TRPM8 Channels Research Articles

A hydrophobic funnel governs monovalent cation selectivity in the ion channel TRPM5

A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting transient receptor potential (TRP) superfamily of ion channels forms a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca2+, TRPM4 and TRPM5 are unique due to their strong monovalent selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent selectivity by in silico electrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity alongside the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance (“hydrophobic funnel”) enables monovalent but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a cooperative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favorably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5.

Search for molecular markers of joint hypermobility

Background: Joint hypermobility (JH) is a condition that can be either benign or accompanied by pain and early development of osteoarthritis. For early verification of the risk of complications, it is necessary to study the molecular pathogenesis of various JH variants. The aim of the study: The aim of this study was to search for candidate genes involved in connective tissue metabolism and causing joint hypermobility in a form isolated or combined with connective tissue dysplasia (CTD). Materials and methods: The study involved young (21,86±0,22 y.o.) people (n=181). A clinical study was conducted for the presence of signs of JH (Beighton scale) and CTD (tables Kadurina T.I.). Further, a molecular genetic study was carried out and the search for associations of polymorphic variants of genes (vitamin D receptor (VDR), lumican (LUM), tenascin (TNXB), growth/differentiation factor 5 (GDF5), magnesium ion channel TRPM6), bone morphogenetic protein type 5 (BMP5)) and JH, CTD in isolated and combined forms. Statistical data processing was carried out by comparing qualitative features using the χ2 test, Fisher's exact test, and the χ2 test with Yates' correction for contingency tables 2x2. The strength of associations was assessed in terms of the odds ratio (Odds Ratio, OR, at p<0.05), the correction for multiple comparisons was carried out using the FDR method (Benjamin- Hochberg). Quantitative data were evaluated in pairs using the Student's t-test, the normality of distribution according to the Kolmogorov-Smirnov test. Results: Associations of the G allele and the GG genotype of the rs3734444 locus of the BMP5 gene with the presence of JH (OR=3.70 and OR=5.10), the A allele of the rs1470527 locus of the BMP5 gene and the presence of JH+CTD- (OR=8.00), the T allele and the TT genotype of the rs11144134 locus of the TRPM6 gene with JH in isolated (OR=3.00 and OR =10.19) and in the forms combined with CTD (OR=3.17 and OR=11.28), the TT genotype was also associated with the isolated form of CTD (OR=3.74). The GT genotype of the rs73611720 locus of the GDF5 gene was associated with isolated CTD (OR=4.15). No statistically significant associations were found for polymorphic variants rs11540149 (VDR), rs2268578 and rs3759222 (LUM), rs3130342 (TNXB). Conclusion: The G allele of locus rs3734444 of the BMP5 gene is a potential risk marker for JH in general, the A allele of locus rs1470527 is an isolated JH, the GT genotype of locus rs73611720 of the GDF5 gene is a marker for isolated CTD, the T allele of locus rs11144134 of the TRPM6 gene is a marker for JH combined with CTD.

A frozen portrait of a warm channel

In order to understand protein function, the field of structural biology makes extensive use of cryogenic electron microscopy (cryo-EM), a technique that enables structure determination at atomic resolution following embedding of protein particles in vitreous ice. Considering the profound effects of temperature on macromolecule function, an important—but often neglected-question is how the frozen particles relate to the actual protein conformations at physiological temperatures. In a recent study, Hu et al. compare structures of the cation channel TRPM4 “frozen” at 4 °C versus 37 °C, revealing how temperature critically affects the binding of activating Ca2+ ions and other channel modulators.

Structural dynamics at cytosolic interprotomer interfaces control gating of a mammalian TRPM5 channel

The transient receptor potential melastatin (TRPM) tetrameric cation channels are involved in a wide range of biological functions, from temperature sensing and taste transduction to regulation of cardiac function, inflammatory pain, and insulin secretion. The structurally conserved TRPM cytoplasmic domains make up >70 % of the total protein. To investigate the mechanism by which the TRPM cytoplasmic domains contribute to gating, we employed electrophysiology and cryo-EM to study TRPM5-a channel that primarily relies on activation via intracellular Ca2+. Here, we show that activation of mammalian TRPM5 channels is strongly altered by Ca2+-dependent desensitization. Structures of rat TRPM5 identify a series of conformational transitions triggered by Ca2+ binding, whereby formation and dissolution of cytoplasmic interprotomer interfaces appear to control activation and desensitization of the channel. This study shows the importance of the cytoplasmic assembly in TRPM5 channel function and sets the stage for future investigations of other members of the TRPM family.

