IP3 Receptors Preferentially Associate with ER-Lysosome Contact Sites and Selectively Deliver Ca2+ to Lysosomes

摘要

class="head no_bottom_margin" id="sec1title">IntroductionIncreases in cytosolic free Ca²⁺ concentration ([Ca²⁺]c) regulate the activities of all cells, allowing them to respond to internal and extracellular signals. Most Ca²⁺ signals are evoked by opening of Ca²⁺-permeable channels within the plasma membrane (PM) or the membranes of intracellular organelles, usually the endoplasmic reticulum (ER). In non-excitable cells, extracellular stimuli typically evoke Ca²⁺ signals by stimulating phospholipase C (PLC), which catalyzes formation of inositol 1,4,5-trisphosphate (IP3). Binding of both IP3 and Ca²⁺ to IP3 receptors (IP3Rs) causes them to open and release Ca²⁺ from the ER (, ).The spatial organization of Ca²⁺ signals allows Ca²⁺ entering the cytosol through different channels to evoke different responses (). Mitochondria, for example, when juxtaposed to ER, selectively sequester Ca²⁺ released by IP3Rs, and this then regulates mitochondrial behavior (). Ca²⁺ released through IP3Rs or ryanodine receptors (RyR) adjacent to the PM regulates membrane potential by selectively activating Ca²⁺-sensitive Cl⁻ or K⁺ channels (, ). Depolarization of cardiac muscle opens voltage-gated Ca²⁺ channels, and the resulting local Ca²⁺ signals are amplified by Ca²⁺-induced Ca²⁺ release (CICR) from RyRs (). Loss of Ca²⁺ from the ER stimulates Ca²⁺ channels in the PM, and the resulting store-operated Ca²⁺ entry (SOCE) selectively regulates adenylyl cyclases, nitric oxide synthase, and nuclear factor of activated T cells (). For each of these examples, and many others, the specificity of the Ca²⁺ signal is conferred by having a channel deliver Ca²⁺ at a high local concentration to closely apposed target proteins.Lysosomes can also sequester Ca²⁺ and they express channels, including TRPML (transient receptor potential mucolipin), TPC2 (two-pore channel 2), and ATP-regulated P2X4 receptors, that allow Ca²⁺ release (). Here, too, cross-talk with the ER is important, and it is facilitated by membrane contact sites (MCSs) between lysosomes and ER, stabilized by scaffold proteins (, , , ). The cytosolic Ca²⁺ signals evoked by TRPML or TPC2 can be amplified by CICR through IP3Rs or RyRs in closely apposed ER (, , href="#bib48" rid="bib48" class=" bibr popnode">Patel et al., 2010). Conversely, Ca²⁺ released by ER channels can be rapidly sequestered by lysosomes. This sequestration attenuates cytosolic Ca²⁺ signals evoked by IP3Rs (href="#bib34" rid="bib34" class=" bibr popnode">López Sanjurjo et al., 2013) and, by loading lysosomes with Ca²⁺, primes TPC2 to respond (href="#bib45" rid="bib45" class=" bibr popnode">Morgan et al., 2013), controls fusion and fission within endolysosomal pathways (href="#bib55" rid="bib55" class=" bibr popnode">Ruas et al., 2010), and regulates autophagy and lysosomal biogenesis through calcineurin activated by TRPML-mediated Ca²⁺ release (href="#bib39" rid="bib39" class=" bibr popnode">Medina et al., 2015). Using pharmacological inhibitors that disrupt lysosomes, perturb their morphology, or block their ability to sequester H⁺, we showed previously that the increase in [Ca²⁺]c evoked by IP3Rs was exaggerated when lysosomes were disrupted, but SOCE-evoked Ca²⁺ signals were unaffected (href="#bib34" rid="bib34" class=" bibr popnode">López-Sanjurjo et al., 2013, href="#bib35" rid="bib35" class=" bibr popnode">López Sanjurjo et al., 2014). We suggested that lysosomes selectively sequester Ca²⁺ released by IP3Rs, while ignoring Ca²⁺ entering cells through SOCE.Using targeted low-affinity Ca²⁺ sensors, we now show that IP3Rs selectively deliver Ca²⁺ to lysosomes. Many long-lived contacts between ER and lysosomes are populated by small clusters of IP3Rs. Increasing lysosomal pH does not immediately prevent Ca²⁺ uptake, but it slowly causes lysosomes to enlarge, redistribute, reduce their affiliation with IP3Rs, and lose their ability to selectively sequester Ca²⁺ released by IP₃Rs. We conclude that the ER, with its IP₃Rs and high-affinity Ca²⁺ pump (SERCA, sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase), can, in “piston-like” fashion, deliver Ca²⁺ from the cytosol with its low [Ca²⁺]_c to the low-affinity uptake system of lysosomes. The involvement of IP₃Rs allows cell-surface receptors, through PLC and IP₃, to regulate this Ca²⁺ transfer and so the behavior of lysosomes.