Potassium Channels In Membrane Research Articles

Zn-ion ultrafluidity via bioinspired ion channel for ultralong lifespan Zn-ion battery.

Rechargeable aqueous Zn-ion batteries have been deemed a promising energy storage device. However, the dendrite growth and side reactions have hindered their practical application. Herein, inspired by the ultrafluidic and K+ ion-sieving flux through enzyme-gated potassium channels (KcsA) in biological plasma membranes, a metal-organic-framework (MOF-5) grafted with -ClO4 groups (MOF-ClO4) as functional enzymes is fabricated to mimic the ultrafluidic lipid-bilayer structure for gating Zn2+ 'on' and anions 'off' states. The MOF-ClO4 achieved perfect Zn2+/SO4 2- selectivity (∼10), enhanced Zn2+ transfer number ([Formula: see text]) and the ultrafluidic Zn2+ flux (1.9×10-3 vs. 1.67mmol m-2 s-1 for KcsA). The symmetric cells based on MOF-ClO4 achieve a lifespan of over 5400h at 10mAcm-2/20mAhcm-2. Specifically, the performance of the PMCl-Zn//V2O5 pouch cell keeps 81% capacity after 2000 cycles at 1Ag-1. The regulated ion transport, by learning from a biological plasma membrane, opens a new avenue towards ultralong lifespan aqueous batteries.

Chloroplast envelope K+/H+ antiporters are involved in cytosol pH regulation.

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K+/H+ antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K+/H+ antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane.

Renal potassium regulation in a pig model of therapeutic hypothermia

Therapeutic hypothermia is increasingly utilized clinically to reduce tissue metabolism and protect against neurological damage in cases of cardiac arrest, traumatic brain injury or perinatal hypoxic ischemia. With even mild exposure to hypothermia, hypokalemia develops purportedly due to transcellular potassium shifts resulting in decreased circulating potassium levels. Renal dysfunction with body temperature cooling has also been postulated to potentially contribute to development of sustained hypokalemia. In this study, we used a pig model of controlled acute hypothermia without confounding hypotension and hypoxemia, to test the hypothesis that renal handling of potassium remains intact in hypothermia and is not the cause of decreased circulating plasma potassium seen with body cooling. Anesthetized, mechanically ventilated Duroc cross young pigs (n=25, body weight = 10.0 ± 0.4 kg, male or female) were catheterized for hemodynamic monitoring, regional blood flow assessment via colored microspheres, and urine collection. After baseline values were obtained, pigs were divided into a control normothermia group (n=12) with body temperature maintained at 38.4 ± 0.1 oC or a hypothermia group (n=13) with core body temperature cooled to 34.5 ± 0.1 oC and hypothermia sustained for 2 hours. Hypothermia reduced tissue metabolism as indicated by a decrease in oxygen consumption (VO2= 5.2 ± 0.2 ml/kg in normothermia vs 3.7 ± 0.3 ml/kg in hypothermia. Mean arterial pressure (68± 3 mm Hg) and cardiac output (199 ± 21 ml/min/kg) were not different between groups. Even with the short duration of cold exposure, plasma potassium was lower (p<0.05) within 2 hours of hypothermia (4.1 ± 0.1 mEq/L) vs control (4.8 ± 0.1 mEq/L). Cold exposure caused redistribution of blood flow to the small intestines from other internal organs. Renal blood flow tended to decrease with cold, but glomerular filtration rate was suffcient to maintain urine flow, sodium excretion and free water clearance. Potassium clearance, however, was lower (p<0.05) in hypothermia (0.70 ± 0.07 ml/min/kg) than in normothermia (1.04 ± 0.07 ml/min/kg), with lower potassium excretion (hypothermia = 2997 ± 331 mEq/min vs control = 4784 ± 393 mEq/min, p<0.05) and fractional excretion of potassium. Reduced renal potassium excretion is consistent with renal compensation for lower circulating potassium induced by hypothermia. Interestingly, with relatively higher blood flow to the small intestines compared to decreased blood flow of other internal organs, potassium secretion in the early colon may contribute to further potassium wasting in hypothermia. The rapid initiation of decreased potassium circulating levels with hypothermia supports the thesis of temperature sensitive cell membrane potassium channels affecting extracellular to intracellular shifts of potassium. Results of this study indicate that renal potassium regulation is maintained in hypothermia and functions to decrease urinary potassium loss in the face of hypokalemia. The views expressed are those of the authors and do not necessarily reflect the offcial policy or position of the Defense Health Agency, Department of the Army, Department of Defense, or the U.S. Government. 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.

