title: Both RyRs and TPCs are required for NAADP-induced intracellular Ca²⁺ release.
abstract: Intracellular Ca(2+) release is mostly mediated by inositol trisphosphate, but intracellular cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are important messengers in many systems. Whereas cADPR generally activates type 2 ryanodine receptors (RyR2s), the NAADP-activated Ca(2+) release mechanism is less clear. Using knockouts and antibodies against RyRs and Two-Pore Channels (TPCs), we have compared their relative importance for NAADP-induced Ca(2+) release from two-photon permeabilized pancreatic acinar cells. In these cells, cholecystokinin-elicited Ca(2+) release is mediated by NAADP. TPC2-KO reduced NAADP-induced Ca(2+) release by 64%, but the combination of TPC2-KO and an antibody against TPC1, significantly reduced Ca(2+) release by 86% (64% vs. 86%, p<0.0002). In RyR3-KO, NAADP-evoked Ca(2+) release reduced by ∼50% but, when combined with antibodies against RyR1, responses were 90% inhibited. Antibodies against RyR2 had practically no effect on NAADP-evoked Ca(2+) release, but reduced release in response to cADPR by 55%. Antibodies to RyR1 inhibited NAADP-induced Ca(2+) liberation by 81%, but only reduced cADPR responses by 30%. We conclude that full NAADP-mediated Ca(2+) release requires both TPCs and RyRs. The sequence of relative importance for NAADP-elicited Ca(2+) release from the all stores is RyR1>TPC2>RyR3>TPC1>>RyR2. However, when assessing NAADP-induced Ca(2+) release solely from the acidic stores (granules/endosomes/lysosomes), antibodies against TPC2 and TPC1 virtually abolished the Ca(2+) liberation as did antibodies against RyR1 and RyR3. Our results indicate that the primary, but very small, NAADP-elicited Ca(2+) release via TPCs from endosomes/lysosomes triggers the detectable Ca(2+)-induced Ca(2+) release via RyR1 and RyR3 occurring from the granules and the ER.
authors.affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.
authors.forename: Julia V
authors.initials: JV
authors.lastname: Gerasimenko
authors.affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Charlesworth
authors.affiliation: Laboratory for Developmental Neurobiology, Riken Brain Science Institute, Wako City, Saitama, Japan.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Sherwood
authors.affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.
authors.forename: Pawel E
authors.initials: PE
authors.lastname: Ferdek
authors.affiliation: Laboratory for Developmental Neurobiology, Riken Brain Science Institute, Wako City, Saitama, Japan; Ca(2+) Oscillation Project, ICORP-SORST, JST, Wako City, Saitama, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mikoshiba
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
authors.forename: John
authors.initials: J
authors.lastname: Parrington
authors.affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.
authors.forename: Ole H
authors.initials: OH
authors.lastname: Petersen
authors.affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK. Electronic address: gerasimenkoov@cardiff.ac.uk.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Gerasimenko
mesh_terms: D061354:Acinar Cells / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D020013:Calcium Signaling; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D046790:Pancreas, Exocrine / Q000378:metabolism* / Q000648:ultrastructure; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D000097903:Two-Pore Channels
chemical_list: D002118:Calcium; D015220:Calcium Channels; D009249:NADP; D019837:Ryanodine Receptor Calcium Release Channel; C546784:TPCN1 protein, mouse; D000097903:Two-Pore Channels; C546785:TPCN2 protein, mouse; C024376:NAADP
keywords: Acidic store; Pancreatic acinar cells; RyR3 knockouts; TPC2 knockouts; cADPR