Zakharian Eleonora, Thyagarajan Baskaran, French Robert J, Pavlov Evgeny, Rohacs Tibor
Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey, United States of America.
PLoS One. 2009;4(4):e5404. doi: 10.1371/journal.pone.0005404. Epub 2009 Apr 30.
Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P(2)). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.
多聚磷酸盐（polyP）是一种由数十至数百个磷酸盐组成的无机聚合物，通过高能磷酸酐键相连。多聚磷酸盐形成复合物并调节包括离子通道在内的许多蛋白质的活性。在此，我们研究了多聚磷酸盐在瞬时受体电位褪黑素8（TRPM8）通道功能中的作用。使用全细胞膜片钳和荧光钙测量，我们证明外切多聚磷酸酶（scPPX1）对多聚磷酸盐的酶促分解抑制了在表达TRPM8的人胚肾细胞和F-11神经细胞中的通道活性。我们证明TRPM8通道蛋白与多聚磷酸盐相关。此外，添加scPPX1改变了电压依赖性并阻断了重构到平面脂质双分子层中的纯化TRPM8通道的活性，在平面脂质双分子层中，通道的活性由冷和薄荷醇在磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P(2)）存在的情况下引发。对TRPM8蛋白的生化分析还发现了聚（R）-3-羟基丁酸酯（PHB）的存在，其经常与多聚磷酸盐相关。我们得出结论，TRPM8蛋白与多聚磷酸盐形成稳定的复合物，其存在对于正常通道活性至关重要。
