BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release.

Article

BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release Highlights

Authors

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Two dileucine sorting motifs determine BK channel lysosomal localization

Qi Cao, Xi Zoe¨ Zhong, ..., Ligia Toro, Xian-Ping Dong

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Activation of lysosomal BK channels hyperpolarizes lysosomal membrane

Correspondence

BK interacts with TRPML1 to regulate lysosomal Ca2+ release and trafficking

In Brief

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BK upregulation rescues the cellular phenotypes of NiemannPick C1 disease

Cao et al., 2015, Developmental Cell 33, 1–15 May 26, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2015.04.010

[email protected]

Defects in lysosomal Ca2+ release are associated with the lysosomal storage disease Niemann-Pick C1 (NPC1). Cao et al. find that the Ca2+-activated potassium channel BK is expressed in lysosomes, where it facilitates Ca2+ release via TRPML1 channels. BK overexpression rescues abnormal lysosomal storage in cells from NPC1 patients.

Please cite this article in press as: Cao et al., BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release, Developmental Cell (2015), http://dx.doi.org/10.1016/j.devcel.2015.04.010

Developmental Cell

Article BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release Qi Cao,1,3 Xi Zoe¨ Zhong,1,3 Yuanjie Zou,1 Zhu Zhang,2 Ligia Toro,2 and Xian-Ping Dong1,* 1Department of Physiology and Biophysics, Sir Charles Tupper Medical Building, Dalhousie University, 5850 College Street, Halifax, NS B3H 4R2, Canada 2Division of Molecular Medicine, Department of Anesthesiology and Department of Molecular & Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-7115, USA 3Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2015.04.010

SUMMARY

Promoting lysosomal trafficking represents a promising therapeutic approach for lysosome storage diseases. Efficient Ca2+ mobilization from lysosomes is important for lysosomal trafficking. Ca2+ release from lysosomes could generate a negative potential in the lumen to disturb subsequent Ca2+ release in the absence of counter ion flux. Here we report that lysosomes express big-conductance Ca2+-activated potassium (BK) channels that form physical and functional coupling with the lysosomal Ca2+ release channel, TRPML1. Ca2+ release via TRPML1 causes BK activation, which in turn facilitates further lysosomal Ca2+ release and membrane trafficking. Importantly, BK overexpression rescues the impaired TRPML1-mediated Ca2+ release and abnormal lysosomal storage in cells from NiemannPick C1 patients. Therefore, we have identified a lysosomal K+ channel that provides a positive feedback mechanism to facilitate TRPML1-mediated Ca2+ release and membrane trafficking. Our findings suggest that upregulating BK may be a potential therapeutic strategy for certain lysosomal storage diseases and common neurodegenerative disorders. INTRODUCTION Lysosome storage diseases (LSDs) are a group of approximately 60 inherited metabolic disorders caused by mutations in acidic hydrolases required for catabolic degradation or mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking (Luzio et al., 2007; Saftig and Klumperman, 2009). New evidence suggests that promoting lysosomal membrane trafficking could be a promising therapeutic approach for LSDs (Chen et al., 2014; Medina et al., 2011; Samie and Xu, 2014; Shen et al., 2012). Lysosomal membrane trafficking is a Ca2+-dependent process (Lloyd-Evans and Platt,

2011; Luzio et al., 2007; Morgan et al., 2011; Pittman, 2011; Pryor et al., 2000), which is initiated by Ca2+ release from the lysosome itself (Christensen et al., 2002; Morgan et al., 2011; Pryor et al., 2000). Defective lysosomal Ca2+ release has been associated with a number of LSDs (Coen et al., 2012; Dong et al., 2008; Kiselyov et al., 2010; Lloyd-Evans et al., 2008; Lloyd-Evans and Platt, 2011; Shen et al., 2012). Transient receptor potential mucolipin-1 (ML1) proteins are ubiquitously expressed and form lysosomal Ca2+/Na+ release channels that regulate lysosomal membrane trafficking. Mutations in the human ML1 gene cause an LSD called mucolipidosis type IV (ML4). Cells from ML4 patients show enlarged lysosomes and abnormal lysosomal storage (Dong et al., 2008; Venkatachalam et al., 2014), implicating the importance of ML1 in lysosomal trafficking and function. Because ML1 channels are strongly inwardly rectifying (Dong et al., 2008), their activation causes a large amount of Ca2+ and Na+ loss from lysosomal lumen, which could collapse the negative potential across the lysosomal membrane, preventing further Ca2+/Na+ release. Thus, either counter cation influx or anion co-release should exist to balance the loss of luminal cations resulting from continuous Ca2+/Na+ release. However, neither the nature of the ion(s) nor the molecular identity of the channel is known. In the plasma membrane (PM), it has been well established that Ca2+ influx through voltage-gated Ca2+ channels activates Ca2+-activated big-conductance K channel (BK; also known as Slo1 or MaxiK) to regulate cell excitability using a feedback mechanism (Berkefeld et al., 2010; Fakler and Adelman, 2008; Salkoff et al., 2006). Because lysosomal membrane potential is sensitive to K+ (Cang et al., 2013), we hypothesized that BK might also function as a lysosomal K+ channel to provide the needed counter cation influx to prevent the collapse of membrane potential due to ML1-mediated Ca2+/ Na+ release. This should facilitate continued Ca2+/Na+ release via activated ML1 channels and in turn lysosomal trafficking and function. Because defects in ML1-mediated lysosomal Ca2+ release have been associated with Niemann-Pick type C1 (NPC1) disease (Kiselyov et al., 2010; Lloyd-Evans and Platt, 2011; Morgan et al., 2011; Neefjes and van der Kant, 2014; Vitner et al., 2010), we speculated that enhancing BK function may be a plausible approach to rescue membrane trafficking defects in NPC1 / cells. Developmental Cell 33, 1–15, May 26, 2015 ª2015 Elsevier Inc. 1


