title: Characterization of two-pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated lysosomes.
abstract: Two-pore channels (TPCNs) have been proposed to form lysosomal Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.
authors.affiliation: Department Pharmazie, Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, D-81377 München, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schieder
authors.forename: Katrin
authors.initials: K
authors.lastname: Rötzer
authors.forename: Andrea
authors.initials: A
authors.lastname: Brüggemann
authors.forename: Martin
authors.initials: M
authors.lastname: Biel
authors.forename: Christian A
authors.initials: CA
authors.lastname: Wahl-Schott
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry*; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D020013:Calcium Signaling; D002460:Cell Line; D004594:Electrophysiology / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D015220:Calcium Channels; C530224:TPCN2 protein, human; C546785:TPCN2 protein, mouse; D049452:Green Fluorescent Proteins; D001619:beta-N-Acetylhexosaminidases; D002118:Calcium