Expression Of Channel Subunits Research Articles

Expression levels of KATP channel subunits and morphological changes in the mouse liver after exposure to radiation.

ATP sensitive K+ (KATP) channels are ubiquitously distributed in various of cells and tissues, including the liver. They play a role in the pathogenesis of myocardial and liver ischemia. To evaluate the radiation-induced changes in the expression of KATP channel subunits in the mouse liver to understand the potential role of KATP channels in radiation injury. Adult C57BL/6 mice were randomly exposed to γ-rays at 0 Gy (control, n = 2), 0.2 Gy (n = 6), 1 Gy (n = 6), or 5 Gy (n = 6). The livers were removed 3 and 24 h after radiation exposure. Hematoxylin and eosin staining was used for morphological observation; immunohistochemical staining was applied to determine the expression of KATP channel subunits in the liver tissue. Compared with the control group, the livers exposed to 0.2 Gy γ-ray showed an initial increase in the expression of Kir6.1 at 3 h, followed by recovery at 24 h after exposure. Exposure to a high dose of 5.0 Gy resulted in decreased expression of Kir6.1 and increased expression of SUR2B at 24 h. However, the expression of Kir6.2, SUR1, or SUR2A had no remarkable changes at 3 and 24 h after exposure to any of these doses. The expression levels of Kir6.1 and SUR2B in mouse liver changed differently in response to different radiation doses, suggesting a potential role for them in radiation-induced liver injury.

Disease-causing Slack potassium channel mutations produce opposite effects on excitability of excitatory and inhibitory neurons

The KCNT1 gene encodes the sodium-activated potassium channel Slack (KCNT1, KNa1.1), a regulator of neuronal excitability. Gain-of-function mutations in humans cause cortical network hyperexcitability, seizures, and severe intellectual disability. Using a mouse model expressing the Slack-R455H mutation, we find that Na+-dependent K+ (KNa) and voltage-dependent sodium (NaV) currents are increased in both excitatory and inhibitory cortical neurons. These increased currents, however, enhance the firing of excitability neurons but suppress that of inhibitory neurons. We further show that the expression of NaV channel subunits, particularly that of NaV1.6, is upregulated and that the length of the axon initial segment and of axonal NaV immunostaining is increased in both neuron types. Our study on the coordinate regulation of KNa currents and the expression of NaV channels may provide an avenue for understanding and treating epilepsies and other neurological disorders.

Pulmonary Circulation Under Pressure: Pathophysiological and Therapeutic Implications of BK Channel.

The large-conductance Ca2+-activated K+ (BK) channel is widely expressed in the pulmonary blood vessels and plays a significant role in regulating pulmonary vascular tonus. It opens under membrane depolarization, increased intracellular Ca+2 concentration, and chronic hypoxia, resulting in massive K+ efflux, membrane hyperpolarization, decreased L-type Ca+2 channel opening, and smooth muscle relaxation. Several reports have demonstrated an association between BK channel dysfunction and pulmonary hypertension (PH) development. Decreased BK channel subunit expression and impaired regulation by paracrine hormones result in decreased BK channel opening, increased pulmonary vascular resistance, and pulmonary arterial pressure being the cornerstone of PH. The resulting right ventricular pressure overload ultimately leads to ventricular remodeling and failure. Therefore, it is unsurprising that the BK channel has arisen as a potential target for treating PH. Recently, a series of selective, synthetic BK channel agonists have proven effective in attenuating the pathophysiological progression of PH without adverse effects in animal models.

Cell-cycle arrest at the G1/S boundary enhances transient voltage-gated ion channel expression in human and insect cells

Heterologous expression of recombinant ion channel subunits in cell lines is often limited by the presence of a low number of channels at the cell surface level. Here, we introduce a combination of two techniques: viral expression using the baculovirus system plus cell-cycle arrest at the G1/S boundary using either thymidine or hydroxyurea. This method achieved a manifold increase in the peak current density of expressed ion channels compared with the classical liposome-mediated transfection methods. The enhanced ionic current was accompanied by an increase in the density of gating charges, confirming that the increased yield of protein and ionic current reflects the functional localization of channels in the plasma membrane. This modified method of viral expression coordinated with the cell cycle arrest will pave the way to better decipher the structure and function of ion channels and their association with ion channelopathies.

The Role of Ion Channels in Functional Gastrointestinal Disorders (FGID): Evidence of Channelopathies and Potential Avenues for Future Research and Therapeutic Targets.

