Activation Of Cation Channels Research Articles

Transient receptor potential vanilloid 4 regulates extracellular matrix composition and mediates load-induced intervertebral disc degeneration in a mouse model

ObjectiveTransient receptor potential vanilloid 4 (TRPV4) is a multi-modally activated cation channel that mediates mechanotransduction pathways by which musculoskeletal tissues respond to mechanical load and regulate tissue health. Using conditional Trpv4 knockout mice, we investigated the role of Trpv4 in regulating intervertebral disc (IVD) health and injury-induced IVD degeneration. MethodsCol2Cre;Trpv4fl/f (Trpv4 KO) mice were used to knockout Trpv4 in all type 2 collagen-expressing cells. Effects of gene targeting alone was assessed in lumbar spines, using vertebral bone length measurement, histological, immunohistochemistry and gene expression analyses, and mechanical testing. Disc puncture was performed on caudal IVDs of wild-type (WT) and Trpv4 KO mice at 2.5- and 6.5-months-of-age. 6 weeks after puncture (4- and 8-months-of-age at sacrifice), caudal spines were assessed using histological analyses. ResultsWhile loss of Trpv4 did not significantly alter vertebral bone length and tissue histomorphology compared to age-matched WT mice, Trpv4 KO mice showed decreased proteoglycan and PRG4 staining in the AF compared to WT. At the gene level, Trpv4 KO mice showed significantly increased expression of Acan, Bgn, and Prg4 compared to WT. Functionally, loss of Trpv4 was associated with significantly increased neutral zone length in lumbar IVDs. Following puncture, both Trpv4 KO and WT mice showed similar signs of degeneration at the site of injury. Interestingly, loss of Trpv4 prevented mechanically-induced degeneration in IVDs adjacent to sites of injury. ConclusionThese studies suggest a role for Trpv4 in regulating extracellular matrix synthesis and mediating the response of IVD tissues to mechanical stress.

Evaluation of 5'-tRF-His-GTG As a Molecular Biomarker in Breast Cancer Diagnoses and Prognosis.

Background: Breast cancer (BC) is the most prevalent cancer among women worldwide. Although advances have been made in the identification of predictive biomarkers, current options for early diagnosis and prognostic analysis are still suboptimal. Recently, transfer-RNA-derived RNA fragments (tRFs) have emerged as a class of small noncoding RNAs that play a role in the cancer progression. The authors aimed to identify a specific class of tRFs as a molecular marker for BC diagnosis and prognosis in clinical management. Methods: The levels of 5'-tRF-His-GTG were quantified in BC tissue (n = 101) and inflammatory normal breast tissue (n = 22) using in situ hybridization. Clinicopathological parameters were obtained, including age, tumor node metastasis stage, hormone receptor status, histopathological grade, lymphovascular invasion, and recurrence. The correlation between the expression of 5'-tRF-His-GTG and these parameters in different BC subtypes was analyzed. Patient death and cancer progression were regarded as clinical endpoints in the survival analysis. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also performed to predict the involvement in pivotal biological process. Results: The expression of 5'-tRF-His-GTG was significantly downregulated in BC tissues and was in connection with T stage in human epidermal growth factor 2-positive and basal-like BC, as well as N stage and histopathological grade in luminal BC. Patients with low expression of 5'-tRF-His-GTG had a poor overall survival rate. Statistics of GO and KEGG pathway revealed that cation channel activity, protein catabolic process, response to temperature stimulus, cell cycle, focal adhesion, and glycerophospholipid metabolism were significantly enriched. Conclusions: This study suggests that the assessment of 5'-tRF-His-GTG expression could serve as a novel biomarker for individual diagnosis and prognosis in BC.

