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Objective: To evaluate the effects of uncoupling protein (UCP) 1, UCP2 and UCP3 gene variants on body composition and metabolic changes in response to chronic overfeeding and the recovery after the period of overfeeding. Subjects and design: Twenty-four normal weight men (21±2 y), who constituted 12 pairs of identical twins, ate a 4.2 MJ/day energy surplus, 6 days a week, during a period of 100 days. The subjects were asked to return to the laboratory for testing at 4 months and for a final examination 5 y after completion of the overfeeding protocol. Methods: Resting metabolic rate (RMR) measurements were performed before and after overfeeding. A 4.2 MJ test meal was consumed, after which calorimetric measurements were continued for 240 min. Total body fat was assessed by hydrodensitometry and total subcutaneous fat by the sum of eight skinfolds. Polymorphisms were typed by PCR and PCR-RFLP-techniques. Thyroid stimulating hormone (TSH) concentrations after a thyrotropin releasing hormone (TRH) injection were measured by radioimmunoassay (RIA). Results: The changes in body weight and adiposity were not different between UCP1 Bcl I, UCP2 alanine to valine (A55V), UCP2 insertion/deletion (I/D) or UCP3 Rsa I genotypes. However, the recovery from overfeeding was worse among G-allele carriers of the UCP1 Bcl I, I allele non-carriers of the UCP2 I/D, AV heterozygote subjects of the UCP2 A55V and CC subjects of the UCP3 Rsa I polymorphisms. RMR was lower both before (P=0.01) and after (P=0.001) overfeeding in subjects with the CC genotype of the UCP3 Rsa I polymorphism. Moreover, after overfeeding, the UCP2 A55V heterozygote and UCP3 Rsa I CC homozygote subjects had significantly higher respiratory quotient (RQ) values at rest (P

Dry eye disease (DED) which affects millions of people worldwide is an ocular surface disease that is strongly associated with pain, discomfort, and visual disturbances. Altered tear film dynamics, hyperosmolarity, ocular surface inflammation, and neurosensory abnormalities are the key contributors to DED pathogenesis. The presence of discordance between signs and symptoms of DED in patients and refractoriness to current therapies in some patients underpin the need for studying additional contributors that can be modulated. The presence of electrolytes or ions including sodium, potassium, chloride, bicarbonate, calcium, and magnesium in the tear fluid and ocular surface cells contribute to ocular surface homeostasis. Ionic or electrolyte imbalance and osmotic imbalance have been observed in DED and feed-forward interaction between ionic imbalances and inflammation alter cellular processes in the ocular surface resulting in DED. Ionic balances in various cellular and intercellular compartments are maintained by dynamic transport via ion channel proteins present in cell membranes. Hence, alterations in the expression and/or activity of about 33 types of ion channels that belong to voltage-gated channels, ligand-gated channels, mechanosensitive ion channel, aquaporins, chloride ion channel, sodium-potassium-chloride pumps or cotransporters have been investigated in the context of ocular surface health and DED in animal and/or human subjects. An increase in the expression or activity of TRPA1, TRPV1, Nav1.8, KCNJ6, ASIC1, ASIC3, P2X, P2Y, and NMDA receptor have been implicated in DED pathogenesis, whereas an increase in the expression or activity of TRPM8, GABAA receptor, CFTR, and NKA have been associated with resolution of DED.

Peripheral neuropathy is one of the major adverse effects that limit the clinical application of bortezomib (BTZ). However, the underlying mechanisms of BTZ-induced peripheral neuropathy (BIPN) remain elusive. To examine cell types potentially involved in the development of BIPN, we used four purified cultures of cells of the peripheral nervous system: Schwann cells (SCs), satellite glial cells (SGCs), macrophages, and dorsal root ganglion (DRG) neurons. Administration of a low BTZ concentration (5 nM; similar to concentrations in clinical use) caused dedifferentiation of cultured SCs, returning mature SCs to an immature state. In cultured SGCs, BTZ increased glial fibrillary acidic protein (GFAP) levels without inducing the release of inflammatory cytokines or chemokines. In macrophages, BTZ caused little inflammatory response. Finally, in DRG neurons, BTZ strongly suppressed the expression levels of sensor and transducer ion channels without affecting cell morphology. Taken together, low concentrations of BTZ can cause SC dedifferentiation (i.e., demyelination), increased GFAP level in SGC, and decreased expression levels of sensor and transducer ion channels in DRG neurons (i.e., numbness feeling). Thus, we have reported, for the first time, specific effects of BTZ on peripheral nervous system cells, thereby contributing to a better understanding of the initiating mechanism of BIPN.

Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently reported as an ion channel in neurons for mechanosensing and pain sensing, is also distributed in diverse non-neuronal tissues, such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in a complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes, we found that TACAN inhibits the channel activity of PKD2 gain-of-function mutant F604P. TACAN fragments containing the first and last transmembrane domains interacted with the PKD2 C- and N-terminal fragments, respectively. The TACAN N-terminus acted as a blocking peptide, and TACAN inhibited the function of PKD2 by the binding of PKD2 with TACAN. By patch clamping in mammalian cells, we found that TACAN inhibits both the single-channel conductance and the open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechanosensitivity of the PKD2-TACAN channel complex. KEY POINTS: TACAN inhibits the function of PKD2 in vitro and in vivo. TACAN N-terminal S1-containing fragment T160X interacts with the PKD2 C-terminal fragment N580-L700, and its C-terminal S6-containing fragment L296-D343 interacts with the PKD2 N-terminal A594X. TACAN inhibits the function of the PKD2 channel by physical interaction. The complex of PKD2 with TACAN, but not PKD2 alone, confers mechanosensitivity.

Inflammation can contribute to the initiation and progression of atrial fibrillation (AF), and pinocembrin can suppress downstream inflammatory cytokine production by inhibiting the inflammation pathway. In our previous studies, pinocembrin was also beneficial in ameliorating cardiac arrhythmia in different models of rats, such as depression, myocardial infarction, and heart failure. This study aims to investigate the effect of pinocembrin on the susceptibility to AF in isoproterenol-induced rats.Rats were randomly divided into four groups. Pinocembrin was injected through the tail vein. Isoproterenol was treated by intraperitoneal injection for one week (5 mg/kg/day). We evaluated the susceptibility to AF by atrial electrophysiological experiments. Masson staining was used to evaluate the fibrosis area. The protein levels of connexin (Cx) 40, Cav1.2, Kv4.2, collagen I, collagen III, α-SMA, transforming growth factor (TGF)-β, NLRP3, caspase 1, and interleukin (IL)-1β were detected by western blot.Our data demonstrated that pinocembrin could prolong the atrial effective refractory period (ERP) and action potential duration (APD), and decrease AF inducibility. Isoproterenol increased the expression of Cav1.2 and Kv4.2 ion channels whereas pinocembrin could alleviate this change. Pinocembrin could reduce the fibrosis area, fibrosis-related protein collagen I, collagen III, α-SMA, and TGF-β and upregulate gap junction protein Cx40. In addition, pinocembrin reduced the expression of NLRP3, caspase 1, and IL-1β.Our study indicated that pinocembrin was beneficial to alleviate atrial electrical remodeling and fibrosis. Accompanied the downregulation of ion channels and upregulation of gap junction protein Cx40. Pinocembrin may produce these effects by inhibiting the NLRP3 pathway.

