Ion Channel Activity Research Articles

Investigation into the restoration of TRPM3 ion channel activity in post-COVID-19 condition: a potential pharmacotherapeutic target.

Recently, we reported that post COVID-19 condition patients also have Transient Receptor Potential Melastatin 3 (TRPM3) ion channel dysfunction, a potential biomarker reported in natural killer (NK) cells from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) patients. As there is no universal treatment for post COVID-19 condition, knowledge of ME/CFS may provide advances to investigate therapeutic targets. Naltrexone hydrochloride (NTX) has been demonstrated to be beneficial as a pharmacological intervention for ME/CFS patients and experimental investigations have shown NTX restored TRPM3 function in NK cells. This research aimed to: i) validate impaired TRPM3 ion channel function in post COVID-19 condition patients compared with ME/CFS; and ii) investigate NTX effects on TRPM3 ion channel activity in post COVID-19 condition patients. Whole-cell patch-clamp was performed to characterize TRPM3 ion channel activity in freshly isolated NK cells of post COVID-19 condition (N = 9; 40.56 ± 11.26 years), ME/CFS (N = 9; 39.33 ± 9.80 years) and healthy controls (HC) (N = 9; 45.22 ± 9.67 years). NTX effects were assessed on post COVID-19 condition (N = 9; 40.56 ± 11.26 years) and HC (N = 7; 45.43 ± 10.50 years) where NK cells were incubated for 24 hours in two protocols: treated with 200 µM NTX, or non-treated; TRPM3 channel function was assessed with patch-clamp protocol. This investigation confirmed impaired TRPM3 ion channel function in NK cells from post COVID-19 condition and ME/CFS patients. Importantly, PregS-induced TRPM3 currents were significantly restored in NTX-treated NK cells from post COVID-19 condition compared with HC. Furthermore, the sensitivity of NK cells to ononetin was not significantly different between post COVID-19 condition and HC after treatment with NTX. Our findings provide further evidence identifying similarities of TRPM3 ion channel dysfunction between ME/CFS and post COVID-19 condition patients. This study also reports, for the first time, TRPM3 ion channel activity was restored in NK cells isolated from post COVID-19 condition patients after in vitro treatment with NTX. The TRPM3 restoration consequently may re-establish TRPM3-dependent calcium (Ca2+) influx. This investigation proposes NTX as a potential therapeutic intervention and TRPM3 as a treatment biomarker for post COVID-19 condition.

Modeling the effect of magnetoelectric nanoparticles on neuronal electrical activity: An analog circuit approach.

This paper introduces a physical neuron model that incorporates magnetoelectric nanoparticles (MENPs) as an essential electrical circuit component to wirelessly control local neural activity. Availability of such a model is important as MENPs, due to their magnetoelectric effect, can wirelessly and noninvasively modulate neural activity, which, in turn, has implications for both finding cures for neurological diseases and creating a wireless noninvasive high-resolution brain-machine interface. When placed on a neuronal membrane, MENPs act as magnetic-field-controlled finite-size electric dipoles that generate local electric fields across the membrane in response to magnetic fields, thus allowing to controllably activate local ion channels and locally initiate an action potential. Herein, the neuronal electrical characteristic description is based on ion channel activation and inhibition mechanisms. A MENP-based memristive Hodgkin-Huxley circuit model is extracted by combining the Hodgkin-Huxley model and an equivalent circuit model for a single MENP. In this model, each MENP becomes an integral part of the neuron, thus enabling wireless local control of the neuron's electric circuit itself. Furthermore, the model is expanded to include multiple MENPs to describe collective effects in neural systems.

Investigating the role of local acidosis in hypothalamic vasopressin cell excitation during hypoxia