Quantitative Proteomic Analysis Identifying and Evaluating TRAF6 and IL-8 as Potential Diagnostic Biomarkers in Neonatal Patients with Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a common gastrointestinal complication in premature infants, resulting in high morbidity and mortality, and its early detection is crucial for accurate treatment and outcome prediction. Extensive research has demonstrated a clear correlation between NEC and extremely low birth weight, degree of preterm, formula feeding, infection, hypoxic/ischemic damage, and intestinal dysbiosis. The development of noninvasive biomarkers of NEC from stool, urine, and serum has attracted a great deal of interest because to these clinical connections and the quest for a deeper knowledge of disease pathophysiology. Therefore, this study aims to identify protein expression patterns in NEC and discover innovative diagnostic biomarkers. In this study, we recruited five patients diagnosed with NEC and paired necrotic segments of intestinal tissue with adjacent normal segments of intestine to form experimental and control groups. Quantitative proteomics tandem mass tagging (TMT) labeling technique was used to detect and quantify the proteins, and the expression levels of the candidate biomarkers in the intestinal tissues were further determined by quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, Immunofluorescence methods and enzyme-linked immunosorbent assay (ELISA). A total of 6880 proteins were identified and quantified in patients with NEC. A significant disparity in protein expression was observed between necrotic and normal segments of intestinal tissue in NEC patients. A total of 55 proteins were found to be upregulated, and 40 proteins were found to be downregulated in NEC patients when using a p-value of < 0.05, and an absolute fold change of > 1.2 for analysis. GO function enrichment analysis showed the positive regulation of significant biological processes such as mitochondrial organization, vasoconstriction, rRNA catabolism, fluid shear stress response, and glycerol ether biosynthesis processes. Enrichment analysis also revealed essential functions such as ligand-gated ion channel activity, potassium channel activity, ligand-gated cation channel activity, ligand-gated ion channel activity, and ligand-gated channel activity, including molecular functions such as ligand-gated ion channel activity and mitotic events in this comparative group. Significant changes were found in endomembrane protein complex, membrane fraction, mitochondrial membrane fraction, membrane components, membrane intrinsic components, and other localized proteins. Additional validation of intestinal tissue and serum revealed a substantial increase in TRAF6 (tumor necrosis factor receptor-associated factor 6) and IL-8(Interleukin-8, CXCL8). The quantitative proteomic TMT method can effectively detect proteins with differential expression in the intestinal tissues of NEC patients. Proteins TRAF6 and CXCL8/IL-8 are significantly upregulated in the intestinal tissues and serum samples of patients and may serve as valuable predictor factors for NEC's early diagnosis.

The role of mechanically sensitive ion channel Piezo1 in bone remodeling.

Piezo1 (2010) was identified as a mechanically activated cation channel capable of sensing various physical forces, such as tension, osmotic pressure, and shear force. Piezo1 mediates mechanosensory transduction in different organs and tissues, including its role in maintaining bone homeostasis. This review aimed to summarize the function and possible mechanism of Piezo1 in the mechanical receptor cells in bone tissue. We found that it is a potential therapeutic target for the treatment of bone diseases.

Piezo1-ERK1/2-YAP Signaling Cascade Regulates the Proliferation of Urine-derived Stem Cells on Collagen Gels.

Urine-derived stem cells (USCs) were considered to be an ideal source of stem cells for repairing urological diseases. However, the proliferative ability of USCs significantly decreased when cultured on plastic dishes, which limited their clinical application. It was found that collagen gels could promote the proliferation of USCs, but the underlying molecular mechanisms were unclear. The study aims to investigate the role of the mechanically activated cation channel Piezo1 and the transcriptional coactivator YAP in the regulation of proliferation of USCs on collagen gels. USCs were cultured on collagen gels (group COL), or plastic dishes (group NON). MTT assay, Scratch assay, EDU staining, and immunofluorescence (IF) of Ki67 were performed to evaluate the proliferation of USCs; IF of YAP was conducted to observe its nuclear localization; calcium imaging experiment was executed to evaluate the function of Piezo1; western blot was used to compare changes in protein expression of YAP, LATS1, ERK1/2, and p-ERK1/2. In addition, the regulatory effect of YAP on the proliferative capacity of USCs was confirmed by intervening YAP with its inhibitor verteporfin (VP); and the inhibitor or activator of Piezo1, GsMTx4 or Yoda1 was used to explore the effect of Piezo1 on the nuclear localization of YAP, the proliferation of USCs and the regeneration of injured bladder. The results showed that cell proliferation was significantly enhanced in USCs in the COL group with the nuclear accumulation of YAP compared with the NON group and VP attenuated these effects. The expression and function of Piezo1 were higher in the COL group compared with the NON group. Blockage of Piezo1 by GsMTx4 decreased nuclear localization of YAP, the proliferation of USCs, and caused the failure of bladder reconstruction. Activation of Piezo1 by Yoda1 increased the nuclear expression of YAP, and the proliferation of USCs, which further improved the regeneration of the injured bladder. Finally, the ERK1/2 rather than LATS1 was revealed to participate in the Piezo1/YAP signal cascades of USCs proliferation. Taken together, Piezo1-ERK1/2-YAP signal cascades were involved in regulating the proliferation ability of USCs in collagen gels which would be beneficial for the regeneration of the bladder.

