Inorganic Polyphosphate Modulates TRPM8 Channels

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.

The TRPM8 Protein Is a Testosterone Receptor

“…It is noteworthy that TRPM8 inhibition with a novel TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride (M8-B) (18), occurred only upon its application onto one side of the channel, presumably extracellular, according to the inhibitory effects of this compound observed previously on intact cells (18). Unlike M8-B, application of polylysine was effective only when applied to the intracellular side of the channel, similar to previously demonstrated menthol-activated TRPM8 channels (11).…”

“…The cells were transfected with the rat TRPM8 cDNA using the Effectene reagent (Qiagen, Chatsworth, CA). The TRPM8 stable cell line was developed with TRPM8 tagged with Myc on the N terminus, as described previously (11).…”

“…Planar Lipid Bilayer Measurements-Planar lipid bilayer measurements were performed as described previously (11,13,14). Planar lipid bilayers were formed from a solution of synthetic 1-palmitoyl-2-oleoyl-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine (POPE; Avanti Polar Lipids, Birmingham, AL) in a 3:1 ratio in n-decane (Sigma-Aldrich).…”

Regulation of the Temperature-dependent Activation of Transient Receptor Potential Vanilloid 1 (TRPV1) by Phospholipids in Planar Lipid Bilayers

“…1. The TRPM8 protein was purified from HEK-293 cells as described previously (13,(17)(18)(19). A, representative single-channel current trace recordings of TRPV1 channels obtained upon heating the bilayer system from 24 to 42°C in the presence of 2.5 M PI(4)P. Representative current trace recordings demonstrate different segments upon heating.…”

Inorganic polyphosphate–an unusual suspect of the mitochondrial permeability transition mystery

“…Examples of the roles of polyP in mammalians include: skeletal mineralization, 1 blood coagulation and inflammation, 2 cell bioenergetics, 3 ion channel function 4 and nuclear transcription. 5 At present, very little is known about the molecular details of polyP metabolism.…”