The supraoptic nucleus (SON) of the hypothalamus plays a key role in the regulation of fluid homeostasis through the synthesis and release of vasopressin (VP). In this population, VP release can occur at both dendritic and axonal sites, allowing for both localized and distal effects such as renal water retention and vasoconstriction. Recent work from our laboratory demonstrated an inverse neurovascular coupling (iNVC) response in which VP cell activation by acute salt loading resulted in dendritic release of VP, leading to localized vasoconstriction and hypoxia. Additionally, we demonstrated that the hypoxic milieu in turn potentiates VP cell activity and increases firing, suggesting that these mechanism constitutes a positive feedback loop that optimized VP activation in order to cope with the homeostatic challenge (Roy et al., Cell Reports 2021). Still, the biophysical mechanism by which the iNVC-hypoxia elicits increased firing in VP neurons has not yet been explored. We hypothesized that hypoxia-induced acidosis, via activation of acid sensitive ion channels (ASICs) (which are present in SON VP neurons; Ohbuchi et al in 2010) is a key mechanism contributing to VP cell excitation in the iNVC positive feedback loop. We investigated this with whole cell patch clamp electrophysiology using eGFP-VP transgenic rats. We recorded from identified SON VP cells and measured deflections in voltage as bath pH was changed from 7.25 to 6.3 (10 mins). Bath solution was either HCL-buffered artificial cerebrospinal fluid (aCSF) aerated with 95% O2/5% CO2 or HEPES-buffered solution (HBS). We observed an excitatory response to the decreases in pH in most SON VP cells recorded (68.75%, n = 11/16). These responses were reversible and, in many cases, repeatable following a second stimulation. To examine cell-type specificity, ongoing studies are focusing on comparing these responses to those obtained from oxytocin cells. Further, we plan to repeat these experiments in the presence of the ASIC-blocker amiloride and determine whether the hypoxia-induced excitatory effect during iNVC is indeed dependent on ASIC-channels. Together, our results support our hypothesis that tissue acidosis (likely via activation of ASIC-channels) contributes to the excitatory feedback loop evoked during salt-induced iNVC in VP neurons. This abstract was supported by the National Heart Lung and Blood Institute of the National Institutes of Health [R01 HL162575-01 (J.E.S.)]. 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.

Neuro-inflammatory interactions as a mediator of the Exaggerated Exercise Pressor Reflex (EPR) in a mouse model of Peripheral Artery Disease

Introduction and rationale: The Exercise pressor reflex (EPR) is a physiological mechanism in which the contraction of skeletal muscle results in a rise in heart rate (HR) and mean arterial pressure (MAP). In patients with Peripheral Artery Disease (PAD), this reflex is exaggerated, leading to abnormally high elevations in blood pressure during exercise. These cardiovascular responses in patients with PAD have been linked to increased morbidity and mortality, thus limiting the prescription of exercise for these patients. Using a novel murine model of the EPR, our laboratory has demonstrated that mice with femoral artery ligation (a well-established model of PAD), exhibit augmented increases in MAP which is mediated by PIEZO2 positive, mechanosensitive afferent neurons. We have demonstrated that inhibition of PIEZO2 expressing channels normalizes the MAP response to exercise. Patients with PAD also display increased macrophage infiltration in their ischemia-injured limbs, indicating a pro-inflammatory environment. Sensory neurons have been reported to detect various signals produced by immune cells including IL-1β and TNFα. The immune-derived signals can sensitize the peripheral terminals of the sensory neurons, thereby reducing their firing threshold and increasing their responsiveness. Previous studies have also reported the enhancement of Piezo2-mediated mechanosensitive currents in the presence of inflammatory signals. Hypothesis: We, therefore, hypothesize that in mice with femoral artery ligation, muscle inflammation increases the expression and activation of resident ion channels (e.g., Piezo2) in mechanosensitive afferent neurons to mediate the exaggerated EPR in PAD.Study objective: To mechanistically investigate the interaction between muscle inflammation and Piezo2 in mechanosensitive afferent neurons.Methods: We performed sham surgery or ligation of the femoral artery ipsilaterally in C57BL/6 mice. After 72 hours, we harvested the gastrocnemius muscle for histological and molecular analyses.Data and summary and results: Our preliminary findings reveal that there is significantly increased expression of pro-inflammatory genes and infiltrating immune cells in the ischemic muscle when compared to non-ischemic muscle.Specifically, H&E staining revealed the presence and accumulation of inflammatory cells, including macrophages, in the ischemic muscle compared to control, along with observed necrosis. Transcript analysis of the ischemic gastrocnemius muscle showed significantly increased IL-1β and TNFα, two pan-proinflammatory genes that encode cytokines released by macrophages. TNFα is known to induce the synthesis of adhesion molecules (E-selectin and ICAM-1) on endothelial cells, allowing leukocytes to adhere and cross the endothelial wall into the injured tissue. Indeed, we observed significantly increased ICAM-1 and E-selectin transcripts in the ischemic muscle compared to controls. MCP1, a well-established chemokine, was also significantly increased, further emphasizing the recruitment of leukocytes to the ischemic muscle. In addition, inducible NOS ( iNOS), which is known to be expressed in IL-1β- and TNFα-rich proinflammatory environment was also significantly increased in our FAL model compared to Sham. These data establish that the ischemic muscle of a femoral-artery ligated mouse has a high proinflammatory phenotype. We are currently exploring the impact of proinflammatory mediators on Piezo2 channel expression and activity, as well as the effect of macrophage depletion on Piezo2 in afferent neurons. The data from these studies will provide novel insights regarding the influence of neuroimmune interactions on cardiovascular responses to exercise in PAD. These data hold the potential to reveal innovative therapies enabling safe prescription of exercise, thus alleviating the burden of PAD, and improving the quality of life for those affected by this condition. Internal sources. 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.