In the almost 70 years since the first hints of its existence, the phosphoinositide, phosphatidyl-D-myo-inositol 4,5-bisphosphate has been found to be central in the biological regulation of plasma membrane (PM) function. Here, we provide an overview of the signaling, transport and structural roles the lipid plays at the cell surface in animal cells. These include being substrate for second messenger generation, direct modulation of receptors, control of membrane traffic, regulation of ion channels and transporters, and modulation of the cytoskeleton and cell polarity. We conclude by re-evaluating PI(4,5)P2’s designation as a signaling molecule, instead proposing a cofactor role, enabling PM-selective function for many proteins.

Organisms of all phyla express mechanosensitive ion channels with a wide range of physiological functions. In recent years, several classes of mechanically gated ion channels have been identified. Some of these ion channels are intrinsically mechanosensitive. Others depend on accessory proteins to regulate their response to mechanical force. The mechanotransduction machinery of cochlear hair cells provides a particularly striking example of a complex force-sensing machine. This molecular ensemble is embedded into a specialized cellular compartment that is crucial for its function. Notably, mechanotransduction channels of cochlear hair cells are not only critical for auditory perception. They also shape their cellular environment and regulate the development of auditory circuitry. Here, we summarize recent discoveries that have shed light on the composition of the mechanotransduction machinery of cochlear hair cells and how this machinery contributes to the development and function of the auditory system.

Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure, as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from patients with PAH, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in patients with pulmonary hypertension, and in lung tissue of monocrotaline (MCT) and sugen-hypoxia-induced PH (SuHx) PAH rat models, as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia, and TGFβ stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT, and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased the expression of vascular smooth muscle markers ACTA2, SM22a, and calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodeling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.NEW & NOTEWORTHY The mechanosensory ion channel Piezo2 is exclusively expressed in lung microvascular endothelial cells (MVECs). Patient MVECs as well as animal models of pulmonary (arterial) hypertension showed lower expression of Piezo2 in the lung. Mechanistically, Piezo2 is required for calcium influx and NO production in response to shear stress, whereas stimuli known to induce endothelial to mesenchymal transition (EndMT) reduce Piezo2 expression in MVECs, and Piezo2 knockdown induces a gene and protein expression pattern consistent with EndMT.

Plasmonic absorption of light can create significant local heat and has become a promising tool for rapid temperature regulation in diverse fields, from biomedical technology to optoelectronics. Current plasmonic heating usually relies on specially designed nanomaterials randomly distributed in the space and barely provides uniform temperature regulation in a wide field. Herein, we report a rapid temperature regulation strategy on a plain gold-coated glass slip using a plasmonic scattering microscopy, which can be referred to as wide-field plasmonic thermal microscopy (W-PTM). We calibrated the W-PTM by monitoring the phase transition of the temperature-sensitive polymer solutions, showing that it can provide a temperature regulation range of 33-80 °C. Moreover, the W-PTM provides imaging capability, thus allowing the statistical analysis of the phase-transitioned polymeric nanoparticles. Finally, we demonstrated that W-PTM can be used for noninvasive and local regulation of the transient receptor potential vanilloid 1 (TRPV1) ion channels in the living cells, which can be monitored by simultaneous fluorescence imaging of the calcium influx. With the nondestructive local temperature-regulating and concurrent fluorescence imaging capability, we anticipate that W-PTM can be a powerful tool to study cellular activities associated with cellular membrane temperature changes.

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid–protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

Mechanical allodynia impinges on the life quality of patients. Hen Egg Lysozyme (HEL) is a substance extracted from eggs that is commonly used to inhibit bacterial activity. The role of HEL in regulating and treating pain is unclear. Here, we find that HEL selectively attenuates static mechanical allodynia of mice induced by complete Freund's adjuvant (CFA), spinal nerve ligation (SNL) and chemotherapeutic agent. RNA-seq screening reveals that CFA significantly reduces the expression of Parkin in dorsal root ganglion (DRG) neurons of mice, while pre-administration of HEL increases the expression of Parkin and remits the static mechanical allodynia induced by Parkin-siRNA. Moreover, HEL increases the interaction between nuclear respiratory factor 1 (NRF1) and histone acetyltransferase P300 and then enhances the NRF1 mediated histone acetylation in prkn promoter region in DRGs of mice. Further, Parkin interacts with mechanotransducing ion channel TACAN (Tmem120a) and knockdown of Parkin significantly increases the membrane trafficking of TACAN in sensory neurons of mice. While pre-administration of HEL inhibits the increased membrane trafficking of TACAN in sensory neurons of mice induced by Parkin-siRNA. In addition, pre-given of HEL also significantly attenuates the static mechanical allodynia induced by overexpression of TACAN in mice, and the effect of HEL can be blocked by Parkin-siRNA. This indicates that HEL increases the expression of Parkin through epigenetic mechanisms and then decreases TACAN membrane trafficking in sensory neurons to relieve static mechanical hypersensitivity. Therefore, we reveal a novel function of HEL, which is a potential substance for the treatment of static mechanical pain.

It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear. Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium. Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis. To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines.

Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl- sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.

Autism spectrum disorder (ASD) is a neurological condition that directly affects brain functions and can culminate in delayed intellectual development, problems in verbal communication, difficulties in social interaction, and stereotyped behaviors. Its etiology reveals a genetic basis that can be strongly influenced by socio-environmental factors. Ion channels controlled by ligand voltage-activated calcium, sodium, and potassium channels may play important roles in modulating sensory and cognitive responses, and their dysfunctions may be closely associated with neurodevelopmental disorders such as ASD. This is due to ionic flow, which is of paramount importance to maintaining physiological conditions in the central nervous system and triggers action potentials, gene expression, and cell signaling. However, since ASD is a multifactorial disease, treatment is directed only to secondary symptoms. Therefore, this research aims to gather evidence concerning the principal pathophysiological mechanisms involving ion channels in order to recognize their importance as therapeutic targets for the treatment of central and secondary ASD symptoms.

Several integral membrane proteins undergo regulated intramembrane proteolysis (RIP), a tightly controlled process through which cells transmit information across and between intracellular compartments. RIP generates biologically active peptides by a series of proteolytic cleavage events carried out by two primary groups of enzymes: sheddases and intramembrane-cleaving proteases (iCLiPs). Following RIP, fragments of both pore-forming and non-pore-forming ion channel subunits, as well as immunoglobulin super family (IgSF) members, have been shown to translocate to the nucleus to function in transcriptional regulation. As an example, the voltage-gated sodium channel

Funder: Cancer Research UK (CRUK); doi: https://doi.org/10.13039/501100000289, Funder: American Lebanese Syrian Associated Charities (ALSAC); doi: https://doi.org/10.13039/100012524, Funder: U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics); doi: https://doi.org/10.13039/100011541, Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI); doi: https://doi.org/10.13039/100000054, Funder: RCUK | MRC | Medical Research Foundation; doi: https://doi.org/10.13039/501100009187, We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.

