NDLI: Membrane Topology of NAADP-sensitive Two-pore Channels and Their Regulation by N-linked Glycosylation*

Content Provider	PubMed Central
Author	Hooper, Robert Churamani, Dev Brailoiu, Eugen Taylor, Colin W. Patel, Sandip
Abstract	Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the Ca2+-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 Ca2+ 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 Ca2+ signals. Our data establish essential features of the topology of two-pore channels.
Related Links	http://dx.doi.org/10.1074/jbc.m110.189985
Ending Page	9149
Page Count	9
Starting Page	9141
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	11
Volume Number	286
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2011-03-18
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Membrane Topology of NAADP-sensitive Two-pore Channels and Their Regulation by N-linked Glycosylation

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