Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators.

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but the molecular mechanism of activation by PI(4,5)P2 is not known. As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PI(4,5)P2via molecular modeling. We identified putative contact residues for PI(4,5)P2 in the pre-S1 segment, the S4-S5 linker, and the proximal C-terminal TRP domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PI(4,5)P2 levels. Changes in ligand-binding affinities via molecular mechanics/generalized Born surface area (MM/GBSA) showed reduced PI(4,5)P2 affinity for the mutants. Mutating PI(4,5)P2-interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate, pointing to an allosteric interaction between PI(4,5)P2 and pregnenolone sulfate. Similarly, mutating residues in the PI(4,5)P2 binding site in TRPM8 resulted in increased sensitivity to PI(4,5)P2 depletion and reduced sensitivity to menthol. Mutations of most PI(4,5)P2-interacting residues in TRPM3 also increased sensitivity to inhibition by Gßγ, indicating allosteric interaction between Gßγ and PI(4,5)P2 regulation. Disease-associated gain-of-function TRPM3 mutations on the other hand resulted in no change of PI(4,5)P2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P2, its relationship to endogenous activators and inhibitors, as well as identify similarities and differences between PI(4,5)P2 regulation of TRPM3 and TRPM8.

Phosphatidic acid is an endogenous negative regulator of PIEZO2 channels and mechanical sensitivity.

Mechanosensitive PIEZO2 ion channels play roles in touch, proprioception, and inflammatory pain. Currently, there are no small molecule inhibitors that selectively inhibit PIEZO2 over PIEZO1. The TMEM120A protein was shown to inhibit PIEZO2 while leaving PIEZO1 unaffected. Here we find that TMEM120A expression elevates cellular levels of phosphatidic acid and lysophosphatidic acid (LPA), aligning with its structural resemblance to lipid-modifying enzymes. Intracellular application of phosphatidic acid or LPA inhibited PIEZO2, but not PIEZO1 activity. Extended extracellular exposure to the non-hydrolyzable phosphatidic acid and LPA analogue carbocyclic phosphatidic acid (ccPA) also inhibited PIEZO2. Optogenetic activation of phospholipase D (PLD), a signaling enzyme that generates phosphatidic acid, inhibited PIEZO2, but not PIEZO1. Conversely, inhibiting PLD led to increased PIEZO2 activity and increased mechanical sensitivity in mice in behavioral experiments. These findings unveil lipid regulators that selectively target PIEZO2 over PIEZO1, and identify the PLD pathway as a regulator of PIEZO2 activity.

Structural mechanism of TRPV5 inhibition by econazole.

The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.

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

TMEM120A (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.

TMEM120A/TACAN inhibits mechanically activated PIEZO2 channels.

PIEZO2 channels mediate rapidly adapting mechanically activated currents in peripheral sensory neurons of the dorsal root ganglia (DRG), and they are indispensable for light touch and proprioception. Relatively little is known about what other proteins regulate PIEZO2 activity in a cellular context. TMEM120A (TACAN) was proposed to act as a high threshold mechanically activated ion channel in nociceptive DRG neurons. Here, we find that Tmem120a coexpression decreased the amplitudes of mechanically activated PIEZO2 currents and increased their threshold of activation. TMEM120A did not inhibit mechanically activated PIEZO1 and TREK1 channels and TMEM120A alone did not result in the appearance of mechanically activated currents above background. Tmem120a and Piezo2 expression in mouse DRG neurons overlapped, and siRNA-mediated knockdown of Tmem120a increased the amplitudes of rapidly adapting mechanically activated currents and decreased their thresholds to mechanical activation. Our data identify TMEM120A as a negative modulator of PIEZO2 channel activity, and do not support TMEM120A being a mechanically activated ion channel.