by the same anion channel.

Conclusions and implications

Our results suggest that OCA2 is an essential component of a melanosome-specific anion channel. Heterologous endolysosomal expression of OCA2 contributes to a chloride channel with biophysical properties similar to an endogenous melanosomal OCA2-mediated channel. Importantly, OCA2 activity controls the melanin content of melanosomes, most likely by regulating organellar pH. We propose that OCA2 contributes to a novel melanosome-specific anion current that modulates melanosomal pH for optimal tyrosinase activity required for melanogenesis.

Materials and methods

Cells and tissue

All cells were grown at 37°C and 5% CO2, and reagents were from Invitrogen/Life Technologies (Grand Island, NY) unless stated otherwise. AD293, HeLa, and NF-SV60 fibroblasts were grown in DMEM, 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA) with or without 1% penicillin/streptomycin (P/S).

Immortalized mouse melanocyte cell lines melan-Ink4a (Sviderskaya et al., 2002) and melan-Oa1 (Palmisano et al., 2008) were grown in RPMI 1640, 10% FBS, 1% P/S, 200 nM phorbol 12-myristate-13-acetate (Sigma, St. Louis, MO) at 37°C and 10% CO2. Primary human epidermal melanocytes and keratinocytes were isolated from neonatal foreskin. Human epidermal melanocytes (Cascade Biologics/Life Technologies) were cultured in Medium 254, Human Melanocytes Growth Supplement, and 1% P/S. Human epidermal keratinocytes (Lifeline Cell Technology, Frederick, MD) were cultured in Dermalife basal medium supplemented with LifeFactors and 1% P/S.

Transfection of cells used for electrophysiology and imaging was carried out using Lipofectamine 2000 (Invitrogen/Life Technologies) according to manufacturer's protocol, unless otherwise stated.

RPE tissue

American Bullfrogs (L. catesbeianus) eyes were hemisected and RPE tissue was removed from the eyes with forceps after removal of the retinas. The dissection was performed in normal room light using light-adapted frogs. The tissue was kept at 4°C in a modified Ringer's solution containing (mM): 111 NaCl, 2.5 KCl, 1 CaCl2, 1.5 MgCl2, 0.02 EDTA, 3 HEPES, pH 7.6. Tissue was used for a maximum of 36 hr.

Molecular biology

OCA2 tagged with GFP or mCherry was generated by inserting human OCA2 (hOCA2, NM_000275) between the BamHI/Xhol sites of pcDNA4/TO (Invitrogen/Life Technologies). K614E and V443I mutations in hOCA2 were made using site-directed mutagenesis and verified by sequencing. 5mut hOCA2 was generated by Genscript (Piscataway, NJ).

siRNA

Mouse OCA2 (mOCA2)-targeted and scrambled siRNAs were designed and expressed using the BLOCK-iT miRNA lentiviral system (Invitrogen/Life Technologies). mOCA2 specific oligos were cloned into pcDNA6.2-GW/EmGFP-miRNA, then recombined into pLenti6/V5-DEST vector and expressed in Oa1−/− mouse melanocytes using viral transduction (Bellono et al., 2013). Stable cell lines of OCA2-targetd or control siRNA-expressing melanocytes were created using fluorescence-activated cell sorting (FACS) and subsequently kept under blasticidin selection (10 μg/ml). For rescue experiments hOCA2 was transfected in mouse melanocytes expressing mOCA2-targeted siRNA.

Quantitative PCR

Total RNA was extracted using the RNeasy Plus kit (QIAGEN) and reverse transcribed using SuperScript III kit (Invitrogen/Life Sciences). mOCA2 mRNA levels were determined using the following primers: F 5′-CAGGAATCGCAGAGAAGCAT-3′ and R 5′-AGATAGCACATCCCAATGGTG-3′. hOCA2 mRNA levels in an array of human tissues (Origene) and in human epidermal skin cells were determined by qPCR with the following primers F 5′-GTGTGCAGGGATTGCAGAAC-3′ and R 5′-ACATCCCAACAGTGCAGGAC-3′. Reactions were prepared according to manufacturer instructions using Power SYBR Green (Applied Biosystems/Life Technologies) and cycled on a 7500 Real-Time PCR System (Applied Biosystems/Life Technologies). Actin was used as an internal control and all reactions were run in triplicate. mRNA levels were quantified by calculating 2−ΔCT values for each condition.

