Sitsapesan John Parrington, Antony Galione and Rebecca Gosain

Molecular Biophysics: TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+ Samantha J. Pitt, Tim M. Funnell, Mano Sitsapesan, Elisa Venturi, Katja Rietdorf, Margarida Ruas, A. Ganesan, Rajendra Gosain, Grant C. Churchill, Michael X. Zhu, Downloaded from John Parrington, Antony Galione and Rebecca Sitsapesan J. Biol. Chem. 2010, 285:35039-35046. doi: 10.1074/jbc.M110.156927 originally published online August 18, 2010 http://www.jbc.org/ Access the most updated version of this article at doi: 10.1074/jbc.M110.156927 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. at Univ of St Andrews on December 16, 2013 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts Supplemental material: http://www.jbc.org/content/suppl/2010/09/03/M110.156927.DC1.html This article cites 46 references, 21 of which can be accessed free at http://www.jbc.org/content/285/45/35039.full.html#ref-list-1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 45, pp. 35039–35046, November 5, 2010 Author’s Choice © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. ,TPC2 Is a Novel NAADP-sensitive Ca2؉ Release Channel ؉ Operating as a Dual Sensor of Luminal pH and Ca2 *□S Received for publication, June 22, 2010, and in revised form, August 17, 2010 Published, JBC Papers in Press, August 18, 2010, DOI 10.1074/jbc.M110.156927 Samantha J. Pitt‡1, Tim M. Funnell§1, Mano Sitsapesan‡, Elisa Venturi‡, Katja Rietdorf§, Margarida Ruas§, A. Ganesan¶, Rajendra Gosain¶, Grant C. Churchill§, Michael X. Zhuʈ, John Parrington§, Antony Galione§2, and Rebecca Sitsapesan‡3 From the ‡School of Physiology and Pharmacology, Medical Sciences Building, and Center for Nanoscience and Quantum Information, University of Bristol, Bristol BS8 1TD, United Kingdom, the §Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom, the ¶School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom, and the ʈDepartment of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas 77030 Nicotinic acid adenine dinucleotide phosphate (NAADP) is Nicotinic acid adenine dinucleotide phosphate (NAADP)4 is a molecule capable of initiating the release of intracellular the most potent Ca2ϩ-releasing second messenger yet identi- Downloaded from Ca2؉ required for many essential cellular processes. Recent fied (1–4). Although originally discovered in sea urchin eggs, it ϩ evidence links two-pore channels (TPCs) with NAADP-in- is becoming increasing clear that NAADP-induced Ca2 duced release of Ca2؉ from lysosome-like acidic organelles; release is crucial to many physiological processes in a diverse however, there has been no direct demonstration that TPCs range of mammalian cell types (5, 6). Many distinctive features ϩ ϩ 2 2 http://www.jbc.org/ can act as NAADP-sensitive Ca2؉ release channels. Contro- of NAADP-induced Ca release indicate that the Ca release versial evidence also proposes ryanodine receptors as the pri- channels involved are functionally and structurally different 2ϩ mary target of NAADP. We show that TPC2, the major lyso- from the well characterized Ca release channels found on somal targeted isoform, is a cation channel with selectivity for sarcoplasmic/endoplasmic reticulum stores (ryanodine recep- ؉ ؉ Ca2 that will enable it to act as a Ca2 release channel in the tors (RyRs) and inositol trisphosphate receptors (IP3Rs) (7, 8)). 2ϩ cellular environment. NAADP opens TPC2 channels in a con- NAADP triggers Ca release from acidic stores, but the iden- at Univ of St Andrews on December 16, 2013 tity of the Ca2ϩ release channel involved has remained a mys- centration-dependent manner, binding to high affinity acti- tery (7). New evidence has shown that NAADP binds with high vation and low affinity inhibition sites. At the core of this affinity to TPC protein complex (9), which is located on acidic, process is the luminal environment of the channel. The sen- ϩ lysosome-like Ca2 stores (10, 11). A recent report also dem- sitivity of TPC2 to NAADP is steeply dependent on the lumi- 2؉ onstrates that activation of cell surface muscarinic receptors nal [Ca ] allowing extremely low levels of NAADP to open requires TPC2 expression to couple Ca2ϩ release from acidic the channel. In parallel, luminal pH controls NAADP affinity stores in mouse bladder smooth muscle (12). The importance for TPC2 by switching from reversible activation of TPC2 at of TPC2 to Ca2ϩ movements across lysosomal membranes was low pH to irreversible activation at neutral pH. Further evi- recently shown in whole lysosome recordings. Mutation of dence earmarking TPCs as the likely pathway for NAADP- TPC2 affected the magnitude of whole lysosomal Ca2ϩ cur- 2؉ induced intracellular Ca release is obtained from the use of rents, highlighting the need to investigate the conductance and Ned-19, the selective blocker