Photoaffinity Labeling of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Targets in Mammalian Cells
Photoaffinity Labeling of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Targets in Mammalian Cells
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DOI：
10.1074/jbc.m111.305813
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发表时间：
2012-01-20
影响因子：
4.8
通讯作者：
Marchant, Jonathan S.
中科院分区：
文献类型：
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作者：
Lin-Moshier, Yaping;Walseth, Timothy F.;Marchant, Jonathan S.
烟酸腺嘌呤二核苷酸磷酸（NAADP）是激动剂产生的第二信使，其从细胞内酸性Ca 2+库释放Ca 2+。最近的证据已经确定了内溶酶体系统内的双孔通道（TPC）作为NAADP调节的Ca 2+通道，其响应于NAADP释放细胞器Ca 2+。然而，很少有人知道耦合NAADP结合钙释放的机制。为了鉴定NAADP结合位点，我们采用了基于5-叠氮基-NAADP（[P-32- 5 N（3）]NAADP）的放射性光探针的光亲和标记方法，该探针对NAADP受体表现出高亲和力结合。在广泛用于研究NAADP诱发的Ca 2+信号传导的几个系统中，包括海胆卵、人细胞系（HEK 293、SKBR 3）和小鼠胰腺，5 N（3）-NAADP选择性标记低分子量位点，这些位点表现出NAADP敏感性Ca 2+释放的诊断药理学。令人惊讶的是，我们无法证明内源性或过表达的TPC的标记。此外，高亲和力NAADP结合位点的标记保留在TPC 1和TPC 2敲除小鼠的胰腺样品中。这些光标记数据表明，一个更大的TPC复合物内的辅助组件负责结合NAADP，这是唯一的核心通道本身。这一观察结果需要严格评估目前的模型NAADP触发激活的TPC家族。
Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca2+ from intracellular acidic Ca2+ stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca2+ channels that release organellar Ca2+ in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([P-32-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca2+ signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca2+ release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knockout mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.