Organellar electrophysiology

Organelle dissection

AD293 cells were transiently transfected 1 day prior to electrophysiological recordings and treated with 1 μM vacuolin-1 (EMD Millipore, Billerica, MA) for 6–12 hr before the patch clamp experiments. Enlarged endolysosomes were individually dissected for patch clamp experiments, as previously described (Dong et al., 2008). In short, a borosilicate patch pipette was used to cut the cell membrane and push out individual organelles. Enlarged dermal melanosomes were dissected from Oa1−/− mouse melanocytes and RPE melanosomes from bullfrog RPE cells using two patch pipettes.

Recording conditions

Organelle patch-clamp recordings were carried out at room temperature using an EPC 10 amplifier (HEKA Instruments, Lambrecht, Germany) with PatchMaster software (HEKA Instruments). Data were filtered at 2.9 kHz and digitized at 10 kHz. Membrane potentials were corrected for liquid junction potentials. Organelle currents were recorded using borosilicate glass pipettes polished to 7–8 MΩ for lysosomes and 9–12 MΩ for melanosomes. Standard pipette/lumen solutions contained (mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 MES, 10 glucose; pH 4.6 for endolysosomes and pH 6.8 for melanosomes, unless otherwise stated. Standard bath/cytoplasmic solution contained (mM): 140 K-gluconate, 5 NaCl, 2 MgCl2, 0.39 CaCl2, 1 EGTA (Ca2+ buffered to 100 nM), 20 HEPES, pH 7.2. NMDG-based solutions were used in some experiments to block endogenous cation permeabilities. The holding potential was 0 mV and currents were measured in response to 500 ms voltage ramps from −100 to +100 mV, unless otherwise stated. Whole-organelle current density was calculated by normalizing to capacitance (0.14–0.5 pF for melanosomes, 0.24–1.2 pF for endolyosomes). Single-channel current amplitudes were measured from the middle of the noise band between closed and open states or calculated from the difference between Gaussian-fitted closed and open peaks on all-points amplitude histograms for each excised patch record. To determine if IOCA2 passes Cl− electrodiffusively, reversal potential (Erev) was measured using 48 mM luminal Cl− and variable cytoplasmic [Cl−] and compared with Erev predicted by the Nernst potential for Cl−: ECl = (RT/zF)ln([Clluminal]/[Clcytoplasmic]). R = gas constant, z = valence (−1 for Cl−), T = absolute temperature, and F = Faraday constant.

Anion permeability

Relative permeability of IOCA2 was determined by measuring the shift in Erev after the substitution of bath/cytoplasmic anions from Cl− to Br−, I−, F−, or Gluc−. Pipette/luminal and bath/cytoplasmic solutions were 48 mM NMDG-X solutions, where X = Cl− for pipette/luminal and X = Cl−, Br−, I−, F−, or Gluc− for bath/cytoplasmic. Permeability ratios were estimated using the Goldman-Hodgkin-Katz (GHK) equation: PX/PCl = exp (ΔErevF/RT), where ΔErev is the difference between Erev in symmetrical Cl (Erev = 0 mV) and that in cytoplasmic X. We did not correct for the possibility of the permeation of symmetrical NMDG+ or H+.

pH imaging

pHluorin-LAMP1

AD293 cells were cotransfected with mCherry-OCA2 and pHluorin-LAMP1 1 day prior to imaging experiments. pHluorin-LAMP1 expression was verified by treatment with bafilomycin A1 (BafA1, 2 μM). pHluorin-LAMP1 fluorescence intensity was quantified in endoysosomes that coexpressed mCherry-OCA2 and pHluorin-LAMP1 by subtracting the initial fluorescence from fluorescence elicited by treatment with BafA1: [(FBafA1 − Fo)/Fo]−1.

