title: Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation.
abstract: Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the Ca(2+)-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, their membrane topology is unknown. Using fluorescence protease protection assays, we show that human TPC1 and TPC2 possess cytosolic N and C termini and therefore an even number of transmembrane regions. Fluorophores placed at position 225 or 347 in TPC1, or 339 in TPC2 were also cytosolic, whereas a fluorophore at position 628 in TPC1 was luminal. These data together with sequence similarity to voltage-gated Ca(2+) and Na(+) channels, and unbiased in silico predictions are consistent with a topology in which two homologous domains are present, each comprising 6 transmembrane regions and a re-entrant pore loop. Immunocytochemical analysis of selectively permeabilized cells using antipeptide antibodies confirmed that the C-terminal tails of recombinant TPCs are cytosolic and that residues 240-254 of TPC2 prior to putative pore 1 are luminal. Both TPC1 and TPC2 are N-glycosylated with residues 599, 611, and 616 contributing to glycosylation of TPC1. This confirms the luminal position of these residues, which immediately precede the putative pore loop of the second domain. Mutation of all three glycosylation sites in TPC1 enhances NAADP-evoked cytosolic Ca(2+) signals. Our data establish essential features of the topology of two-pore channels.
authors.affiliation: Department of Cell and Developmental Biology, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Hooper
authors.forename: Dev
authors.initials: D
authors.lastname: Churamani
authors.forename: Eugen
authors.initials: E
authors.lastname: Brailoiu
authors.forename: Colin W
authors.initials: CW
authors.lastname: Taylor
authors.forename: Sandip
authors.initials: S
authors.lastname: Patel
mesh_terms: D020013:Calcium Signaling / Q000502:physiology*; D045744:Cell Line, Tumor; D005260:Female; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D009154:Mutation; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D046188:Protein Modification, Translational / Q000502:physiology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D050052:TRPC Cation Channels; C491859:TRPC2 protein, human; C096351:transient receptor potential cation channel, subfamily C, member 1; D009249:NADP; C024376:NAADP