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RESULTS Functional BK Channels Are Expressed in Lysosomes In order to investigate the role of BK in lysosomal Ca2+ regulation, we first examined lysosomal expression of endogenous BK by immunofluorescence staining of human fibroblast cells loaded with dextran Texas Red, a lysosome marker, using a BK antibody. All human fibroblasts displayed strong BK immunofluorescence that colocalized with the dextran puncta (Figure 1A). To further define the subcellular locations of BK, cellular fractions from Cos1 cells were prepared with lysosomes enriched using density-gradient centrifugation (Wang et al., 2012). Immunoblotting of these fractions revealed abundant presence of endogenous BK proteins in lysosomal fractions, as indicated by the expression of lysosomal-associated membrane protein 1 (Lamp1) (Figure 1B). Similarly, the majority of heterologously expressed c-Myc-BK-GFP (Toro et al., 2006) also colocalized with Lamp1-mCherry coexpressed in Cos1 cells (Figure 1C), and abundant c-Myc-BK-GFP protein was found in lysosomal fractions (Figure 1D). Furthermore, we detected colocalization between endogenous BK and Lamp1 in mouse embryo fibroblast (MEF) cells by double immunocytochemical staining with respective antibodies (Figure 1E). The BK antibody stained Lamp1-positve puncta in wild-type but not BK-knockout MEF cells (Meredith et al., 2004) (Figures 1E and S1A), demonstrating its specificity. Therefore, both endogenous and heterologously expressed BK channels are expressed in lysosomes. Typically, dileucine-based motifs, such as [D/E]XXXL[L/I] and [D/E]XXL[L/I], are involved in targeting of lysosomal membrane proteins (Bonifacino and Traub, 2003). Our inspection of BK protein sequence revealed two potential dileucine motifs, D(485) ACLI and D(731)PLLI, located in the regulator of conductance for K+ (RCK) domains in the large cytoplasmic carboxy-terminus (Figure 1F). Substitution of LI(488,489) with MM and LI(734,735) with VV eliminated lysosomal localization of the BK channel (Figure 1G), suggesting that two putative sorting motifs are indeed important for BK targeting to lysosomes.

Next, to test whether the lysosome-localized BK channels were functional, we performed whole-lysosome patch-clamp recordings directly on enlarged vacuoles representing lysosome vesicles (Dong et al., 2008) (Figure S1D) isolated from Cos1 cells that expressed c-Myc-BK-GFP. In c-Myc-BK-GFP-positive lysosomes, small basal outwardly rectifying currents were detected in a bath solution that contained 100 nM free Ca2+, which were dramatically increased by bath application (to cytosolic side) of 100 mM Ca2+ (Figure 1H). Consistent with what is known for BK channels studied on the PM, increasing the free [Ca2+]c shifted the voltage dependence of channel activation to more negative potentials (Figure 1I); from 100 nM to 10 mM free Ca2+, V1/2 shifted negatively by >100 mV (Figure 1I, inset). Similar to the heterologously expressed c-Myc-BK-GFP, vacuoles isolated from human skin fibroblasts also displayed sizable endogenous BK-like currents when challenged with 100 mM Ca2+ from the cytosolic side (Figure 1J). With increasing [Ca2+]c, the voltage dependence was also negatively shifted, with the V1/2 value reduced by >100 mV from 100 nM to 10 mM free Ca2+ (Figure 1K). This shift of voltage dependence provides the mechanistic basis for BK channel activation in lysosomes; that is, BK becomes more easily activated in response to a local [Ca2+]c increase because of the opening of Ca2+ release channels (Berkefeld et al., 2010; Fakler and Adelman, 2008; Salkoff et al., 2006). Likewise, vacuoles from wild-type MEFs also exhibited BK-like currents activated by 100 mM Ca2+, which was completely blocked by the selective BK channel blocker paxilline (1 mM) (Berkefeld et al., 2010; Fakler and Adelman, 2008; Salkoff et al., 2006) and the Ca2+-sensitive current was undetectable in vacuoles isolated from BK-knockout MEFs (Figure 1L). Similar currents were also detected in vacuoles from human fibroblasts, and these currents were eliminated by the knockout of BK gene using the CRISPR/Cas9 system (Cong et al., 2013) from these cells (Figures S1B, S1C, S1E, and S1F). By using immunofluorescence staining, lysosomal patch clamping, and western blotting, endogenous BK was also detected in lysosomes from several other cell types, including neurons, beta cells (Min6), primary

Figure 1. Lysosomal Localization of Functional BK Channels (A) Co-staining of endogenous BK (Slo1) with Texas Red dextran, which labels lysosomes, in human skin fibroblast cells. (B) Presence of endogenous BK in lysosome fractions (bands 1–6) prepared by density gradient ultracentrifugation from Cos1 cells. Lamp1, a lysosomal marker; Complex II, a mitochondrial marker. (C) Colocalization of heterologously expressed c-Myc-BK-GFP with Lamp1-mCherry in Cos1 cells. (D) Presence of heterologously expressed c-Myc-BK-GFP in lysosome fractions (bands 1–6) in Cos1 cells. (E) BK immunofluorescence was detected in Lamp1-positive puncta in wild-type but not BK-knockout MEFs. (F) Topology and domain structure of human BK channel. Two dileucine motifs located in the RCK domains in the large cytoplasmic carboxy-terminus are indicated. (G) Substitution of two dileucine motifs inhibited lysosomal targeting of BK. (H) BK currents at 105 mV induced by bath (cytosolic side) application of 100 mM Ca2+ to vacuoles isolated from a Cos1 cell expressing c-Myc-BK-GFP. (I) G-V curves obtained from steady-state currents evoked by [Ca2+]c indicated in vacuoles from Cos1 cells expressing c-Myc-BK-GFP showing the shift of G-V curves to negative voltages by increasing [Ca2+]c. Data points (means ± SEM, n = 4) were fitted with Boltzmann function, and V1/2 values obtained for different [Ca2+]c are plotted in the inset. (J) Bath application of Ca2+ (100 mM) elicited robust endogenous BK-like currents in vacuole isolated from human skin fibroblasts. (K) G-V curves for steady-state endogenous BK-like currents in vacuoles from human fibroblasts showing the negative shift of the voltage dependence by increasing [Ca2+]c. Data points (means ± SEM, n = 3) were fitted with Boltzmann function, and V1/2 values obtained for different [Ca2+]c are plotted in the inset. (L) BK-like currents were induced by Ca2+ (100 mM) in vacuoles isolated from wild-type but not BK-knockout MEFs. Currents were evoked by voltage ramps. Currents measured at 105 mV were normalized to the basal value under 100 nM Ca2+. (M) Membrane potential was strongly hyperpolarized by bath application of Ca2+ (100 mM) in vacuoles isolated form Cos1 cells expressing c-Myc-BK-GFP. Paxilline and Na+ replacement eliminated the membrane potential change, suggesting that lysosomal membrane potential was regulated by K+ flux into lysosomes through BK channels. (N) Ca2+ (100 mM) induced hyperpolarization of vacuoles isolated from human fibroblasts, which was suppressed by application of paxilline.