Overexpressed KCNK1 regulates potassium channels affecting molecular mechanisms and biological pathways in bladder cancer

BackgroundThis study aimed to explore the expression, molecular mechanism and its biological function of potassium two pore domain channel subfamily K member 1 (KCNK1) in bladder cancer (BC).MethodsWe integrated large numbers of external samples (n = 1486) to assess KCNK1 mRNA expression levels and collected in-house samples (n = 245) for immunohistochemistry (IHC) experiments to validate at the KCNK1 protein level. Single-cell RNA sequencing (scRNA-seq) analysis was performed to further assess KCNK1 expression and cellular communication. The transcriptional regulatory mechanisms of KCNK1 expression were explored by ChIP-seq, ATAC-seq and ChIA-PET data. Highly expressed co-expressed genes (HECEGs) of KCNK1 were used to explore potential signalling pathways. Furthermore, the immunoassay, clinical significance and molecular docking of KCNK1 were calculated.ResultsKCNK1 mRNA was significantly overexpressed in BC (SMD = 0.58, 95% CI [0.05; 1.11]), validated at the protein level (p < 0.0001). Upregulated KCNK1 mRNA exhibited highly distinguishing ability between BC and control samples (AUC = 0.82 [0.78–0.85]). Further, scRNA-seq analysis revealed that KCNK1 expression was predominantly clustered in BC epithelial cells and tended to increase with cellular differentiation. BC epithelial cells were involved in cellular communication mainly through the MK signalling pathway. Secondly, the KCNK1 transcription start site (TSS) showed promoter-enhancer interactions in three-dimensional space, while being transcriptionally regulated by GRHL2 and FOXA1. Most of the KCNK1 HECEGs were enriched in cell cycle–related signalling pathways. KCNK1 was mainly involved in cellular metabolism–related pathways and regulated cell membrane potassium channel activity. KCNK1 expression was associated with the level of infiltration of various immune cells. Immunotherapy and chemotherapy (docetaxel, paclitaxel and vinblastine) were more effective in BC patients in the high KCNK1 expression group. KCNK1 expression correlated with age, pathology grade and pathologic_M in BC patients.ConclusionsKCNK1 was significantly overexpressed in BC. A complex and sophisticated three-dimensional spatial transcriptional regulatory network existed in the KCNK1 TSS and promoted the upregulated of KCNK1 expression. The high expression of KCNK1 might be involved in the cell cycle, cellular metabolism, and tumour microenvironment through the regulation of potassium channels, and ultimately contributed to the deterioration of BC.

Diacylglycerol kinase is downregulated in the Drosophila Seizure Mutant during Spaceflight

Abstract Accelerated aging in space is detrimental to long-term space missions. The environmental conditions in space (e.g., microgravity and radiation) cause harmful effects similar to those seen during aging. As the mechanistic pathways underlying accelerated aging in spaceflight are not fully understood, the identification of critical targets for promoting longevity in spaceflight remains challenging. We analyzed genomics data from the GLDS-207 project to identify potential targets related to longevity. Analysis of RNA-seq data from four Drosophila variants using the GeneLab Galaxy platform indicated that spaceflight significantly affected differential gene expression in the heads of flies, specifically in the seizure (sei) mutant, which alters the voltage gated potassium channels in the cell membrane. Spaceflight induced a significant decrease in the expression of the retinal degeneration A gene (rdgA) in mutant flies that survived the 30-day space mission. This gene encodes for the protein diacylglycerol kinase (DGK), which modulates the activation of the mechanistic target of the rapamycin (mTOR) signaling pathway, known to negatively regulate aging. Therefore, DGK may be a potential target for promoting longevity in space conditions. Further investigation of the effects of decreased rdgA expression on the lifespan of other organisms under spaceflight conditions will clarify the role of DGK in promoting longevity.

Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments.

Verrucomicrobia are a group of microorganisms that have been proposed to be deeply rooted in the Tree of Life. Some are methanotrophs that oxidize the potent greenhouse gas methane and are thus important in decreasing atmospheric concentrations of the gas, potentially ameliorating climate change. They are widespread in various environments including soil and fresh or marine waters. Recently, a clade of extremely acidophilic Verrucomicrobia, flourishing at pH < 3, were described from high-temperature geothermal ecosystems. This novel group could be of interest for studies about the emergence of life on Earth and to astrobiologists as homologs for possible extraterrestrial life. In this paper, we describe predicted mechanisms for survival of this clade at low pH and suggest its possible evolutionary trajectory from an inferred neutrophilic ancestor. Extreme acidophiles are defined as organisms that thrive in extremely low pH environments (≤ pH 3). Many are polyextremophiles facing high temperatures and high salt as well as low pH. They are important to study for both providing fundamental insights into biological mechanisms of survival and evolution in such extreme environments and for understanding their roles in biotechnological applications such as industrial mineral recovery (bioleaching) and mitigation of acid mine drainage. They are also, potentially, a rich source of novel genes and pathways for the genetic engineering of microbial strains. Acidophiles of the Verrucomicrobia phylum are unique as they are the only known aerobic methanotrophs that can grow optimally under acidic (pH 2–3) and moderately thermophilic conditions (50–60°C). Three moderately thermophilic genera, namely Methylacidiphilum, Methylacidimicrobium, and Ca. Methylacidithermus, have been described in geothermal environments. Most of the investigations of these organisms have focused on their methane oxidizing capabilities (methanotrophy) and use of lanthanides as a protein cofactor, with no extensive study that sheds light on the mechanisms that they use to flourish at extremely low pH. In this paper, we extend the phylogenetic description of this group of acidophiles using whole genome information and we identify several mechanisms, potentially involved in acid resistance, including “first line of defense” mechanisms that impede the entry of protons into the cell. These include the presence of membrane-associated hopanoids, multiple copies of the outer membrane protein (Slp), and inner membrane potassium channels (kup, kdp) that generate a reversed membrane potential repelling the intrusion of protons. Acidophilic Verrucomicrobia also display a wide array of proteins potentially involved in the “second line of defense” where protons that evaded the first line of defense and entered the cell are expelled or neutralized, such as the glutamate decarboxylation (gadAB) and phosphate-uptake systems. An exclusive N-type ATPase F0-F1 was identified only in acidophiles of Verrucomicrobia and is predicted to be a specific adaptation in these organisms. Phylogenetic analyses suggest that many predicted mechanisms are evolutionarily conserved and most likely entered the acidophilic lineage of Verrucomicrobia by vertical descent from a common ancestor. However, it is likely that some defense mechanisms such as gadA and kup entered the acidophilic Verrucomicrobia lineage by horizontal gene transfer.

Endogenous VIP VPAC1 Receptor Activation Modulates Hippocampal Theta Burst Induced LTP: Transduction Pathways and GABAergic Mechanisms.