Roles of Chemokines in Intervertebral Disk Degeneration.

Intervertebral disc degeneration is the primary etiology of low back pain and radicular pain. This review examines the roles of crucial chemokines in different stages of degenerative disc disease, along with interventions targeting chemokine function to mitigate disc degeneration. The release of chemokines from degenerated discs facilitates the infiltration and activation of immune cells, thereby intensifying the inflammatory cascade response. The migration of immune cells into the venous lumen is concomitant with the emergence of microvascular tissue and nerve fibers. Furthermore, the presence of neurogenic factors secreted by disc cells and immune cells stimulates the activation of pain-related cation channels in the dorsal root ganglion, potentially exacerbating discogenic and neurogenic pain and intensifying the degenerative cascade response mediated by chemokines. Gaining a deeper comprehension of the functions of chemokines and immune cells in these processes involving catabolism, angiogenesis, and injury detection could offer novel therapeutic avenues for managing symptomatic disc disease.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) regulates toll-like receptor 4 signaling in macrophages

MARCKS (myristoylated alanine-rich C-kinase substrate) is a membrane-associated protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (toll-like receptor 4).The presence of MARCKS and the formation of phospho-MARCKS in various cell types have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. We investigated the role of MARCKS in inflammation. Confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation.CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated the production of TNF and IL6, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes including innate immune response, inflammatory response, cytokine production, and molecular functions such as extracellularly ATP-gated cation channel activity, electron transfer activity and oxidoreductase activity, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory diseases.

A new method to measure aquaporin-facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells.

Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H2 O2 ) and, possibly, cations, essential signalling molecules in many physiological processes. While the determination of the channel activity generally depends on heterologous expression of AQPs in Xenopus oocytes or yeast cells, we established a genetic tool to determine whether they facilitate the diffusion of H2 O2 through the plasma membrane in living plant cells. We designed genetic constructs to co-express the fluorescent H2 O2 sensor HyPer and AQPs, with expression controlled by a heat shock-inducible promoter in Nicotiana tabacum BY-2 suspension cells. After induction of ZmPIP2;5 AQP expression, a HyPer signal was recorded when the cells were incubated with H2 O2 , suggesting that ZmPIP2;5 facilitates H2 O2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H2 O2 channel. ZmPIP2;1, ZmPIP2;4and AtPIP2;1 also facilitated H2 O2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H2 O2 accumulation in BY-2 cells expressing ZmPIP2;5. We also monitored cation channel activity of ZmPIP2;5 using a novel fluorescent photo-switchable Li+ sensor in BY-2 cells. BY-2 suspension cells engineered for inducible expression of AQPs as well as HyPer expression and the use of Li+ sensors constitute a powerful toolkit for evaluating the transport activity and the molecular determinants of PIPs in living plant cells.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability in Working Memory Circuits.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels co-immunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

A high-throughput electrophysiology assay to study the response of PIEZO1 to mechanical stimulation.