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astrocytes, primary myoblasts, C2C12 cells, macrophages (raw264.7), and HEK293T cells (data not shown). The ion selectivity of lysosomal BK channel was investigated by replacing normal bath solution with isotonic Na+ (150 mM, containing 100 mM Ca2+), Ca2+ (105 mM), and K+ (150 mM, containing 100 mM Ca2+) solutions, respectively. Although no outward current was observed when isotonic Na+ or isotonic Ca2+ was perfused, application of isotonic K+ solution induced a large outward current in the same vacuole, suggesting that the Ca2+activated currents in the lysosome expressing c-Myc-BK-GFP are mediated solely by K+ (Figure S1G). In addition, the lysosomal BK currents from transfected Cos1 cells were selectively inhibited by the BK channel blocker paxilline (1 mM) but not the IK channel blockers TRAM-34 (10 mM) and clotrimazole (10 mM) (Wulff et al., 2001) (Figure S1H). Similarly, the endogenous BKlike currents in lysosomes from human fibroblasts also displayed high K+ selectivity (Figure S1I) and were inhibited by paxilline (1 mM) but not by TRAM34 (10 mM) or clotrimazole (10 mM) (Figure S1J). These data indicate that BK channels in the lysosome function in the same manner as their PM counterparts. BK Channels Regulate Lysosomal Membrane Potential The PM BK channels regulate the PM potential and thereby the driving force for Ca2+ entry. Lysosomal BK channels have electrophysiological properties similar to the PM BK channels and therefore may also regulate lysosomal membrane potential (Dc, Dc defined as Vcytosol Vlumen; Vlumen set at 0 mV) and in turn influence lysosomal Ca2+ efflux. To directly measure the contribution of BK channels to lysosomal membrane potential, vacuoles isolated from c-Myc-BK-GFP-expressing Cos1 cells were recorded under current-clamp mode (Cang et al., 2013, 2014). As shown in Figure 1M, the lysosomal membrane potential decreased from 11.9 ± 4.7 mV (n = 14) (cytosolic side more negative than luminal side) to 60.3 ± 3.0 mV (n = 11) in response to the application of 100 mM Ca2+. This decrease in Dc was eliminated by either paxilline (1 mM) or replacement of cytosolic K+

with Na+. Similarly, activation of endogenous BK in vacuoles isolated from human skin fibroblasts by 100 mM Ca2+ reduced lysosomal Dc from 9.8 ± 4.0 mV (n = 9) to 28.0 ± 3.9 mV (n = 8), and this response was suppressed by paxilline (Figure 1N). These results indicate that activation of either heterologous or endogenous BK in lysosomes hyperpolarizes the lysosomal membrane. BK Forms a Macromolecular Complex with ML1 to Regulate ML1-Mediated Ca2+ Release The Ca2+ sensitivity of BK and the ability of BK to further enhance the driving force for Ca2+ efflux through release channels, especially the strongly inwardly rectifying ML1 channels, suggest that the two channel types may be coupled to create a reinforcing loop for sustained lysosomal Ca2+ release. To test this hypothesis, Cos1 cells were transfected with cMyc-BK-GFP, ML1-HA, or both as indicated in Figure 2A. BK was coimmunoprecipitated with ML1, suggesting that BK and ML1 are in the same complex. Consistently, double immunofluorescence staining revealed colocalization of the two proteins in lysosomes (Figure 2B). To determine the functional interaction between ML1 and BK, we measured whole-lysosome currents elicited by the ML1 agonist mucolipin synthetic agonist 1 (ML-SA1; 10 mM), using a protocol with 300 ms step pulses to potentials from 15 to 85 mV with 20 mV increments with or without a pre-step to 135 mV (Figure 2C). The 135 mV pre-pulse served to not only reveal the ML1 currents but also to drive the Ca2+ efflux from the lysosome to induce BK activation. As shown in Figure 2C (top), the addition of ML-SA1 to lysosomes coexpressing cMyc-hSlo1-GFP and ML1-mCherry evoked robust inward currents, through ML1, during the pre-pulse, which were followed by marked outward currents upon switching to the step pulse. The outward currents were eliminated by omitting the pre-pulse (Figure 2C, middle), removing Ca2+ from the pipette solution (Figure 2C, top right), or applying paxilline (1 mM) to the bath (Figure S2), indicating that they were Ca2+ dependent and mediated

Figure 2. BK Exists in the Same Macromolecular Complex with ML1, Where It Strongly Facilitates ML1-Mediated Lysosome Ca2+ Release (A) Co-immunoprecipitation of ML1 and BK in lysosome fractions from Cos1 cells. At 24 hr after transfection, lysosomes were collected and lysed by adding 0.5% Triton X-100. Samples were immunoprecipitated with protein A/G-anti-HA and blotted with antibodies indicated. (B) Colocalization of c-Myc-BK-GFP and ML1-mCherry. (C) Functional coupling of heterologously expressed ML1 and BK channels. Top: representative currents recorded in a lysosome from a Cos1 cell expressing BK and ML1 in response to a pre-step of 135 mV followed by individual voltage steps (from 15 to 85 mV with 20 mV increments) before and during application of ML-SA1 (10 mM). The inward currents at 135 mV represent ML-SA1-induced ML1 activity, while the outward currents during the voltage steps represent summation of basal (the sustained currents) and ML1-activated (the transient currents that decay with time) BK currents. The pipette (lumen) contained 20 mM Ca2+ except for the 0 Ca2+ group. Note that no transient outward current was observed when the Ca2+ free pipette solution (0 Ca2+) was used. Middle: representative currents recorded from the same lysosome as at the top, but without the pre-step. Note the small outward basal BK currents and the lack of inward in response to ML-SA1 in the absence of the pre-step. Bottom: ML1-activated BK currents by subtracting currents recorded without the pre-step (middle) from that recorded with the 135 mV pre-step (top). (D) I-V relation of ML1-activated BK currents in lysosomes isolated from Cos1 cells expressing BK and ML1, individually or in combination. Large lysosomal BK currents were only observed with the coexpression of both BK and ML1 and when Ca2+ was present in the pipette. (E) Functional coupling of native ML1 and BK channels in human fibroblasts. Representative currents recorded in a lysosome in response to the indicated voltage protocol before and during application of ML-SA1 (10 mM) ± paxilline (1 mM). (F) ML1-activated BK currents in lysosomes isolated from human fibroblasts in the absence and presence of paxilline. (G) ML-SA1-induced GECO-ML1 responses in HEK293T cells without or with BK overexpression. (H) BK overexpression reduced GECO-ML1 response to GPN, which empties lysosomal Ca2+ stores. (I) BK overexpression did not alter GECO-ML1 response to ionomysin (1 mM). (J) Paxilline (1 mM) markedly decreased ML-SA1-induced GECO-ML1 response in HEK293T cells. (K) BK-knockout MEFs exhibited smaller GECO-ML1 response to ML-SA1 than wild-type MEFs. (L) BK knockout did not alter GECO-ML1 response to ionomysin (1 mM) in MEFs. Values are mean ± SEM. NS, not significant. *p < 0.05, **p < 0.01.



