Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells

Rapamycin (rapa)-induced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) enzyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5)P2 as monitored by the PLCδ1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca2+ signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5)P2 was associated with a complete blockade of transferrin uptake and inhibition of epidermal growth factor internalization. None of these changes were observed upon rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. These data demonstrate that rapid inducible depletion of PM PtdIns(4,5)P2 is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5)P2 depletion

Structural insights on TRPV5 gating by endogenous modulators.

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery

Inorganic Polyphosphate Modulates TRPM8 Channels

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

TMEM120A/TACAN: A putative regulator of ion channels, mechanosensation, and lipid metabolism

ABSTRACTTMEM120A (TACAN) is an enigmatic protein with several seemingly unconnected functions. It was proposed to be an ion channel involved in sensing mechanical stimuli, and knockdown/knockout experiments have implicated that TMEM120A may be necessary for sensing mechanical pain. TMEM120A’s ion channel function has subsequently been challenged, as attempts to replicate electrophysiological experiments have largely been unsuccessful. Several cryo-EM structures revealed TMEM120A is structurally homologous to a lipid modifying enzyme called Elongation of Very Long Chain Fatty Acids 7 (ELOVL7). Although TMEM120A’s channel function is debated, it still seems to affect mechanosensation by inhibiting PIEZO2 channels and by modifying tactile pain responses in animal models. TMEM120A was also shown to inhibit polycystin-2 (PKD2) channels through direct physical interaction. Additionally, TMEM120A has been implicated in adipocyte regulation and in innate immune response against Zika virus. The way TMEM120A is proposed to alter each of these processes ranges from regulating gene expression, acting as a lipid modifying enzyme, and controlling subcellular localization of other proteins through direct binding. Here, we examine TMEM120A’s structure and proposed functions in diverse physiological contexts