A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores

Abstract

The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane's selective permeability to K⁺ We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na⁺ and H⁺, but not K⁺, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca²⁺ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K⁺-selective conductance (LysoK_VCa). LysoK_VCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoK_VCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca²⁺ stores may be refilled from endoplasmic reticulum (ER) Ca²⁺ via ER-lysosome membrane contact sites. We propose that LysoK_VCa serves as the perilysosomal Ca²⁺ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoK_VCa, or abolition of its Ca²⁺ sensitivity, blocks refilling and maintenance of lysosomal Ca²⁺ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.

Figure 1.

Ca²⁺-activated K⁺ currents were detected in lysosomes, but not in early endosomes. (A) Whole-endolysosome patch-clamp configuration. The pipette (extracellular/luminal) solution was a standard Na⁺-based external solution (Tyrode’s) adjusted to pH 4.6 to mimic the acidic environment of the lysosome lumen. The bath (internal/cytoplasmic) solution was a K⁺-based solution (140 mM K⁺) with free [Ca²⁺] ranging from 0.1 to 1,000 µM as indicated in each experiment. The arrow indicates the direction of K⁺ ion flow into the endolysosome (defined as the outward current). (B) Bath application of 100 or 1,000 µM Ca²⁺ activated outwardly rectifying currents in an enlarged vacuole from a Cos-1 cell pretreated with vacuolin-1. The currents were elicited by repeated voltage ramps (−100 to 140 mV; 400 ms; only partial voltage ranges are shown) with a 4-s interramp interval. (C) Time courses of current activation by Ca²⁺ in an enlarged vacuole isolated from a Cos-1 cell. (D and E) In isolated early endosomes that were enlarged by Rab5-Q79L overexpression (D) or vicenistatin (E), Ca²⁺ (500 µM) failed to activate measurable whole-endosome outward currents. PI(3,5)P₂ (0.5 µM) or ML-SA1 (20 µM) readily activated TPC-like (D) or ML1-like (E) currents, respectively. (F) Representative traces of Ca²⁺-activated outward currents with a pipette/luminal solution containing low [Cl⁻] (11 mM versus 156 mM in G. (G) Substitution of cytoplasmic K⁺ with Na⁺ abolished Ca²⁺-activated outward currents.

Figure 2.

SLO1 mediates Ca²⁺-activated K⁺ currents in the lysosomes of excitable and nonexcitable cells. (A) Whole-endolysosome Ca²⁺-activated outward currents in a WT MEF. (B) Whole-endolysosome Ca²⁺-activated outward currents in a KCNMA1 KO (Kcnma1^−/− or Slo1^−/−) MEF. Note the lack of background outward currents typically seen in whole-cell recordings. (C) Whole-endolysosome Ca²⁺-activated currents in a SLO1-YFP–expressing Cos-1 cell (Lyso-SLO1). (D) Summary of whole-endolysosome Ca²⁺- and voltage-activated K⁺ currents (LysoK_VCa) in a variety of cell types, including Cos-1 cells, HEK293T cells, A7r5 smooth muscle cell lines, mouse BECs, cultured mouse cortical neurons, INS-1 pancreatic cell lines, WT and KCNMA1 KO MEFs, WT and KCNMA1 KO parietal cells, and SLO1-YFP-expressing Cos-1 cells. Both individual (blue) and mean (red; ± SEM) current densities are shown for each cell type (n = 3–15 patches). (E) Subcellular fractionation analysis revealed enrichment of SLO1 proteins in organellar fractions containing Lamp-1 or Complex-II (a mitochondrial marker). Subcellular fractionations (1–9) were obtained by gradient-based ultracentrifugation. Cell lysates were included as controls (fraction 0). (F and G) Colocalization analyses of SLO1-YFP with Lamp1, MitoTracker, EEA1 (an early endosomal marker), and DAPI (a nuclear marker). Bar, 10 µm. Error bars indicate SEM.

Figure 3.