Dehydrated hereditary stomatocytosis (DHS) (MIM#194380) is a rare autosomal dominant disorder of red blood cell permeability, characterized by a partially or fully compensated nonimmune hemolytic anemia. PIEZO1 is the major gene involved with hundreds of families described, some of which present transient perinatal edema of varying severity. A smaller subset of individuals harbors pathogenic variants in KCNN4, sometimes referred as "Gardos channelopathy." Up to now, only six pathogenic variants in KCNN4 have been reported in 13 unrelated families. Unlike PIEZO1-DHS, neither perinatal edema nor fetal loss has ever been observed linked to KCNN4-DHS. We report the first fetal loss due to non-immune hydrops fetalis related to a pathogenic 28 bp deletion (NM_002250.2: c.1109_1119+17del) in KCNN4. This observation underlies the need for very close monitoring of pregnancies when one parent is affected by DHS regardless of genotype (PIEZO1 or KCNN4).

Regulation of genetic activity in single cells and tissues is pivotal to determine key cellular functions in current biomedicine, yet the conventional biochemical activators lack spatiotemporal precision due to the diffusion-mediated slow kinetics and nonselectivity. Here, we describe a magnetogenetic method for target-specific activation of a clustered regularly interspaced short palindromic repeats (CRISPR) system for the regulation of intracellular proteins. We used magnetomechanical force generated by the magnetic nanostructure to activate pre-encoded Piezo1, the mechanosensitive ion channel, on the target cell. The activated Piezo1 further triggers the intracellular Ca

Plants deploy extracellular and intracellular immune receptors to sense and restrict pathogen attacks. Rapidly evolving pathogen effectors play crucial roles in suppressing plant immunity but are also monitored by intracellular nucleotide-binding, leucine-rich repeat immune receptors (NLRs), leading to effector-triggered immunity (ETI). Here, we review how NLRs recognize effectors with a focus on direct interactions and summarize recent research findings on the signalling functions of NLRs. Coiled-coil (CC)-type NLR proteins execute immune responses by oligomerizing to form membrane-penetrating ion channels after effector recognition. Some CC-NLRs function in sensor–helper networks with the sensor NLR triggering oligomerization of the helper NLR. Toll/interleukin-1 receptor (TIR)-type NLR proteins possess catalytic activities that are activated upon effector recognition-induced oligomerization. Small molecules produced by TIR activity are detected by additional signalling partners of the EDS1 lipase-like family (enhanced disease susceptibility 1), leading to activation of helper NLRs that trigger the defense response.

Primary fatty acid amides (PFAMs) are interesting lipid-derived signaling molecules with wide-ranging actions, including bioelectric. In this perspective, we evaluate a possible triangular relationship: PFAMs-ion channels-cancer. The primary emphasis is on oleamide and its metabolic associate, oleic acid (OA). As ion channels, we have focused on voltage-gated sodium channels (VGSCs) and voltage-gated and inward rectifier potassium channels in a variety of cell types, including cancer cells. As cancers, we evaluate the evidence for breast and colorectal cancers. Cis-oleamide blocked VGSCs and potassium channels. In parallel, but not always concurrently, oleamide has been shown to produce antiproliferative and anti-invasive effects, consistent with the known control of these cellular behaviors by potassium channels and VGSCs, respectively. Importantly, there is also evidence that oleamide can potentiate chemotherapy. In contrast to oleamide, the effects of OA on the ion channels of interest and, correspondingly, cancer cell behaviors are much less consistent. The perspective is concluded with three short sections. First, we outline the actions of some other PFAMs, especially anandamide and linoleamide. Second, we give an overview of some other targets that have been associated with oleamide, especially cannabinoid receptors and gap junctions. Finally, we cover the anticancer effects of oleamide on some other neoplasms.

Piezo1 is a recently discovered Ca

Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The organization of stem cells in 3D culture affects cell microenvironment and their mechanical properties. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. Electrophysiological data on the features of ion channels functioning in 3D cell culture models are currently very limited in the literature. This gap of knowledge may be associated with technical difficulties that exist when researchers try to apply the standard patch clamp method for the registration of ion channels in cells aggregated in spheroids. In this regard, our study focuses on solving emerging technical difficulties and presents an example of their successful solution. Here, we developed a specific approach and have recorded the activity of mechanosensitive stretch-activated ion channels (SACs) in endometrial MSCs (eMSCs) assembled in spheroids. Moreover, we observed functional interplay of SACs with potassium channels of big conductance (BK) in the plasma membrane of eMSC spheroids consistently to revealed earlier in routine 2D cultured cells. Additionally, we observed a significant decrease in the frequency of SACs activation in spheroids that may indicate the differences in the level of functional expression of channels in 3D culture comparing to 2D culture of eMSCs.

Transmembrane protein 16F (TMEM16F) is a ubiquitously expressed Ca

Background: Epicardial adipose tissue (EAT) accumulation is associated with cardiac arrhythmias. The effect of EAT secretome (EATs) on cardiac electrophysiology remains largely unknown. Objective: The purpose of this study was to investigate the arrhythmogenicity of EATs and its underlying molecular and electrophysiological mechanisms. Methods: We collected atrial EAT and subcutaneous adipose tissue (SAT) from 30 patients with atrial fibrillation (AF), and EAT from 3 donors without AF. The secretome was collected after a 24-hour incubation of the adipose tissue explants. We cultured neonatal rat ventricular myocytes (NRVMs) with EATs, subcutaneous adipose tissue secretome (SATs), and cardiomyocytes conditioned medium (CCM) for 72 hours. We implemented the electrophysiological changes observed after EATs incubation into a model of human left atrium and tested arrhythmia inducibility. Results: Incubation of NRVMs with EATs decreased expression of the potassium channel subunit Kcnj2 by 26% and correspondingly reduced the inward rectifier K+ current IK1 by 35% compared to incubation with CCM, resulting in a depolarized resting membrane of cardiomyocytes. EATs decreased expression of connexin43 (29% mRNA, 46% protein) in comparison to CCM. Cells incubated with SATs showed no significant differences in Kcnj2 or Gja1 expression in comparison to CCM, and their resting potential was not depolarized. Cardiomyocytes incubated with EATs showed reduced conduction velocity and increased conduction heterogeneity compared to SATs and CCM. Computer modeling of human left atrium revealed that the electrophysiological changes induced by EATs promote sustained reentrant arrhythmias if EAT partially covers the myocardium. Conclusion: EAT slows conduction, depolarizes the resting potential, alters electrical cell–cell coupling, and facilitates reentrant arrhythmias.