by BK. Subtraction of currents without the pre-pulse from those with the pre-pulse yielded ML-SA1-evoked BK currents, which had a current-voltage (I-V) relationship similar that elicited by Ca2+ (Figure 2D). This response was minimal when Ca2+ was omitted from the pipette solution and in lysosomes that expressed either cMyc-hSlo1-GFP or ML1-mCherry alone (Figures 2C, 2D, and S2). Furthermore, ML1-meidated BK activation were eliminated by paxilline (1 mM) application (Figure S2). Notably, the BK currents evoked by ML-SA1 quickly ran down. This is likely attributed to the fast diffusion of Ca2+ released through ML1, because BK currents induced by bath application of 100 mM Ca2+ did not show any reduction in the same lysosome (Figure S2). Importantly, functional coupling between endogenous BK and ML1 was also observed in vacuoles isolated from human skin fibroblasts (Figures 2E and 2F). Taken together, our data suggest that ML1-mediated Ca2+ release is coupled to BK activation in lysosomes. We hypothesized that K+ influx through BK may be important for maintaining the lysosomal membrane potential disrupted by the ML1-mediated loss of Ca2+ (and Na+) from the lysosomal lumen and thereby facilitating the driving force for continued Ca2+ release. To test this possibility, we measured ML1mediated lysosomal Ca2+ release using an ML1 construct that contained GECO, a single-wavelength genetically encoded Ca2+ indicator (Zhao et al., 2011), at its cytoplasmic amino terminus (Figure S3A). When transfected into HEK293T or Cos1 cells, GECO-ML1 maintained a primarily lysosomal localization that allowed preferential detection of juxta-lysosomal [Ca2+] changes at the cytosolic side, representing mainly the ML1 activity in intact cells (Figures S3B–S3H) (Shen et al., 2012). As shown in Figures 2G and 2H, ML-SA1 induced increases in GECO-ML1 fluorescence signal in cells bathed in a low Ca2+ (free Ca2+ < 10 nM) solution. Although BK overexpression led to a moderate increase in the ML-SA1-evoked fluorescence change, it did not reach statistical significance (Figure 2G). However, the lysosomal Ca2+ content, as assessed using glycyl-phenylalanine 2-naphthylamide (GPN; 200 mM), a substrate of the lysosomal exopeptidase cathepsin C that induces lysosome osmolysis to dump all Ca2+ out (Shen et al., 2012), was significantly reduced in HEK293T cells that overexpressed BK, suggesting that BK facilitated lysosomal Ca2+ release even before ML1 was stimulated by the exogenous ligand. Presumably, ML1 activation by its endogenous ligands, for example, PI(3,5)P2 (Dong et al., 2010), is required for regular cell function, and this could have already been facilitated by BK. We confirmed that GECO-ML1 expression, as estimated from the maximal fluorescence increase induced by ionomycin (1 mM, with 2 mM Ca2+ in the bath) (Shen et al., 2012), was comparable between control and BK overexpression cells (Figure 2I). Further supporting the role of BK in facilitating ML1-mediated lysosomal Ca2+ release, the GECO-ML1 response to ML-SA1 (10 mM) in HEK293T cells was markedly reduced by paxilline (1 mM) (Figure 2J), and that in MEFs was suppressed by BK knockout (Figure 2K), while the lysosomal Ca2+ content was actually increased (see Figures 3C and 3D) and GECO-ML1 expression were unaffected by the knockout of BK (Figure 2L). Altogether, these data demonstrated the physical and functional coupling between BK and ML1 channels and the importance of BK in maintaining sustained lysosomal Ca2+ release through ML1. 6 Developmental Cell 33, 1–15, May 26, 2015 ª2015 Elsevier Inc.

