Membrane topology of XXXX -sensitive two-pore channels and their regulation by N-linked glycosylation.

@entity1 @entity15293 @entity55217 @entity141 @entity16688
Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the @entity141 -mobilizing messenger, @entity16688 ( @entity16688 ). However, their membrane topology is unknown. Using fluorescence protease protection assays, we show that @entity1 @entity15293 and @entity55217 possess cytosolic N and C termini and therefore an even number of transmembrane regions. Fluorophores placed at position 225 or 347 in @entity15293 , or 339 in @entity55217 were also cytosolic, whereas a fluorophore at position 628 in @entity15293 was luminal. These data together with sequence similarity to voltage-gated @entity141 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 @entity55217 prior to putative pore 1 are luminal. Both @entity15293 and @entity55217 are N-glycosylated with residues 599, 611, and 616 contributing to glycosylation of @entity15293 . 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 @entity15293 enhances @entity16688 -evoked cytosolic @entity141 signals. Our data establish essential features of the topology of two-pore channels.