Dorsal and ventral medial entorhinal cortex (mEC) regions have distinct neural network firing patterns to differentially support functions such as spatial memory. Accordingly, mEC layer II dorsal stellate neurons are less excitable than ventral neurons. This is partly because the densities of inhibitory conductances are higher in dorsal than ventral neurons. Here, we report that T-type Ca2+ currents increase 3-fold along the dorsal-ventral axis in mEC layer II stellate neurons, with twice as much CaV3.2 mRNA in ventral mEC compared with dorsal mEC. Long depolarizing stimuli trigger T-type Ca2+ currents, which interact with persistent Na+ currents to elevate the membrane voltage and spike firing in ventral, not dorsal, neurons. T-type Ca2+ currents themselves prolong excitatory postsynaptic potentials (EPSPs) to enhance their summation and spike coupling in ventral neurons only. These findings indicate that T-type Ca2+ currents critically influence the dorsal-ventral mEC stellate neuron excitability gradient and, thereby, mEC dorsal-ventral circuit activity.

Nociception is a protective mechanism alerting an organism to noxious stimuli and potential harm. However, dysregulation of the nociceptive system can result in chronic pain, which has a prevalence of approximately 40 % in the adult population. Current therapeutics are often inefficacious, and the growing opioid crisis demonstrates a clear need to develop new analgesics and improved pain management strategies. A common feature of inflammation is tissue acidosis, and the raised extracellular proton concentration can activate acid-sensing ion channel 3 (ASIC3), which is most highly expressed in those sensory neurones tuned to detect noxious stimuli, i.e., nociceptors. To date, the most potent non-peptide ASIC3 inhibitors act at micromolar concentrations *in vitro* on rat-ASIC3 (rASIC3), but are non-selective, thus inhibiting other ASIC subunits. Intriguingly, certain non-steroidal anti-inflammatory drugs (NSAIDs) directly inhibit rASIC3 but are neither potent ligands nor is it understood precisely how they interact with ASIC3. Here, I aimed to use *in silico* modelling to not only predict the plausible binding mode of some known ASIC3 modulators to this channel, but to further identify new ligands that can modulate ASIC3. Homology models of rASIC3 were constructed based on published 3D structures of chicken ASIC1a solved at various states. These models were then used for blind docking with some known small molecule modulators of ASIC3 that notably included the NSAID diclofenac. The resultant poses of these ligands were then subjected to further refinement using a focused docking approach. Altogether, this led to a prediction of a potential binding site and mode of binding for the ASIC3 selective NSAID inhibitors near the acidic pocket domain of rASIC3. A 2D-ligand similarity approach was undertaken to identify scaffolds possessing key functional groups and physico-chemical properties that were similar to those known ASIC3 modulating NSAIDs, and subsequently docked to predict binding interactions. Using these criteria, three molecules (diflunisal, fenbufen and tolmetin) were chosen from a number of hits and were then tested for their ability to modulate the function of rASIC3 transiently transfected in Chinese hamster ovary cells using whole-cell patch-clamp electrophysiology. This *in silico* approach was also conducted for pro-inflammatory mediators known to activate/enhance ASIC3 activity, which identified potential physiological modulators. Upon activation, the ASIC3 current showed two characteristic phases: a rapid transient phase followed by a prolonged and smaller sustained phase in the presence of continued stimulation, and this was in complete agreement with existing literature. Diclofenac significantly inhibited the sustained, but not the transient phase of the current at pH 4, but no effect on either phase was observed at pH 5. Conversely, the three hits identified *in silico* showed a varying degree of inhibition on the sustained phase at pH 4 and 5. Finally, site-directed mutagenesis was conducted to validate those amino acids computationally predicted to be involved in NSAID modulation of ASIC3. This thesis outlines a method to predict binding regions of ASIC3 ligands and identifies a possible functional region of ASIC3 by which these ligands interact. These results provide a workflow for identifying novel modulators of ASIC3, which may be of analgesic application.

Atrial fibrillation (AF) is the most frequent persistent arrhythmia. Many genes have been reported as a genetic background for AF. However, most transcriptome analyses of AF are limited to the atrial samples and have not been evaluated by multiple cardiac regions. In this study, we analyzed the expression levels of protein-coding and long noncoding RNAs (lncRNAs) in six cardiac regions by RNA-seq. Samples were donated from six subjects with or without persistent AF for left atria, left atrial appendages, right atria, sinoatrial nodes, left ventricles, right ventricles, and pulmonary veins (PVs), and additional four right atrial appendages samples were collected from patients undergoing mitral valve replacement. In total, 23 AF samples were compared to 23 non-AF samples. Surprisingly, the most influenced heart region in gene expression by AF was the PV, not the atria. The ion channel-related gene set was significantly enriched upon analysis of these significant genes. In addition, some significant genes are cancer-related lncRNAs in PV in AF. A co-expression network analysis could detect the functional gene clusters. In particular, the cancer-related lncRNA, such as SAMMSON and FOXCUT, belong to the gene network with the cancer-related transcription factor FOXC1. Thus, they may also play an aggravating role in the pathogenesis of AF, similar to carcinogenesis. In the least, this study suggests that (1) RNA alteration is most intense in PVs and (2) post-transcriptional gene regulation by lncRNA may contribute to the progression of AF. Through the screening analysis across the six cardiac regions, the possibility that the PV region can play a role other than paroxysmal triggering in the pathogenesis of AF was demonstrated for the first time. Future research with an increase in the number of PV samples will lead to a novel understanding of the pathophysiology of AF.

Normal and abnormal cardiac rhythms are of key physiological and clinical interest. This introductory article begins from Sylvio Weidmann's key historic 1950s microelectrode measurements of cardiac electrophysiological activity and Singh & Vaughan Williams's classification of cardiotropic targets. It then proceeds to introduce the insights into cardiomyocyte function and its regulation that subsequently emerged and their therapeutic implications. We recapitulate the resulting view that surface membrane electrophysiological events underlying cardiac excitation and its initiation, conduction and recovery constitute the final common path for the cellular mechanisms that impinge upon this normal or abnormal cardiac electrophysiological activity. We then consider progress in the more recently characterized successive regulatory hierarchies involving Ca 2+ homeostasis, excitation–contraction coupling and autonomic G-protein signalling and their often reciprocal interactions with the surface membrane events, and their circadian rhythms. Then follow accounts of longer-term upstream modulation processes involving altered channel expression, cardiomyocyte energetics and hypertrophic and fibrotic cardiac remodelling. Consideration of these developments introduces each of the articles in this Phil. Trans. B theme issue. The findings contained in these articles translate naturally into recent classifications of cardiac electrophysiological targets and drug actions, thereby encouraging future iterations of experimental cardiac electrophysiological discovery, and testing directed towards clinical management. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.

Started as an academic curiosity more than two decades ago, the idea that ion channels can regulate cellular processes in ways that do not depend on their conducting properties (non-ionic functions) gained traction and is now a flourishing area of research. Channels can regulate physiological processes including actin cytoskeletal remodeling, cell motility, excitation-contraction coupling, non-associative learning and embryogenesis, just to mention some, through non-ionic functions. When defective, non-ionic functions can give rise to channelopathies involved in cancer, neurodegenerative disease and brain trauma. Ion channels exert their non-ionic functions through a variety of mechanisms that range from physical coupling with other proteins, to possessing enzymatic activity, to assembling with signaling molecules. In this article, we take stock of the field and review recent findings. The concept that emerges, is that one of the most common ways through which channels acquire non-ionic attributes, is by assembling with integrins. These integrin-channel complexes exhibit broad genotypic and phenotypic heterogeneity and reveal a pleiotropic nature, as they appear to be capable of influencing both physiological and pathological processes.