BK Channels Regulate Lysosomal Ca2+ Contents Reduced ML1-mediated Ca2+ release in BK deficient cells is likely to cause Ca2+ accumulation in lysosomes. To directly monitor intraluminal Ca2+ levels in the lysosomes, we loaded endolysosomes with a membrane-impermeant Ca2+ indicator, Oregon Green 488 BAPTA-5N (OG-BAPTA-5N), and a Ca2+insensitive probe, Texas Red-conjugated dextran, through endocytosis, and compared fluorescence intensities between green and red signals. Because OG-BAPTA-5N has only a negligible sensitivity to pH, its green fluorescence intensity is indicative of luminal Ca2+ levels in lysosomes (Lelouvier and Puertollano, 2011; Lo´pez-Sanjurjo et al., 2013). As shown in Figures 3A and 3B, although dye loading was comparable among different groups (Figure S4A), paxilline treatment (3 mM, 5 hr) caused a significant increase in OG-BAPTA-5N signal in human fibroblasts, suggesting that BK inhibition suppressed lysosomal Ca2+ release and thereby causing Ca2+ accumulation in lysosomes. This effect was due to lysosomal BK channels because inhibiting BK on the PM with a membrane-impermeable BK channel blocker, tetraethylammonium (TEA; 5 mM), did not mimic the effect of paxilline (Figures 3A and 3B). Interestingly, the paxilline-induced lysosomal Ca2+ accumulation was rescued by the co-treatment with ML-SA1 (10 mM, 5 hr) (Figures 3A and 3B), indicating that paxilline acted by suppressing ML1-mediated lysosomal Ca2+ release. The role of BK in lysosomal Ca2+ handling was further studied by comparing lysosomal Ca2+ contents between wild-type and BK-knockout MEFs. The BK deficient MEFs displayed markedly higher OG-BAPTA-5N fluorescence than wild-type cells (Figures 3C and 3D), and this was rescued by overexpression of either BK or ML1, but not Lamp1, in the BK-knockout MEFs (Figures 3C and 3E). The dye loading was comparable between wild-type and BK-knockout MEFs (Figures S4B and S4C). ML1 deficiency also causes lysosomal Ca2+ accumulation (Wong et al., 2012). If BK controls lysosome Ca2+ release exclusively through ML1, then BK overexpression should not be able to rescue lysosomal Ca2+ accumulation in ML1 / cells. Indeed, we observed an increase in lysosomal Ca2+ contents in ML1 / human fibroblasts (ML4) compared with wild-type cells, which was not rescued by BK overexpression (Figures 3F and 3G; see Figure S4D for dye loading controls). Altogether, our results indicate that BK regulates lysosomal Ca2+ homeostasis by modulating ML1-mediated Ca2+ release from lysosomes. BK Channels Regulate Lysosomal Membrane Trafficking Lysosomal Ca2+ release is essential for lysosomal membrane trafficking (Lloyd-Evans and Platt, 2011; Luzio et al., 2007; Morgan et al., 2011; Pittman, 2011; Pryor et al., 2000). The above results suggest that by affecting Ca2+ homeostasis, BK may also play a key role in regulating lysosomal membrane trafficking. As shown in Figures 4A and 4B, treatment with paxilline, but not TEA, markedly increased lysosomal size in Cos1 cells, and the effect was rescued by co-treatment with ML-SA1 (Figures 4A and 4B) or overexpression of ML1, but not Lamp1 or ML1DD/KK, a non-conducting pore mutation (Dong et al., 2008) (Figures 4C and 4D). Enlarged lysosomes were also consistently observed by electron microscopy in human fibroblasts treated with paxilline, but not TEA, and ML-SA1 again rescued this effect


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Figure 3. BK Deficiency Causes Lysosomal Ca2+ Accumulation, which Can Be Corrected by Upregulating ML1 (A and B) Paxilline (3 mM, 5 hr) but not TEA (5 mM, 5 hr) treatment increased lysosomal Ca2+ levels in human fibroblasts, as indicated by the enhanced OG-BAPTA5N staining in dextran-positive puncta. The paxilline-induced increase was abolished by ML-SA1. In (B), 52 cells were analyzed for each condition. (C–E) BK-knockout MEFs exhibited higher lysosomal Ca2+ levels than wild-type MEFs, and this effect was alleviated by overexpressing ML1 or BK, but not Lamp1. In (D) and (E), 44 cells were analyzed for each condition. (F and G) Lysosomal Ca2+ accumulation in ML4 cells was not rescued by BK overexpression. Fifty-three cells were analyzed for each condition. Values are mean ± SEM. NS, not significant. *p < 0.05, **p < 0.01.

(Figures 4E and 4F). Furthermore, we detected lysosome enlargement in BK-knockout MEFs, which was also rescued by ML-SA1 (Figures 4G and 4H). Interestingly, in both paxilline-treated fibroblasts and BK-deficient MEFs, the enlarged lysosomes displayed the buildup of membranous and electrondense inclusions, closely resembling those found in patients with LSDs (Lloyd-Evans and Platt, 2011). As in most LSDs, Lamp1 expression was upregulated in BK-deficient MEFs (Figure S5). Overall, these results confirmed the importance of endogenous BK channels in membrane trafficking through regulating ML1 activity. BK deficiency results in defective membrane trafficking, leading to abnormal lysosomal storage.

Impaired lysosomal trafficking and accumulation of lysosomal contents often result in the formation of autofluorescent lipofuscin, as seen in ML1 / and NPC1 / cells (Dong et al., 2008; Shen et al., 2012). Not surprisingly, dramatically increased lipofuscin fluorescence was also observed in BK-knockout MEFs compared with wild-type controls (Figures 4I and 4J). Importantly, the increase of lipofuscin in BK-knockout MEFs was eliminated by treatment with ML-SA1 or overexpressing ML1 or BK, but not overexpressing ML-DD/KK or Lamp1. These data suggest that the loss of BK channels is sufficient to induce lysosomal storage. Consistently, paxilline (3 mM overnight) also induced lipofuscin accumulation in human fibroblasts, which was rescued Developmental Cell 33, 1–15, May 26, 2015 ª2015 Elsevier Inc. 7


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Figure 4. BK Deficiency Impairs Lysosome Trafficking and Causes Lysosome Storage (A and B) Paxilline (3 mM, 3 hr), but not TEA (5 mM, 3 hr), caused lysosome enlargement in Cos1 cells, which was prevented by ML-SA1 (10 mM). Lysosomes were labeled with dextran Texas Red. (C and D) Paxilline-induced lysosome enlargement was ablated by overexpressing ML1, but not ML1-DD/KK or Lamp1, in Cos1 cells. The images were from a single representative experiment, and the experiments were repeated three times. n = 225 for (B), and n = 250 for (D). (E and F) TEM images of enlarged lysosomes in paxilline-treated human fibroblasts and inhibition of this effect by ML-SA1. Percentage of lysosomes with diameters < 0.5 mm or in a range of 0.5 to 1 mm per TEM field was quantified from ten randomly selected fields each time, with each sample counted at least three times from two independently prepared sample grids. (legend continued on next page)