Simple SummaryRegulation of synaptic plasticity through control of disinhibition is an important process in the prevention of excessive plasticity in both physiological and pathological conditions. Interneuron-selective interneurons, such as the ones expressing VIP in the hippocampus, may play a crucial role in this process. In this paper we showed that endogenous activation of VPAC1—not VPAC2 receptors—exerts an inhibitory control of long-term potentiation (LTP) induced by theta-burst stimulation (TBS) in the hippocampus, through a mechanism dependent on GABAergic transmission. This suggests that VPAC1-mediated modulation of synaptic transmission at GABAergic synapses to interneurons will ultimately influence NMDA-dependent LTP expression by modulating inhibitory control of pyramidal cell dendrites and postsynaptic depolarization during LTP induction. Accordingly, the transduction pathways mostly involved in this effect were the ones involved in TBS-induced LTP expression like NMDA receptor activation and CaMKII activity. In addition, the actions of endogenous VIP through VPAC1 receptors may indirectly influence the control of dendritic excitability by Kv4.2 channels.Vasoactive intestinal peptide (VIP), acting on both VPAC1 and VPAC2 receptors, is a key modulator of hippocampal synaptic transmission, pyramidal cell excitability and long-term depression (LTD), exerting its effects partly through modulation GABAergic disinhibitory circuits. Yet, the role of endogenous VIP and its receptors in modulation of hippocampal LTP and the involvement of disinhibition in this modulation have scarcely been investigated. We studied the modulation of CA1 LTP induced by TBS via endogenous VIP release in hippocampal slices from young-adult Wistar rats using selective VPAC1 and VPAC2 receptor antagonists, evaluating its consequence for the phosphorylation of CamKII, GluA1 AMPA receptor subunits and Kv4.2 potassium channels in total hippocampal membranes obtained from TBS stimulated slices. Endogenous VIP, acting on VPAC1 (but not VPAC2) receptors, inhibited CA1 hippocampal LTP induced by TBS in young adult Wistar rats and this effect was dependent on GABAergic transmission and relied on the integrity of NMDA and CaMKII-dependent LTP expression mechanisms but not on PKA and PKC activity. Furthermore, it regulated the autophosphorylation of CaMKII and the expression and Ser438 phosphorylation of Kv4.2 potassium channels responsible for the A-current while inhibiting phosphorylation of Kv4.2 on Thr607. Altogether, this suggests that endogenous VIP controls the expression of hippocampal CA1 LTP by regulating disinhibition through activation of VPAC1 receptors in interneurons. This may impact the autophosphorylation of CaMKII during LTP, as well as the expression and phosphorylation of Kv4.2 K+ channels at hippocampal pyramidal cell dendrites.

Augmented KCa2.3 Channel Feedback Regulation of Oxytocin Stimulated Uterine Strips from Nonpregnant Mice.

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca2+) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K+) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca2+-activated K+ (KCa2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit KCa2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice were suppressed by CyPPA and, in the presence of CyPPA, oxytocin failed to augment contractions. In tissues exposed to oxytocin, depletion of internal Ca2+ stores with cyclopiazonic acid (CPA) impaired CyPPA relaxation, whereas blockade of nonselective cation channels (NSCC) using gadolinium (Gd3+) had no significant effect. Immunofluorescence revealed close proximity of KCa2.3 channels and ER inositol trisphosphate receptors (IP3Rs) within myometrial smooth muscle cells. The findings suggest internal Ca2+ stores play a role in KCa2.3-dependent feedback control of uterine contraction and offer new insights for tocolytic therapies.

Role of Potassium Ions Quantum Tunneling in the Pathophysiology of Phantom Limb Pain.

(1) Background: multiple theories were proposed to explain the phenomenon of phantom limb pain (PLP). Nevertheless, the phenomenon is still shrouded in mystery. The aim of this study is to explore the phenomenon from a new perspective, where quantum tunneling of ions, a promising field in medical practice, might play a major role. (2) Methods: investigators designed a quantum mathematical model based on the Schrödinger equation to examine the probability of potassium ions quantum tunneling through closed membrane potassium channels to the inside of phantom axons, leading to the generation of action potential. (3) Results: the model suggests that the probability of action potential induction at a certain region of the membrane of phantom neurons, when a neuron of the stump area is stimulated over 1 mm2 surface area of the membrane available for tunneling is 1.04 × 10−2. Furthermore, upon considering two probabilities of potassium channelopathies, one that decreased the energy of the barrier by 25% and another one by 50%, the tunneling probability became 1.22 × 10−8 and 3.86 × 10−4, respectively. (4) Conclusion: quantum models of potassium ions can provide a reliable theoretical hypothesis to unveil part of the ambiguity behind PLP.

