title: Expression of Ca²⁺-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.
abstract: The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Margarida
authors: M
authors: Ruas
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Lianne C
authors: LC
authors: Davis
authors: Center for Integrated Protein Science CIPS-M and Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany.
authors: Cheng-Chang
authors: CC
authors: Chen
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Anthony J
authors: AJ
authors: Morgan
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Kai-Ting
authors: KT
authors: Chuang
authors: Pharmacology Department, University of Minnesota, Minneapolis, MN, USA.
authors: Timothy F
authors: TF
authors: Walseth
authors: Center for Integrated Protein Science CIPS-M and Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany.
authors: Christian
authors: C
authors: Grimm
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Clive
authors: C
authors: Garnham
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Trevor
authors: T
authors: Powell
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Nick
authors: N
authors: Platt
authors: Department of Pharmacology, University of Oxford, Oxford, UK.
authors: Frances M
authors: FM
authors: Platt
authors: Center for Integrated Protein Science CIPS-M and Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany.
authors: Martin
authors: M
authors: Biel
authors: Center for Integrated Protein Science CIPS-M and Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany.
authors: Christian
authors: C
authors: Wahl-Schott
authors: Department of Pharmacology, University of Oxford, Oxford, UK john.parrington@pharm.ox.ac.uk antony.galione@pharm.ox.ac.uk.
authors: John
authors: J
authors: Parrington
authors: Department of Pharmacology, University of Oxford, Oxford, UK john.parrington@pharm.ox.ac.uk antony.galione@pharm.ox.ac.uk.
authors: Antony
authors: A
authors: Galione
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D005071:Evoked Potentials / Q000187:drug effects; D015870:Gene Expression / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000097903:Two-Pore Channels
chemical_list: D002118:Calcium; D015220:Calcium Channels; D009249:NADP; D020033:Protein Isoforms; C546784:TPCN1 protein, mouse; D000097903:Two-Pore Channels; C546785:TPCN2 protein, mouse; C024376:NAADP
keywords: Ca2+; NAADP; TPC; electrophysiology; endo‐lysosome