by overexpressing ML1, but not Lamp1 (Figure S6). On the other hand, the lipofuscin accumulation in ML4 cells was not rescued by BK overexpression (Figures 4K and 4L). Collectively, these data demonstrate that BK deficiency impairs lysosomal function in an ML1-dependent manner. Upregulation of BK Channels Rescued NPC1–/– Phenotypes Membrane trafficking defects are common in LSDs (Kiselyov et al., 2010; Lloyd-Evans and Platt, 2011; Morgan et al., 2011; Samie and Xu, 2014; Vitner et al., 2010). Fibroblasts from NPC1 patients exhibited defective lysosome Ca2+ release and impaired lysosome membrane trafficking (Kiselyov et al., 2010; Lloyd-Evans et al., 2008; Shen et al., 2012; Vitner et al., 2010) characterized by cholesterol and lipofuscin accumulation in lysosomes (Shen et al., 2012). Previously, these defects were shown to be alleviated by stimulating ML1 function with ML-SA1 (Shen et al., 2012). We reasoned that because BK facilitates ML1mediated lysosome Ca2+ release, enhancing BK function might also help rescue the trafficking defects of NPC1 / cells. As expected, NPC1 / cells displayed markedly increased cholesterol, as indicated by filipin staining, compared with wild-type human fibroblasts (Lloyd-Evans et al., 2008; Shen et al., 2012), and this was significantly decreased by the overexpression of BK, but not Lamp1 (Figures 5A and 5B). In addition, the rescue effect of BK on filipin signal was abolished by coexpressing ML1-DD/KK, which acts as a dominant-negative (DN) form of ML1 (Yamaguchi et al., 2011; Zeevi et al., 2010), indicating that BK overexpression prevented cholesterol accumulation in NPC1 / cells through facilitating ML1. Consistent with the findings on cholesterol, BK overexpression also prevented the abnormal lipofuscin accumulation in NPC1 / cells (Shen et al., 2012), and the rescue effect of BK was suppressed by the coexpression of ML1-DD/KK (Figures 5C and 5D). Collectively, these data support the notion that enhancing BK expression or function represents a plausible approach to correct membrane trafficking defects and reduce cholesterol and lipofuscin accumulation in NPC1 / cells. To confirm that the beneficial effect of BK overexpression indeed occurred through increasing ML1 function in NPC1 / cells, we measured ML1-mediated Ca2+ release using GECOML1. As shown previously (Lloyd-Evans et al., 2008; Shen et al., 2012), the ML-SA1-evoked Ca2+ release, indicated by the GECO signal, was strongly reduced in NPC1 / fibroblasts compared with wild-type fibroblasts (Figure 5E). BK overexpression in NPC1 / cells partially rescued this defect (Figure 5E) without significantly affecting the lysosomal Ca2+ content (Figure 5F) and GECO-ML1 expression (Figure 5G). Altogether, our results suggest that upregulating BK promotes ML1-mediated Ca2+ release in NPC1 / fibroblasts, through which it corrects the lysosome trafficking and storage defects in these cells.

BK Channel Inhibition Eliminated the ML1 Rescue Effect on NPC1–/– Phenotypes ML1 activation has been shown to alleviate defects in NPC1 / fibroblasts (Shen et al., 2012). Given the importance of BK in sustaining ML1-mediated lysosomal Ca2+ release, it was quite plausible that the rescue by ML1 activation also required BK function. Indeed, inhibiting all BK by paxilline, but not just the PM BK by TEA, eliminated the rescue effect by either ML-SA1 (10 mM) or ML1 overexpression on both cholesterol (seen by filipin signals; Figures 6A–6D) and lipofuscin (autofluorescence; Figures 6E–6G) accumulation in NPC1 / fibroblasts. Therefore, BK is essential for correcting the defective lysosome Ca2+ release and membrane trafficking in NPC1 disease by ML1 upregulation. BK and ML1 Rescue NPC1–/– Phenotypes by Promoting Lysosomal Exocytosis Lipofuscin represents non-degradable materials accumulated in lysosomes. Therefore, the rescue of lipofuscin by BK or ML1 activation cannot be explained by enhancing lysosomal degradation. Recent studies have suggested that lysosome fusion with the PM (lysosomal exocytosis) can release lysosomal storage. Increasing lysosomal exocytosis has been suggested to be a promising therapeutic approach for LSDs (Chen et al., 2014; Medina et al., 2011; Samie and Xu, 2014; Shen et al., 2012). The importance of ML1 for lysosomal exocytosis has been suggested (Cheng et al., 2014; LaPlante et al., 2006; Medina et al., 2011; Samie et al., 2013), and hence the rescue effects on lipofuscin accumulation in NPC1 / fibroblasts seen with ML1 and/or BK upregulation could be due to increased lysosomal exocytosis. To investigate this possibility, BK was coexpressed with a DN form of synaptotagmin-VII (Syt-VII-DN) (Reddy et al., 2001; Samie et al., 2013), which blocks lysosomal exocytosis. As shown in Figures 7A and 7B, Syt-VII-DN prevented the rescue effect of BK overexpression on lipofuscin accumulation in NPC1 / fibroblasts. Likewise, Syt-VII-DN also abolished the effect of ML-SA1 (10 mM) stimulation and ML1 overexpression on eliminating lipofuscin in these cells (Figures 7C and 7D). These results support the idea that BK and ML1 upregulation promotes lipofuscin clearance from NPC1 / fibroblasts by enhancing lysosomal exocytosis. To directly evaluate lysosomal exocytosis, we measured the level of lysosomal enzyme b-hexosaminidase in the culture media. BK overexpression in NPC1 / fibroblasts significantly increased the release of b-hexosaminidase, suggesting an elevation of lysosomal exocytosis, and this was eliminated by the coexpression with Syt-VII-DN (Figure 7E). ML1 overexpression (Figure 7E) and stimulating ML1 with ML-SA1 (10 mM) (Figure 7G) also significantly increased lysosomal exocytosis in NPC1 / fibroblasts, and both effects were eliminated by SytVII-DN overexpression. The above treatments could result in cell death, which could in turn lead to lysosomal b-hexosaminidase release into the culture media. To exclude this possibility, cell death was monitored by measuring the levels of lactate

(G and H) TEM images of enlarged lysosomes and lysosomal storage in BK-knockout but not wild-type MEFs. ML-SA1 partially rescued lysosomal enlargement in BK-knockout MEFs. (I and J) Lysosomal accumulation of lipofuscin in BK-knockout MEFs and its suppression by ML-SA1 treatment or overexpression of BK or ML1, but not Lamp1 or ML1-DD/KK. Fifty-five cells were analyzed for each condition. (K and L) BK overexpression did not rescue abnormal lipofuscin accumulation in ML1 / human fibroblasts. More than 45 cells were analyzed for each condition. Values are mean ± SEM. NS, not significant. *p < 0.05, **p < 0.01.