The exercise pressor reflex (EPR), a neurocirculatory control mechanism, is exaggerated in hypertensive humans and rats. Disease-related abnormalities within the afferent arm of the reflex loop, including mechano- and metabosensitive receptors located at the terminal end of group III/IV muscle afferents, may contribute to the dysfunctional EPR in hypertension. Using control (WKY) and spontaneous hypertensive (SHR) rats, we examined dorsal root ganglion (DRG) gene and protein expression of molecular receptors recognized as significant determinants of the EPR. Twelve lumbar DRGs (6 left, 6 right) were harvested from each of 10 WKY [arterial blood pressure (MAP): 96 ± 9 mmHg] and 10 SHR (MAP: 144 ± 9 mmHg). DRGs from the left side were used for protein expression (Western blotting; normalized to GAPDH), whereas right-side DRGs (i.e., parallel structure) were used to determine mRNA levels (RNA-sequencing, normalized to TPM). Analyses focused on metabosensitive (ASIC3, Bradykinin receptor B2, EP4, P2X3, TRPv1) and mechanosensitive (Piezo1/2) receptors. Although Piezo1 was similar in both groups ( P = 0.75), protein expression for all other receptors was significantly higher in SHR compared with WKY. With the exception of a greater Bradykinin-receptor B2 in SHR ( P < 0.05), mRNA expression of all other receptors was not different between groups ( P > 0.18). The higher protein content of these sensory receptors in SHR indirectly supports the previously proposed hypothesis that the exaggerated EPR in hypertension is, in part, due to disease-related abnormalities within the afferent arm of the reflex loop. The upregulated receptor content, combined with normal mRNA levels, insinuates that posttranscriptional regulation of sensory receptor protein expression might be impaired in hypertension.

Mutations ofThe trimeric intracellular cation (TRIC) channel TRIC-B is an ER-resident potassium channel that mediates potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium ion release. Using TRIC-B as a tool, we examined the function of ER-localized polycystin-2 and its role in ADPKD pathogenesis in cultured cells, zebrafish, and mouse models.Agonist-induced ER calcium ion release was defective in cells lacking polycystin-2 and reversed by exogenous expression of TRIC-B.Polycystin-2 in the ER appears to be critical for anticystogenesis and likely functions as a potassium ion channel to facilitate potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium release. The results advance the understanding of ADPKD pathogenesis and provides proof of principle for pharmacotherapy by TRIC-B activators.

Before advancing to clinical trials, new drugs are screened for their pro-arrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the mis-classification of beneficial drugs as pro-arrhythmic.An in silico-in vitro pipeline was developed to circumvent these shortcomings. A computational human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used as part of a genetic algorithm to design experiments, specifically electrophysiological voltage clamp (VC) protocols, to identify which of several cardiac ion channels were blocked during in vitro drug studies. Such VC data, along with dynamically clamped action potentials (AP), were acquired from iPSC-CMs before and after treatment with a control solution or a low- (verapamil), intermediate- (cisapride or quinine) or high-risk (quinidine) drug.Significant AP prolongation (a pro-arrhythmia marker) was seen in response to quinidine and quinine. The VC protocol identified block of IWe developed an in silico-in vitro pipeline that simultaneously identifies pro-arrhythmia risk and mechanism of ion channel-blocking drugs. The approach offers a new tool for evaluating cardiotoxicity during preclinical drug screening.

The extravasation of leukocytes is a critical step during inflammation that requires the localized opening of the endothelial barrier. This process is initiated by the close interaction of leukocytes with various adhesion molecules such as ICAM-1 on the surface of endothelial cells. Here we reveal that mechanical forces generated by leukocyte-induced clustering of ICAM-1 synergize with fluid shear stress exerted by the flowing blood to increase endothelial plasma membrane tension and to activate the mechanosensitive cation channel PIEZO1. This leads to increases in [Ca2+]i and activation of downstream signaling events including phosphorylation of tyrosine kinases sarcoma (SRC) and protein tyrosine kinase 2 (PYK2), as well as of myosin light chain, resulting in opening of the endothelial barrier. Mice with endothelium-specific Piezo1 deficiency show decreased leukocyte extravasation in different inflammation models. Thus, leukocytes and the hemodynamic microenvironment synergize to mechanically activate endothelial PIEZO1 and subsequent downstream signaling to initiate leukocyte diapedesis.

This retrospective on the voltage-sensing mechanisms and gating models of ion channels begins in 1952 with the charged gating particles postulated by Hodgkin and Huxley, viewed as charges moving across the membrane and controlling its permeability to Na

Mature circulating red blood cells (RBCs) are classically viewed as passive participants in circulatory function, given erythroblasts eject their organelles during maturation. Endogenous production of nitric oxide (NO) and its effects are of particular significance; however, the integration between RBC sensation of the local environment and subsequent activation of mechano-sensitive signaling networks that generate NO remain poorly understood. The present study investigated endogenous NO production via the RBC-specific nitric oxide synthase isoform (RBC-NOS), connecting membrane strain with intracellular enzymatic processes. Isolated RBCs were obtained from apparently healthy humans. Intracellular NO was compared at rest and following shear (cellular deformation) using semiquantitative fluorescent imaging. Concurrently, RBC-NOS phosphorylation at its serine

Piezo1 and Piezo2 are recently discovered mechanosensory ion channels. Piezo channels transduce mechanical stimulation into cellular signaling in a variety of tissues and organ systems. The functional roles of Piezo1 and Piezo2 have been revealed in both developmental and physiological scenarios by using mouse genetic models. Mechanotransduction by Piezo1 channels regulates osteoblast/osteocyte activity and, thus, strengthens the skeleton enabling it to adapt to a wide range of mechanical loadings. Deletion of the Piezo1 gene in the developing skeleton causes bone malformations that lead to spontaneous bone fractures, while inactivity of Piezo1 in adulthood results in osteoporosis. Furthermore, Piezo2 channels in sensory neurons might provide another route of skeletal regulation. Piezo channels also regulate the proliferation and differentiation of various types of stem cells. PIEZO1 and PIEZO2 mutations and channel malfunctions have been implicated in an increasing number of human diseases, and PIEZO channels are currently emerging as potential targets for disease treatment. This review summarizes the important findings of Piezo channels for skeletal development and homeostasis using the mouse genetic model system.

Over the last decades, photopharmacology has gone far beyond its proof-of-concept stage to become a bona fide approach to study neural systems in vivo. Indeed, photopharmacological control has expanded over a wide range of endogenous targets, such as receptors, ion channels, transporters, kinases, lipids, and DNA transcription processes. In this review, we provide an overview of the recent progresses in the in vivo photopharmacological control of neuronal circuits and behavior. In particular, the use of small aquatic animals for the in vivo screening of photopharmacological compounds, the recent advances in optical modulation of complex behaviors in mice, and the development of adjacent techniques for light and drug delivery in vivo are described.