Balancing sodium and potassium: Implication of mTORC2-mediated ENaC activation in DCT2 and early CNT for potassium balance in high dietary potassium

Background: The activity of the epithelial sodium ion channel, ENaC, and ENaC-dependent potassium (K+) secretion through ROMK are influenced by both aldosterone-dependent and independent mechanisms in response to dietary potassium (K+). While ENaC activity in the late connecting tubule (CNT) and cortical collecting duct (CCD) depends on aldosterone, it is aldosterone-independent in the late distal convoluted tubule (DCT2) and early CNT (eCNT). Aldosterone-stimulated ENaC activity relies on SGK1, transcriptionally upregulated by aldosterone and subsequently phosphorylated and activated by mTOR complex-2 (mTORC2). Recent evidence suggests that high potassium can rapidly activate mTORC2/SGK1 signaling to stimulate ENaC. In this study, we explore mTORC2’s role in K+ secretion and aldosterone-independent ENaC regulation. Methods: Rictor, a core component of mTORC2, was selectively knocked out (KO'd) in DCT2, CNT and CD by utilizing Calbindin as Cre-driver (CRKO mice) or late CNT and CD, by employing AQP2 as the Cre-driver (ARKO mice). Both wild-type (WT) and KO mice were subjected to a high potassium (HK) diet for short-term (4 hours) or medium-term (48 hours) periods, enabling the monitoring of both the early response and prolonged adaptation to the HK diet. Parameters assessed include urinary and blood electrolyte levels, renal transporter expression, activity and localization, and mTORC2 target phosphorylation. Results: On a normal K+ diet, both KO mice maintained Na+ and K+ balance but CRKO mice had elevated aldosterone levels. After short-term (4-hour) HK intake, CRKO mice had disrupted Na+ and K+ balance, reduced K+ excretion and hyperkalemia. In contrast, ARKO mice maintained Na+ and K+ balance with elevated aldosterone. On a prolonged HK diet (48 hours), CRKO mice had severe hyperkalemia, reduced GFR and significantly elevated aldosterone level. Immunofluorescence studies indicated markedly reduced apical localization of active ENaC in DCT2/eCNT of CRKO mice. Conversely, ENaC apical localization in the late CNT and CCD was mostly unaffected. Phosphorylation of mTORC2 targets involved in ENaC regulation, such as SGK1, and SGK1’s target, Nedd4-2, was also reduced in CRKO mice. ARKO mice maintained ion balance with normal K+ levels but significantly increased aldosterone levels and maintained ENaC apical localization in the late CNT and CCD. Conclusions: The data suggest that high aldosterone can compensate mTORC2 deficiency and maintain ENaC activity in the late CNT and CCD. However, mTORC2 activity is crucial for maintaining ENaC activity in the late DCT and early CNT (aldosterone-independent) and preserving potassium balance during high dietary potassium intake. National Institutes of Health (R01- DK56695), James Hilton Manning and Emma Austin Manning Foundation. 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.

Deletion of Piezo2 from renal Foxd1+ stromal progenitors and descendent cells results in abnormal mesangial development and sex-specific differences in kidney function

Piezo2 is a mechanically activated ion channel recently identified in glomerular mesangial cells and renin+ juxtaglomerular (JG) cells. Despite several studies which confirm this localization of Piezo2 in the kidney, the function of Piezo2 in these cells remains ambiguous. In the current study, we hypothesized that loss of Piezo2 in mesangial and JG cells would result in abnormal glomerular development during nephrogenesis and impaired salt and water balance in adult mice. To test this hypothesis, we generated mutant mice lacking Piezo2 in Foxd1+ stromal progenitors and their cell lineages, which include mesangial and JG cells. Kidney morphology was assessed by immunofluorescent staining and z-stack confocal microscopy during late nephrogenesis (post-natal day 0, P0) and following maturation of the nephron (P20) in wild-type (Piezo2fl/fl, n=3), heterozygous (Foxd1GC/+Piezo2fl/+, n=3), and mutant (Foxd1GC/+Piezo2fl/fl, n=3) mice. In adult male and female mice (n=2/genotype/sex), blood pressure was measured by tail cuff (CODA Systems) between 1-4pm following 3 days of acclimation. In a separate group of control and mutant male and female mice (8-10 weeks, n=3-5/group/sex), a 4-hour saline challenge (i.p. 10% body weight) was performed to assess diuresis and natriuresis. At P0, we found that deletion of Piezo2 from Foxd1+ stromal progenitors and their cell lineages resulted in the abnormal distribution of mesangial cells within glomeruli, such that the mesangial area appears compressed compared to control or heterozygous mice. This phenotype was exacerbated in mutant mice by P20. In adult mice, female mutants had an increase in daytime mean arterial pressure (~10mmHg, mean 79 vs 88) compared to female controls. Cumulative urine excretion (4 hours, percent injected) was decreased in females compared to males, regardless of genotype (p<0.001). Further, female mutant mice had a delayed diuresis response compared to control (2 hour, mean 16% vs 28% cumulative urine volume). However by 4 hours following a saline bolus both groups were similar. Taken together, these data indicate that deletion of Piezo2 in Foxd1+ stromal progenitors results in impairments in mesangial cell development during nephrogenesis and alterations in water and salt balance in adult female mice. Understanding the sex-specific roles of Piezo2 in kidney development and physiology is critical to identify the role of mechanosensation on renal homeostasis. NIH R01DK121014 to LO. 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.