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Figure 5. BK Upregulation Reduces Cholesterol and Lipofuscin Accumulation in NPC1–/– Cells in a Manner that Requires ML1 Activity (A and B) Accumulation of unesterified cholesterol (filipin staining) in NPC1 / human fibroblasts and its reduction by overexpressing BK, but not Lamp1. The effect of BK on filipin signals was eliminated by coexpressing ML1-DD/KK. More than 38 cells were analyzed for each condition. (C and D) Abnormal accumulation of lipofuscin (detected by autofluorescence) in NPC1 / human fibroblasts and its rescue by overexpressing BK, but not Lamp1. Coexpression of ML1-DD/KK reversed the effect of BK upregulation. More than 52 cells were analyzed for each condition. (E) NPC1 / human fibroblasts exhibited compromised GECO-ML1 response to ML-SA1, which was reversed by BK overexpression. (F and G) BK overexpression did not alter GECO-ML1 responses to GPN (F) or ionomycin (G), suggesting no change in lysosomal Ca2+ content or GECO-ML1 expression level, respectively. Values are mean ± SEM. NS, not significant. *p < 0.05, **p < 0.01.



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Figure 6. BK Activity Is Required for ML1 to Rescue the Abnormal Cholesterol and Lipofuscin Accumulation in NPC1–/– Cells (A and B) ML-SA1 (10 mM, 16 hr) abolished filipin staining in NPC1 / human fibroblasts and its inhibition by paxilline (3 mM, 16 hr), but not TEA (5 mM, 16 hr). More than 62 cells were analyzed for each condition. (C and D) ML1 overexpression reduced filipin staining in NPC1 / human fibroblasts and its inhibition by paxilline (3 mM, 16 hr), but not TEA (5 mM, 16 hr). More than 55 cells were analyzed for each condition. (E–G) Abnormal lipofuscin accumulation in NPC1 / human fibroblasts was rescued by ML-SA1 treatment and ML1 overexpression, both of which were inhibited by paxilline. More than 41 cells were analyzed for each condition. Values are mean ± SEM. NS, not significant. *p < 0.05, **p < 0.01.



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Figure 7. BK and ML1 Rescue the Abnormal Lipofuscin Accumulation in NPC1–/– Cells by Enhancing Lysosomal Exocytosis

(A and B) Coexpression of Syt-VII-DN, which inhibits lysosomal exocytosis suppressed the rescue effect of BK overexpression on abnormal lipofuscin accumulation in NPC1 / human fibroblasts. More than 32 cells were analyzed for each condition. (C and D) Coexpression of Syt-VII-DN suppressed the rescue effect of ML-SA1 (10 mM) and ML1 overexpression on abnormal lipofuscin accumulaC G tion in NPC1 / human fibroblasts. More than 34 cells were analyzed for each condition. (E) Lysosomal exocytosis was indicated by the elevation of b-hexosaminidase in cell culture supernatant. BK overexpression increased the release of b-hexosaminidase in the culture media, indicative of enhanced lysosomal exocytosis, and this effect was prevented by the coexpression of Syt-VII-DN or treatment with ML-SI1, an ML1 channel inhibitor. ML1 overexpression also increased b-hexosaminidase release, and this was inhibited by Syt-VII-DN coexpression or paxilline E F H treatment. (F) LDH activities in the culture media under conditions indicated were comparable. Values are mean ± SD from three independent experiments with triplicates. (G) ML-SA1 treatment increased b-hexosaminidase release into culture media, which was suppressed by paxilline (1 mM) or Syt-VII-DN expression. (H) LDH activities in the culture media under conditions indicated were similar. The data represent mean ± SEM from three experiments, each perI formed in triplicates. (I) A diagram illustrating the role of BK in ML1mediated lysosomal Ca2+ release and a potential therapeutic strategy for NPC1 disease. ML1 and BK form a macromolecular complex in the lysosome. (a) Under normal conditions, ML1 activation depolarizes lysosomal membrane (Dc, defined as Vcytosol Vlumen; Vlumen set at 0 mV) due to massive Ca2+ (and Na+) efflux. This would retard subsequent Ca2+ (and Na+) release without a counter-ion to compensate the disruption of electrical gradient. Lysosomal BK, which is activated by the very Ca2+ released from lysosomes through ML1, provides a counter-ion shunt to dissipate the trans-membrane potential generated by Ca2+ (Na+) release. Therefore, ML1 and BK form a positive feedback loop to ensure efficient Ca2+ release for membrane trafficking. (b) BK deficiency breaks the positive feedback cycle, leading to a reduction in ML1-mediated Ca2+ release and the subsequent lysosomal storage. (c) Boosting ML1 activity increases lysosomal Ca2+ release and thus rescues lysosomal storage in BK deficient cells. (d) Decreased ML1 function results in lysosomal storage in NPC1 / cells. (e) Increasing BK activity facilitates ML1-mediated Ca2+ release by strengthening the positive feedback, which corrects the trafficking defects and lysosome storage in NPC1 / cells. Therefore, activation of BK may provide a therapeutic approach for NPC1 and many other disease-bearing defects in lysosomal Ca2+ release and membrane trafficking.

dehydrogenase (LDH) in the culture media (Cao et al., 2014). Neither overexpression of BK or ML1 nor ML-SA1 treatment induced significant increase in the LDH level. Taken together, our data suggest that upregulation of either BK or ML1 rescues abnormal substance accumulation in NPC1 / fibroblasts via promoting lysosomal exocytosis. DISCUSSION ML1 has been well established to play important roles in mediating lysosomal Ca2+ release and in turn regulating lysosomal 12 Developmental Cell 33, 1–15, May 26, 2015 ª2015 Elsevier Inc.