The KATP Channel is a Potential Target for Natural Plant Products in Type 2 Diabetes Mellitus

Diabetes mellitus (DM) is a chronic metabolic disorder affecting a vast number of people all around the world. Diabetes is mainly characterized by chronic hyperglycemia, resulting from defects in insulin secretion or insulin action, or both. To date, several glucose-lowering drugs have been used clinically. These drugs exert their anti-diabetic effect by stimulating insulin secretion, increasing peripheral absorption of glucose, delaying the absorption of carbohydrates from the intestine and reducing hepatic gluconeogenesis. The concern over effectiveness and side effects of currently available therapies in the treatment of Type 2 DM (T2DM) has promoted interest in discovery and development of natural antidiabetic drugs. Plants used in folk medicine to treat DM represent a viable alternative for the control of this disease. Several plants with antidiabetic properties exert their effects by inhibiting ATP-sensitive potassium (KATP) channels in the β-cell plasma membrane, resulting in membrane depolarization and stimulation of insulin release from insulin stores by exocytosis. In this article, we examined the results of earlier studies on the actions of natural compounds on pancreatic KATP channels. It is likely that these natural compounds can be employed as lead structures for the synthesis of therapeutic agents to treat T2DM in future clinical studies

Defining the Kv2.1–syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.

Importance of the Choice of a Recombinant System to Produce Large Amounts of Functional Membrane Protein hERG.

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.

Intestinal epithelial potassium channels and CFTR chloride channels activated in ErbB tyrosine kinase inhibitor diarrhea.

Diarrhea is a major side effect of ErbB receptor tyrosine kinase inhibitors (TKIs) in cancer chemotherapy. Here, we show that the primary mechanism of ErbB TKI diarrhea is activation of basolateral membrane potassium (K+) channels and apical membrane chloride (Cl-) channels in intestinal epithelia and demonstrate the efficacy of channel blockers in a rat model of TKI diarrhea. Short-circuit current in colonic epithelial cells showed that the TKIs gefitinib, lapatinib, and afatinib do not affect basal secretion but amplify carbachol-stimulated secretion by 2- to 3-fold. Mechanistic studies with the second-generation TKI afatinib showed that the amplifying effect on Cl- secretion was Ca2+ and cAMP independent, was blocked by CF transmembrane conductance regulator (CFTR) and K+ channel inhibitors, and involved EGFR binding and ERK signaling. Afatinib-amplified activation of basolateral K+ and apical Cl- channels was demonstrated by selective membrane permeabilization, ion substitution, and channel inhibitors. Rats that were administered afatinib orally at 60 mg/kg/day developed diarrhea with increased stool water from approximately 60% to greater than 80%, which was reduced by up to 75% by the K+ channel inhibitors clotrimazole or senicapoc or the CFTR inhibitor (R)-BPO-27. These results indicate a mechanism for TKI diarrhea involving K+ and Cl- channel activation and support the therapeutic efficacy of channel inhibitors.

Mitochondrial potassium channels - an overview.

Mitochondria play a fundamental role in ATP synthesis within the majority of mammalian cells. Potassium channels present in the inner mitochondrial membrane are fine regulators of mitochondrial function, based on inner membrane K+ permeability. These channels are regulated by a plethora of factors and conditions in a way similar to plasma membrane potassium channels. Regulators of mitochondrial potassium channels include the membrane potential, calcium ions, free fatty acids and ATP levels within the cells. Recently, it was shown that these channels are regulated by the respiratory chain, stretching of the membrane and phosphorylation. The essential interest that has driven studies of mitochondrial potassium channels for nearly 25 years is their role in cytoprotection and in cell death. Mitochondrial potassium channels have been described in neurons, astrocytoma, cardiac and skeletal muscles, fibroblasts, keratinocytes and endothelial cells. In this overview, we summarize the current knowledge of mitochondrial potassium channels. This summary will be done with a special focus on studies performed over the last 20 years in the Laboratory of Intracellular Ion Channels at the Nencki Institute. These include studies on the electrophysiological and pharmacological properties of mitochondrial potassium channels and on their regulation by endogenous intracellular substances. Additionally, the regulation of mitochondrial potassium channels by the respiratory chain and by stretching of the inner mitochondrial membrane will be reviewed. Properties of mitochondrial potassium channels in various organisms will also be summarized.