Piezo2 sensory innervation during kidney development

Piezo2 is a mechanically activated ion channel expressed in a subset of sensory neurons, including in the bladder. Our lab has recently mapped innervation of the developing kidney and found that renal nerve development begins at embryonic day 13.5 (E13.5) and is tightly aligned with smooth muscle actin (SMA+) arterial development. However, the presence and spatiotemporal innervation of Piezo2+ sensory nerves in the kidney remains unknown. In the current study, we hypothesized that Piezo2+ sensory nerves would be present early in kidney development adjacent developing nephrons and that these axons innervating the kidney could be traced back to Piezo2+ cell bodies in the dorsal root ganglia (DRGS). To test this hypothesis, we performed immunofluorescent staining to label all nerves (Tubb3+, pan-neuronal marker) and Piezo2 in kidneys from wildtype mice at early (E13.5), mid (E15.5) and late (post-natal day 0, P0) nephrogenesis. To determine the signaling pathways of Piezo2+ nerves in the kidney to the peripheral nervous system (PNS), we utilized retrograde tracing with a Cre-driven fluorescently (pAAV-Flex-tdTomato) labeled virus injected into the kidney of heterozygous Piezo2Cre/+ adult mice (n=4, 10 weeks). The injected kidney, the contralateral control, and cervical to sacral DRGs were collected and fluorescent imaging was performed with confocal microscopy. We found that by E15.5, Piezo2+ sensory nerves were present in the developing kidney which constitute a small subset of all nerves (Tubb3+). These Tubb3+Piezo2+ sensory nerves persist through nephrogenesis (P0) and in the adult mouse kidney. Piezo2+ sensory nerves were found in nerve bundles running along SMA+ vasculature as well as observed adjacent glomeruli (Nphs2+) and proximal tubules (Lrp2+). Importantly, we also identified Piezo2+ sensory nerves in the renal cortex from human kidney samples. Retrograde tracing experiments revealed Piezo2+ sensory nerves in the kidney originate from lower thoracic and lumbar DRGs. Taken together, these data suggest that Piezo2+ sensory nerves are present in the mouse and human kidney and are capable of transmitting signals back to the PNS. As Piezo2 is known to be expressed in juxtaglomerular cells, Piezo2+ sensory nerves observed in close proximity suggests a previously unrecognized kidney cell-nerve crosstalk. Future studies will investigate Piezo2+ sensory activity and feedback in the kidney to understand the role of Piezo2 in renal mechanosenstion. NIH R01DK121014 to LO. 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.

Piezo1 mediated calcium-calcineurin signaling pathway regulates gastrointestinal smooth muscle cell phenotype in a stiffness dependent manner

