Abstract
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)P2). 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.
Highlights
transient receptor potential melastatin 8 (TRPM8) is a member of the transient receptor potential (TRP) channel family of the melastatin subgroup, which is thought to be a major sensor for a wide range of cold temperatures in the peripheral nervous system [1,2,3]
We demonstrate that TRPM8 expressed in HEK-293 and F-11 neuronal cells is associated with polyP and PHB, and that polyP serves as crucial regulator of TRPM8 channel function
The activity of TRPM8 channels is regulated by a plethora of factors including temperature, ligand binding, voltage, pH, etc. reflecting diverse stimuli and mechanisms of these regulators
Summary
TRPM8 is a member of the transient receptor potential (TRP) channel family of the melastatin subgroup, which is thought to be a major sensor for a wide range of cold temperatures in the peripheral nervous system [1,2,3]. TRPM8 is activated by low temperatures in the range of 8–26uC and a number of chemical compounds such as menthol, icilin, eucalyptol, geraniol and linalool [4,5,6]. Several other factors, such as voltage [7,8], pH [8], lysophospholipids and fatty acids [9,10] modulate TRPM8 activity. It has been shown that TRPA1 channels can be activated by pungent chemicals only in the presence of inorganic polyphosphates [23]
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