Latest Insights into the In Vivo Studies in Murine Regarding the Role of TRP Channels in Wound Healing-A Review.

Wound healing involves physical, chemical and immunological processes. Transient receptor potential (TRP) and other ion channels are implicated in epidermal re-epithelization. Ion movement across ion channels can induce transmembrane potential that leads to transepithelial potential (TEP) changes. TEP is present in epidermis surrounding the lesion decreases and induces an endogenous direct current generating an epithelial electric field (EF) that could be implicated in wound re-epithelialization. TRP channels are involved in the activation of immune cells during mainly the inflammatory phase of wound healing. The aim of the study was to review the mechanisms of ion channel involvement in wound healing in in vivo experiments in murine (mice, rats) and how can this process be influenced. This review used the latest results published in scientific journals over the last year and this year to date (1 January 2023-31 December 3000) in order to include the in-press articles. Some types of TRP channels, such as TRPV1, TRPV3 and TRPA1, are expressed in immune cells and can be activated by inflammatory mediators. The most beneficial effects in wound healing are produced using agonists of TRPV1, TRPV4 and TRPA1 channels or by inhibiting with antagonists, antisense oligonucleotides or knocking down TRPV3 and TRPM8 channels.

TRPM4-Inspired Polymeric Nanochannels with Preferential Cation Transport for High-Efficiency Salinity-Gradient Energy Conversion.

Biological ion channels exhibit switchable cation transport with ultrahigh selectivity for efficient energy conversion, such as Ca2+-activated TRPM4 channels tuned by cation-π interactions, but achieving an analogous highly selective function is challenging in artificial nanochannels. Here, we design a TRPM4-inspired cation-selective nanochannel (CN) assembled by two poly(ether sulfone)s, respectively, with sulfonate acid and indole moieties, which act as cation-selective activators to manage Na+/Cl- selectivity via ionic and cation-π interactions. The cation selectivity of CNs can be activated by Na+, and thereby the Na+ transference number significantly improves from 0.720 to 0.982 (Na+/Cl- selectivity ratio from 2.6 to 54.6) under a 50-fold salinity gradient, surpassing the K+ transference number (0.886) and Li+ transference number (0.900). The TRPM4-inspired nanochannel membrane enabled a maximum output power density of 5.7 W m-2 for salinity-gradient power harvesting. Moreover, a record energy conversion efficiency of up to 46.5% is provided, superior to most nanochannel membranes (below 30%). This work proposes a novel strategy to biomimetic nanochannels for highly selective cation transport and high-efficiency salinity-gradient energy conversion.

History of TRP channels and lower urinary tract function and dysfunction

The history of TRP channels and bladder started in the 1980-ies with the studies of Maggi and collaborators, who created the basis for using capsaicin and other vanilloids in LUT disorders. This narrative review gives a brief history of bladder TRP channels, from their discovery, distribution, and function in the bladder to the rationale for and current status of the use of agents acting on them in treating LUT dysfunction. The most studied TRP channels related to physiological and pathological bladder conditions are the TRPV1, TRPA1, TRPV4, and TRPM8 channels Their preclinical rationale and potential as targets for effective therapies are attractive. However, even if the results with agents blocking these channels have been promising in experimental models of LUT disorders, this has not resulted in any clinically useful treatments. Thus, the history of TRP channels, their involvement in the physiology and pathophysiology of the LUT, and the search for TRP channel-active drugs for treating LUT disorders are ongoing. Further research will reveal if the promises of these agents will be fulfilled.

Roles of Thermosensitive Transient Receptor Channels TRPV1 and TRPM8 in Paclitaxel-Induced Peripheral Neuropathic Pain.

Paclitaxel, a microtubule-stabilizing chemotherapy drug, can cause severe paclitaxel-induced peripheral neuropathic pain (PIPNP). The roles of transient receptor potential (TRP) ion channel vanilloid 1 (TRPV1, a nociceptor and heat sensor) and melastatin 8 (TRPM8, a cold sensor) in PIPNP remain controversial. In this study, Western blotting, immunofluorescence staining, and calcium imaging revealed that the expression and functional activity of TRPV1 were upregulated in rat dorsal root ganglion (DRG) neurons in PIPNP. Behavioral assessments using the von Frey and brush tests demonstrated that mechanical hyperalgesia in PIPNP was significantly inhibited by intraperitoneal or intrathecal administration of the TRPV1 antagonist capsazepine, indicating that TRPV1 played a key role in PIPNP. Conversely, the expression of TRPM8 protein decreased and its channel activity was reduced in DRG neurons. Furthermore, activation of TRPM8 via topical application of menthol or intrathecal injection of WS-12 attenuated the mechanical pain. Mechanistically, the TRPV1 activity triggered by capsaicin (a TRPV1 agonist) was reduced after menthol application in cultured DRG neurons, especially in the paclitaxel-treated group. These findings showed that upregulation of TRPV1 and inhibition of TRPM8 are involved in the generation of PIPNP, and they suggested that inhibition of TRPV1 function in DRG neurons via activation of TRPM8 might underlie the analgesic effects of menthol.

TRPM2 channels are essential for regulation of cytokine production in lung interstitial macrophages.

Interstitial macrophages (IMs) are essential for organ homeostasis, inflammation, and autonomous immune response in lung tissues, which are achieved through polarization to a pro-inflammatory M1 and an M2 state for tissue repair. Their remote parenchymal localization and low counts, however, are limiting factors for their isolation and molecular characterization of their specific role during tissue inflammation. We isolated viable murine IMs in sufficient quantities by coculturing them with stromal cells and analyzed mRNA expression patterns of transient receptor potential (TRP) channels in naïve and M1 polarized IMs after application of lipopolysaccharide (LPS) and interferon γ. M-RNAs for the second member of the melastatin family of TRP channels, TRPM2, were upregulated in the M1 state and functional channels were identified by their characteristic currents induced by ADP-ribose, its specific activator. Most interestingly, cytokine production and secretion of interleukin-1α (IL-1α), IL-6 and tumor necrosis factor-α in M1 polarized but TRPM2-deficient IMs was significantly enhanced compared to WT cells. Activation of TRPM2 channels by ADP-ribose (ADPR) released from mitochondria by ROS-produced H2O2 significantly increases plasma membrane depolarization, which inhibits production of reactive oxygen species by NADPH oxidases and reduces cytokine production and secretion in a negative feedback loop. Therefore, TRPM2 channels are essential for the regulation of cytokine production in M1-polarized murine IMs. Specific activation of these channels may promote an anti-inflammatory phenotype and prevent a harmful cytokine storm often observed in COVID-19 patients.

Physiological temperature drives TRPM4 ligand recognition and gating

Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca2+-activated ion channel3–7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a ‘warm’ conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca2+-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)8 and ATP (an inhibitor)9, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.

Loss of the TRPM4 channel in humans causes immune dysregulation with defective monocyte migration

BackgroundTransient receptor potential melastatin subfamily member 4 (TRPM4) is a broadly expressed, calcium-activated, monovalent cation channel that regulates immune cell function in mice and cell lines. Clinically however, partial loss or gain of function mutations in TRPM4 lead to arrythmia and heart disease, with no documentation of immunological disorders. ObjectiveTo characterize the functional cellular mechanisms underlying the immune dysregulation phenotype in a proband with a mutated trpm4 gene. MethodsWe employ a combination of biochemical, cell biological, imaging, omics analyses, flow cytometry, and gene editing approaches. ResultsWe report the first human cases with complete loss of the TRPM4 channel leading to immune dysregulation with frequent bacterial and fungal infections. Single cell and bulk RNAseq point to altered expression of genes affecting cell migration, specifically in monocytes. Inhibition of TRPM4 in T cells and the THP1 monocyte cell line reduces migration. More importantly primary T cells and monocytes from the TRPM4 patients migrate poorly. Finally, CRISPR knockout of TRPM4 in THP1 cells greatly reduces their migration potential. ConclusionOur results demonstrate that TRPM4 plays a critical role in regulating immune cell migration, leading to increased susceptibility to infections.

Brain‐Targeted 9‐Phenanthrol‐Loaded Lipid Nanoparticle Prevents Brain Edema after Cerebral Ischemia‐Reperfusion Injury by Inhibiting the Trpm4 Channel in Mice

AbstractBrain edema robustly increases mortality and hinders functional recovery after acute ischemic stroke. However, there are currently no effective therapies available for treating or preventing it. The unchecked opening of the transient receptor potential M4 (TRPM4) channel results in an excessive influx of Na+ and water, which contributes significantly to the formation of brain edema after ischemic stroke. 9‐phenanthrol (9‐Phe), a potent TRPM4 inhibitor, has limited clinical applicability due to its potential cytotoxicity and poor solubility. A brain‐targeting T7 (HAIYPRH)‐modified lipid nanoparticle (LNP) encapsulated 9‐Phe (9‐Phe@T7‐LNP) is designed and synthesized to improve the physicochemical properties and pharmacokinetic properties of 9‐Phe for treating brain edema in vivo. These results demonstrated that 9‐Phe@T7‐LNP can penetrate the intact blood‐brain barrier (BBB) in normal mice and target the brain parenchyma. Moreover, 9‐Phe@T7‐LNP effectively reduced infarct volume and brain edema, prevented neuronal loss and BBB disruption, improved survival, and facilitated neurological function recovery after transient middle cerebral artery occlusion in mice. Additionally, 9‐Phe@T7‐LNP scavenged oxygen‐free radicals and prevented neuronal apoptosis in cultured neurons subjected to oxygen and glucose deprivation/reperfusion. In summary, these findings showed that 9‐Phe@T7‐LNP holds strong potential as a promising targeted therapy for brain edema after stroke, providing superior pharmacological neuroprotection against brain edema.

The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

TRPM3-mediated inhibition of active Na-K transport by the ocular lens epithelium

Purpose: The TRPM3 channel is selectively expressed in the lens epithelium, a monolayer at the anterior lens surface, but undetectable in fiber cells that make up the lens bulk. A point mutation in TRPM3 gene (substitution of isoleucine by methionine at codon 65; I65M) causes early onset autosomal dominant cataract in humans and in mice. From studies on transgenic mice, it is apparent the I65M TRPM3 mutation disrupts lens Na-K homeostasis. However, the normal role of lens TRPM3 in the lens is currently unknown. Here, we explore the lens response to TRPM3 activation. Methods: Studies were carried out using intact mouse lenses, cultured mouse lens epithelial cells and a human lens epithelium cell line (HLEB3). Active Na-K transport was studied by using rubidium (Rb) as a tracer for potassium entry. Rb was detected by atomic absorption spectrophotometry. Intracellular calcium was measured in nonconfluent single cells by Fura-2 ratiometric imaging. Results: Rb uptake was measured over a period of 10 min in intact mouse lenses exposed to a TRPM3 agonist CIM0216 (10 μM). CIM0216 reduced the rate of Rb uptake by ~20% (Ctrl 6.4±0.4 vs CIM 4.5±0.4 mmoles/kg lens dry weight/10 min, n=7, p<.01). The concentration dependence of the response to CIM0216 (1 to 50 μM) was examined in primary cultured mouse lens epithelium and Rb uptake was reduced by CIM0216 concentrations > 10 μM. The TRPM3 agonist reduced Rb uptake to a similar extent (~20%) in mouse lens epithelium, HLEB3 cells and HLEB3 cells that had been stably transfected with the I65M mutant TRPM3. However, the mutant cells responded to CIM0216 at a 10-fold lower concentration than WT mouse and HLEB3 cells. The Rb uptake response to CIM0216 was significantly inhibited by a TRPM3 antagonist, primidone (50 μM), and was abolished when cells were bathed in calcium-free solution. To study calcium entry, Fura2-loaded mouse lens epithelial cells were exposed to the TRPM3 agonist pregnenolone (100 μM). Pregnenolone caused an immediate increase in cytoplasmic calcium that was inhibited by primidone (Peak calcium concentration Ctrl 201±37 vs primidone 137±8 nM, n=5, p<.001). In cultures lens epithelium, agents that increase cellular cAMP, including IBMX, forskolin and 8p-CPT-cAMP, all caused a marked reduction of Rb uptake. Conclusions: TRPM3 activation causes a significant reduction in the rate of active Na-K transport and the response is associated with calcium entry. Because most lens Rb uptake is ouabain-sensitive, the response points to Na,K-ATPase inhibition. Studies are under way to test whether the Na-K transport response to TRPM3 activation involves cAMP signaling. This work was supported by NIH grants R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A comparative study of carotid body chemosensitivity in lean and obese mice: Implication for obesity induced hypertension

Obesity is associated with increased circulating level of the hormone leptin. Previously we have shown in mice that, leptin induces hypertension acting on transient receptor potential melastatin 7 (Trpm7) channels in the carotid body (CB), a multi-modal peripheral chemoreceptor organ that controls breathing and drives sympathetic tone. Further, in mice leptin augments hypoxic ventilatory response which is abolished by CB denervation. This suggests a potential involvement of CB chemosensitivity. However, no comparative study of CB chemosensory responses has been conducted in lean and obese mice to support this notion. Based on these premises, we hypothesize that obese mice should have higher leptin induced CB chemosensory responses during normoxia and hypoxia compared to lean mice, and Trpm7 receptor antagonist should abolish/reduce these responses. To prove this, we measured afferent carotid sinus nerve (CSN) activity, using a novel ex vivo perfused mouse (male) CB preparation from lean (b.w.; 35 ± 0.5 gm) and diet induced obese (DIO) mice (b.w.; 53 ± 1.5gm) with C57BL/6J background. We found that compared to lean mice; (a) CSN response to hypoxia (PO2 =60 Torr) was 50% more in obese mice, (b) in presence of leptin (50nM), CSN responses to normoxia (PO2=100Torr) and hypoxia were augmented 50% and 150% more respectively in obese mice. Finally, leptin induced increase in CSN responses were partially reduced by Trpm7 antagonist FTY720 (3μM) in obese compared to lean mice. In conclusion, CB chemosensitivity is enhanced in obese compared to lean mice and CB Trpm7 channel could be a therapeutic target for obesity related hypertension. Grant support: NIH R01 HL1331000. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pharmacological and genetic inhibition of transient receptor potential melastatin-subfamily member 7 (Trpm7) channel in the carotid body attenuated intermittent hypoxia-induced hypertension

RATIONALE: Obesity causes hypertension and sleep disordered breathing (SDB). Intermittent hypoxia (IH), a cardinal feature of SDB, also causes hypertension contributing to cardiovascular sequelae of obesity. The carotid body plays a role in the IH-induced hypertension. We have previously shown that obesity increases expression of the transient receptor potential melastatin-subfamily member 7 (TRPM7) channel in CB glomus cells and that knockdown of TRPM7 in CB treats hypertension in diet-induced obese mice. Our data in cell culture indicate that hypoxia increases TRPM7 expression. Therefore, we hypothesized that IH increases TRPM7 expression in CB and that pharmacological and genetic inhibition of Trpm7 in CB will treat IH-induced hypertension. Methods: 1) Pharmacological inhibition: We exposed male C57BL/6J mice to IH (n = 9) or air (n = 9) for 5 days and obtained continuous BP telemetry recording prior and after the exposure. Then we applied a long-acting TRPM7 inhibitor FTY720 or placebo to the CB area and repeated BP measurements before and after the 5-day IH or air exposure.2) Genetic inhibition: In another experiment, we knocked out Trpm7 gene expression in CB of male Trpm7 flox/flox mice by administering adenovirus carrying Cre-recombinase to the CB followed by BP recording during the 5-day IH or air exposure. RESULTS: We found that IH induced > 20-fold increase in Trpm7 mRNA expression in CB. IH increased BP from 102.9 ± 1.0 mmHg to 111.7 ± 1.1 mmHg. FTY720 hydrogel abolished the BP increase induced by IH. In Trpm7 flox/flox mice, IH also markedly increased BP prior to the adenoviral treatment from 101.9 ± 1.6 mmHg to 110.2 ± 4.0 mmHg. CB-specific Trpm7 knockout abolished IH-induced hypertension. CONCLUSION: We conclude that IH-induced upregulation of Trpm7 in CB contributes to the development of hypertension in IH and that IH-induced hypertension can be treated by pharmacological and genetic inhibition of Trpm7. 19CDA34700025, R01HL133100, R01HL128970. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanosensor channels of the lens and ciliary body: patch clamp studies

Epithelia from the lens and the ciliary body possess mechanosensors, including Piezo1 and transient receptor potential (TRP) channels, that may partake in the regulation of lens and aqueous humor volume. Although these mechanosensor channels commonly allow calcium entry, the outcomes of elevated intracellular calcium vary with the type of channel activated. For instance, separate activation of TRPV4 and TRPV1 increases Na-K-ATPase and Na/K/2Cl cotransporter activities, respectively. To further understand the role of mechanosensitivity in lens and ciliary body, we are currently using patch clamp techniques to characterize the response of primary cultured cells to chemical agonists and antagonists, as well as direct mechanical stimulation. Previously, we found that in mouse lens epithelial cells (mLEC), TRPV4 activation by agonist GSK1016790A (GSK) is followed by disappearance of a fast, transient K+-like outward current (Ipeak), decrease of a steady state outward current (Iss), fast depolarization of resting membrane potential (RMP) values from near -50 mV to near 0 mV, increase of Cx43 hemichannel-like events, and increase of overall membrane conductance ( gm) at holding potential (HP) = -50mV. More recently, we found that in mLEC, shear stress (streaming of external solution from a pipette tip over the cell membrane) reduced Ipeak but did not cause large RMP changes. Porcine non-pigmented epithelial cells (pNPE) in normal conditions display no Ipeak, small Iss, small RMP near -20 mV and spontaneous Cx43 hemichannel-like event activity. Nevertheless, GSK caused further depolarization of pNPE cells, and while no gm changes were seen when GSK was applied while holding cells at -50 mV, gm did increase when GSK exposure occurred during repeated ±80 mV pulses. Preliminary experiments show that activation of TRPM3 by its agonist pregnenolone causes a minimal hyperpolarization in mLEC. In comparison, in a line of human LEC (HLE B3) expressing TRPM3 channels, and which appear generally depolarized, pregnenolone caused a proportionally larger shift toward more negative RMP values; more intriguing, GSK has no measurable effect on the gm of HLE B3 cells. Taken together, and apart from considerations of cell- and species-specific differences, the data suggest that mechanosensor channels have different and/or opposite effects on the regulation of membrane ion channel activity, and thus on cell function. R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evolutionary trajectory of TRPM2 channel activation by adenosine diphosphate ribose and calcium

Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance. TRPM2, a typical ancient ion channel, is activated by adenosine diphosphate ribose (ADPR) and calcium and its activation has evolved from a simple mode in invertebrates to a more complex one in vertebrates, but the evolutionary process is still unknown. Molecular evolutionary analysis of TRPM2s from more than 280 different animal species has revealed that, the C-terminal NUDT9-H domain has evolved from an enzyme to a ligand binding site for activation, while the N-terminal MHR domain maintains a conserved ligand binding site. Calcium gating pattern has also evolved, from one Ca2+-binding site as in sea anemones to three sites as in human. Importantly, we identified a new group represented by olTRPM2, which has a novel gating mode and fills the missing link of the channel gating evolution. We conclude that the TRPM2 ligand binding or activation mode evolved through at least three identifiable stages in the past billion years from simple to complicated and coordinated. Such findings benefit the evolutionary investigations of other channels and proteins.

Magnesium Ions Promote the Induction of Immunosuppressive Bone Microenvironment and Bone Repair through HIF-1α-TGF-β Axis in Dendritic Cells.

The effect of immunoinflammation on bone repair during the recovery process of bone defects needs to be further explored. It is reported that Mg2+ can promote bone repair with immunoregulatory effect, but the underlying mechanism on adaptive immunity is still unclear. Here, by using chitosan and hyaluronic acid-coated Mg2+ (CSHA-Mg) in bone-deficient mice, it is shown that Mg2+ can inhibit the activation of CD4+ T cells and increase regulatory T cell formation by inducing immunosuppressive dendritic cells (imDCs). Mechanistically, Mg2+ initiates the activation of the MAPK signaling pathway through TRPM7 channels on DCs. This process subsequently induces the downstream HIF-1α expression, a transcription factor that amplifies TGF-β production and inhibits the effective T cell function. In vivo, knock-out of HIF-1α in DCs or using a HIF-1α inhibitor PX-478 reverses inhibition of bone inflammation and repair promotion upon Mg2+-treatment. Moreover, roxadustat, which stabilizes HIF-1α protein expression, can significantly promote immunosuppression and bone repair in synergism with CSHA-Mg. Thus, the findings identify a key mechanism for DCs and its HIF-1α-TGF-β axis in the induction of immunosuppressive bone microenvironment, providing potential targets for bone regeneration.

Anti-Nociceptive Effects of Sphingomyelinase and Methyl-Beta-Cyclodextrin in the Icilin-Induced Mouse Pain Model.

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.