Background: Gastrointestinal (GI) disorders like chronic constipation are more prevalent with aging. Aging GI tract becomes stiffer and exhibits delayed GI transit. Smooth muscle cells (SMCs) are involved in controlling GI transit and show reduced contractility with age. Piezo1 is a mechanically activated ion channel that responds to membrane tension by opening its pore to allow flow of ions such as calcium (Ca2+) and regulates various cellular processes through Ca2+-calcineurin signaling pathway. We wondered GI stiffness changes might control SMC contractile to non-contractile phenotype through Piezo1. Objective: To determine how substrate stiffness regulates GI SMC contractile phenotype through Piezo1. Hypothesis: SMC Piezo1 senses GI tissue stiffness changes and alters SMC phenotype. Methods: Pathway analysis on human colon single cell sequencing data was performed using Gene Set Enrichment Analysis (GSEA) pre-ranked module with publicly available gene-sets from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Immortalized human jejunal SMCs (HuSMCs) and freshly dissociated SMCs from control (CTRL, Myh11-creERT2) and SMC specific Piezo1 knockout [Myh11-creERT2xPiezo1 Flox (Piezo1-KO)] mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrates to model age-dependent shifts in intestinal stiffness for 48 hours either in the presence/absence of Piezo1 siRNA, Piezo1 agonist (Yoda1, 1μM), and calcineurin blocker (Cyclosporin A, CsA, 1μM) and used for Ca2+ imaging, RT-qPCR and immunostaining. Results: GSEA showed Piezo1+ve SMCs were significantly enriched in pathways involved in the regulation of Ca2+ signaling compared to Piezo1−ve SMCs (p<0.05). HuSMCs and primary SMCs showed increased Piezo1 expression (HuSMCs:1.1±0.2-fold, n=8, p<0.05; primary SMCs:2.1±0.1-fold, n=3, p<0.05) and cytoplasmic Ca2+ (3.6-fold, n=10, p<0.05) in response to Yoda1 on stiff substrates compared to soft. HuSMCs on stiff substrate showed decreased expression of contractile SMC markers smoothelin ( SMTN:0.7±0.2-fold, n=7, p<0.05) and transgelin ( TAGLN:0.6±0.1-fold, n=7, p<0.05) which was rescued by Piezo1 siRNA treatment ( SMTN:1.2±0.1-fold, n=2-7, p<0.05; TAGLN:1.15±0.02-fold, n=2-7, p<0.05) (Piezo1 is necessary). Stimulation by Yoda1 of HuSMCs on the soft substrate resulted in decreased expression of SMTN (0.7±0.3-fold, n=2-7, p<0.05) and TAGLN (0.7±0.1-fold, n=2-7, p<0.05) (Piezo1 is suffcient). CTRL primary SMCs on stiff substrate showed decreased expression of Smtn (0.4±0.1-fold, n=3, p<0.05) and Tagln (0.3±0.2-fold, n=3, p<0.05) compared to SMCs on soft substrate which was reversed when SMCs from Piezo1-KO mice were cultured on these substrates ( Smtn:1.8±0.5-fold, n=3, p<0.05; Tagln:2.4±0.07-fold, n=3, p<0.05). Treatment of CsA in HuSMCs cultured on stiff substrate also rescued from loss of SMTN (1.3±0.1-fold, n=3, p<0.05) and TAGLN (1.7±0.3-fold, n=3, p<0.05) expression and prevented nuclear factor of activated T cells (NFAT) nuclear translocation. Summary and Conclusion: GI SMC Piezo1 senses stiffness changes and regulates SMC phenotype through Ca2+-calcineurin signaling pathway. Targeting Piezo1 might be protective from age induced reduced GI SMC contractility. NIH DK052766, DK123549. 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.

Modulation of Endothelial Cell Mitochondrial Phenotype and Angiogenesis by TRPV4

Transient receptor potential vanilloid type 4 (TRPV4) is a mechanically activated ion channel implicated in the regulation of endothelial cell (EC) proliferation, migration, and angiogenesis. However, the molecular mechanism(s) by which TRPV4 regulates EC functions remains underexplored. Here, we investigated if TRPV4 channels mediate endothelial function via modulation of mitochondria. Confocal microscopy showed a peri-nuclear localization of mitochondria in normal EC (NEC), while they were localized throughout the cell in TRPV4 knockout EC (KOEC). Furthermore, transmission electron microscopy confirmed clear round mitochondria in NEC compared to elongated mitochondria with distinct cristae in KOEC. Importantly, we found increased distribution of mitochondria in KOEC with increasing stiffness, when cultured on extracellular matrix (ECM) gels of varying stiffness that mimic the stiffness of matrix in pathophysiological conditions such as tumor or heart failure (0.2, 8, and 50 kPa). Mechanistically, western blot analysis showed increased fusion/fission protein ratio (Optic Atrophy 1 (OPA1)/mitochondrial fission factor (MFF)), in KOEC compared to NEC. Seahorse flux analyzer analysis demonstrated increased basal oxygen consumption rate (OCR), maximal OCR, ATP-linked OCR, and spare capacity in KOEC compared to NEC, which were significantly attenuated by a small molecule inhibitor of OPA1, MYLS22. Finally, MYLS22 normalized TRPV4-knockout mediated abnormal proliferation, migration, and angiogenesis ex vivo. Taken together, these findings indicate that mechanosensitive TRPV4 channels regulate mitochondrial phenotype and function during angiogenesis through OPA1. This work was supported by the National Institutes of Health (and R01HL148585 and AHA-TPA-971237) to CKT. 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.