PIEZO1 channels are mechanically activated cation channels that play a pivotal role in sensing mechanical forces in various cell types. Their dysfunction has been associated with numerous pathophysiological states, including generalized lymphatic dysplasia, varicose vein disease, and hereditary xerocytosis. Given their physiological relevance, investigating PIEZO1 is crucial for the pharmaceutical industry, which requires scalable techniques to allow for drug discovery. In this regard, several studies have used high-throughput automated patch clamp (APC) combined with Yoda1, a specific gating modifier of PIEZO1 channels, to explore the function and properties of PIEZO1 in heterologous expression systems, as well as in primary cells. However, a combination of solely mechanical stimulation (M-Stim) and high-throughput APC has not yet been available for the study of PIEZO1 channels. Here, we show that optimization of pipetting parameters of the SyncroPatch 384 coupled with multihole NPC-384 chips enables M-Stim of PIEZO1 channels in high-throughput electrophysiology. We used this approach to explore differences between the response of mouse and human PIEZO1 channels to mechanical and/or chemical stimuli. Our results suggest that applying solutions on top of the cells at elevated pipetting flows is crucial for activating PIEZO1 channels by M-Stim on the SyncroPatch 384. The possibility of comparing and combining mechanical and chemical stimulation in a high-throughput patch clamp assay facilitates investigations on PIEZO1 channels and thereby provides an important experimental tool for drug development.

Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

Enhancement of muscarinic receptor-mediated excitation in spontaneously hypertensive rat adrenal medullary chromaffin cells.

One of the mechanisms for hypertension is an increase in blood catecholamines due to increased secretion from sympathetic nerve terminals and adrenal medullary chromaffin (AMC) cells. Spontaneously hypertensive rats (SHRs) are used as an animal model of hypertension. Catecholamine secretion in AMC cells occurs in response to humoral factors and neuronal inputs from the sympathetic nerve fibres. Acetylcholine (ACh) released from the nerve terminals activates nicotinic as well as muscarinic ACh receptors. The present experiment aimed to elucidate whether muscarinic receptor-mediated excitation is altered in SHR AMC cells and, if it is, how. Compared with normotensive rat AMC cells, muscarinic stimulation induced greater catecholamine secretion and larger depolarising inward currents in SHR AMC cells. In contrast to normotensive rat AMC cells, the muscarine-induced current consisted of quinine-sensitive and quinine-insensitive components. The former and the latter are possibly ascribed to nonselective cation channel activation and TWIK-related acid-sensitive K+ (TASK) channel inhibition, as noted in guinea pig AMC cells. In fact, immunoreactive material for TASK1 and several isoforms of transient receptor potential canonical (TRPC) channels was detected in SHR AMC cells. Stromal interaction molecule 1 (STIM1), which plays an essential role for heteromeric TRPC1-TRPC4 channel formation and is not expressed in normotensive rat AMC cells, was detected in the cytoplasm and co-localised with TRPC1. The expression of muscarinic M1 receptors was enhanced in SHR AMC cells compared with normotensive rats. The results indicate that muscarinic excitation is enhanced in SHR AMC cells, probably through facilitation of TRPC channel signalling.

Abstract EC.09: Single Nuclei RNA Sequencing Analysis Of Mybpc-3- Associated HCM To Identify Common Pathological Mechanisms In Human, Feline, And Murine Hearts