of cellular NAADP-induced gating properties of TPC2 at the single-channel level (13). ؉ Ca2 release. Ned-19 antagonizes NAADP-activation of TPCs are thus named because their close sequence homology TPC2 in a non-competitive manner at 1 ␮M but potentiates with voltage-gated cation channels predicts a monomeric NAADP activation at nanomolar concentrations. This single- structure containing two pore loops (7, 14). It is proposed that channel study provides a long awaited molecular basis for the two homodimers form the pore (10, 15, 16); however, it is not peculiar mechanistic features of NAADP signaling and a known whether TPC2 can actually function as an ion channel ϩ framework for understanding how NAADP can mediate key with all the properties necessary to fulfill the role of a Ca2 physiological events. release channel activated by NAADP. Many characteristics of NAADP-induced Ca2ϩ release distinguish it from Ca2ϩ release triggered by other signaling molecules, and these indicate un- usual underlying mechanisms of ion channel function. For the first time, we describe here the fundamental single- * This work was supported by the British Heart Foundation (to R. S.) and the Wellcome Trust (to A. G. and J. P.). channel gating and conductance properties of purified human Author’s Choice—Final version full access. TPC2 and demonstrate that TPC2 exhibits many unique func- □ S The on-line version of this article (available at http://www.jbc.org) contains tional characteristics that can explain the idiosyncrasies of cel- supplemental text and references and Figs. 1–3. 1 Both authors contributed equally to this work. 2 To whom correspondence may be addressed. Fax: 44-1865-271-853; E-mail: [email protected]. 4 The abbreviations used are: NAADP, nicotinic acid adenine dinucleotide 3 To whom correspondence may be addressed. Fax: 44-117-331-2288; E-mail: phosphate; TPC, two-pore channel; RyR, ryanodine receptor; IP3R, inositol [email protected]. trisphosphate receptor; IP, immunoprecipitation; pS, picosiemens. NOVEMBER 5, 2010•VOLUME 285•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 35039 TPC2 Is an NAADP-sensitive Ca2؉ Release Channel lular NAADP signaling characteristics, whereas cardiac (RyR2) Pfalz Germany). Measurements of single-channel current and skeletal (RyR1) RyRs do not. A preliminary report of these amplitudes were made using the single-channel analysis soft- data has been presented in abstract form (17). ware WinEDR version 2.5.9 (University of Strathclyde, Glas- gow, UK). The closed and open current levels were assessed MATERIALS AND METHODS manually using cursors. Amplitude histograms were obtained Purification of Human, Recombinant TPC2—cDNA for hTPC2 from single-channel data using Clampfit version 10.2 (Molecu- (GenBankTM accession number AY029200) was cloned from lar Devices, Sunnyvale, CA) and fitted with Gaussian curves. In HEK293 cells by rapid amplification of cDNA ends-PCR us- all records, amplitude histograms were best fit with a single ing primers designed against the expressed sequence tag Gaussian function representing the main conductance level AA309878 (10). Overexpressing HA-HsTPC2 HEK293 cells (300 pS; see Fig. 2b). Subconductance state gating was too brief were harvested and centrifuged at 2000 rpm for 5 min at 4 °C. to be detected by this method of analysis. The channel open The cell pellet was resuspended in hypotonic buffer (20 mM probability (Po) was determined over 3 min of continuous re- HEPES, 1 mM EGTA, pH 7.2) and kept for 60 min on ice before cording, using the method of 50% threshold analysis (19). Con- homogenization. The lysate was centrifuged for 5 min at centration-response curves were fit using a four-parameter 2000 ϫ g at 4 °C, and the supernatant was centrifuged for1hat logistical equation in GraphPad Prism (GraphPad Software 100,000 ϫ g at 4 °C. The membrane pellet was resuspended in Inc). The Ca2ϩ/Kϩ permeability ratio (PCa2ϩ/PKϩ) was calcu- M M IP buffer (150 m NaCl, 20 m HEPES, pH 7.4) and kept on ice lated using the equation given by Fatt and Ginsborg (1958) (20). Downloaded from for 1 h, after which insoluble material was pelleted by centrifu- 2ϩ ϩ ϭ ͓ ϩ͔ ϫ ͓ 2ϩ͔ ϫ ͑ ͒ gation at 14,000 ϫ g for 30 min at 4 °C. The supernatant was PCa /PK K /4 Ca exp ErevF/RT precleared by adding non-immune rabbit serum coupled ϫ ͓exp͑ErevF/RT͒ ϩ 1͔ (Eq. 1) covalently to protein-A-Sepharose for1hat4°C.Thepre- cleared supernatant was incubated overnight at 4 °C with either The value of RT/F used in our calculations was 25.4 mV based http://www.jbc.org/ non-immune rabbit serum (control) or polyclonal anti-TPC2 on the recording temperature of 22 °C. The zero current antibody (10), both covalently coupled to protein-A-Sepharose reversal potential (Erev) was obtained under bi-ionic condi- beads.

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