Genomic and non-genomic action of vitamin D on ion channels – Targeting mitochondria

Recent studies revealed that mitochondria are not only a place of vitamin D3 metabolism but also direct or indirect targets of its activities. This review summarizes current knowledge on the regulation of ion channels from plasma and mitochondrial membranes by the active form of vitamin D3 (1,25(OH)2D3). 1,25(OH)2D3, is a naturally occurring hormone with pleiotropic activities; implicated in the modulation of cell differentiation, and proliferation and in the prevention of various diseases, including cancer. Many experimental data indicate that 1,25(OH)2D3 deficiency induces ionic remodeling and 1,25(OH)2D3 regulates the activity of multiple ion channels. There are two main theories on how 1,25(OH)2D3 can modify the function of ion channels. First, describes the involvement of genomic pathways of response to 1,25(OH)2D3 in the regulation of the expression of the genes encoding channels, their auxiliary subunits, or additional regulators. Interestingly, intracellular ion channels, like mitochondrial, are encoded by the same genes as plasma membrane channels. Therefore, the comprehensive genomic regulation of the channels from these two different cellular compartments we analyzed using a bioinformatic approach. The second theory explores non-genomic pathways of vitamin D3 activities. It was shown, that 1,25(OH)2D3 indirectly regulates enzymes that impact ion channels, change membrane physical properties, or directly bind to channel proteins. In this article, the involvement of genomic and non-genomic pathways regulated by 1,25(OH)2D3 in the modulation of the levels and activity of plasma membrane and mitochondrial ion channels was investigated by an extensive review of the literature and analysis of the transcriptomic data using bioinformatics.

Targeting TRPV4 Channels for Cancer Pain Relief.

Despite the unique and complex nature of cancer pain, the activation of different ion channels can be related to the initiation and maintenance of pain. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel broadly expressed in sensory afferent neurons. This channel is activated by multiple stimuli to mediate pain perception associated with inflammatory and neuropathic pain. Here, we focused on summarizing the role of TRPV4 in cancer etiology and cancer-induced pain mechanisms. Many studies revealed that the administration of a TRPV4 antagonist and TRPV4 knockdown diminishes nociception in chemotherapy-induced peripheral neuropathy (CIPN). Although the evidence on TRPV4 channels' involvement in cancer pain is scarce, the expression of these receptors was reportedly enhanced in cancer-induced bone pain (CIBP), perineural, and orofacial cancer models following the inoculation of tumor cells to the bone marrow cavity, sciatic nerve, and tongue, respectively. Effective pain management is a continuous problem for patients diagnosed with cancer, and current guidelines fail to address a mechanism-based treatment. Therefore, examining new molecules with potential antinociceptive properties targeting TRPV4 modulation would be interesting. Identifying such agents could lead to the development of treatment strategies with improved pain-relieving effects and fewer adverse effects than the currently available analgesics.

MiR-153 promotes neural differentiation by activating the cell adhesion/Ca2+ signaling pathway and targeting ion channel activity in HT-22 cells by bioinformatic analysis

MicroRNAs have been studied extensively in neurodegenerative diseases. In a previous study, miR-153 promoted neural differentiation and projection formation in mouse hippocampal HT-22 cells. However, the pathways and molecular mechanism underlying miR-153-induced neural differentiation remain unclear. To explore the molecular mechanism of miR-153 on neural differentiation, we performed RNA sequencing on miR-153-overexpressed HT-22 cells. Based on RNA sequencing, differentially expressed genes (DEGs) and pathways in miR-153-overexpressed cells were identified. The Database for Annotation, Visualization and Integrated Discovery and Gene Set Enrichment Analysis were used to perform functional annotation and enrichment analysis of DEGs. Targetscan predicted the targets of miR-153. The Search Tool for the Retrieval of Interacting Genes and Cytoscape, were used to construct protein-protein interaction networks and identify hub genes. Q-PCR was used to detect mRNA expression of the identified genes. The expression profiles of the identified genes were compared between embryonic days 9.5 (E9.5) and E11.5 in the embryotic mouse brain of the GDS3442 dataset. Cell Counting Kit-8 assay was used to determine cell proliferation and cellular susceptibility to amyloid β-protein (Aβ) toxicity in miR-153-overexpressed cells. The results indicated that miR-153 increased cell adhesion/Ca2+ (Cdh5, Nrcam, and P2rx4) and Bdnf/Ntrk2 neurotrophic signaling pathway, and decreased ion channel activity (Kcnc3, Kcna4, Clcn5, and Scn5a). The changes in the expression of the identified genes in miR-153-overexpressed cells were consistent with the expression profile of GDS3442 during neural differentiation. In addition, miR-153 overexpression decreased cellular susceptibility to Aβ toxicity in HT-22 cells. In conclusion, miR-153 overexpression may promote neural differentiation by inducing cell adhesion and the Bdnf/Ntrk2 pathway, and regulating electrophysiological maturity by targeting ion channels. MiR-153 may play an important role in neural differentiation; the findings provide a useful therapeutic direction for neurodegenerative diseases.