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease characterized by unexplained hypertrophy, often associated with left ventricular outflow tract (LVOT) obstruction, ventricular tachyarrhythmias, diastolic heart failure, and sudden cardiac death. Mutations in mybpc3 are the most common genetic mutations associated with human and feline HCM. To understand how mutations in mybc3 causing sarcomere dysfunction result in the development of non-myocyte phenotypes such as fibrosis, mitral valve anomalies, and microvascular occlusion across species, we generated single nuclei RNA-sequencing datasets from feline, human and murine heart tissue carrying mybpc3 variants. We analyzed these datasets for differential gene expression by genotype for each cell type and assessed changes in cell-cell communication by ligand-receptor analysis. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in feline cardiomyocytes revealed alteration in regulation of calcium ion transmembrane transporter activity, regulation of cation channel activity, and extracellular matrix structure and organization, consistent with known phenotypic aspects of HCM such as calcium dyshomeostasis and fibrosis. DEGs in cardiomyocytes of human tissues revealed novel obstruction of cGMP-mediated signaling, protein O-linked glycosylation and negative regulation of cAMP-mediated signaling. DEGs in murine cardiomyocytes affected cardiac muscle thin filament assembly, CDP-diacylglycerol biosynthesis and cell adhesion molecule production. Analysis of intercellular communication in feline and murine hearts revealed an overall loss of ligands broadcast from all cell types, with notable losses from fibroblast cells and cardiomyocytes. These findings are consistent with recent studies implicating altered intercellular communication between cardiomyocytes and non-myocyte cell types as a potential disease mechanism in human HCM. We will further identify common and species-specific disease-promoting pathways in HCM tissue that will allow identification of conserved disease mechanisms that may be targeted in both human and feline disease and will facilitate an understanding of the relevance of mouse models.

Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer

Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca2+, ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP2) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (Po) shows little voltage dependence and is stable over several minutes. Po is high without addition of exogenous PIP2, but is largely blunted by treatment with poly-l-Lysine, a polycation that masks PIP2 headgroups. These results indicate that PIP2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP2 binding affinity of nvTRPM2 under physiological conditions.

Identification of Novel Gene Regulatory Networks for Dystrophin Protein in Vascular Smooth Muscle Cells by Single-Nuclear Transcriptome Analysis.

Duchenne muscular dystrophy is an X-linked recessive disease caused by mutations in dystrophin proteins that lead to heart failure and respiratory failure. Dystrophin (DMD) is not only expressed in cardiomyocytes and skeletal muscle cells, but also in vascular smooth muscle cells (VSMCs). Patients with DMD have been reported to have hypotension. Single nuclear RNA sequencing (snRNA-seq) is a state-of-the-art technology capable of identifying niche-specific gene programs of tissue-specific cell subpopulations. To determine whether DMD mutation alters blood pressure, we compared systolic, diastolic, and mean blood pressure levels in mdx mice (a mouse model of DMD carrying a nonsense mutation in DMD gene) and the wide-type control mice. We found that mdx mice showed significantly lower systolic, diastolic, and mean blood pressure than control mice. To understand how DMD mutation changes gene expression profiles from VSMCs, we analyzed an snRNA-seq dataset from the muscle nucleus of DMD mutant (DMDmut) mice and control (Ctrl) mice. Gene Ontology (GO) enrichment analysis revealed that the most significantly activated pathways in DMDmut-VSMCs are involved in ion channel function (potassium channel activity, cation channel complex, and cation channel activity). Notably, we discovered that the DMDmut-VSMCs showed significantly upregulated expression of KCNQ5 and RYR2, whereas the most suppressed pathways were transmembrane transporter activity (such as anion transmembrane transporter activity, inorganic anion transmembrane transporter activity, import into cell, and import across plasma membrane). Moreover, we analyzed metabolic pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) using "scMetabolism" R package. DMDmut-VSMCs exhibited dysregulation of pyruvate metabolism and nuclear acid metabolism. In conclusion, via the application of snRNA-seq, we (for the first time) identify the potential molecular regulation by DMD in the upregulation of the expression of KCNQ5 genes in VSMCs, which helps us to understand the mechanism of hypotension in DMD patients. Our study potentially offers new possibilities for therapeutic interventions in systemic hypotension in DMD patients with pharmacological inhibition of KCNQ5.

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

Simulated microgravity leads to altered intracellular calcium homeostasis and protein expression in C2C12 cultures.