Inspired by mechanosensitive potassium channels found in nature, we developed a fluorinated amphiphilic cyclophane composed of fluorinated rigid aromatic units connected via flexible hydrophilic octa(ethylene glycol) chains. Microscopic and emission spectroscopic studies revealed that the cyclophane could be incorporated into the hydrophobic layer of the lipid bilayer membranes and self-assembled to form a supramolecular transmembrane ion channel. Current recording measurements using cyclophane-containing planer lipid bilayer membranes successfully demonstrated an efficient transmembrane ion transport. We also demonstrated that the ion transport property was sensitive to the mechanical forces applied to the membranes. In addition, ion transport assays using pH-sensitive fluorescence dye revealed that the supramolecular channel possesses potassium ion selectivity. We also performed all-atom hybrid quantum-mechanical/molecular mechanical simulations to assess the channel structures at atomic resolution and the mechanism of selective potassium ion transport. This research demonstrated the first example of a synthetic mechanosensitive potassium channel, which would open a new door to sensing and manipulating biologically important processes and purification of key materials in industries.

Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

Incorporating mechanical cues into cellular responses allows us to experience our direct environment. Specialized cells can perceive and discriminate between different physical properties such as level of vibration, temperature, or pressure. Mechanical forces are abundant signals that also shape general cellular responses such as cytoskeletal rearrangement, differentiation, or migration and contribute to tissue development and function. The molecular structures that perceive and transduce mechanical forces are specialized cytoskeletal proteins, cell junction molecules, and membrane proteins such as ion channels and metabotropic receptors. G protein-coupled receptors (GPCRs) have attracted attention as metabotropic force receptors as they are among the most important drug targets. This review summarizes the function of mechano-sensitive GPCRs, specifically, the angiotensin II type 1 receptor and adrenergic, apelin, histamine, parathyroid hormone 1, and orphan receptors, focusing particularly on the advanced knowledge gained from adhesion-type GPCRs. We distinguish between shear stress and cell swelling/stretch as the two major types of mechano-activation of these receptors and contemplate the potential contribution of the force-from-lipid and force-from-tether models that have previously been suggested for ion channels.

Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit. Historically, the effects of compounds on ion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed by using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased in throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods for processing the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.

Proper mitochondrial function and adequate cellular ATP are necessary for normal pancreatic protein synthesis and sorting, maintenance of intracellular organelles and enzyme secretion. Inorganic phosphate is required for generating ATP and its limited availability may lead to reduced ATP production causing impaired Ca(2+) handling, defective autophagy, zymogen activation, and necrosis, which are all features of acute pancreatitis. We hypothesized that reduced dietary phosphate leads to hypophosphatemia and exacerbates pancreatitis severity of multiple causes. We observed that mice fed a low-phosphate diet before the induction of pancreatitis by either repeated caerulein administration or pancreatic duct injection as a model of pressure-induced pancreatitis developed hypophosphatemia and exhibited more severe pancreatitis than normophosphatemic mice. Pancreatitis severity was significantly reduced in mice treated with phosphate. In vitro modeling of secretagogue- and pressure-induced pancreatic injury was evaluated in isolated pancreatic acini using cholecystokinin and the mechanoreceptor Piezo1 agonist, Yoda1, under low and normal phosphate conditions. Isolated pancreatic acini were more sensitive to cholecystokinin- and Yoda1-induced acinar cell damage and mitochondrial dysfunction under low-phosphate conditions and improved following phosphate supplementation. Importantly, even mice on a normal phosphate diet exhibited less severe pancreatitis when treated with supplemental phosphate. Thus, hypophosphatemia sensitizes animals to pancreatitis and phosphate supplementation reduces pancreatitis severity. These appear to be direct effects of phosphate on acinar cells through restoration of mitochondrial function. We propose that phosphate administration may be useful in the treatment of acute pancreatitis. NEW & NOTEWORTHY Impaired ATP synthesis disrupts acinar cell homeostasis and is an early step in pancreatitis. We report that reduced phosphate availability impairs mitochondrial function and worsens pancreatic injury. Phosphate supplementation improves mitochondrial function and protects against experimental pancreatitis, raising the possibility that phosphate supplementation may be useful in treating pancreatitis.

Odontoblasts, terminally differentiated dentin-forming cells with their processes that penetrate into dentin, have been considered potential sensory cells. Current research suggests that odontoblasts sense external stimuli and transmit pain signals. PIEZO1, as a specific mechanically activated ion channel, may play an important role in mechanical transduction in odontoblasts. In this study, we devoted to investigating the functions and underlying molecular mechanisms of PIEZO1 ion channels in odontoblast mechanotransduction.Human dental pulp stem cells were cultured in vitro and induced to differentiate into odontoblast-like cells (OLCs). The expression of PIEZO1 protein in pulp, dental pulp stem cells, and OLCs was detected by immunohistochemistry or immunofluorescence. The mechanical sensitivity of OLCs was detected by a constructed fluid shear stress model and examined by calcium fluorescence intensity. A single-cell mechanical stimulation model was used to detect the PIEZO1 electrophysiological properties of OLCs. Yoda1 (a PIEZO1-specific agonist), GsMTx4 (a PIEZO1 antagonist), and non-calcium ion extracellular solution were utilized to confirm PIEZO1 mechanotransduction in OLCs in both fluid shear stress and single-cell mechanical stimulation assays. The amount of ATP released by OLCs was measured under stimulation with Yoda1 and GsMTx4. Rat trigeminal ganglion neurons were cultured in vitro and detected by whole-cell patch-clamp recording under ATP stimulation.PIEZO1 ion channels were positively expressed in OLCs and odontoblastic bodies and processes but weakly expressed in dental pulp cells. After the treatment of OLCs with shearing stress or Yoda1, the fluorescence intensity of intracellular calcium ions increased rapidly but did not noticeably change after treatment with GsMTx4 or the non-calcium ion extracellular solution. When single-cell mechanical stimuli were applied to OLCs, the evoked inward currents were recorded by patch-clamp electrophysiology. The inward currents increased and current inactivation became slower after Yoda1 treatment, but these currents almost completely disappeared after the addition of GsMTx4. The amount of ATP released by OLCs increased significantly after Yoda1 stimulation, while GsMTx4 reversed the release of ATP. Whole-cell patch-clamp detection showed that ATP evoked slow inward currents and increased the frequency of action potentials of trigeminal ganglion neurons.Taken together, these findings indicated that odontoblasts evoked a fast inward current via PIEZO1 ion channels after the application of external mechanical stimuli and released ATP to transmit signals to adjacent cells. Thus, PIEZO1 ion channels in odontoblasts mediate mechanotransduction under various pathophysiological conditions in dentin.