Several gastrointestinal (GI) tract abnormalities, including visceral hypersensitivity, motility, and intestinal permeability alterations, have been implicated in functional GI disorders (FGIDs). Ion channels play a crucial role in all the functions mentioned above. Hormones and natural molecules modulate these channels and represent targets of drugs and bacterial toxins. Mutations and abnormal functional expression of ion channel subunits can lead to diseases called channelopathies. These channelopathies in gastroenterology are gaining a strong interest, and the evidence of co-relationships is increasing. In this review, we describe the correlation status between channelopathies and FGIDs. Different findings are available. Among others, mutations in the ABCC7/CFTR gene have been described as a cause of constipation and diarrhea. Mutations of the SCN5A gene are instead associated with irritable bowel syndrome. In contrast, mutations of the TRPV1 and TRPA genes of the transient receptor potential (TRP) superfamily manifest hypersensitivity and visceral pain in sensory nerves. Recently, mice and humans affected by Cantu syndrome (CS), which is associated with the mutations of the KCNJ8 and ABCC9 genes encoding for the Kir6.1 and SUR2 subunits, showed dysfunction of contractility throughout the intestine and death in the mice after the weaning on solid food. The discovery of a correlation between channelopathies and FIGD opens new avenues for discovering new direct drug targets for specific channelopathies, leading to significant implications for diagnosing and treating functional GI diseases.

Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Effects of diazoxide on streptozotocin induced β cell damage via HSP70/HSP90/TLR4/AMPK signaling pathways

ABSTRACT I investigated the effects of diazoxide, a mitochondrial potassium channel opener, on streptozotocin (STZ) induced pancreatic β cell damage via the HSP70/HSP90/TLR4/AMPK signaling pathways in vitro. I used the pancreatic β cell line, 1.1B4, to create four groups: control, STZ treated, diazoxide treated, STZ + diazoxide treated. The STZ treated cells were exposed to 20 µM STZ for 2 h with or without 100 µM diazoxide for 24 h. Total antioxidant status (TAS), total oxidant status (TOS), cell viability and mitochondrial membrane potential (MMP) were measured. Expression of ATP-sensitive potassium channel (KATP) subunits, heat shock protein-70 (HSP70), heat shock protein-90 (HSP90), toll-like receptor 4 (TLR4), AMP-activated protein kinase (AMPK) and some apoptotic proteins were detected using western blotting. Apoptosis was assessed using TUNEL staining. STZ increased TOS and OSI in the pancreatic β cells; however, diazoxide failed to improve oxidative stress. Also, STZ increased tunnel positive cells in the pancreatic β cells. Diazoxide decreased the tunnel positive cells in the STZ treated β cell. STZ decreased MMP; however, diazoxide did not normalize MMP in the STZ induced β cells. Diazoxide increased the HSP70:HSP90 protein expression ratio. STZ decreased expression of AMPK and subunits of KATP channel and increased the expression of caspase-3 and TLR4 protein; diazoxide normalized the expression of all proteins studied. KATP channel opening by diazoxide protects pancreatic β cells against STZ toxicity via HSP70/HSP90/TLR4/AMPK signaling.

MiR-9a-5p expression is decreased in the hippocampus of rats resistant to lamotrigine: A behavioural, molecular and bioinformatics assessment

The major obstacle in developing new treatment strategies for refractory epilepsy is the complexity and poor understanding of its mechanisms. Utilizing the model of lamotrigine-resistant seizures, we evaluated whether changes in the expression of sodium channel subunits are responsible for the diminished responsiveness to lamotrigine (LTG) and if miRNAs, may also be associated.Male rats were administered LTG (5 mg/kg) before each stimulation during kindling acquisition. Challenge stimulation following LTG exposure (30 mg/kg) was performed to confirm resistance in fully kindled rats. RT-PCR was used to measure the mRNA levels of sodium channel subunits (SCN1A, SCN2A, and SCN3A) and miRNAs (miR-155-5p, miR-30b-5p, miR-137–3p, miR-342–5p, miR-301a-3p, miR-212–3p, miR-9a-5p, and miR-133a-3p). Western blot analysis was utilized to measure Nav1.2 protein, and bioinformatics tools were used to perform target prediction and enrichment analysis for miR-9a-5p, the only affected miRNA according to the responsiveness to LTG.Amygdala kindling seizures downregulated Nav1.2, miR-137–3p, miR-342–5p, miR-155–5p, and miR-9a-5p as well as upregulated miR-212–3p. miR-9a-5p was the only molecule decreased in rats resistant to LTG. The bioinformatic assessment and disease enrichment analysis revealed that miR-9a-5p targets expressed with high confidence in the hippocampus are the most significantly associated with epilepsy. Due to the miR-9a-5p dysregulation, major pathways affected are neurotrophic processes, neurotransmission, inflammatory response, cell proliferation and apoptosis. Interaction network analysis identified LTG target SCN2A as interacting with highest number of genes regulated by miR-9-5p. Further studies are needed to propose specific genes and miRNAs responsible for diminished responsiveness to LTG. miR-9a-5p targets, like KCNA4, KCNA2, CACNB2, SCN4B, KCNC1, should receive special attention in them.

Motor Dysfunction of Gastric Antral Smooth Muscle in Diabetic Rats: Contribution of ATP-Dependent Potassium Channels.

The goal of the current research was to further elucidate the role of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels in the motility and contractility force of gastric smooth muscle of diabetic rats. Male Wistar rats (190-230 g) were grouped into control and streptozotocin (STZ)-induced diabetes (55 mg/kg) rats. Thirty days later, gastric muscle contractility was measured using a myograph and a force transducer of antral segments immersed in a tissue bath. Gastric emptying response was measured through feeding of standard pellet. Furthermore, the expression of KATP channel subunits in antral smooth muscle was determined by western blot technique. The amplitude of KCl-evoked twitch contractions of diabetic antral strips was about 25% more than control (P < 0.05). Application of minoxidil, a KATP channel opener, dose dependently decreased the force of twitch contractions in both normal and diabetic antral strips. Application of 10 μM glibenclamide, a KATP channel blocker, did not antagonize the minoxidil-induced relaxation of antral strips. Diabetic gastric emptying was faster than normal, although not significant. Despite the relaxant effect of minoxidil on gastric emptying rate in normal rats (P < 0.05), this effect was not observed in diabetic rats. Also, glibenclamide increased gastric emptying and antagonized minoxidil-induced relaxation in normal rats (P < 0.05). Furthermore, the expression of KATP Kir6.1 and SUR2B subunits was substantially reduced in antral smooth muscle in diabetic condition (P < 0.01). These results propose that KATP channels may contribute to the development of gastric motility disorders in diabetes.

Impact of type-2 diabetes mellitus on expression of atp-sensitive potassium channel subunits in human bypass grafts

Background and Aims : Prevalence of type-2 diabetes mellitus (T2DM) among patients that require coronary artery bypass graft (CABG) surgery increased over the years. It is well knowing that diabetes has impact on endothelial function and that cause impaired vasodilation function of blood vessels. However, vasorelaxation can be, also, achieved through opening of potassium (K) channels on vascular smooth muscle. Thus, the aim of our study was to detect the differences in the expression of ATP-sensitive K (KATP) channel subunits in bypass grafts (saphenous vein, SV and internal mammary artery, IMA) obtained from patients with and without T2DM.

KATP channels in lymphatic function.

KATP channels function as negative regulators of active lymphatic pumping and lymph transport. This review summarizes and critiques the evidence for the expression of specific KATP channel subunits in lymphatic smooth muscle and endothelium, the roles that they play in normal lymphatic function, and their possible involvement in multiple diseases, including metabolic syndrome, lymphedema, and Cantú syndrome. For each of these topics, suggestions are made for directions for future research.

KATP Channels Function in the Peripheral and Central Nervous Systems in a Mouse Model of Type 2 Diabetes