LysoK_VCa is a lysosomal large-conductance K⁺ channel that is dually activated by Ca²⁺ and voltage. (A) LysoK_VCa currents were activated by cytoplasmic Ca²⁺ ([Ca²⁺]_C; 0.1, 100, and 1,000 µM). After a 20-ms prepulse at −60 mV, currents were elicited by voltage steps from −80 to 180 mV in 20-mV increments. (B) Activation of Lyso-SLO1 by Ca²⁺ and voltage steps. (C) [Ca²⁺]_C-dependent single-channel LysoK_VCa currents from a cytoplasmic-side-out patch. The open and closed states are indicated by O and C, respectively. (D) [Ca²⁺]_C dependence of macroscopic Lyso-SLO1 currents. (E) Voltage-dependent openings of single LysoK_VCa channels at [Ca²⁺]_C of 100 µM in a cytoplasmic-side-out patch with symmetric (luminal/cytoplasmic) K⁺ (140 mM) solutions. (F) Unitary single-channel chord conductances of LysoK_VCa and Lyso-SLO1. (G) LysoK_VCa and Lyso-SLO1 currents were blocked by the BK-specific inhibitor paxilline (1 µM). (H) Quinidine (500 µM), a SLO1/SLO3 channel inhibitor, blocked LysoK_VCa and Lyso-SLO1 currents. (I) Single LysoK_VCa currents were inhibited by bath application of 100 nM IBTX to a luminal-side-out patch.

Figure 4.

LysoK_VCa is preferentially expressed in the lysosome. (A) Step currents of Lyso-SLO1 (left) and Lyso-SLO1+ β2 (right) in the presence of 100 µM Ca²⁺. (B) Localization of β2-GFP in the Lamp1-positive compartments. Bar, 10 µm. (C) Whole-cell K⁺ currents sensitive to paxilline (1 µM) in nontransfected Cos-1 cells. The pipette solution contained 100 µM Ca²⁺. (D) Densities (mean ± SEM) of plasma membrane BK-like currents vs. LysoK_VCa in Cos-1 cells and MEFs. Statistical comparisons were made with variance analysis (Student’s t test). ***, P < 0.001.

Figure 5.

Regulation of lysosomal membrane potential by lysosomal K⁺ and LysoK_VCa. (A) When the voltage or potential (ψ) of the extracellular solution is set to 0 mV by conventional definition, the ψ of the cytosol (ψ_Cytosol) is approximately −70 mV. Hence the resting membrane potential (Δψ) of the cell is approximately −70 mV (cytoplasmic-side negative). At resting conditions, ψ_Lumen is −100 mV and lysosomal Δψ (ψ_Cytosol − ψ_Lumen) is 30 mV. Upon activation of LysoK_VCa, depending on the extent of LysoK_VCa activation, lysosomal Δψ is reversed to −30 to −60 mV. (B) When ψ_Cytosol is set to 0 mV, lysosomal Δψ at rest is 30 mV. Assuming >10-fold concentration gradients across lysosomal membranes for Na⁺ and K⁺ ([Na⁺]_L >> [Na⁺]_C and [K⁺]_L << [K⁺]_C), E_Na is >57 mV and E_K is <−57 mV. Likewise, E_H is >149 mV. The resting Δψ is determined primarily by Na⁺ and H⁺ permeabilities. Upon Ca²⁺ activation of LysoK_VCa, lysosomal Δψ is changed to −30 to −60 mV. (C) Effects of K⁺ (valinomycin) and H⁺ (niclosamide) ionophores on lysosomal Δψ in a Cos-1 cell in a current-clamp vacuole. (D) Summary of lysosomal Δψ under different ionic compositions in the luminal and cytoplasmic sides. Lysosomal Δψ was 20 – 30 mV under control conditions (lumen, 145 mM Na⁺, pH 4.6; cytosol, 140 mM K⁺, pH 7.4). Replacement of luminal Na⁺ with NMDG⁺ or increasing luminal pH from 4.6 to 7.4 led to a reduction in Δψ. (E) Resting lysosomal Δψ with or without ATP in the bath/cytoplasmic solutions. (F) Current-clamp recordings of lysosomal Δψ in a Cos-1 cell. (G) [Ca²⁺]_C (100 µM) induced voltage transients in a current-clamped vacuole. Single-channel LysoK_VCa currents from the same vacuole under the voltage-clamp configuration are shown in Fig. S5 H. (H and I) Lysosomal Δψ in enlarged vacuoles from SLO1-YFP-expressing (H) and SLO1^R207Q-expressing (I) cells. LysoK_VCa currents from the same patches under the voltage-clamp configuration are shown in Fig. S5, K and L. (J) Summary of lysosomal Δψ in various conditions. In D, E, and J, means ± SEM are shown.

Figure 6.

