Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes*

Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca2+ selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

• Publication year

  2010

• Venue

  Journal of Biological Chemistry

• Publication date

  2010-05-21

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.C110.143123PMID 20495006PMCID PMC2898409
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• TPCs: Endolysosomal channels for Ca2+ mobilization from acidic organelles triggered by NAADP

  2010cited by this paper
• NAADP mobilizes calcium from acidic organelles through two-pore channels

  2009cited by this paper
• The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores

  2009cited by this paper
• The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

  2008cited by this paper
• The small chemical vacuolin‐1 alters the morphology of lysosomes without inhibiting Ca2+‐regulated exocytosis

  2005cited by this paper
• Loss of the chloride channel ClC‐7 leads to lysosomal storage disease and neurodegeneration

  2005cited by this paper
• The small chemical vacuolin‐1 inhibits Ca2+‐dependent lysosomal exocytosis but not cell resealing

  2004cited by this paper
• Outer Pore Architecture of a Ca2+-selective TRP Channel*

  2004cited by this paper
• Molecular Determinants of Permeation through the Cation Channel TRPV4*

  2002cited by this paper
• Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.

  2001cited by this paper
• Preparation of microsomes with calcium.

  1978cited by this paper
• [6] Preparation of microsomes with calcium

  1978cited by this paper

• Circulating miR-4532 is associated with loss of ambulation in dysferlinopathy

  2026cites this paper
• TPCs: From plant to human.

  2025cites this paper
• Lysosomal Ion Channels and Transporters: Recent Findings, Therapeutic Potential, and Technical Approaches

  2025cites this paper
• Two-pore channel regulators - Who is in control?

  2025cites this paper
• Lysosomal TPC2 channel as a new target of chlorpromazine and Clomipramine to induce protective autophagy in L-BMAA-induced neurodegeneration.

  2025cites this paper
• Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes

  2024cites this paper
• Using automated patch clamp electrophysiology platforms in ion channel drug discovery: an industry perspective

  2024cites this paper
• Convergent activation of Ca2+ permeability in two-pore channel 2 through distinct molecular routes

  2023cites this paper
• Pharmacological inhibition of lysosomal two-pore channel 2 (TPC2) confers neuroprotection in stroke via autophagy regulation.

  2023influential citation
• Characterization of Endo-Lysosomal Cation Channels Using Calcium Imaging.

  2023cites this paper
• Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling.

  2023cites this paper
• Enhancement of lysosome biogenesis as a potential therapeutic approach for neurodegenerative diseases

  2023cites this paper
• Two-pore channels: going with the flows

  2022cites this paper
• Lysosomal Ion Channels: What Are They Good For and Are They Druggable Targets?

  2022cites this paper
• Diversity of two-pore channels and the accessory NAADP receptors in intracellular Ca2+ signaling.

  2022cites this paper
• Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters

  2022cites this paper
• Proton-gated anion transport governs macropinosome shrinkage

  2022cites this paper
• Lysosomal potassium channels.

  2022cites this paper
• Neurophysiological functions and pharmacological tools of acidic and non-acidic Ca2+ stores.

  2022cites this paper
• NAADP-Mediated Ca2+ Signalling.

  2022cites this paper
• Activation of endo-lysosomal two-pore channels by NAADP and PI(3,5)P2. Five things to know.

  2022cites this paper
• NAADP-Dependent TPC Current.

  2022cites this paper
• Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account

  2022influential citation
• Endolysosomal cation channels point the way towards precision medicine of cancer and infectious diseases.

  2022cites this paper
• NAADP: From Discovery to Mechanism

  2021cites this paper
• Membrane Transport Proteins in Osteoclasts: The Ins and Outs

  2021cites this paper
• Endolysosomal Ca2+ signaling in cardiovascular health and disease.

  2021cites this paper
• Two-pore and TRPML cation channels: Regulators of phagocytosis, autophagy and lysosomal exocytosis.

  2020cites this paper
• Automated patch clamp in drug discovery: major breakthroughs and innovation in the last decade

  2020cites this paper
• Lsm12 is an NAADP receptor and a two-pore channel regulatory protein required for calcium mobilization from acidic organelles

  2020cites this paper
• Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function

  2020cites this paper
• Gene fusions characterize a subset of uterine cellular leiomyomas

  2020cites this paper
• Discovery of lipophilic two‐pore channel agonists

  2020cites this paper
• Pyridine Nucleotide Metabolites and Calcium Release from Intracellular Stores.

  2019cites this paper
• Readily Releasable Stores of Calcium in Neuronal Endolysosomes: Physiological and Pathophysiological Relevance.

  2019cites this paper
• Functional characterization of intracellular ion channels with the endolysosomal patch-clamp technique

  2019cites this paper
• ApoE4 pathology in Alzheimer's disease from the perspective of organelle dysfunction in astrocytes

  2019cites this paper
• Involvement of organelles and inter-organellar signaling in the pathogenesis of HIV-1 associated neurocognitive disorder and Alzheimer's disease.

  2019cites this paper
• NAADP Receptors.

  2019cites this paper
• 5-Azido-8-ethynyl-NAADP: A bifunctional, clickable photoaffinity probe for the identification of NAADP receptors.

  2019cites this paper
• Cell-wide web of cytoplasmic nanocourses coordinates calcium signalling

  2018cites this paper
• Endolysosomal Ca2+ Signalling and Cancer Hallmarks: Two-Pore Channels on the Move, TRPML1 Lags Behind!