Over 50% of individuals with type 2 diabetes eventually develop diabetic neuropathy. There is a need for increased knowledge in how diabetes affects structural and molecular changes in the nervous system, and how current diabetes interventions affect the progression of chronic pain. Loss of KATP channel activity or function could cause nerve fiber hypersensitivity and consequently increased pain sensitivity due to neuron depolarization. Diabetic neuropathy was induced in male and female C57Bl6 mice over a total of 16 weeks by feeding the animals a high-fat diet (HFD) or corresponding control diet (12330 and 12328, Research Diets Inc., New Brunswick, NJ) for over 16 weeks. Mice on the HFD for at longer than 8 weeks have significantly lower mechanical thresholds and compared to control diet mice. Decreased expression of KATP channel subunits in the spinal cord, and dorsal root ganglia were correlated with decreased mechanical paw withdrawal thresholds. Mice fed a HFD also have decreased analgesia to systemic morphine (0-20 mg/kg, s.c.), which is exacerbated after systemic treatment with glyburide or nateglinide for four weeks, (KATP channel antagonist, glyburide or nateglinide, 50 mg/kg/day, IP or vehicle, 5% DMSO + 0.5% Tween). Upregulation of KATP channels using a viral vector strategy increased paw withdrawal thresholds to systemic morphine. Diabetes affects many adults including those with prediabetes and new-onset diabetes. It is clear that novel biomarkers and pharmaceutical targets are needed in order to (1) confront the demand for new treatments that better manage and (2) identify patients that may develop diabetic neuropathy. Further investigation of KATP channel expression and function during chronic pain syndromes, including diabetic neuropathy, may help to find sufficient treatment options for patients. This work was supported through a University of Minnesota Academic Health Center Faculty Development Grant to AHK and MLG. Funding also provided by the NIH: K01 DA042902, R01 DA051876, and UL1TR002494 (AHK).

Na+-Retaining Action of COX-2 (Cyclooxygenase-2)/EP1 Pathway in the Collecting Duct via Activation of Intrarenal Renin-Angiotensin-Aldosterone System and Epithelial Sodium Channel.

The collecting duct (CD) is a major site of both biosynthesis and action of prostaglandin E2 as highlighted by the predominant expression of COX-2 (cyclooxygenase-2) and some E-prostanoid (EP) subtypes at this nephron site. The purpose of this study was to determine the relevance and mechanism of CD COX-2/prostaglandin E2/EP1 signaling for the regulation of Na+ hemostasis during Na+ depletion. Mice with Aqp2Cre-driven deletion of COX-2 (COX-2fl/flAqp2Cre+) or the EP1 subtype (EP1fl/flAqp2Cre+) were generated and the Na+-wasting phenotype of these mice during low-salt (LS) intake was examined. EP subtypes responsible for prostaglandin E2-induced local renin response were analyzed in primary cultured mouse inner medullary CD cells. Following 28-day LS intake, COX-2fl/flAqp2Cre+ mice exhibited a higher urinary Na+ excretion and lower cumulative Na+ balance, accompanied with suppressed intrarenal renin, AngII (angiotensin II), and aldosterone, expression of CYP11B2 (cytochrome P450 family 11 subfamily B member 2), and blunted expression of epithelial sodium channel subunits compared to floxed controls (COX-2fl/flAqp2Cre-), whereas no differences were observed for indices of systemic renin-angiotensin-aldosterone system. In cultured CD cells, exposure to prostaglandin E2 stimulated release of soluble (pro)renin receptor, prorenin/renin and aldosterone and the stimulation was more sensitive to antagonism of EP1 as compared other EP subtypes. Subsequently, EP1fl/flAqp2Cre+ mice largely recapitulated Na+-wasting phenotype seen in COX-2fl/flAqp2Cre+ mice. The study for the first time reports that CD COX-2/EP1 pathway might play a key role in maintenance of Na+ homeostasis in the face of Na+ depletion, at least in part, through activation of intrarenal renin-angiotensin-aldosterone-system and epithelial sodium channel.

Resurgent Na+ currents promote ultrafast spiking in projection neurons that drive fine motor control

The underlying mechanisms that promote precise spiking in upper motor neurons controlling fine motor skills are not well understood. Here we report that projection neurons in the adult zebra finch song nucleus RA display robust high-frequency firing, ultra-narrow spike waveforms, superfast Na+ current inactivation kinetics, and large resurgent Na+ currents (INaR). These properties of songbird pallial motor neurons closely resemble those of specialized large pyramidal neurons in mammalian primary motor cortex. They emerge during the early phases of song development in males, but not females, coinciding with a complete switch of Na+ channel subunit expression from Navβ3 to Navβ4. Dynamic clamping and dialysis of Navβ4’s C-terminal peptide into juvenile RA neurons provide evidence that Navβ4, and its associated INaR, promote neuronal excitability. We thus propose that INaR modulates the excitability of upper motor neurons that are required for the execution of fine motor skills.

Type-2 diabetes mellitus decreases expression of Kir6.1 subunit of ATP-sensitive potassium channels in human internal Mammary artery grafts

Background and Aims: Previously, we have shown that pinacidil, known as potassium (K) channels opener, relaxed human internal mammary artery (HIMA) obtained from patients with type-2 diabetes mellitus (T2DM). The mechanism of vasodilator action of pinacidil includes interaction with smooth muscle ATP-sensitive K (KATP) channels, but our results on HIMA showed that KATP channels are not involved in this process. Thus, the aim of our study was to detect the differences in the expression of KATP channel subunits in HIMA obtained from patients with and without T2DM.

