JCB_201612123 1715..1730

The precise delivery of hydrolases and cargoes to lysosomes for degradation and the timely removal of lysosomal catabolites require the establishment of luminal ionic homeostasis, ionic membrane gradients, and a membrane potential (Δψ; Morgan et al., 2011; Mindell, 2012; Xu and Ren, 2015). The lysosomal membrane maintains 1,000to 5,000-fold concentration gradients for H+ and Ca2+ (Xu and Ren, 2015). It has been established that lysosomal H+ homeostasis is required for hydrolase activation (Mindell, 2012) and that lysosomal Ca2+ efflux mediates signals integral to lysosomal membrane trafficking; however, the lysosomal effectors on which Ca2+ acts are largely unknown (Kiselyov et al., 2010; Shen et al., 2012). Several specific ion-dependent channels/transporters have been identified in lysosomes, including the V-ATPase H+ pump and transient receptor potential mucolipin channels (TRP MLs), the principle Ca2+ release channels in the lysosome (Medina et al., 2015; Wang et al., 2015; Xu and Ren, 2015). H+ channels and Ca2+ transporters in the lysosomes, however, remain to be molecularly identified (Xu and Ren, 2015; Garrity et al., 2016). Much less is understood about the roles of Na+ and K+ in lysosomal physiology. Although manipulations of lysosomal Na+ and K+ with ionophores can affect several lysosomal functions (Morgan et al., 2011), it was not recognized until recently that, based on ionic composition analysis of isolated lysosomes, there may exist large concentration gradients (>10-fold) across lysosomal membranes for both ions ([Na]Lumen >> [Na]Cytosol, ENa > 57 mV; [K]L << [K]C, EK < −57 mV; Wang et al., 2012; Xu and Ren, 2015). With these ionic concentration gradients, and based on knowledge extrapolated from plasma membrane studies, resting lysosomal Δψ has been expected to be set by the membrane’s relative permeability to K+ over Na+ ions (PK/PNa; Cang et al., 2015; Xu and Ren, 2015). Lysosomal Δψ, considered essential for lysosome function (Mindell, 2012), is thought to be negative (VLumen = 30 mV; VCytosol is defined to 0 mV; Δψ = VCytosol − VLumen = −VLumen = ∼−30 mV), which is suggestive of high PK/PNa in rest conditions. Considering that lysosomal H+ permeability may also contribute a positive value to lysosomal Δψ, background PK at resting conditions is expected to be even higher to maintain a negative lysosomal Δψ. Note that depending on the ratio of [Cl]L versus [Cl]C, background PCl would also contribute a small positive or negative value to lysosomal Δψ (Mindell, 2012). Recently, we identified two-pore channels (TPCs) as the major Na+-selective channels in the lysosome that affect Δψ (Wang et al., 2012). Although still controversial, TPCs may also mediate lysosomal Ca2+ or H+ conductance (Patel, 2015). The 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 (LysoKVCa). LysoKVCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoKVCa 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 LysoKVCa serves as the perilysosomal Ca2+ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoKVCa, or abolition of its Ca2+ sensitivity, blocks refilling and maintenance of lysosomal Ca2+ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype. A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores