Permeable Cation Channel Research Articles

Polycystin-2 (TRPP2) regulates primary cilium length in LLC-PK1 renal epithelial cells.

Polycystin-2 (PC2, TRPP2) is a Ca2+ permeable nonselective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. However, little is known as to whether PC2 contributes to the primary cilium structure. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild-type LLC-PK1 renal epithelial cells. Confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated α-tubulin antibody to identify primary cilia and measure their length. Although primary cilia length measurements did not follow a Normal distribution, the data were normalized by Box-Cox transformation rendering statistical differences under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 μM) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length compared to their respective scrambled RNA transfected cells. The data indicate that conditions that regulate PC2 function or gene expression modify the length of primary cilia in renal epithelial cells. Blocking of PC2 mitigates the effects of elevated external Ca2+ concentration on primary cilia length. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

Transient receptor potential melastatin-7 in the rat dorsal root ganglion

AimsTransient receptor potential melastatin-7 (TRPM7) is a selective cation permeable channel which plays important roles in cellular and developmental biology such as cell proliferation, survival, differentiation and migration. This channel is also known to be necessary for transmitter release in the peripheral nervous system. In this study, immunohistochemistry for TRPM7 was conducted in the rat lumbar dorsal root ganglion (DRG). MethodsTriple immunofluorescence methods were used to demonstrate distribution of TRPM7 and its relationship to other TRP channels in the DRG. Retrograde tracing and double immunofluorescence methods were also performed to know peripheral targets of DRG neurons containing TRPM7 and TRP vanilloid 1 (TRPV1). In addition, transection of the sciatic nerve was conducted to demonstrate an effect of the nerve injury on TRPM7expression in the DRG. ResultsTRPM7-immunoreactivity was expressed by 53.9% of sensory neurons in the 4th lumbar DRG. TRPM7-immunoreactive (-IR) DRG neurons mostly had small (<600 µm²) and medium-sized (600–1200 µm²) cell bodies. By triple and double immunofluorescence methods, approximately 70% of TRPM7-IR DRG neurons contained TRPV1-immunoreactivity. Although the number of DRG neurons co-expressing TRPM7 and TRPM8 was small in the DRG, almost all of TRPM8-IR DRG neurons co-expressed TRPM7-immunoreactivity. By combination of retrograde tracing method and immunohistochemistry, TRPM7 was expressed by half of DRG neurons innervating the plantar skin (61.9%) and gastrocnemius muscle (51.2%), and 79.6% of DRG neurons innervating the periosteum. Co-expression of TRPM7 and TRPV1 among periosteum DRG neurons (75.7%) was more abundant than among cutaneous (53.2%) and muscular (40.4%) DRG neurons. DRG neurons which co-expressed these ion channels in the periosteum had smaller cell bodies compared to the skin and muscle. In addition, the sciatic nerve transection decreased the number of TRPM7-IR neurons in the DRG (approximately 60% reduction). The RT-qPCR analysis also demonstrated reduction of TRPM7 mRNA in the injured DRG. ConclusionThe present study suggests that TRPM7 is mainly located in small nociceptors in the DRG. The content of TRPM7 in DRG neurons is probably different among their peripheral targets. TRPM7 in DRG neurons may be able to respond to noxious stimulation from their peripheral tissues. The nerve injury can decrease the level of TRPM7 mRNA and protein in DRG neurons.

Pathological Mechanisms Induced by TRPM2 Ion Channels Activation in Renal Ischemia-Reperfusion Injury.

Renal ischemia-reperfusion (IR) injury triggers a cascade of signaling reactions involving an increase in Ca2 + charge and reactive oxygen species (ROS) levels resulting in necrosis, inflammation, apoptosis, and subsequently acute kidney injury (AKI).Transient receptor potential (TRP) channels include an essential class of Ca2+ permeable cation channels, which are segregated into six main channels: the canonical channel (TRPC), the vanilloid-related channel (TRPV), the melastatin-related channel (TRPM), the ankyrin-related channel (TRPA), the mucolipin-related channel (TRPML) and polycystin-related channel (TRPP) or polycystic kidney disease protein (PKD2). TRP channels are involved in adjusting vascular tone, vascular permeability, cell volume, proliferation, secretion, angiogenesis and apoptosis.TRPM channels include eight isoforms (TRPM1-TRPM8) and TRPM2 is the second member of this subfamily that has been expressed in various tissues and organs such as the brain, heart, kidney and lung. Renal TRPM2 channels have an important role in renal IR damage. So that TRPM2 deficient mice are resistant to renal IR injury. TRPM2 channels are triggered by several chemicals including hydrogen peroxide, Ca2+, and cyclic adenosine diphosphate (ADP) ribose (cADPR) that are generated during AKI caused by IR injury, as well as being implicated in cell death caused by oxidative stress, inflammation, and apoptosis.

Calcium channels and transporters: Roles in response to biotic and abiotic stresses.

Calcium (Ca2+) serves as a ubiquitous second messenger by mediating various signaling pathways and responding to numerous environmental conditions in eukaryotes. Therefore, plant cells have developed complex mechanisms of Ca2+ communication across the membrane, receiving the message from their surroundings and transducing the information into cells and organelles. A wide range of biotic and abiotic stresses cause the increase in [Ca2+]cyt as a result of the Ca2+ influx permitted by membrane-localized Ca2+ permeable cation channels such as CYCLIC NUCLEOTIDE-GATE CHANNELs (CNGCs), and voltage-dependent HYPERPOLARIZATION-ACTIVATED CALCIUM2+ PERMEABLE CHANNELs (HACCs), as well as GLUTAMATE RECEPTOR-LIKE RECEPTORs (GLRs) and TWO-PORE CHANNELs (TPCs). Recently, resistosomes formed by some NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT RECEPTORs (NLRs) are also proposed as a new type of Ca2+ permeable cation channels. On the contrary, some Ca2+ transporting membrane proteins, mainly Ca2+-ATPase and Ca2+/H+ exchangers, are involved in Ca2+ efflux for removal of the excessive [Ca2+]cyt in order to maintain the Ca2+ homeostasis in cells. The Ca2+ efflux mechanisms mediate the wide ranges of cellular activities responding to external and internal stimuli. In this review, we will summarize and discuss the recent discoveries of various membrane proteins involved in Ca2+ influx and efflux which play an essential role in fine-tuning the processing of information for plant responses to abiotic and biotic stresses.

Radiosynthesis and Evaluation of a C-11 Radiotracer for Transient Receptor Potential Canonical 5 in the Brain.

TRPC5 belongs to the mammalian superfamily of transient receptor potential (TRP) Ca2+-permeable cationic channels and it has been implicated in various CNS disorders. As part of our ongoing interest in the development of a PET radiotracer for imaging TRPC5, herein, we explored the radiosynthesis, and in vitro and in vivo evaluation of a new C-11 radiotracer [11C]HC070 in rodents and nonhuman primates. [11C]HC070 was radiolabeled utilizing the corresponding precursor and [11C]CH3I via N-methylation protocol. Ex vivo biodistribution study of [11C]HC070 was performed in Sprague-Dawley rats. In vitro autoradiography study was conducted for the rat brain sections to characterize the radiotracer distribution in the brain regionals. MicroPET brain imaging studies of [11C]HC070 were done for 129S1/SvImJ wild-type mice and 129S1/SvImJ TRPC5 knockout mice for 0-60-min dynamic data acquisition after intravenous administration of the radiotracer. Dynamic PET scans (0-120min) for the brain of cynomolgus male macaques were performed after the radiotracer injection. [11C]HC070 was efficiently prepared with good radiochemical yield (45 ± 5%, n = 15), high chemical and radiochemical purity (> 99%), and high molar activity (320.6 ± 7.4GBq/μmol, 8.6 ± 0.2Ci/μmol) at the end of bombardment (EOB). Radiotracer [11C]HC070 has good solubility in the aqueous dose solution. The ex vivo biodistribution study showed that [11C]HC070 had a quick rat brain clearance. Autoradiography demonstrated that [11C]HC070 specifically binds to TRPC5-enriched regions in rat brain. MicroPET study showed the peak brain uptake (SUV value) was 0.63 in 129S1/SvImJ TRPC5 knockout mice compared to 1.13 in 129S1/SvImJ wild-type mice. PET study showed that [11C]HC070 has good brain uptake with maximum SUV of ~ 2.2 in the macaque brain, followed by rapid clearance. Our data showed that [11C]HC070 is a TRPC5-specific radiotracer with high brain uptake and good brain washout pharmacokinetics in both rodents and nonhuman primates. The radiotracer is worth further investigating of its suitability to be a PET radiotracer for imaging TRPC5 in animals and human subjects in vivo.

Chemical priming of natural killer cells with branched polyethylenimine for cancer immunotherapy

BackgroundDue to their powerful immune surveillance activity and ability to kill and clear cancer cells, natural killer (NK) cells are an emerging anticancer immunotherapeutic agent. Therefore, there is much interest...

Identification of OSCA gene family in Solanum habrochaites and its function analysis under stress

BackgroundOSCA (hyperosmolality-gated calcium-permeable channel) is a calcium permeable cation channel protein that plays an important role in regulating plant signal transduction. It is involved in sensing changes in extracellular osmotic potential and an increase in Ca2+ concentration. S. habrochaites is a good genetic material for crop improvement against cold, late blight, planthopper and other diseases. Till date, there is no report on OSCA in S. habrochaites. Thus, in this study, we performed a genome-wide screen to identify OSCA genes in S. habrochaites and characterized their responses to biotic and abiotic stresses.ResultsA total of 11 ShOSCA genes distributed on 8 chromosomes were identified. Subcellular localization analysis showed that all members of ShOSCA localized on the plasma membrane and contained multiple stress-related cis acting elements. We observed that genome-wide duplication (WGD) occurred in the genetic evolution of ShOSCA5 (Solhab04g250600) and ShOSCA11 (Solhab12g051500). In addition, repeat events play an important role in the expansion of OSCA gene family. OSCA gene family of S. habrochaites used the time lines of expression studies by qRT-PCR, do indicate OSCAs responded to biotic stress (Botrytis cinerea) and abiotic stress (drought, low temperature and abscisic acid (ABA)). Among them, the expression of ShOSCAs changed significantly under four stresses. The resistance of silencing ShOSCA3 plants to the four stresses was reduced.ConclusionThis study identified the OSCA gene family of S. habrochaites for the first time and analyzed ShOSCA3 has stronger resistance to low temperature, ABA and Botrytis cinerea stress. This study provides a theoretical basis for clarifying the biological function of OSCA, and lays a foundation for tomato crop improvement.

BTDAzo: A Photoswitchable TRPC5 Channel Activator.

Photoswitchable reagents can be powerful tools for high‐precision biological control. TRPC5 is a Ca2+‐permeable cation channel with distinct tissue‐specific roles, from synaptic function to hormone regulation. Reagents giving spatiotemporally‐resolved control over TRPC5 activity may be key to understanding and harnessing its biology. Here we develop the first photoswitchable TRPC5‐modulator, BTDAzo, to address this goal. BTDAzo can photocontrol TRPC5 currents in cell culture, as well as controlling endogenous TRPC5‐based neuronal Ca2+ responses in mouse brain slices. BTDAzos are also the first reported azo‐benzothiadiazines, an accessible and conveniently derivatised azoheteroarene with strong two‐colour photoswitching. BTDAzo′s ability to control TRPC5 across relevant channel biology settings makes it suitable for a range of dynamically reversible photoswitching studies in TRP channel biology, with the aim to decipher the various biological roles of this centrally important ion channel.

BTDAzo: A Photoswitchable TRPC5 Channel Activator**

Photoswitchable reagents can be powerful tools for high-precision biological control. TRPC5 is a Ca2+-permeable cation channel with distinct tissue-specific roles, from synaptic function to hormone regulation. Reagents giving spatiotemporally-resolved control over TRPC5 activity may be key to understanding and harnessing its biology. Here we develop the first photoswitchable TRPC5-modulator, BTDAzo, to address this goal. BTDAzo can photocontrol TRPC5 currents in cell culture, as well as controlling endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. BTDAzos are also the first reported azo-benzothiadiazines, an accessible and conveniently derivatised azoheteroarene with strong two-colour photoswitching. BTDAzo′s ability to control TRPC5 across relevant channel biology settings makes it suitable for a range of dynamically reversible photoswitching studies in TRP channel biology, with the aim to decipher the various biological roles of this centrally important ion channel.

Knockdown of Acid-sensing Ion Channel 1a in the PVN Promotes Metabolic Disturbances in Male Mice.

Increasing incidence of metabolic disturbances has become a severe public healthcare problem. Ion channels and receptors in the paraventricular nucleus (PVN) of the hypothalamus serve vital roles in modulating neuronal activities and endocrine functions, which are linked to the regulation of energy balance and glucose metabolism. In this study, we found that acid-sensing ion channel 1a (ASIC1a), a Ca2+-permeable cationic ion channel was localized in the PVN. Knockdown of ASIC1a in this region led to significant body weight gain, glucose intolerance, and insulin resistance. Pharmacological inhibition of ASIC1a resulted in an increase in food intake and a decrease in energy expenditure. Our findings suggest ASIC1a in the PVN as a potential new target for the therapeutic intervention of metabolic disorders.

TRPA1 modulation by Sigma-1 receptor prevents oxaliplatin-induced painful peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy is a frequent, disabling side effect of anticancer drugs. Oxaliplatin, a platinum compound used in the treatment of advanced colorectal cancer, often leads to a form of chemotherapy-induced peripheral neuropathy characterized by mechanical and cold hypersensitivity. Current therapies for chemotherapy-induced peripheral neuropathy are ineffective, often leading to the cessation of treatment. Transient receptor potential ankyrin 1 (TRPA1) is a polymodal, non-selective cation-permeable channel expressed in nociceptors, activated by physical stimuli and cellular stress products. TRPA1 has been linked to the establishment of chemotherapy-induced peripheral neuropathy and other painful neuropathic conditions. Sigma-1 receptor is an endoplasmic reticulum chaperone known to modulate the function of many ion channels and receptors. Sigma-1 receptor antagonist, a highly selective antagonist of Sigma-1 receptor, has shown effectiveness in a phase II clinical trial for oxaliplatin chemotherapy-induced peripheral neuropathy. However, the mechanisms involved in the beneficial effects of Sigma-1 receptor antagonist are little understood. We combined biochemical and biophysical (i.e. intermolecular Förster resonance energy transfer) techniques to demonstrate the interaction between Sigma-1 receptor and human TRPA1. Pharmacological antagonism of Sigma-1R impaired the formation of this molecular complex and the trafficking of functional TRPA1 to the plasma membrane. Using patch-clamp electrophysiological recordings we found that antagonists of Sigma-1 receptor, including Sigma-1 receptor antagonist, exert a marked inhibition on plasma membrane expression and function of human TRPA1 channels. In TRPA1-expressing mouse sensory neurons, Sigma-1 receptor antagonists reduced inward currents and the firing of actions potentials in response to TRPA1 agonists. Finally, in a mouse experimental model of oxaliplatin neuropathy, systemic treatment with a Sigma-1 receptor antagonists prevented the development of painful symptoms by a mechanism involving TRPA1. In summary, the modulation of TRPA1 channels by Sigma-1 receptor antagonists suggests a new strategy for the prevention and treatment of chemotherapy-induced peripheral neuropathy and could inform the development of novel therapeutics for neuropathic pain.

Overexpression of GsCNGC20-f from Glycine soja confers submergence tolerance to hairy-root composite soybean plants and Arabidopsis seedlings by enhancing anaerobic respiration

Cyclic nucleotide-gated channels (CNGCs) in plants are nonselective Ca 2+ -permeable cationic channel proteins, that are part of the signal transduction cascade system and are involved in the physiological regulation of plant responses to adverse environments. This study firstly showed that the stronger submergence tolerance of G. soja P18B seedlings compared to the G. max cultivar KF1, might be related to the enhanced processes of Ca 2+ signal cascade (higher Ca 2+ content), anaerobic metabolism (higher activities of PDC and ADH), and energy reserves (higher ATP level) in the leaves during submergence. Then, the potential submergence-responsive differentially expressed genes (DEGs) enriched in voltage-gated potassium channel activity, such as GsCNGC20-f and GsCNGC20-g , and those enriched in calcium ion binding, i.e. GsCaML, GsCAM4, GsCML41, and GsCML45 were identified through leaf transcriptomic analysis of P18B and KF1 under submergence. The qRT-PCR, tobacco leaf transient expression and GUS staining displayed a more obviously induced upregulation of GsCNGC20-f and GsCNGC20-g by submergence stress. BiFC and Y2H assays showed that plasma membrane-located GsCNGC-f could interact with GsCaM4 or GsCaML. Finally, overexpression of GsCNGC20-f could alleviate submergence injury in hairy-root composite soybean plants or transgenic Arabidopsis seedlings by maintaining a higher content of Ca 2+ and activities of PDC and ADH (associated with upregulated gene expression of PDCs and ADHs ), and thereafter enhancing ATP levels in the leaves via improved anaerobic respiration under hypoxic conditions. The functions of GsCNGC20-f involving in the adaptation and survival of G. soja P18B seedlings under submergence stress can provide new perspectives for the molecular breeding of flooding-tolerant soybeans or other crops. • Two potential CNGC genes, GsCNGC20-f and GsCNGC20-g, were identified from the submergence-tolerant G. soja accession (P18B). • The plasma membrane-located GsCNGC-f could interact with GsCaM4 or GsCaML. • GsCNGC20-f could alleviate submergence stress on transgenic soybean or Arabidopsis plants by enhancing anaerobic respiration.

Involvement of mechano-sensitive Piezo1 channel in the differentiation of brown adipocytes

Brown adipocytes expend energy via heat production and are a potential target for the prevention of obesity and related metabolic disorders. Piezo1 is a Ca2+-permeable non-selective cation channel activated by mechanical stimuli. Piezo1 is reported to be involved in mechano-sensation in non-sensory tissues. However, the expression and roles of Piezo1 in brown adipocytes have not been well clarified. Here, we generated a brown adipocyte line derived from UCP1-mRFP1 transgenic mice and showed that Piezo1 is expressed in pre-adipocytes. Application of Yoda-1, a Piezo1 agonist, suppressed brown adipocyte differentiation, and this suppression was significantly attenuated by treatment with a Piezo1 antagonist and by Piezo1 knockdown. Furthermore, the suppression of brown adipocyte differentiation by Yoda-1 was abolished by co-treatment with a calcineurin inhibitor. Thus, these results suggest that activation of Piezo1 suppresses brown adipocyte differentiation via the calcineurin pathway.

Abstract 5201: Enhanced production of IL-2 from anti CD3-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis

Abstract Objectives: It has been reported that Brazilian green propolis (BGP) possesses anti-tumor and chemo-preventive properties against prostate cancer (Szliszaka E, 2011). And bioactive components from propolis have been shown to have anti-cancerous effects upon hepatocellular carcinoma (Liu X, 2021). These observations revealed that propolis can be an anti-cancer drugs, however the immunological effects upon tumor microenvironments of these naturally occurring components has not been elucidated. In this report, we attempt to clarify immune modulatory effects of BGP and the major component, artepillin C, by observing cytokine production from activated T cells. Methods: The mouse spleen cell stimulated by anti-CD3 monoclonal antibody were co-cultured with BGP, artepillin C and HC030031, an antagonist of TRPA1 Ca2+ channel. The productions of IFN-γ, IL-6, IL-17, IL-4, IL-10 and IL-2 was assayed by ELISA. Results: Our results demonstrated the production of IL-2 was markedly enhanced, while the production of IL-4 and IL-10 was not significantly affected; by contrast, the production of IFN-γ, IL-6 and IL-17 were markedly reduced, in the highly viable stimulated spleen cells treated with BGP. These effects were also reproduced in the cells treated with artepillin C, a major component of BGP. The enhancement of IL-2 production by artepillin C was significantly alleviated by adding 10 HC030031, an antagonist of TRPA1 Ca2+ channel, suggesting the enhancement of the cytokine production by artepillin C could be mediated by Ca2+ influx. Conclusions: The artepillin C, a major component of BGP, is an important regulator of activated T lymphocytes and specifically augmented the expression of IL-2 vie Ca2+-permeable cation channel, TRPA1. These effects may exert immunomodulatory effects upon tumor microenvironments. Citation Format: Nobuo Kondoh, Masako Kamiya, Hanemi Tsuruta, Moe Takahashi, Kumiko Ikeno, Kyohei Ueno, Naoki Umemura, Eiji Takayama, Harumi Kawaki, Genjiro Nakamura, Toru Nikaido, Yasunori Muramatsu, Megumi Ando. Enhanced production of IL-2 from anti CD3-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5201.

Enhanced production of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis

ObjectivesIn this report, we attempt to clarify the immune modulatory effects of Brazilian green propolis (BGP) and its major component, artepillin C, on the cytokine production of anti-CD3 antibody-stimulated mouse spleen cells. We also estimate the physiological mechanism affecting artepillin C's upon the cells. MethodsMale C3H/HeN mouse spleen cells stimulated by antiCD3 monoclonal antibody were co-cultured with BGP, artepillin C, and HC030031, a transient receptor potential ankyrin 1 (TRPA1) Ca2+ channel antagonist. The synthesis of interferon (IFN)-γ, interleukin (IL)-6, IL-17, IL-4, IL-10, and IL-2 was assayed by enzyme-linked immunoassay. The expression of IL-2 mRNA and the protein product were examined by reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively. ResultsThe production of IL-2 was markedly enhanced, while that of IL-4 and IL-10 was not significantly affected; by contrast, the production of IFN-γ, IL-6, and IL-17 was significantly reduced in the antibody-stimulated spleen cells treated with BGP at a non-cytostatic concentration. These effects were reproduced in the cells treated with artepillin C. The expression of IL-2 mRNA was unaffected; however, that of the protein was significantly enhanced in the artepillin C-treated cells compared to untreated control cells. The enhancement of protein expression and the production of IL-2 by artepillin C was significantly alleviated by adding HC030031. ConclusionsArtepillin C is an important regulator of cytokine synthesis from activated spleen cells. The agent specifically augmented the expression of IL-2 via the Ca2+-permeable cation channel, TRPA1, at least in part, at the translational or secretion levels.

Structures of a mammalian TRPM8 in closed state

Transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable non-selective cation channel that acts as the primary cold sensor in humans. TRPM8 is also activated by ligands such as menthol, icilin, and phosphatidylinositol 4,5-bisphosphate (PIP2), and desensitized by Ca2+. Here we have determined electron cryo-microscopy structures of mouse TRPM8 in the absence of ligand, and in the presence of Ca2+ and icilin at 2.5–3.2 Å resolution. The ligand-free state TRPM8 structure represents the full-length structure of mammalian TRPM8 channels with a canonical S4-S5 linker and the clearly resolved selectivity filter and outer pore loop. TRPM8 has a short but wide selectivity filter which may account for its permeability to hydrated Ca2+. Ca2+ and icilin bind in the cytosolic-facing cavity of the voltage-sensing-like domain of TRPM8 but induce little conformational change. All the ligand-bound TRPM8 structures adopt the same closed conformation as the ligand-free structure. This study reveals the overall architecture of mouse TRPM8 and the structural basis for its ligand recognition.

Polycystin-2 (TRPP2): Ion channel properties and regulation.

Polycystin-2 (TRPP2, PKD2, PC2) is the product of the PKD2 gene, whose mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). PC2 belongs to the superfamily of TRP (Transient Receptor Potential) proteins that generally function as Ca2+-permeable nonselective cation channels implicated in Ca2+ signaling. PC2 localizes to various cell domains with distinct functions that likely depend on interactions with specific channel partners. Functions include receptor-operated, nonselective cation channel activity in the plasma membrane, intracellular Ca2+ release channel activity in the endoplasmic reticulum (ER), and mechanosensitive channel activity in the primary cilium of renal epithelial cells. Here we summarize our current understanding of the properties of PC2 and how other transmembrane and cytosolic proteins modulate this activity, providing functional diversity and selective regulatory mechanisms to its role in the control of cellular Ca2+ homeostasis.

Structural snapshots of the mechanism of TRPV2 channel activation by small-molecule agonists

Transient receptor potential (TRP) channels are polymodal sensors that play critical roles in various physiological processes in living organisms. These cation-permeable channels respond to a variety of physical and chemical stimuli, including cold and hot temperatures, acidic pH, and mechanical stress, often determining a sensory frontier of defense against hostile environments. Vanilloid (V) subfamily is the most studied category of TRP channels that includes six closely related members: highly calcium-selective TRPV5-6 and non-selective TRPV1-4. A remarkable feature of TRPV1-4 is their ability to sense heat, which makes them temperature-sensitive TRP channels or thermo-TRPs. TRPV channels are associated with a multitude of human diseases, including cancers, chronic pain, cardiovascular, neurological and nociceptive disorders. Despite the great clinical interest, pharmacology of TRPV channels remains largely undeveloped because of insufficient knowledge about the mechanisms of their regulation. For instance, activation of TRPV channels by small molecules or heat remains poorly understood. Numerous identified TRPV channel agonists, while effective in physiological experiments, appear limited in their ability to act in the conditions of structural biology experiments. In this regard, the recent study by Pumroy et al. [1] makes a significant contribution towards our understanding of TRPV2 structural dynamics that leads to opening of this channel in physiological conditions.

Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

Skin diseases are common human illnesses that occur in all cultures, at all ages, and affect between 30% and 70% of individuals globally. TRPV3 is a cation-permeable TRP channel predominantly expressed in skin keratinocytes, implicated in cutaneous sensation and associated with numerous skin diseases. TRPV3 is inhibited by the local anesthetic dyclonine, traditionally used for topical applications to relieve pain and itch. However, the structural basis of TRPV3 inhibition by dyclonine has remained elusive. Here we present a cryo-EM structure of a TRPV3-dyclonine complex that reveals binding of the inhibitor in the portals which connect the membrane environment surrounding the channel to the central cavity of the channel pore. We propose a mechanism of TRPV3 inhibition in which dyclonine molecules stick out into the channel pore, creating a barrier for ion conductance. The allosteric binding site of dyclonine can serve as a template for the design of new TRPV3-targeting drugs.

Inhibition of TRPC4 channel activity in colonic myocytes by tricyclic antidepressants disrupts colonic motility causing constipation

Tricyclic antidepressants (TCAs) have been used to treat depression and were recently approved for treating irritable bowel syndrome (IBS) patients with severe or refractory IBS symptoms. However, the molecular mechanism of TCA action in the gastrointestinal (GI) tract remains poorly understood. Transient receptor potential channel canonical type 4 (TRPC4), which is a Ca2+‐permeable nonselective cation channel, is a critical regulator of GI excitability. Herein, we investigated whether TCA modulates TRPC4 channel activity and which mechanism in colonic myocytes consequently causes constipation. To prove the clinical benefit in patients with diarrhoea caused by TCA treatment, we performed mechanical tension recording of repetitive motor pattern (RMP) in segment, electric field stimulation (EFS)‐induced and spontaneous contractions in isolated muscle strips. From these recordings, we observed that all TCA compounds significantly inhibited contractions of colonic motility in human. To determine the contribution of TRPC4 to colonic motility, we measured the electrical activity of heterologous or endogenous TRPC4 by TCAs using the patch clamp technique in HEK293 cells and murine colonic myocytes. In TRPC4‐overexpressed HEK cells, we observed TCA‐evoked direct inhibition of TRPC4. Compared with TRPC4‐knockout mice, we identified that muscarinic cationic current (mI cat) was suppressed through TRPC4 inhibition by TCA in isolated murine colonic myocytes. Collectively, we suggest that TCA action is responsible for the inhibition of TRPC4 channels in colonic myocytes, ultimately causing constipation. These findings provide clinical insights into abnormal intestinal motility and medical interventions aimed at IBS therapy.