Efficient expression of a cnidarian peptide-gated ion channel in mammalian cells

ABSTRACT Hydra Na+ channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the Hydra nervous system. They have previously been successfully characterized in Xenopus oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca2+ imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.

The Pharmacology of ATP-Sensitive K+ Channels (KATP).

ATP-sensitive K+ channels (KATP) are inwardly-rectifying potassium channels, broadly expressed throughout the body. KATP is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels thus playing an important physiological role by coupling cellular metabolism with membrane excitability. The hetero-octameric channel complex is formed of 4 pore-forming inward rectifier Kir6.x subunits (Kir6.1 or Kir6.2) and 4 regulatory sulfonylurea receptor subunits (SUR1, SUR2A, or SUR2B). These subunits can associate in various tissue-specific combinations to form functional KATP channels with distinct electrophysiological and pharmacological properties. KATP channels play many important physiological roles and mutations in channel subunits can result in diseases such as disorders of insulin handling, cardiac arrhythmia, cardiomyopathy, and neurological abnormalities. The tissue-specific expression of KATP channel subunits coupled with their rich and diverse pharmacology makes KATP channels attractive therapeutic targets in the treatment of endocrine and cardiovascular diseases.

Ефекти глутатіону на експресію АТФ-чутливих калієвих каналів, мітохондріальну пору і окисний стрес у серці старих щурів

The effects of a single injection of the reduced form of glutathione on the expression of the KCNJ8 and KCNJ11 genes encoding Kir6.1 and Kir6.2 subunits of ATP-sensitive potassium (KATP) channels and on Ca2+-induced nonspecific mitochondrial permeability transition pores (mPTP) opening in the heart of old rats were studied. Changes in biochemical parameters characterizing the intensity of oxidative processes in organelles at the action of glutathione were also studied. The expression levels of Kir6.1 і Kir6.2 subunits KATP channels were determined using reverse transcription and quantitative PCR. It was shown that after the administration of glutathione to old rats, the expression of KATP channel subunits significantly increased, namely for Kir6.1 9.3 times, for Kir6.2 2.6 times. The use of glutathione in older animals inhibited mPTP opening: reduced the amplitude of spontaneous and Ca2+-induced swelling of mitochondria. An important consequence of the action of glutathione during aging was a decrease in the rate of generation of superoxide (.О2-) and hydroxyl (.ОН) radicals, as well as the content of hydrogen peroxide respectively in 1.8, 2.5 and 3.2 times compared with these figures in older animals without treatment. Glutathione also reduced lipid peroxidation, particularly pools of diene conjugates (2.5-fold) and malonic dialdehyde (1.8-fold). Thus, glutathione significantly increases the expression of KCNJ8 and KCNJ11 genes encoding Kir6.1 and Kir6.2 subunits of KATP channels in the heart of rats, regulates MP, preventing its opening, and reduces oxidative stress, indicating its important role in myocardial protection.

Bisphenol-S and Bisphenol-F alter mouse pancreatic β-cell ion channel expression and activity and insulin release through an estrogen receptor ERβ mediated pathway

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic β-cells from wild type (WT) and estrogen receptor β (ERβ) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in β-cell function. BPS or BPF rapidly increased insulin release and diminished ATP-sensitive K+ (KATP) channel activity. Similarly, 48 h treatment with BPS or BPF enhanced insulin release and decreased the expression of several ion channel subunits in β-cells from WT mice, yet no effects were observed in cells from BERKO mice. PaPE-1, a ligand designed to preferentially trigger extranuclear-initiated ER pathways, mimicked the effects of bisphenols, suggesting the involvement of extranuclear-initiated ERβ pathways. Molecular dynamics simulations indicated differences in ERβ ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERβ whose activation alters three key cellular events in β-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

Heterologous Expression of Ion Channels in Mammalian Cell Lines.

Heterologous expression of recombinant ion channel subunits in mammalian cell lines allows for characterization of their functional properties and pharmacological regulation. In this chapter, we describe methods for thawing, refreezing, passaging, cell culture, and transfection of tsA201 cells suitable for electrophysiology and imaging experiments. Furthermore, we discuss the strengths and limitations of using these methods.