  2018cites this paper
• On the structure and mechanism of two‐pore channels

  2018cites this paper
• mTORC1 controls lysosomal Ca2+ release through the two-pore channel TPC2

  2018cites this paper
• The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin

  2017cites this paper
• Exome-wide association study identifies four novel loci for systemic lupus erythematosus in Han Chinese population

  2017cites this paper
• Investigating ion dyshomeostasis in Niemann-Pick diseasetype C, both in vitro and in vivo

  2017cites this paper
• On the Structure and Mechanism of Two-Pore Channels

  2017cites this paper
• The human two-pore channel 1 is modulated by cytosolic and luminal calcium

  2017cites this paper
• Podocyte Dedifferentiation and Glomerular Injury Mediated by Lysosome Dysfunction: Role of Acid Ceramidase

  2017cites this paper
• Identification of epigenetic interactions between miRNA and DNA methylation associated with gene expression as potential prognostic markers in bladder cancer

  2017cites this paper
• Tuning the ion selectivity of two-pore channels

  2017cites this paper
• Two-Pore Channel Function Is Crucial for the Migration of Invasive Cancer Cells.

  2017cites this paper
• High resolution structural evidence suggests the Sarcoplasmic Reticulum forms microdomains with Acidic Stores (lysosomes) in the heart

  2017cites this paper
• Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions

  2017cites this paper
• Nanojunctions of the Sarcoplasmic Reticulum Deliver Site- and Function-Specific Calcium Signaling in Vascular Smooth Muscles.

  2017cites this paper
• Identification of Sidt2 as a lysosomal cation‐conducting protein

  2017cites this paper
• Methods for monitoring Ca2+ and ion channels in the lysosome.

  2017influential citation
• Genetic Variants of TPCN2 Associated with Type 2 Diabetes Risk in the Chinese Population

  2016influential citation
• Two-pore Channels Enter the Atomic Era: Structure of Plant TPC Revealed

  2016cites this paper
• The roles of sphingosine in calcium signaling and Niemann-Pick disease type C

  2016cites this paper
• Deviant lysosomal Ca2+ signalling in neurodegeneration. An introduction.

  2016cites this paper
• Lysosomal Calcium in Neurodegeneration.

  2016cites this paper
• Exploring the biophysical evidence that mammalian two‐pore channels are NAADP‐activated calcium‐permeable channels

  2016influential citation
• Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells.

  2016cites this paper
• Modulation of Calcium Entry by the Endo-lysosomal System.

  2016cites this paper
• Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

  2016cites this paper
• A primer of NAADP-mediated Ca(2+) signalling: From sea urchin eggs to mammalian cells.

  2015cites this paper
• Reconstitution of lysosomal ion channels into artificial membranes.

  2015cites this paper
• Two-pore channels at the intersection of endolysosomal membrane traffic.

  2015cites this paper
• Function and dysfunction of two-pore channels

  2015cites this paper
• Poring over two-pore channel pore mutants.

  2015influential citation
• Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

  2015influential citation
• TPC: the NAADP discovery channel?

  2015cites this paper
• Departure gate of acidic Ca2+ confirmed

  2015cites this paper
• Mechanism and role of calcium in excitation-mucus secretion coupling in human colonic epithelium

  2015cites this paper
• Coupling acidic organelles with the ER through Ca²⁺ microdomains at membrane contact sites.

  2015cites this paper
• Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion

  2015cites this paper
• Nanopore-Based Methods for Characterizing Single Proteins.

  2015cites this paper
• Evolution of acidic Ca²⁺ stores and their resident Ca²⁺-permeable channels.

  2015cites this paper
• Funktionelle Charakterisierung des Two-Pore-Loop-Kanals 2 (TPC2) im endolysosomalen System

  2014influential citation
• Two-pore channels function in calcium regulation in sea star oocytes and embryos

  2014cites this paper
• A Non-inactivating High-voltage-activated Two-Pore Na+ Channel that Supports Ultra-long Action Potentials and Membrane Bistability

  2014cites this paper
• The voltage-gated sodium channel TPC1 confers endolysosomal excitability.

  2014cites this paper
• Identification of a Novel Gene for Diabetic Traits in Rats, Mice, and Humans

  2014cites this paper
• NAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells.

  2014cites this paper
• High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

  2014cites this paper
• Convergent regulation of the lysosomal two‐pore channel‐2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases

  2014cites this paper
• A computational model of lysosome–ER Ca2+ microdomains

  2014influential citation
• A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

  2014cites this paper
• Reconstituted Human TPC1 Is a Proton-Permeable Ion Channel and Is Activated by NAADP or Ca2+

  2014cites this paper
• Organelle-specific Subunit Interactions of the Vertebrate Two-pore Channel Family*

  2014cites this paper
• The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation

  2014influential citation
• TPC1 Has Two Variant Isoforms, and Their Removal Has Different Effects on Endo-Lysosomal Functions Compared to Loss of TPC2

  2014cites this paper
• Two-pore channels provide insight into the evolution of voltage-gated Ca2+ and Na+ channels

  2014cites this paper
• TRPML Channels and Mucolipidosis Type IV

  2014cites this paper
• Actions of NAADP and other agents in cardiac myocytes

  2014cites this paper
• Two‐pore channels (TPCs): Current controversies

  2014cites this paper
• DEV113563 1..12

  2014cites this paper
• Ca2+ releasing process and nicotinic acid adenine dinucleotide phosphate

  2013cites this paper