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.

Molecular details of ruthenium red pore block in TRPV channels.

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

Phosphoinositide Regulation of TRP Channels: A Functional Overview in the Structural Era.

Transient receptor potential (TRP) ion channels have diverse activation mechanisms including physical stimuli, such as high or low temperatures, and a variety of intracellular signaling molecules. Regulation by phosphoinositides and their derivatives is their only known common regulatory feature. For most TRP channels, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] serves as a cofactor required for activity. Such dependence on PI(4,5)P2 has been demonstrated for members of the TRPM subfamily and for the epithelial TRPV5 and TRPV6 channels. Intracellular TRPML channels show specific activation by PI(3,5)P2. Structural studies uncovered the PI(4,5)P2 and PI(3,5)P2 binding sites for these channels and shed light on the mechanism of channel opening. PI(4,5)P2 regulation of TRPV1-4 as well as some TRPC channels is more complex, involving both positive and negative effects. This review discusses the functional roles of phosphoinositides in TRP channel regulation and molecular insights gained from recent cryo-electron microscopy structures.

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.

Structural basis of the activation of TRPV5 channels by long-chain acyl-Coenzyme-A.

Long-chain acyl-coenzyme A (LC-CoA) is a crucial metabolic intermediate that plays important cellular regulatory roles, including activation and inhibition of ion channels. The structural basis of ion channel regulation by LC-CoA is not known. Transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6) are epithelial calcium-selective ion channels. Here, we demonstrate that LC-CoA activates TRPV5 and TRPV6 in inside-out patches, and both exogenously supplied and endogenously produced LC-CoA can substitute for the natural ligand phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in maintaining channel activity in intact cells. Utilizing cryo-electron microscopy, we determined the structure of LC-CoA-bound TRPV5, revealing an open configuration with LC-CoA occupying the same binding site as PI(4,5)P2 in previous studies. This is consistent with our finding that PI(4,5)P2 could not further activate the channels in the presence of LC-CoA. Our data provide molecular insights into ion channel regulation by a metabolic signaling molecule.

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.

Gain-of-function variants in the ion channel gene TRPM3 underlie a spectrum of neurodevelopmental disorders.

TRPM3 is a temperature- and neurosteroid-sensitive plasma membrane cation channel expressed in a variety of neuronal and non-neuronal cells. Recently, rare de novo variants in TRPM3 were identified in individuals with developmental and epileptic encephalopathy, but the link between TRPM3 activity and neuronal disease remains poorly understood. We previously reported that two disease-associated variants in TRPM3 lead to a gain of channel function . Here, we report a further 10 patients carrying one of seven additional heterozygous TRPM3 missense variants. These patients present with a broad spectrum of neurodevelopmental symptoms, including global developmental delay, intellectual disability, epilepsy, musculo-skeletal anomalies, and altered pain perception. We describe a cerebellar phenotype with ataxia or severe hypotonia, nystagmus, and cerebellar atrophy in more than half of the patients. All disease-associated variants exhibited a robust gain-of-function phenotype, characterized by increased basal activity leading to cellular calcium overload and by enhanced responses to the neurosteroid ligand pregnenolone sulfate when co-expressed with wild-type TRPM3 in mammalian cells. The antiseizure medication primidone, a known TRPM3 antagonist, reduced the increased basal activity of all mutant channels. These findings establish gain-of-function of TRPM3 as the cause of a spectrum of autosomal dominant neurodevelopmental disorders with frequent cerebellar involvement in humans and provide support for the evaluation of TRPM3 antagonists as a potential therapy.

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.

What structures did, and did not, reveal about the function of the epithelial Ca2+ channels TRPV5 and TRPV6.

Transient Receptor Potential Vanilloid 5 and 6 (TRPV5 and TRPV6) are Ca2+ selective epithelial ion channels. They are the products of a relatively recent gene duplication in mammals, and have high sequence homology to each other. Their functional properties are also much more similar to each other than to other members of the TRPV subfamily. They are both constitutively active, and this activity depends on the endogenous cofactor phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Both channels undergo Ca2+-induced inactivation, which is mediated by direct binding of the ubiquitous Ca2+ binding protein calmodulin (CaM) to the channels, and by a decrease in PI(4,5)P2 levels by Ca2+ -induced activation of phospholipase C (PLC). Recent cryo electron microscopy (cryo-EM) and X-ray crystallography structures provided detailed structural information for both TRPV5 and TRPV6. This review will discuss this structural information in the context of the function of these channels focusing on the mechanism of CaM inhibition, activation by PI(4,5)P2 and binding of pharmacological modulators.

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.

Structural basis of TRPV5 regulation by physiological and pathophysiological modulators.