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.

Activating Ion Channels in Collapsed Hydrogel Derived Densified MXene Films with Cellulose Nanofibers to Overcome the Areal Versus Volumetric Capacitance Trade-Off.

Concomitant achievement of all three performance pillars of a supercapacitor device, namely gravimetric, areal, and volumetric capacitance is a grand challenge. Nevertheless, its fulfilment is indispensable for commercial usage. Although, high compactness is the fundamental requirement to achieve high volumetric performance, it severely affects ion transportation in thick electrodes. Such trade-off makes it extremely challenging to realize very high areal and volumetric performance simultaneously. Here, a collapsed hydrogel strategy is introduced to develop MXene/cellulose nanofiber (CNF) based densified electrodes that offer excellent ion transportation despite a massive increase in areal mass loading (>70mg cm-2). Quasi-oriented MXene/CNF (MXCF) hydrogels are produced through an electric field-guided co-assembly technique. Ambient dehydration of these hydrogels incorporates numerous pores in the resultant compact electrodes due to crumpling of the MXene sheets, while CNF ensures connectivity among the locally blocked pores in different length scales. The resultant collapsed MXCF densified electrode shows a remarkably high areal capacitance of 16 F cm-2 while simultaneously displaying a high volumetric capacitance of 849.8 F cm-3 at an ultrahigh mass loading of up to 73.4mg cm-2. The universality of strategy, including the co-assembly of hydrogel and its collapse, is further demonstrated to develop high-performance asymmetric and wearable devices.

Selective activation of photoactivatable fluorescent protein based on binary holography

The ability to deliver laser doses to different target locations with high spatial and temporal resolution has been a long-sought goal in photo-stimulation and optogenetics research via, for example, photoactivatable proteins. These light-sensitive proteins undergo conformational changes upon photoactivation, serving functions such as triggering fluorescence, modulating ion channel activities, or initiating biochemical reactions within cells. Conventionally, photo-stimulation on light-sensitive proteins is performed by serially scanning a laser focus or via 2D projection, which is limited by relatively low spatiotemporal resolution. In this work, we present a programmable two-photon stimulation method based on a digital micromirror device (DMD) and binary holography to perform the activation of photoactivatable green fluorescent protein (PAGFP) in live cells. This method achieved grayscale and 3D selective PAGFP activation with subcellular resolution. In the experiments, we demonstrated the 3D activation capability and investigated the diffusion dynamics of activated PAGFP on the cell membrane. A regional difference in cell membrane diffusivity was observed, indicating the great potential of our approach in interrogating the spatiotemporal dynamics of cellular processes inside living cells.

Cholestane-3β,5α,6β-Triol Inhibits Acid-Sensing Ion Channels and Reduces Acidosis-Mediated Ischemic Brain Injury.

Activation of the acid-sensing ion channels (ASICs) by tissue acidosis, a common feature of brain ischemia, contributes to ischemic brain injury, while blockade of ASICs results in protection. Cholestane-3β,5α,6β-triol (Triol), a major cholesterol metabolite, has been demonstrated as an endogenous neuroprotectant; however, the mechanism underlying its neuroprotective activity remains elusive. In this study, we tested the hypothesis that inhibition of ASICs is a potential mechanism. The whole-cell patch-clamp technique was used to examine the effect of Triol on ASICs heterogeneously expressed in Chinese hamster ovary cells and ASICs endogenously expressed in primary cultured mouse cortical neurons. Acid-induced injury of cultured mouse cortical neurons and middle cerebral artery occlusion-induced ischemic brain injury in wild-type and ASIC1 and ASIC2 knockout mice were studied to examine the protective effect of Triol. Triol inhibits ASICs in a subunit-dependent manner. In Chinese hamster ovary cells, it inhibits homomeric ASIC1a and ASIC3 without affecting ASIC1β and ASIC2a. In cultured mouse cortical neurons, it inhibits homomeric ASIC1a and heteromeric ASIC1a-containing channels. The inhibition is use-dependent but voltage- and pH-independent. Structure-activity relationship analysis suggests that hydroxyls at the 5 and 6 positions of the A/B ring are critical functional groups. Triol alleviates acidosis-mediated injury of cultured mouse cortical neurons and protects against middle cerebral artery occlusion-induced brain injury in an ASIC1a-dependent manner. Our study identifies Triol as a novel ASIC inhibitor, which may serve as a new pharmacological tool for studying ASICs and may also be developed as a potential drug for treating stroke.