LysoSensor DND-160

AD293 cells were transfected 1 day prior to imaging experiments with mCherry-OCA2 or LAMP1 to identify endolysosomes, and incubated with 1 μM LysoSensor-ND160 for 5 min. Lysosensor was excited at 405 nm and its emission detected at 417–483 nm (W1) and 490–530 nm (W2). The ratio of emissions (W1/W2) in endolysosomes expressing mCherry-tagged OCA2 or LAMP1 was assigned to a pH value based on a calibration curve generated prior to each experiment using solutions containing 125 mM KCl, 25 mM NaCl, 24 μM Monensin, and varying concentrations of MES to adjust the pH to 3.5, 4.5, 5, 5.5, 6.5, 7, 7.5. The fluorescence ratio was linear for pH 5.0–7.0.

Immunofluorescence microscopy

Melan-ink4a melanocytes were seeded on coverslips coated with Matrigel (BD Biosciences, San Jose, CA) at 3–4 × 104 cells/well in a 24-well plate, transfected the next day with 0.8 μg of plasmid DNA; HeLa cells were seeded on glass coverslips at 105 cells/well in a six well plate, transfected the next day using GeneJuice (EMD Millipore) as recommended by the manufacturer, with 0.1 µg of plasmid DNA. Both cell types were analyzed 48 hr post-transfection. Cells were fixed in HBSS (Invitrogen)/2% paraformaldehyde (Sigma) for 20 min at roo temperature, washed once with PBS and labeled with primary and secondary antibodies diluted in PBS with 0.2% (wt/vol) saponin, 0.1% (wt/vol) bovine serum albumin, 0.02% (wt/vol) sodium azide as described (Calvo et al., 1999). Nuclei were labeled with 500 ng/µl Hoescht 33342 (Sigma). Antibodies used were: mouse anti-TYRP1 (TA99/mel-5, ATCC); mouse anti-human LAMP1 (H4A3) and rat anti-mouse LAMP2 (GL2A7; both from Developmental Studies Hybridoma Bank, Iowa City, IA). Donkey antibodies specific to mouse or rat immunoglobulin and conjugated to Dylight 594 or 488 were from Jackson Immunoresearch (West Grove, PA). Cells were imaged using a 100× HCX PL APO Lens on a Leica DM IRBE microscope equipped with a Retiga Exi Fast 1394 digital camera (Qimaging, Surrey, Canada) and Improvision Openlab software (Perkin–Elmer, Waltham, MA). Sequential z-stack images separated by 0.2 µm were acquired and deconvolved using the OpenLab Volume Deconvolution module. Images from single stacks are shown. Final images were generated and insets magnified using Photoshop (Adobe, San Jose, CA). Colocalization analyses were performed using OpenLab software, as described (Setty et al., 2007). Briefly, images of individual cropped cells were rendered binary using the ‘Density slice’ module and the densely labeled perinuclear region was excluded from analysis. Pixel overlap between binary red and green images in the remaining cell regions was defined using the ‘Boolean operations’ module and further analyzed using Excel (Microsoft, Redmond, WA).

Melanin quantification

Melanin from Oa1−/− melanocytes expressing scrambled or OCA2-targeted siRNA was quantified as previously described (Oancea et al., 2009). Briefly, the soluble and insoluble fractions of melanocytes were separated after cell lysis with 1% Triton X-100 (Sigma) in PBS pH 7.4. Total protein was measured in the soluble fraction using a Bradford Assay (Bio-Rad Laboratories, Waltham, MA). The insoluble fraction was dissolved in 1 N NaOH by incubation for 30 min at 80°C and used to quantify melanin by measuring the optical density of each sample at 405 nm, then fit with a standard curve generated using synthetic melanin (Sigma). Average cellular melanin values were quantified as the ratio between total melanin and total protein from the same dish.

Data analysis

All data are shown as mean ± s.e.m. Data were considered significant if p < 0.05 using unpaired two-tailed Student t test or one-way ANOVA.

Acknowledgements

We thank Drs C Cang and D Ren, who were instrumental for assisting us with the endolysosomal patch-clamp technique, V Yorgan and Dr SB Lizarraga for help with pH measurements and analyses, Dr TJ Roberts and D Sleboda for help with frogs, and Dr AL Zimmerman for advice and suggestions. We are also thankful to Dr JS Orange for providing the pHluorin-LAMP1 construct. We thank Drs AL Zimmerman, JA Kauer, and D Ren for critical reading of the manuscript. This work was supported by NIAMS (R01 AR066318 to EO and R01 AR048155 to MSM), NEI (R01 EY015625 to MSM), Brown University (EO), NIGMS training grant T32 GM077995 (NWB), a NSF Graduate Research Fellowship (NWB), and NIGMS training grant T32 GM007229 (AJL).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

  • National Science Foundation to Nicholas W Bellono.

