Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation.
Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation.
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NAADP敏感的两孔通道的膜拓扑及其通过N-连接的糖基化调节。
DOI：
10.1074/jbc.m110.189985
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发表时间：
2011-03-18
期刊：
影响因子：
--
通讯作者：
Patel S
中科院分区：
文献类型：
--
作者：
Hooper R;Churamani D;Brailoiu E;Taylor CW;Patel S
双孔通道（TPC）定位于内溶酶体系统，最近出现的Ca 2+动员信使，烟酸腺嘌呤二核苷酸磷酸（NAADP）的目标。然而，它们的膜拓扑结构是未知的。使用荧光蛋白酶保护试验，我们表明，人类TPC 1和TPC 2具有胞质N和C末端，因此，偶数跨膜区。位于TPC 1中225或347位或TPC 2中339位的荧光团也是胞质的，而TPC 1中628位的荧光团是管腔的。这些数据连同与电压门控Ca 2+和Na+通道的序列相似性以及无偏的计算机模拟预测与其中存在两个同源结构域的拓扑结构一致，每个结构域包含6个跨膜区和一个凹孔环。使用抗肽抗体的选择性透化细胞的免疫细胞化学分析证实，重组TPC的C-末端尾部是胞质的，并且推定孔1之前的TPC 2的残基240-254是管腔的。TPC 1和TPC 2都是N-糖基化的，残基599、611和616有助于TPC 1的糖基化。这证实了这些残基的腔位置，其紧接在第二结构域的推定孔环之前。TPC 1中所有三个糖基化位点的突变增强NAADP诱发的胞质Ca 2+信号。我们的数据建立的拓扑结构的两个孔通道的基本特征。
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.