TPC2 is a novel NAADP-sensitive Ca-release channel, operating as a dual sensor of luminal pH and Ca

School of Physiology and Pharmacology, Medical Sciences Building, and Center for Nanoscience and Quantum Information, University of Bristol, Bristol BS8 1TD, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2010; 285(45):35039-46. DOI: 10.1074/jbc.M110.156927
Source: PubMed

ABSTRACT Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca(2+) required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca(2+) from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca(2+) release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca(2+) that will enable it to act as a Ca(2+) release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca(2+)] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca(2+) release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca(2+) release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

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Available from: Elisa Venturi, Feb 21, 2014
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    • "Loss of function of this channel causes mucolipidosis type IV, a disease in which lysosomal pH is increased and the cellular pathology includes defective autophagy and prominent autolysosome accumulations (Vergarajauregui et al., 2008; Curcio-Morelli et al., 2010). A second lysosomal Ca 2+ channel, two pore segment channel 2 (TPC2), may also regulate lysosomal pH via a nicotinic acid adenine dinucleotide phosphate (NAADP)-dependent Ca 2+ –pH feedback mechanism, whereby calcium regulation and acidification of the lysosome are intimately linked through the activity of this two-pore outwardly rectifying channel (Pitt et al., 2010). Additionally, the lysosomal-specific inwardly rectifying voltage-dependent chloride channel (CLC)-7 has been identified as potentially providing the negative counter-ion flux necessary to maintain lysosomal pH, although there is some disagreement as to the overall magnitude of the contribution of CLC-7 and chloride in this process (Graves et al., 2008; Pressey et al., 2010). "
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    ABSTRACT: Autophagy is a lysosomal degradative process which recycles cellular waste and eliminates potentially toxic damaged organelles and protein aggregates. The important cytoprotective functions of autophagy are demonstrated by the diverse pathogenic consequences that may stem from autophagy dysregulation in a growing number of neurodegenerative disorders. In many of the diseases associated with autophagy anomalies, it is the final stage of autophagy-lysosomal degradation that is disrupted. In several disorders, including Alzheimer's disease (AD), defective lysosomal acidification contributes to this proteolytic failure. The complex regulation of lysosomal pH makes this process vulnerable to disruption by many factors, and reliable lysosomal pH measurements have become increasingly important in investigations of disease mechanisms. Although various reagents for pH quantification have been developed over several decades, they are not all equally well suited for measuring the pH of lysosomes. Here, we evaluate the most commonly used pH probes for sensitivity and localisation, and identify LysoSensor yellow/blue-dextran, among currently used probes, as having the optimal profile of properties for measuring lysosomal pH. In addition, we review evidence that lysosomal acidification is defective in AD and extend our original findings, of elevated lysosomal pH in presenilin 1 (PS1)-deficient blastocysts and neurons, to additional cell models of PS1 and PS1/2 deficiency, to fibroblasts from AD patients with PS1 mutations, and to neurons in the PS/APP mouse model of AD.
    European Journal of Neuroscience 06/2013; 37(12):1949-61. DOI:10.1111/ejn.12169 · 3.18 Impact Factor
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    • "Moreover, TPCs (or complexes containing TPCs) exhibited many of the properties associated with NAADP receptors such as NAADP binding (Calcraft et al., 2009; Ruas et al., 2010; Walseth et al., 2012a), a bell-shaped NAADP concentration-response curve (Calcraft et al., 2009; Pitt et al., 2010; Zong et al., 2009), sensitivity to the NAADP antagonist, Ned-19 (Brailoiu et al., 2010b; Pitt et al., 2010) as well as coupling to ER amplification via IP 3 Rs/RyRs (Brailoiu et al., 2009; Brailoiu et al., 2010b; Calcraft et al., 2009; Ogunbayo et al., 2011; Ruas et al., 2010; Zong et al., 2009). That TPCs were pore-forming channels permeable to Ca 2+ (or a Ca 2+ surrogate) was further evidenced by electrophysiological recordings of TPCs in in artificial bilayers (Pitt et al., 2010; Rybalchenko et al., 2012), planar patch clamp (Schieder et al., 2010) or patch clamp of TPCs re-targeted to the plasma membrane (Brailoiu et al., 2010b). "
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    ABSTRACT: The pyridine nucleotide second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), is widely accepted to release Ca2+ from acidic Ca2+-storing organelles (e.g., endo-lysosomes, lysosome-related organelles, exocytotic vesicles) (Guse, 2012; Morgan et al., 2011; Zhu et al., 2010) but the identity of its molecular target channel has been a hotly debated topic and two recent papers in Cell (Cang et al., 2013; Wang et al., 2012) suggest that this discussion shows little sign of abating. This commentary aims to briefly discuss the background issues and to place the latest data in context.
    01/2013; 2(2). DOI:10.1166/msr.2013.1026
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    • "With an average membrane capacitance of less than 0.4 pF, enlarged lysosomes are not suited to the detection of transporter-mediated currents, which have typical current densities of 10–20 pA pF −1 (Hille, 2001). Incorporation into liposomes or artificial membranes, as done for TPCN2 (Pitt et al. 2010), requires the purification of sufficient amounts of recombinant protein and lacks control of the protein orientation within the membrane. Finally, by manipulation of N-terminal sorting signals in CLC-6 (Neagoe et al. 2010) and CLC-7 (Leisle et al. 2011), these endosomal CLC proteins were partially targeted to the plasma membrane and became accessible to patch-clamp experiments. "
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    ABSTRACT: Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride–proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl(-)/1H(+) exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7(E245A) is non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells.
    The Journal of Physiology 05/2012; 590(Pt 15):3421-30. DOI:10.1113/jphysiol.2012.230227 · 5.04 Impact Factor
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