18 p.-6 fig.-1 tab., Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing LmnaG609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the KV7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify KV7 channels as a candidate target for the treatment of HGPS., Work supported by Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) (MCIN/AEI/https://doi.org/10.13039/501100011033 grants SAF2016-79490-R and PID2019-108489RB-I00), with co-funding from Fondo Social Europeo (“El FSE invierte en tu futuro”), and a donation from Asociación Progeria Alexandra Peraut. Microscopy was conducted at the Microscopy & Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/https://doi.org/10.13039/501100011033. R.M.N was supported by the Ministerio de Educación, Cultura y Deporte (pre-doctoral contract FPU16/05027), and I.B. is supported by the Comunidad Autónoma de Madrid (grants 2017-T1/BMD-5247 and 2021-5A/BMD-20944), and the Ramón y Cajal contract (RYC2021-033805-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union “NextGenerationEU”/PRTR. The CNIC is supported by the MCIN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant number CEX2020-001041-S funded by MCIN/AEI/https://doi.org/10.13039/501100011033).

Influx of sodium ions through voltage-gated sodium channels in cardiomyocytes is essential for proper electrical conduction within the heart. Both acquired conditions associated with sodium channel dysfunction (myocardial ischaemia, heart failure) as well as inherited disorders secondary to mutations in the gene SCN5A encoding for the cardiac sodium channel Nav1.5 are associated with life-threatening arrhythmias. Research in the last decade has uncovered the complex nature of Nav1.5 distribution, function, in particular within distinct subcellular subdomains of cardiomyocytes. Nav1.5-based channels furthermore display previously unrecognized non-electrogenic actions and may impact on cardiac structural integrity, leading to cardiomyopathy. Moreover, SCN5A and Nav1.5 are expressed in cell types other than cardiomyocytes as well as various extracardiac tissues, where their functional role in, e.g. epilepsy, gastrointestinal motility, cancer and the innate immune response is increasingly investigated and recognized. This review provides an overview of these novel insights and how they deepen our mechanistic knowledge on SCN5A channelopathies and Nav1.5 (dys)function. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.

In this note, the author proposes a definition for neurobiological consciousness. The definition is based on a thought experiment wherein theactivity of neuronal ion pumps is intervened with, leading to a modulationin information processing. The view is thus a bottom-up view where thephysical layer of the system influences the data layer.

Skozi zgodovino so ljudje različnih kultur uporabljali psihoaktivne snovi za rituale, da bi razširili svoje dojemanje sveta, zdravili ter nadzorovali svoj vpliv na družbo. Psihoaktivna snov je katerakoli kemična snov, ki lahko s svojim farmakološkim delovanjem vpliva na razpoloženje, zaznavanje ali zavest. S konvencijo Združenih narodov o psihotropnih snoveh iz leta 1971 so te snovi pod mednarodnim nadzorom. Nasprotno pa nove psihoaktivne snovi (NPS) tvorijo širok spekter snovi, ki jih mednarodna zakonodaja o drogah ne nadzoruje. Opredelitev snovi, ki zahteva nadzor, je običajno sestavljena iz dveh od treh naslednjih elementov: snov mora biti psihoaktivna, prisoten mora biti motiv zlorabe ali zastrupitve in mora obstajati morebitna škoda ali nevarnost za zdravje. Izraz „novo“ se nanaša na „novo“ prisotne snovi na tržišču drog pred kratkim spremenjene kemijske strukture, ki omogočajo podobne učinke kot že nadzorovane prepovedane droge, hkrati pa se zato izognejo zakonski prepovedi. Te nove psihoaktivne snovi lahko glede na spekter njihovih psihoaktivnih učinkov razvrstimo v: stimulanse, ki zavirajo prenos ali spodbujajo sproščanje dopamina in noradrenalina opioide, ki so agonisti opioidnih receptorjev centralnega živčnega sistema (CŽS) klasične halucinogene, ki so neposredni serotoninergični agonisti receptorjev 5-HT2A disociativi, ki delujejo kot antagonisti NMDA in sedativi / hipnotiki, ki so agonisti GABA. Razvrstiti jih je mogoče tudi glede na njihovo kemijsko družino: fenetilamini, amfetamini, katinoni, piperazini, pipradroli / piperidini, aminoindani, benzofurani in triptamini. Obstaja nekaj splošnih toksičnih učinkov NPS, kot so: akutni serotoninski sindrom, hipertermija, epileptični napadi in hiponatriemija za serotoninergične snovi dolgotrajna stimulacija, nespečnost, vznemirjenost, psihoza in velika odvisnost pri dopaminergičnih snoveh medtem ko sintetični kanabinoidi povzročajo vznemirjenost, tesnobo, paranojo, hipertenzijo, miokardni infarkt in ledvično odpoved. Zdi se, da se največ uporabljajo skupine sintetičnih kanabinoidov in katinonov, v zadnjem času pa opažamo tudi porast uporabe sintetičnih opioidov, zaradi česar jih v podrobnejši pregled v uvodu prav tako vključujemo. Nove psihoaktivne snovi predstavljajo velik izziv na področju javnega zdravja, saj jih hitro in enostavno razvijejo ter zamenjajo z nedovoljenim analogom, hkrati pa so lahko nepredvidljive v farmakološkem smislu, učinkih in toksikologiji. Nekatere študije in poročila o akutnih primerih so pokazale povezavo sintetičnih katinonov in kanabinoidov s škodljivimi učinki na CNS in kardiovaskularni sistem. Farmakologija sintetičnih kanabinoidov je v glavnem posledica interakcije s kanabinoidnimi receptorji, vendar so nekatere študije odkrile tudi več SC, ki interagirajo tudi z ne-kanabinoidnimi receptorji, med drugim blokirajo Na+ kanal izmenjevalca Na+/Ca2 +, zavirajo hERG kanal in izooblike napetostnega natrijevega kanala NaV (NaV1.2 1.3 1.4 1.5 1.7 1.8). Študije vezav sintetičnih katinonov (SCT) na ionske kanale so redkejše, večinoma obsegajo študije na serotoninske in dopaminske receptorje centralnega živčnega sistema. Namen magistrske naloge je bil zagotoviti dodatne podatke o možnih interakcijah raztopin 18 novih sintetičnih katinonov in kanabinoidov s srčnimi napetostnimi ionskimi kanali Kv1.4, hERG, NaV1.5, v srčnem in živčnem sistemu izražen 'pacemaker' (HCN), ter GABAA receptorjem in nikotinskim acetilholinskim receptorjem 7. Eksperimente smo izvedli z raztopinami spojin 1 μM koncentracije, saj glede na nekatere študije določene NPS izkazujejo delovanje že v nanomolarnem razponu, kar smo želeli raziskati tudi pri naših spojinah. Raziskavo smo izvedeli z uporabo pogoste elektrofiziološke metode 'dvo elektrodne napetostne sponke' (two electrode voltage clamp, TEVC), kjer aktivnost kanala merimo z vbodom dveh mikroelektrod v merjeno celico (v našem primeru smo izbrane ionske kanale izrazili v oocitih žabe krempljičarke Xenopus laevis), ki jo lahko izpostavimo določeni napetosti ob znanih ionskih pogojih in s tem umetno nadziramo membranski potencial (Vm). Preko mikroelektrodnega zapisa odčitamo spremembe v prevodnostikanalov ob dodajanju raziskovanih spojin, s čimer določimo morebitni vpliv na ionske kanale. Od 18 katinonov in kanabinoidov, ki so bili načrtovani za raziskavo, je bilo med seboj različnih 10, ostali so bili podvojeni in zgolj drugače poimenovani, kar so raziskovalci v laboratoriju potrdili z masno spektroskopijo pred opravljanjem elektrofizioloških raziskav. Prav tako nismo mogli izraziti dveh kanalov - GABAA in nikotinskih acetilholinskih receptorjev 7. Rezultati vpliva 1 μM raztopin 10 sintetičnih katinonov in kanabinoidov na splošno ne kažejo pomembnega vpliva na preiskovane kanale, razen treh spojin, kjer bi bile priporočljive nadaljnje študije. Dodatek1 μM 5F-AMB je povzročil povprečno 5,5 % inhibicijo kanala hERG, vendar je bilo povprečje računano na podlagi dveh meritev, saj tretje nismo uspeli kvalitetno izvesti, bodisi zaradi nestabilnih celic bodisi zaradi neuspelega izražanja kanala. Dodatek 1 μM 4-Cl-PVP je povzročil povprečno 6,3 % inhibicijo hERG kanala. Po dodajanju 1 μM 5F-ADB smo pri vseh treh meritvah opazili povprečno 7,1 % zvišanje prevodnosti Kv1.4 kanalov. Šlo je za preliminarne preizkuse spojin na omenjene tarče, zato - razen povprečnih vrednosti procentualne spremembe v treh poizkusih na spojino - statistike nismo opravljali. Zaradi pogoste nestabilnosti celic in nejasnih krivulj meritev bi bilo za nedvoumni vpliv preiskovanih spojina ionske kanale meritve potrebno ponoviti, izmeriti še potencialne spremembe prevodnosti pri višjih koncentracijah substanc ter določiti morebitno polovično maksimalno inhibitorno koncentracijo (IC50). Throughout history, different cultures have used various kinds of substances to expand their perception for healing or social rituals. Psychoactive substance is any chemical substance which affects mood, perception or consciousness through different mechanisms of action in the brain. The United Nations Convention on Psychotropic Substances in 1971 placed them under control, but new psycho¬active substances have emerged to circumvent international drug laws while producing effects similar to illicit substances. These new substances can be classified based on their psychoactive effects into: stimulants, opioids, classic hallucinogens, dissociatives and sedatives/hipnotics or according to their chemical family: phenethylamines, amphetamines, cathinones, piperazines, pipradrols/piperidines, aminoindanes, benzofurans, and tryptamines. They present a challenge to public health as they are unfamiliar molecules that can be - despite the similar chemical structure to the ones they try to mimic - unpredictable in terms of pharmacology and toxicology. There have been reports of adverse effects on CNS and cardiovascular system. The most popular groups seem to be synthetic cannabinoids and cathinones, but lately we observe rise in synthetic opioids as well, therefore detailed representation of this group is also included in the introduction. The aim of this master thesis was search for possible interactions of 18 new synthetic cathinones and cannabinoids with cardiac voltage-gated ion channels K1.4, hERG, Na1.5, pacemaker (HCN), and GABAA and nicotine acetylcholine receptor. The experiments were done using 1 μM solutions of analyzed substances, as certain studies suggest that some NPS exhibit effects already in nanomolar concentrations to verify such possible potency of our substances was also a part of our study. The research was done using two-electrode voltage clamp technique. Out of 18 analyzed cathinones and cannabinoids only 10 were what they advertised them for, while the remaining 8 were doubled as some of the ten already identified the researchers at the laboratory performed mass spectrometry for each substance to confirm their identities prior to our TEVC experiments. Expression of GABAA and nicotine acetylcholine receptor 7 were unsuccessful, HCN proved difficult as well, resulting in non-sufficient data acquisition. The results in general show no significant impact of 1 μM solutions of selected substances on analyzed ion channels, yet there are 3 cases, where further studies would be recommended. There was an average 5,5 % inhibition of hERG channel after the addition of 1μM 5F-AMB but only within 2 measurements, as we were not able to obtain the third one. 1μM 4-Cl-PVP caused an average 6,3 % inhibition of hERG channel. We have also observed an increase in Kv1.4 channel conductivity after the addition of 1 μM 5F-ADB in all 3 measurements with average increase of 7,1 %. As these were preliminary studies, statistical analysis was not committed. In order to unambiguously confirm the effect of the compounds on selected ion channels, measurements should be performed at even higher concentrations and half-maximum inhibitory concentration (IC50) determined.