membrane trafficking. We show here that BK channels form a macromolecular complex with ML1 in lysosomes in which BK is activated by ML1-mediated Ca2+ release, and such activity provides the counter cation flux to maintain the negative membrane potential needed for continued Ca2+ release from lysosomes through ML1. Therefore, a positive feedback loop is formed between ML1 and BK to ensure sufficient Ca2+ release to support lysosomal membrane trafficking, the deficiency of which has been linked to LSDs. Supporting the importance of ML1-BK coupling in lysosomal functions, either ML1 or BK deficiency resulted in lysosomal Ca2+ accumulation, defective


lysosomal membrane trafficking, and lysosomal storage. We further show that upregulation of either ML1 or BK corrected the impaired lysosomal Ca2+ release and lysosomal membrane trafficking in NPC1 / human fibroblasts. Therefore, in certain types of LSDs, such as NPC, NPA, NPB, and mild cases of ML4 (Chen et al., 2014; Dong et al., 2008; Shen et al., 2012), when decreased ML1 function is a main contributor of the disease phenotype, upregulating BK channels may help strengthen this positive feedback loop and alleviate the disease phenotype (Figure 7I). Our study has strong and broad therapeutic implications. Defects in lysosomal Ca2+ release and membrane trafficking have been associated with not only many types of LSDs (Chen et al., 2014; Dong et al., 2008; Kiselyov et al., 2010; Lloyd-Evans et al., 2008; Lloyd-Evans and Platt, 2011; Medina et al., 2011; Samie and Xu, 2014; Shen et al., 2012) but also classic forms of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (Bae et al., 2014; Coen et al., 2012; Funk and Kuret, 2012; Jeyakumar et al., 2005; Neefjes and van der Kant, 2014; Pan et al., 2008; Wang et al., 2013; Zhang et al., 2009). In addition, ML1 has also been implicated in large-particle phagocytosis (Samie et al., 2013) and muscular dystrophy (Cheng et al., 2014). Given the importance of lysosomal BK in facilitating ML-mediated lysosomal Ca2+ release, our data also suggest that lysosomal BK channels may play important roles in these conditions. Therefore, upregulating lysosomal BK function may be a good therapeutic strategy for not only LSDs but also some common neurodegenerative diseases as well as large particle phagocytosis and muscular dystrophy. EXPERIMENTAL PROCEDURES Lysosomal Electrophysiology Lysosomal electrophysiology was performed in isolated enlarged late endosome/lysosome vacuoles using a modified patch-clamp method, as described previously (Dong et al., 2008). Briefly, cells were treated for >2 hr with 1 mM vacuolin-1, a lipophilic polycyclic triazine that can selectively increase the size of endosomes and lysosomes. Large vacuoles/lysosomes were observed in most vacuolin-1-treated cells. Whole-lysosome recordings were performed on manually isolated enlarged vacuoles. In brief, a patch pipette was pressed against a cell and quickly pulled away to slice the cell membrane. This allowed enlarged lysosomes to be released into the recording chamber and identified by monitoring enhanced GFP fluorescence. After the formation of a gigaseal between the patch pipette and an enlarged lysosome, capacitance transients were compensated. Voltage steps of several hundred millivolts with millisecond duration(s) were then applied to break the patched membrane and establish the whole-lysosome configuration. Unless otherwise stated, bath (cytoplasmic) solution contained 140 mM K-gluconate, 4 mM NaCl, 1 mM EGTA, 2 mM MgCl2, 0.39 mM CaCl2, and 20 mM HEPES (pH was adjusted with KOH to 7.2; free [Ca2+] was 100 nM). For determination of channel activation at defined free [Ca2+], the amount of CaCl2 required was calculated using the Maxchelator software (http://maxchelator.stanford.edu). The pipette (luminal) solution was a standard extracellular solution (modified Tyrode’s: 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose; pH was adjusted with HCl to 4.6). Data were collected using an Axopatch 2A patch-clamp amplifier, Digidata 1440, and pClamp 10.2 software (Axon Instruments). Whole-lysosome currents were digitized at 10 kHz and filtered at 2 kHz. All experiments were conducted at room temperature (21 C–23 C), and all recordings were analyzed with pClamp 10.2 and Origin 8.0 (OriginLab). Liquid junction potential was corrected. BK currents were evoked by either a ramp protocol (from 125 to 125 mV within 2 s, Vholding = 0 mV), or voltage steps (from 95 to 215 mV in 15-mV in-

crements, Vholding = 80 mV) unless indicated in the text. Conductance-voltage (G-V) curves were generated from steady-state currents evoked by voltage steps, fitted to a Boltzmann equation: G/Gmax = 1/(1 + exp[(V V1/2)/k]), where G is the conductance of the channel, Gmax is the maximal G, V is the holding potential, and V1/2 is the voltage for half-maximal activation. Lysosomal membrane potential was measured as previously reported (Cang et al., 2013, 2014). Briefly, after the formation of whole-lysosome configuration, recording mode was switched from voltage clamp to current clamp. Lysosomal membrane potential was continuously recorded while bath solutions containing different chemicals were applied. Lysosomal [Ca2+] Measurement Lysosomal Ca2+ was evaluated on the basis of an Oregon Green 488 BAPTA5N and Texas Red 10 kD-conjugated dextran fluorescence assay. Fibroblasts were loaded with membrane-impermeant Oregon Green 488 BAPTA-5N (a pH-insensitive Ca2+ indicator; 10 mM) and Texas Red 10 kD-conjugated dextran (Ca2+ insensitive; 1 mg/ml) for 2 hr and chased for an hour with indicated chemicals to allow lysosomal accumulation of dyes through endocytosis. The fluorescence intensity of Oregon Green 488 BAPTA-5N is indicative of luminal Ca2+ concentration in lysosomes. The fluorescence intensity of dextran is used as a dye loading control that is indicative of comparable endocytosis ability of cells. GECO Ca2+ Imaging At 20 to 30 hr after transfection with GECO-ML1, HEK293T cells, human fibroblasts, or MEFs were trypsinized and plated onto glass coverslips. Most experiments were carried out within 0.5 to 2 hr after plating, when cells still exhibited a round morphology. The fluorescence intensity at 470 nm (F470) was monitored using the EasyRatioPro system. Lysosomal Ca2+ release was measured under a ‘‘low’’ external Ca2+ solution, which contained 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA, and 20 mM HEPES (pH 7.4). Ca2+ concentration in the nominally free Ca2+ solution is estimated to be 1 to 10 mM. With 1 mM EGTA, the free Ca2+ concentration is estimated to be

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