  • National Institute of General Medical Sciences to Ariel J Lefkovith, Nicholas W Bellono.

  • National Institute of Arthritis and Musculoskeletal and Skin Diseases to Michael S Marks.

  • National Eye Institute to Michael S Marks.

  • Brown University to Elena Oancea.

Additional information

Competing interests

The authors declare that no competing interests exist.

Author contributions

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

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

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

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

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

Ethics

Animal experimentation: All mouse melanocytes were obtained from the Wellcome Trust Functional Genomics Cell Bank as described in the Methods. American bullfrog eyes were obtained as discarded tissue from Dr Thomas Roberts' laboratory at Brown University (protocol number 1303990009) and used in agreement with all the ethics rules and regulations.

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eLife. 2014 Dec 16;3:e04543. doi: 10.7554/eLife.04543.020

Decision letter

Editor: Christopher Miller1

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “An intracellular anion channel critical for pigmentation” for consideration at eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor) and Chris Miller, who is serving as a guest editor, as well as two other reviewers, one of whom, David Clapham, has agreed to reveal his identity. Chris Miller has been in touch with you about the steps required for submission of a revised manuscript. This is a more formal letter that summarizes the decision.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

This is a satisfying study that identifies a novel ion channel involved in regulation of acidification of melanosomes, which are lysosome-like intracellular organelles specialized for melanin pigment-producing cells. The authors start with a guess – that OCA2, an unknown-function membrane protein whose genetic disruption leads to albinism – might contribute to melanosome physiology. Accordingly, they heterologously express OCA2 in mammalian cells, showing that the protein is directed to the endolysosomes, and in exceedingly challenging patch-recording experiments document a new current appearing in dissected lysosomes. They carry out a thorough characterization of this voltage-dependent, Cl- selective current, showing that it is reduced by certain mutations associated with albinism. Moreover, they detect a similar current endogenous to melanosomes from a melanocyte cell line. The biological function of this current is addressed by experiments showing that heterologous expression of OCA2 leads to pH regulation in lysosomes similar to that known for melanosomes in melanocytes, and effect that albinism mutants fail to produce. Moreover, they show that proper pH regulation in the melanocyte line is suppressed by OCA2 siRNA.

These difficult experiments are carefully done, the analysis is rigorous, and the conclusions compelling (but see below). Moreover, the system offers novel insight into an unusual cellular physiology: pH regulation of melanin-producing organelles in melanocytes and retinal pigment epithelial cells.

The reviewers had several criticisms that will need to be addressed in a revised manuscript.

1) The conclusion that the OCA2 currents observed are mediated by an electrodiffusive ion channel mechanism is not well enough supported by the single-channel recordings of Figure 6. It is clear that these recordings are unusually difficult to make, but the absence of a statistically serious argument that these appear only upon OCA2 expression is lacking here. Since OCA2 is a member of the SLC13 family, for which only transporters are known, makes it all the more important to establish the identity of the single channels observed.

2) The use of Lysotracker to put a precise value on the lumenal pH of these lysosome-like compartments is questioned. Lysosensors are weak bases that accumulate in acidic organelles, but since this accumulation is pH dependent, as is the molecules' fluorescence it is very difficult to be confident of assessments of pH change. The calibrations are questionable because monensin alone is unlikely to allow full pH equilibration with the bathing medium.

3) The authors have given no plausible picture of why expression of a Cl- conductance should lead to alkalinization of the melanosomal lumen via the V-ATPAse, rather than acidification, and so the physiological rationale of the study needs sharpening in the Discussion (or Introduction).

4) The family of voltage-pulse-evoked currents (Figure 4F) are far noisier than the ramp-evoked IV curves of Figure 4E, and this discrepancy raises concerns. In addition, the currents of Figure 4F are rather redundant to those of 4E, and could be removed, but the large difference on primary data quality should be explained in the authors' response.

