Because these findings are consistent with studies in yeast showing that the Ca 2+/H + exchangers establish the vacuolar Ca 2+ gradient ( Morgan et al., 2011), this 'pH hypothesis' has been widely accepted ( Calcraft et al., 2009 Christensen et al., 2002 Lloyd-Evans et al., 2008 Morgan et al., 2011 Shen et al., 2012). These findings have been interpreted to mean that the proton gradient in the lysosome is responsible for actively driving Ca 2+ into the lysosome via an unidentified H +-dependent Ca 2+ transporter ( Morgan et al., 2011). In various cell types, when the lysosomal pH gradient is dissipated, either by inhibiting the V-ATPase or by alkalizing reagents such as NH 4Cl, free luminal Ly was found to drop drastically ( Calcraft et al., 2009 Christensen et al., 2002 Dickson et al., 2012 Lloyd-Evans et al., 2008 Shen et al., 2012), with no or very small concomitant increase in cytosolic Ca 2+ ( Christensen et al., 2002 Dickson et al., 2012). However, most Ca 2+ taken up through endocytosis is lost quickly during the initial course of endosomal acidification prior to reaching lysosomes during endosome maturation ( Gerasimenko et al., 1998). The endocytic pathway may theoretically deliver extracellular Ca 2+ to lysosomes. However, depletion of lysosomal Ca 2+ stores does not induce extracellular Ca 2+ entry ( Haller et al., 1996b). Upon store depletion, the luminal sensor protein STIM1 oligomerizes to activate the highly Ca 2+-selective ORAI/CRAC channels on the plasma membrane, refilling the ER Ca 2+ store via the SERCA pump ( Clapham, 2007 Lewis, 2007 Berridge, 2012). The most well understood Ca 2+ store in the cell is the ER. How the 5000-fold Ca 2+ concentration gradient across the lysosomal membrane is established and maintained is poorly understood. Whereas human mutations of TRPML1 cause type IV Mucolipidosis, pathogenic inhibition of ML1 underlies several other LSDs ( Shen et al., 2012). Consistently, the principal Ca 2+ channel in the lysosome, Mucolipin TRP channel 1 (TRPML1 or ML1), as well as lysosomal Ca 2+ sensors such as the C2 domain–containing synaptotagmin VII, are also required for these functions ( Steen et al., 2007 Lewis, 2007 Kinnear et al., 2004). Using the fast Ca 2+ chelator BAPTA, Ca 2+ release from the lysosome has been shown to be required for late endosome-lysosome fusion ( Pryor et al., 2000), lysosomal exocytosis, phagocytosis, membrane repair, and signal transduction ( Reddy et al., 2001 Lewis, 2007 Kinnear et al., 2004). A reduction in Ly is believed to be the primary pathogenic cause for some LSDs and common neurodegenerative diseases ( Lloyd-Evans et al., 2008 Coen et al., 2012). Like the Endoplasmic Reticulum (ER) ( Clapham, 2007 Berridge, 2012), lysosomes are also intracellular Ca 2+ stores with free Ly ~0.4–0.6 mM ( Christensen et al., 2002 Lloyd-Evans et al., 2008), which is 3–4 orders of magnitude higher than the cytosolic (~100 nM). By closely apposing each other, the ER may serve as a direct and primary source of Ca 2+for the lysosome.Ī vacuolar-type H +-ATPase (V-ATPase) on the membrane of the lysosome maintains the acidic lumen (pH Ly ~ 4.6), and improper acidification of lysosomes may lead to lysosomal storage diseases (LSDs) ( Mindell, 2012). Furthermore, reducing ER Ca 2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca 2+ refilling of lysosomes, but not in cells lacking IP3Rs. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca 2+ prevented lysosomal Ca 2+ stores from refilling. In contrast to the prevailing view that lysosomal acidification drives Ca 2+ into the lysosome, inhibiting the V-ATPase H + pump did not prevent Ca 2+ refilling. We developed a physiological assay to monitor lysosomal Ca 2+ store refilling using specific activators of lysosomal Ca 2+ channels to repeatedly induce lysosomal Ca 2+ release. Impaired homeostasis of lysosomal Ca 2+ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca 2+ are not known.
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