A phytosphingosine derivative mYG-II-6 inhibits histamine-mediated TRPV1 activation and MRGPRX2-dependent mast cell degranulation

BackgroundPhytosphingosine and its derivative are known for their skin-protective properties. While mYG-II-6, a phytosphingosine derivative, has shown anti-inflammatory and antipsoriatic effects, its potential antipruritic qualities have yet to be explored. This study aimed to investigate mYG-II-6′s antipruritic properties. MethodsThe calcium imaging technique was employed to investigate the activity of ion channels and receptors. Mast cell degranulation was confirmed through the β-hexosaminidase assay. Additionally, in silico molecular docking and an in vivo mouse scratching behavior test were utilized. ResultsUsing HEK293T cells transfected with H1R and TRPV1, we examined the impact of mYG-II-6 on histamine-induced intracellular calcium rise, a key signal in itch-mediating sensory neurons. Pretreatment with mYG-II-6 significantly reduced histamine-induced calcium levels and inhibited TRPV1 activity, suggesting its role in blocking the calcium influx channel. Additionally, mYG-II-6 suppressed histamine-induced calcium increase in primary cultures of mouse dorsal root ganglia, indicating its potential antipruritic effect mediated by histamine. Interestingly, mYG-II-6 exhibited inhibitory effects on human MRGPRX2, a G protein-coupled receptor involved in IgE-independent mast cell degranulation. However, it did not inhibit mouse MrgprB2, the ortholog of human MRGPRX2. Molecular docking analysis revealed that mYG-II-6 selectively interacts with the binding pocket of MRGPRX2. Importantly, mYG-II-6 suppressed histamine-induced scratching behaviors in mice. ConclusionsOur findings show that mYG-II-6 can alleviate histamine-induced itch sensation through dual mechanisms. This underscores its potential as a versatile treatment for various pruritic conditions.

Piezo1 stretch-activated channel activity differs between murine bone marrow-derived and cardiac tissue-resident macrophages.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28days after cryoinjury compared to sham operated mice.

Mechanism of noise induced hidden hearing loss based on proteomics

Objective: To explore the mechanism of noise-induced hidden hearing loss by proteomics. Methods: In October 2022, 64 SPF male C57BL/6J mice were divided into control group and noise exposure group with 32 mice in each group according to random sampling method. The noise exposure group was exposed to 100 dB sound pressure level, 2000-16000 Hz broadband noise for 2 h, and the mouse hidden hearing loss model was established. Auditory brainstem response (ABR) was used to test the change of hearing threshold of mice on the 7th day after noise exposure, the damage of basal membrane hair cells was observed by immunofluorescence, and the differentially expressed proteins in the inner ear of mice in each group were identified and analyzed by 4D-Label-free quantitative proteomics, and verified by Western blotting. The results were statistically analyzed by ANOVA and t test. Results: On the 7th day after noise exposure, there was no significant difference in hearing threshold between the control group and the noise exposure group at click and 8000 Hz acoustic stimulation (P>0.05) . The hearing threshold in the noise exposure group was significantly higher than that in the control group under 16000 Hz acoustic stimulation (P<0.05) . Confocal immunofluorescence showed that the basal membrane hair cells of cochlear tissue in noise exposure group were arranged neatly, but the relative expression of C-terminal binding protein 2 antibody of presynaptic membrane in middle gyrus and basal gyrus was significantly lower than that in control group (P<0.05) . GO enrichment analysis showed that the functions of differentially expressed proteins were mainly concentrated in membrane potential regulation, ligand-gated channel activity, and ligand-gated ion channel activity. KEGG pathway enrichment analysis showed that differentially expressed proteins were significantly enriched in phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling pathway, NOD-like receptor signaling pathway, calcium signaling pathway, etc. Western blotting showed that the expression of inositol 1, 4, 5-trisphosphate receptor 3 (Itpr3) was increased and the expression of solute carrier family 38 member 2 (Slc38a2) was decreased in the noise exposure group (P<0.05) . Conclusion: Through proteomic analysis, screening and verification of the differential expression proteins Itpr3 and Slc38a2 in the constructed mouse noise-induced hidden hearing loss model, the glutaminergic synaptic related pathways represented by Itpr3 and Slc38a2 may be involved in the occurrence of hidden hearing loss.