5) Albinism mutations. All three reported mutations presumably cause albinism, but the K614E mutation seems to generate completely normal currents. The authors never address this discrepancy, which is difficult to reconcile with the claimed function of the protein.

eLife. 2014 Dec 16;3:e04543. doi: 10.7554/eLife.04543.021

Author response

1) The conclusion that the OCA2 currents observed are mediated by an electrodiffusive ion channel mechanism is not well enough supported by the single-channel recordings of Figure 6. It is clear that these recordings are unusually difficult to make, but the absence of a statistically serious argument that these appear only upon OCA2 expression is lacking here. Since OCA2 is a member of the SLC13 family, for which only transporters are known, makes it all the more important to establish the identity of the single channels observed.

Our argument that single channels are only present in cells transfected with OCA2 is based on a negative control (endolysosomes dissected from mock-transfected cells) of only two patches. As the reviewer indicates, this is because of the extreme difficulty of the experiments. We agree that increasing the n for the negative control would make our claim statistically stronger. In response to this, we added more recordings from cytoplasmic-side-out patches from dissected mock-transfected endolysosomes, included in a new supplemental figure (Figure 6–figure supplement 1). In this figure we show all six negative control recordings, none of which has currents similar to those measured in all eight of the OCA2-expressing cells and dermal melanosomes. We also plotted all-points amplitude histograms for each recording and graphed the average maximal amplitudes obtained from Gaussian fits of each amplitude histogram for each recording. These data are very consistent and, we believe, convincing that the single channels recorded require OCA2 expression.

2) The use of Lysotracker to put a precise value on the lumenal pH of these lysosome-like compartments is questioned. Lysosensors are weak bases that accumulate in acidic organelles, but since this accumulation is pH dependent, as is the molecules' fluorescence it is very difficult to be confident of assessments of pH change. The calibrations are questionable because monensin alone is unlikely to allow full pH equilibration with the bathing medium.

We agree that LysoSensor quantification is not flawless and we altered the text and figures to reflect that. However, because the LysoSensor measurements complement the pHluorin experiments, we feel that it would be useful to keep them in supplemental data (Figure 3–figure supplement 1) and adjusting the wording for the quantification of luminal pH accordingly.

3) The authors have given no plausible picture of why expression of a Cl- conductance should lead to alkalinization of the melanosomal lumen via the V-ATPAse, rather than acidification, and so the physiological rationale of the study needs sharpening in the Discussion (or Introduction).

We don’t understand the mechanism by which OCA2 expression makes the endolysosomal lumen more alkaline, but can offer some of our hypotheses or speculations.

There are two determinant factors for our hypothesis/model: the organelle membrane potential (Vm) and the Nernst potential for endolysosomal/melanosomal Cl− (ECl).

If Vm is more positive than ECl, then Cl− will flow from the lumen into the cytosol. Negative charges leaving the lumen will make the inside more positive and decrease the electrogenic pumping of V-ATPase. In addition, OCA2-mediated Cl− efflux will also decrease the luminal Cl− concentration, decreasing the driving force for CLC antiporters, which will bring less H+ into the lumen. A decrease in H+ pumped by V-ATPases and transported by CLC would result in a more basic luminal pH.

Vm > ECl → ↑ pH lumen

If Vm is more negative than ECl, then Cl− will flow from the cytosol into the lumen. The Cl− influx would shunt the positive-inside membrane potential, which in turn would increase the electrogenic pumping of V-ATPase, leading to H+ accumulation in the lumen. In this case OCA2-mediated Cl− transport would acidify the lumen of the endolysosome or melanosome.