Skeletal muscle has the role to maintain body posture against a constant gravitational load. During aging, in low physical activity or even in spaceflight, the muscle mass is decreasing, resulting in impaired myogenesis and regeneration. The cytoskeleton, mechanosensitive ion channels, and calcium homeostasis play crucial role in maintaining normal myogenic processes. Simulated microgravity (SM) with 3D clinorotation is one of the accepted techniques to model gravitational unloading in vitro. C2C12 mouse myoblast cell line is a verified in vitro model system of myogenesis. Using RPM 2.0, a Random Positioning Machine as a partial g simulator, by randomly rotating the accommodated experiment package around the Earth's gravity vector, we are able to investigate alteration of physiological processes caused by SM. In our experiments, territorial gravity is compared to 0 g, respectively. However, fusion of myoblasts was preserved with SM, the formation of myotubes was different as compared to the control environment. Expression of Myosin Heavy Chain 2 (MYH2) protein, which is an important structural protein and a marker molecule of myotube differentiation program, was significantly reduced in samples kept in RPM. Immunocytochemistry of myogenic cultures revealed decreased size and average nuclei content in differentiated myotubes. The expression level of Piezo1 channels, which are mechanically activated cation channels are also decreased under SM. Altered calcium transients evoked by KCl have been observed in myotubes formed under simulated microgravity. Our results are in coherence with the recently published data. Our future goal is to analyze the architecture of cytoskeletal Septin7 protein and investigate how intracellular calcium concentrations and mechanotransduction is regulated by Piezo1 channels under microgravity condition.

A point mutation turns an inactivated potassium channel into a hyperpolarization activated cation channel.

In potassium channels, the slow inactivation (C-type) process decreases the ionic conduction by affecting the selectivity filter. The W434F mutation in Shaker potassium channel stabilizes it in the inactivated conformation. The recent determination of the structure of the Shaker W434F channel shows a dilated selectivity filter, compared to Shaker WT structure, reminiscent to the hyperpolarization activated cyclic nucleotide gated channel (HCN). This suggests a common link between inactivation and hyperpolarization gating, which is explored in this work. In Shaker the P475D (Sh-P475D) is a mutation in the bundle crossing region that stabilizes the activation gate in the open conformation over a wide voltage range (−140 mV to +100). Sh-P475D was expressed in Xenopus oocytes and inactivation was favored by removing external potassium. Under these conditions, the channel displays robust hyperpolarization activated currents reminiscent of HCN. (Similar currents are observed when the W434F mutation is added in Sh-P475D background.) The currents elicited by hyperpolarizing voltages in the Sh-P475D mutant are non-selective (PK/PNa∼1) and even N-methyl-D-glucamine and calcium are permeable in this mutant. We analyzed the role of residues known to affect pore inactivation, the contribution of the selectivity filter, and the influence of the voltage sensor movement on the kinetics and steady state characteristics of the P475D-induced hyperpolarization activated currents. Our results suggest a mechanism of hyperpolarization gating and inactivation at the selectivity filter that seems to be common among voltage gated cation channels. Supported by NIH GM03076.

Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties

Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris, and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His6D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.

Sequence Similarity among Structural Repeats in the Piezo Family of Mechanosensitive Ion Channels

Members of the Piezo family of mechanically activated cation channels are involved in multiple physiological processes in higher eukaryotes, including vascular development, cell differentiation, touch perception, hearing, and more, but they are also common in single-celled eukaryotic microorganisms. Mutations in these proteins in humans are associated with a variety of diseases, such as colorectal adenomatous polyposis, dehydrated hereditary stomatocytosis, and hereditary xerocytosis. Available 3D structures for Piezo proteins show nine regions of four transmembrane segments each that have the same fold. Despite the remarkable similarity among the nine characteristic structural repeats in the family, no significant sequence similarity among them has been reported. Using bioinformatics approaches and the Transporter Classification Database (TCDB) as reference, we reliably identified sequence similarity among repeats based on four lines of evidence: (1) hidden Markov model-profile similarities across repeats at the family level, (2) pairwise sequence similarities between different repeats across Piezo homologs, (3) Piezo-specific conserved sequence signatures that consistently identify the same regions across repeats, and (4) conserved residues that maintain the same orientation and location in 3D space.