Transient receptor potential vanilloid 5 (TRPV5) is a kidney-specific Ca2+-selective ion channel that plays a key role in Ca2+ homeostasis. The basal activity of TRPV5 is balanced through activation by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and inhibition by Ca2+-bound calmodulin (CaM). Parathyroid hormone (PTH), the key extrinsic regulator of Ca2+ homeostasis, increases the activity of TRPV5 via protein kinase A (PKA)-mediated phosphorylation. Metabolic acidosis leads to reduced TRPV5 activity independent of PTH, causing hypercalciuria. Using cryoelectron microscopy (cryo-EM), we show that low pH inhibits TRPV5 by precluding PI(4,5)P2 activation. We capture intermediate conformations at low pH, revealing a transition from open to closed state. In addition, we demonstrate that PI(4,5)P2 is the primary modulator of channel gating, yet PKA controls TRPV5 activity by preventing CaM binding and channel inactivation. Our data provide detailed molecular mechanisms for regulation of TRPV5 by two key extrinsic modulators, low pH and PKA.

How Many Cell Types Are in the Kidney and What Do They Do?

The kidney maintains electrolyte, water, and acid-base balance, eliminates foreign and waste compounds, regulates blood pressure, and secretes hormones. There are at least 16 different highly specialized epithelial cell types in the mammalian kidney. The number of specialized endothelial cells, immune cells, and interstitial cell types might even be larger. The concerted interplay between different cell types is critical for kidney function. Traditionally, cells were defined by their function or microscopical morphological appearance. With the advent of new single-cell modalities such as transcriptomics, epigenetics, metabolomics, and proteomics we are entering into a new era of cell type definition. This new technological revolution provides new opportunities to classify cells in the kidney and understand their functions.

Methods to study phosphoinositide regulation of ion channels.

Ion channel are embedded in the lipid bilayers of biological membranes. Membrane phospholipids constitute a barrier to ion movement, and they have been considered for a long time as a passive environment for channel proteins. Membrane phospholipids, however, do not only serve as a passive amphipathic environment, but they also modulate channel activity by direct specific lipid-protein interactions. Phosphoinositides are quantitatively minor components of biological membranes, and they play roles in many cellular functions, including membrane traffic, cellular signaling and cytoskeletal organization. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is mainly found in the inner leaflet of the plasma membrane. Its role as a potential ion channel regulator was first appreciated over two decades ago and by now this lipid is a well-established cofactor or regulator of many different ion channels. The past two decades witnessed the steady development of techniques to study ion channel regulation by phosphoinositides with progress culminating in recent cryoEM structures that allowed visualization of how PI(4,5)P2 opens some ion channels. This chapter will provide an overview of the methods to study regulation by phosphoinositides, focusing on plasma membrane ion channels and PI(4,5)P2.

The newest TRP channelopathy: Gain of function TRPM3 mutations cause epilepsy and intellectual disability.

Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+ permeable nonselective cation channel, activated by heat and chemical agonists, such as the endogenous neuro-steroid Pregnenolone Sulfate (PregS) and the chemical compound CIM0216. TRPM3 is expressed in peripheral sensory neurons of the dorsal root ganglia (DRG), and its role in noxious heat sensation in mice is well established. TRPM3 is also expressed in a number of other tissues, including the brain, but its role there has been largely unexplored. Recent reports showed that two mutations in TRPM3 are associated with a developmental and epileptic encephalopathy, pointing to an important role of TRPM3 in the human brain. Subsequent reports found that the two disease-associated mutations increased basal channel activity, and sensitivity of the channel to activation by heat and chemical agonists. This review will discuss these mutations in the context of human diseases caused by mutations in other TRP channels, and in the context of the biophysical properties and physiological functions of TRPM3.

Dual regulation of TRPV1 channels by phosphatidylinositol via functionally distinct binding sites.

Regulation of the heat- and capsaicin-activated transient receptor potential vanilloid 1 (TRPV1) channel by phosphoinositides is complex and controversial. In the most recent TRPV1 cryo-EM structure, endogenous phosphatidylinositol (PtdIns) was detected in the vanilloid binding site, and phosphoinositides were proposed to act as competitive vanilloid antagonists. This model is difficult to reconcile with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being a well-established positive regulator of TRPV1. Here we show that in the presence of PtdIns(4,5)P2 in excised patches, PtdIns, but not PtdIns(4)P, partially inhibited TRPV1 activity at low, but not at high capsaicin concentrations. This is consistent with PtdIns acting as a competitive vanilloid antagonist. However, in the absence of PtdIns(4,5)P2, PtdIns partially stimulated TRPV1 activity. We computationally identified residues, which are in contact with PtdIns, but not with capsaicin in the vanilloid binding site. The I703A mutant of TRPV1 showed increased sensitivity to capsaicin, as expected when removing the effect of an endogenous competitive antagonist. I703A was not inhibited by PtdIns in the presence of PtdIns(4,5)P2, but it was still activated by PtdIns in the absence of PtdIns(4,5)P2 indicating that inhibition, but not activation by PtdIns proceeds via the vanilloid binding site. In molecular dynamics simulations, PtdIns was more stable than PtdIns(4,5)P2 in this inhibitory site, whereas PtdIns(4,5)P2 was more stable than PtdIns in a previously identified, nonoverlapping, putative activating binding site. Our data indicate that phosphoinositides regulate channel activity via functionally distinct binding sites, which may explain some of the complexities of the effects of these lipids on TRPV1.

Crowding-induced opening of the mechanosensitive Piezo1 channel in silico.