Vm < ECl → ↓ pH lumen

Our experimental evidence suggests that the first possibility applies to our data. But neither of the two values, Vm nor ECl, has been unequivocally determined. In support of the assumption that Vm > ECl, the lysosomal membrane potential has been estimated to be in the -30 – -10 mV range (Koivusalo et al., Traffic 2011; Steinberg et al., JCB 2010; Sonawane et al., JBC 2002, Cang et al., Cell 2013) and ECl is likely to be in the range of the typical plasma membrane value of -60mV, based on estimates of Cl− concentrations in lysosomes and the cytosol (Steinberg et al., JCB 2010). Under our normal experimental conditions ([Cl−]in = 150 mM, [Cl−]out = 10 mM) ECl = -68 mV. In support of the second scenario (Vm < ECl), the Brilliant laboratory found the melanosomes of OCA2-deficient mice to be less acidic compared with WT using DAMP and anti-DNP staining (Puri et al., J. Invest. Dermatol. 2000). However, this change in pH cannot be directly attributed to the absence of a Cl− conductance mediated by OCA2 because these melanosomes have biogenesis and maturation defects and are likely to lack other proteins important for pH and membrane voltage regulation.

Our ability to come up with a clear model is hindered by the fact that there are many unknown variables that could contribute to the regulation of organellar pH and Vm and that might be different between melanosomes and endolysosomes. The effect of the Cl− conductance on the pH of the lumen could be a more complex mechanism mediated by additional channels and transporters. It is possible that melanosomes may have voltage-sensitive channels or enzymes, similar to endolysosomes (Cang et al., Nature Chem. Biol. 2014), which could be affected by OCA2 to regulate pH. In endolysosomes expressing OCA2, other ion channels might become regulated by heterologous OCA2 (TRPML, TPC, etc.) to indirectly modulate pH. In melanosomes, there are a number of membrane proteins with predicted ion transport function (SLC45A2, SLC24A5, etc.) that could contribute to this pathway by directly transporting H+ or by modulating H+ transport mechanisms. Because endolysosomes and melanosomes express different proteins that contribute to ion transport and might have different Vm, in addition to the hypothesized different luminal pH, it is also possible that OCA2 expression does not have the same effect on the pH of endolysosomes compared to the pH of melanosomes. Thus, until we better understand melanosome membrane conductance and signaling, our model depicting pH regulation by OCA2 remains highly speculative.

We now include a brief paragraph discussing our hypothetical model.

4) The family of voltage-pulse-evoked currents (Figure 4F) are far noisier than the ramp-evoked IV curves of Figure 4E, and this discrepancy raises concerns. In addition, the currents of Figure 4F are rather redundant to those of 4E, and could be removed, but the large difference on primary data quality should be explained in the authors' response.

We agree that the melanosomal recordings from Figure 4F are lower quality than the currents recorded in response to ramps. We included them with the intention of showing current kinetics in response to voltage steps. We think these recordings look so noisy because whole-melanosome recordings, in particular from RPE melanosomes, are especially unstable in response to prolonged voltage pulses, compared with ramps. We also recognize the redundancy in the data and removed Figure 4F, as suggested. In place, we have added summary data from the ramp-elicited currents in RPE melanosomes (new Figure 4F).

5) Albinism mutations. All three reported mutations presumably cause albinism, but the K614E mutation seems to generate completely normal currents. The authors never address this discrepancy, which is difficult to reconcile with the claimed function of the protein.

We were also surprised by the lack of localization or whole-organelle current phenotype for the K614E variant, and do not have a definitive answer regarding why it is associated with oculocutaneous albinism. This mutation was found in a German patient with albinism who had an additional OCA2 mutation (W679C), perhaps each mutation inherited from one parent (Passmore et al., Hum Genet 1999). A similar mutation (K614N) was found in a Tanzanian patient who also had a deletion in the second allele of the gene (Spritz et al., Am J Hum Genet 1995). In contrast, the V443I mutation is very common – it has been described in more than 10 studies (http://www.ifpcs.org/albinism/oca2mut.html) – and is, by itself, sufficient to cause oculocutaneous albinism.

Therefore, it is possible that the K614E mutation has a mild localization or functional phenotype that we are not able to detect using overexpression. Endogenous OCA2 is present at low amounts, thus even “modest” exogenous expression by viral transduction will likely increase the endogenous OCA2 levels by at least 10 fold. We speculate that the high expression levels could mask a mild defect in localization to melanosomes or in protein folding in the endoplasmic reticulum. It is also possible that the K614E mutation, found in a conserved cytoplasmic loop, affects the regulation of OCA2 function via an unknown mechanism present in the cytoplasm, which is lost during organelle dissection. But we have no way of addressing this concern in a more effective way.

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