Fil: Belkacemi, Thabet. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Niermann, Alexander. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Hofmann, Laura. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Wissenbach, Ulrich. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Research Triangle Park. Neurobiology Laboratory; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Leidinger, Petra. Universität des Saarlandes. Institut für Humangenetik; Alemania Fil: Backes, Christina. Universität des Saarlandes. Klinische Bioinformatik; Alemania Fil: Meese, Eckart. Universität des Saarlandes. Institut für Humangenetik; Alemania Fil: Keller, Andreas. Universität des Saarlandes. Klinische Bioinformatik; Alemania Fil: Bai, Xianshu. Universität des Saarlandes. Molekulare Physiologie; Alemania Fil: Scheller, Anja. Universität des Saarlandes. Molekulare Physiologie; Alemania Fil: Kirchhoff, Frank. Universität des Saarlandes. Molekulare Physiologie; Alemania Fil: Philipp, Stephan E. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Weissgerber, Petra. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Flockerzi, Veit. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Beck, Andreas. Universität des Saarlandes. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Beck, Andreas. Universität des Saarlandes. Molekulare Physiologie; Alemania Abstract: Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.

Background Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. Results To overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed. Conclusions Our strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.

VDAC (Voltage-Dependent Anion-selective Channel) is the primary metabolite pore in the mitochondrial outer membrane (OM). Atomic structures of VDAC, consistent with its physiological “open” state, are β-barrels formed by 19 transmembrane (TM) β-strands and an N-terminal segment (NTERM) that folds inside the pore lumen. However, structures are lacking for VDAC’s partially “closed” states. To provide clues about possible VDAC conformers, we used the RoseTTAFold neural network to predict structures for human and fungal VDAC sequences modified to mimic removal from the pore wall or lumen of “cryptic” domains, i.e., segments buried in atomic models yet accessible to antibodies in OM-bound VDAC. Predicted in vacuo structures for full-length VDAC sequences are 19-strand β-barrels similar to atomic models, but with weaker H-bonding between TM strands and reduced interactions between NTERM and the pore wall. Excision of combinations of “cryptic” subregions yields β-barrels with smaller diameters, wide gaps between N- and C-terminal β-strands, and in some cases disruption of the β-sheet (associated with strained backbone H-bond registration). Tandem repeats of modified VDAC sequences also were explored, as was domain swapping in monomer constructs. Implications of the results for possible alternative conformational states of VDAC are discussed.