Lysosomal K⁺ homeostasis and cytosolic Ca²⁺ increase are both required for lysosomal Ca²⁺ store refilling. (A) Lysosome-targeted genetically encoded Ca²⁺ indicator GCaMP3-ML1. (B) In HEK293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells), lysosomal Ca²⁺ release, as indicated by elevated GCaMP3 fluorescence (F₄₇₀), was induced by bath application of ML-SA1 (25 µM), a membrane-permeable ML1-specific agonist, in a 0 Ca²⁺ (<10 nM) external solution. Averaged responses of 15–30 cells in one coverslip are shown. (C) Short-term (5-min) application of valinomycin (20 µM) in the refilling phase abolished the second ML-SA1 response. (D) Effect of valinomycin on lysosome Ca²⁺ refilling. (E and F) Effect of BAPTA-AM treatment on recovery of lysosomal Ca²⁺ content upon ML-SA1 treatment in ML1-expressing HEK293 cells that were loaded with OG-BAPTA dextran dyes. Statistical comparisons were made with variance analysis (Student’s t test). **, P < 0.01. Error bars indicate SEM.

Figure 7.

LysoK_VCa and its Ca²⁺ sensitivity regulate the refilling of lysosomal Ca²⁺ stores. (A and B) Acute application of paxilline (A) and quinidine (B) abolished the second ML-SA1-induced responses. Prolonged washout for 10–15 min led to a partial recovery of the responses. (C) Lysosome Ca²⁺ refilling in HEK-GCaMP3-ML1 cells treated with paxilline, quinidine, and IBTX. (D) Compared with WT MEFs, ML-SA1–stimulated refilled responses were reduced in GCaMP7-ML1-expressing KCNMA1 KO MEFs. (E) Lyso-SLO1^M513I/D898A currents at different concentrations of Ca²⁺ (0.1 and 10 µM). (F and G) Lysosomal refilling in GCaMP7-ML1–expressing KCNMA1 KO MEFs that were transfected WT SLO-mCherry and SLO1^M513I/D898A-mCherry. (H) GPN-induced refilled (the second) Ca²⁺ response, measured with Fura-2 imaging, was reduced in KCNMA1 KO MEFs. (I) Paxilline effects in lysosomal refilling in WT MEF cells. (J) Mean refilling responses in WT and KCNMA1 KO MEF cells. Statistical comparisons were made with variance analysis (Student’s t test). **, P < 0.01; ***, P < 0.001. Error bars indicate SEM.

Figure 8.

LysoK_VCa is required for the maintenance of lysosomal Ca²⁺ contents. (A and B) Effects of paxilline on ML-SA1–induced changes in lysosomal Ca²⁺ contents in WT and KCNMA1 KO MEFs. (C) Effects of overexpressing WT SLO-mCherry and SLO1^M513I/D898A-mCherry on lysosomal Ca²⁺ contents in HEK293 cells. (D) Effects of pretreatment with paxilline on lysosomal Ca²⁺contents in WT and KCNMA1 KO MEFs. Statistical comparisons were made with variance analysis (Student’s t test). **, P < 0.01; ***, P < 0.001; N.S., not significant. Error bars indicate SEM.

Figure 9.

LysoK_VCa is required for lysosome function. (A) Western blot analysis of Lamp1 expression in WT and KCNMA1 KO MEFs. (B) LysoTracker staining in WT and KCNMA1 KO MEFs. Bar, 10 µm. (C) Confocal imaging of DQ-red-BSA in starved Cos-1 cells (amino acid + serum starvation) in the presence of paxilline (10 µM), quinidine (500 µM), or IBTX (100 nM). Bar, 10 µm. (D) Normalized proteolytic index for starved Cos-1 cells treated with paxilline or quinidine. (E) Normalized proteolytic index for starved WT and KCNMA1 KO MEFs treated with paxilline. (F) Cholesterol levels, detected with filipin staining, in WT, KCNMA1 KO, and NPC1 KO MEFs. Bar, 50 µm. (G) Normalized filipin density in WT, KCNMA1 KO, and NPC1 KO MEFs. Means ± SEM are shown in A, D, E, and G. Statistical comparisons were made using variance analysis (ANOVA for D, E, and G). *, P < 0.05; **, P < 0.01.

Figure 10.

A working model of the potential roles of LysoK_VCa-mediated K⁺ flux in lysosomal dynamics and Ca²⁺ store refilling. Endolysosomes have a positive Δψ (Fig. 5, A and B) at rest. Upon juxta-lysosomal Ca²⁺ increase, LysoKVCa-mediated K⁺ flux causes reduction and reversal of Δψ toward E_K (−57 mV, assuming a 10-fold concentration gradient). Changes in lysosomal Δψ may initiate the refilling of lysosomal Ca²⁺ stores via yet-to-be-defined mechanisms, such as formation of ER–lysosome membrane contact sites.