Mechanosensitive Piezo1 channels are essential mechanotransduction proteins in eukaryotes. Their curved transmembrane domains, called arms, create a convex membrane deformation, or footprint, which is predicted to flatten in response to increased membrane tension. Here, using a hyperbolic tangent model, we show that, due to the intrinsic bending rigidity of the membrane, the overlap of neighboring Piezo1 footprints produces a flattening of the Piezo1 footprints and arms. Multiple all-atom molecular dynamics simulations of Piezo1 further reveal that this tension-independent flattening is accompanied by gating motions that open an activation gate in the pore. This open state recapitulates experimentally obtained ionic selectivity, unitary conductance, and mutant phenotypes. Tracking ion permeation along the open pore reveals the presence of intracellular and extracellular fenestrations acting as cation-selective sites. Simulations also reveal multiple potential binding sites for phosphatidylinositol 4,5-bisphosphate. We propose that the overlap of Piezo channel footprints may act as a cooperative mechanism to regulate channel activity.

TRPM3 Channels Play Roles in Heat Hypersensitivity and Spontaneous Pain after Nerve Injury.

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel in primary sensory neurons of the dorsal root ganglia (DRGs). Pharmacological and genetic studies implicated TRPM3 in various pain modalities, but TRPM3 inhibitors were not validated in TRPM3-/- mice. Here we tested two inhibitors of TRPM3 in male and female wild-type and TRPM3-/- mice in nerve injury-induced neuropathic pain. We found that intraperitoneal injection of either isosakuranetin or primidone reduced heat hypersensitivity induced by chronic constriction injury (CCI) of the sciatic nerve in wild-type, but not in TRPM3-/- mice. Primidone was also effective when injected locally in the hindpaw or intrathecally. Consistently, intrathecal injection of the TRPM3 agonist CIM0216 reduced paw withdrawal latency to radiant heat in wild-type, but not in TRPM3-/- mice. Intraperitoneal injection of 2 mg/kg, but not 0.5 mg/kg isosakuranetin, inhibited cold and mechanical hypersensitivity in CCI, both in wild-type and TRPM3-/- mice, indicating a dose-dependent off-target effect. Primidone had no effect on cold sensitivity, and only a marginal effect on mechanical hypersensitivity. Genetic deletion or inhibitors of TRPM3 reduced the increase in the levels of the early genes c-Fos and pERK in the spinal cord and DRGs in CCI mice, suggesting spontaneous activity of the channel. Intraperitoneal isosakuranetin also inhibited spontaneous pain related behavior in CCI in the conditioned place preference assay, and this effect was eliminated in TRPM3-/- mice. Overall, our data indicate a role of TRPM3 in heat hypersensitivity and in spontaneous pain after nerve injury.SIGNIFICANCE STATEMENT Neuropathic pain is a major unsolved medical problem. The heat-activated TRPM3 ion channel is a potential target for novel pain medications, but the pain modalities in which it plays a role are not clear. Here we used a combination of genetic and pharmacological tools to assess the role of this channel in spontaneous pain, heat, cold, and mechanical hypersensitivity in a nerve injury model of neuropathic pain in mice. Our findings indicate a role for TRPM3 in heat hyperalgesia, and spontaneous pain, but not in cold and mechanical hypersensitivity. We also find that not only TRPM3 located in the peripheral nerve termini, but also TRPM3 in the spinal cord or proximal segments of DRG neurons are important for heat hypersensitivity.

The TRPM3 ion channel mediates nociception but not itch evoked by endogenous pruritogenic mediators.

During the molecular transduction of itch, the stimulation of pruriceptors on sensory fibers leads to the activation or sensitization of ion channels, which results in a consequent depolarization of the neurons. These ion channels mostly belong to the transient receptor potential (TRP) channels, which are involved in nociception and thermosensation. In particular, TRPV1 and TRPA1 were described in the transduction of both thermal nociception as well as histaminergic and non-histaminergic itch. The thermosensitive TRPM3 plays an indispensable role in heat nociception together with TRPV1 and TRPA1. However, the role of TRPM3 in the development of pruritus has not been studied yet. Therefore, in this study we aimed at investigating the potential role of TRPM3 in the transduction of pruritus and pain by investigating itch- and nociception-related behavior of Trpm3+/+ and Trpm3-/- mice, and by studying the activation of somatosensory neurons isolated from trigeminal ganglia upon application of algogenic and pruritogenic substances. Activators of TRPM3 evoked only nocifensive responses, but not itch in Trpm3+/+ animals, and these nocifensive responses were abolished in the Trpm3-/- strain. Histamine and endogenous non-histaminergic pruritogens induced itch in both Trpm3+/+ and Trpm3-/- mice to a similar extent. Genetic deletion or pharmacological blockade diminished TRPM3 mediated Ca2+ responses of sensory neurons, but did not affect responses evoked by pruritogenic substances. Our results demonstrate that, in contrast to other thermosensitive TRP channels, TRPM3 selectively mediates nociception, but not itch sensation, and suggest that TRPM3 is a promising candidate to selectively target pain sensation.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.