Chronic Stress Causes Projection-Specific Adaptation of Amygdala Neurons via Small-Conductance Calcium-Activated Potassium Channel Downregulation

BackgroundThe role of the amygdala in mediating stress coping has been long appreciated. However, basolateral amygdala (BLA) projection neurons (PNs) are organized into discrete output circuits, and it remains unclear whether stress differentially impacts these circuits. MethodsMice were exposed to acute restraint stress or chronic restraint stress (CRS), and c-fos expression was measured as a proxy for neuronal activation in Retrobead retrogradely labeled dorsomedial prefrontal cortex–targeting PNs (BLA→dmPFC) and non-dmPFC-targeting PNs (BLA↛dmPFC). Next, the effects of CRS on neuronal firing and membrane potassium channel current were examined via ex vivo electrophysiology in these neuronal populations and correlated with anxiety-like behavior, as measured in the elevated plus maze and novel open field tests. Lastly, the ability of virus-mediated overexpression of subtype 2 of small-conductance, calcium-activated potassium (SK2) channel in BLA↛dmPFC PNs to negate the anxiety-related effects of CRS was assessed. ResultsBLA→dmPFC PNs were transiently activated after CRS, whereas BLA↛dmPFC showed sustained c-fos expression and augmented firing to external input. CRS led to a loss of SK2 channel–mediated currents in BLA↛dmPFC PNs, which correlated with heightened anxiety-like behavior. Virus-mediated maintenance of SK2 channel currents in BLA↛dmPFC PNs prevented CRS-induced anxiety-like behavior. Finally, CRS produced persistent activation of BLA PNs targeting the ventral hippocampus, and virally overexpressing SK2 channels in this projection population were sufficient to prevent CRS-induced anxiety-like behavior. ConclusionsThe current data reveal that chronic stress produces projection-specific functional adaptations in BLA PNs. These findings offer new insight into the neural circuits that contribute to stress-induced psychopathology.

Binding of sulphonylureas to plasma proteins - A KATP channel perspective.

Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic β-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic β-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug’s therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion.

Widening control of fin inter-rays in zebrafish and inferences about actinopterygian fins.

The amputation of a teleost fin rapidly triggers an intricate maze of hierarchically regulated signalling processes which ultimately reconstruct the diverse tissues of the appendage. Whereas the generation of the fin pattern along the proximodistal axis brings with it several well-known developmental regulators, the mechanisms by which the fin widens along its dorsoventral axis remain poorly understood. Utilizing the zebrafish as an experimental model of fin regeneration and studying more than 1000 actinopterygian species, we hypothesized a connection between specific inter-ray regulatory mechanisms and the morphological variability of inter-ray membranes found in nature. To tackle these issues, both cellular and molecular approaches have been adopted and our results suggest the existence of two distinguishable inter-ray areas in the zebrafish caudal fin, a marginal and a central region. The present work associates the activity of the cell membrane potassium channel kcnk5b, the fibroblast growth factor receptor 1 and the sonic hedgehog pathway to the control of several cell functions involved in inter-ray wound healing or dorsoventral regeneration of the zebrafish caudal fin. This ray-dependent regulation controls cell migration, cell-type patterning and gene expression. The possibility that modifications of these mechanisms are responsible for phenotypic variations found in euteleostean species, is discussed.

Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation.

The conductance of KATP channel activity can be regulated by gating and/or surface expression. Gating analysis is usually performed by electrophysiological recording. Analysis of surface KATP channel expression levels requires cell fractionation, protein separation, and quantification. Cell fractionation involves time-consuming high-speed centrifugation steps and skilled techniques for taking out specific layers. Moreover, contamination of intracellular membranes can confound results. Although commercial kits have been developed to make it easier for scientists, qualities of these kits vary which can give rise to variable results. Detection of membrane proteins using surface biotinylation technique consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pulldown. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes, and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control). This alternative method for detecting expression of surface protein is relatively easy in steps and more economical in comparison to other methods such as cell fractionation.

Yeast Tok1p channel is a major contributor to membrane potential maintenance under chemical stress

Tok1p is a highly specific yeast plasma membrane potassium channel with strong outward directionality. Its opening is induced by membrane depolarization. Although the biophysical properties of Tok1p are well-described, its potentially important physiological role is currently largely unexplored. To address this issue, we examined the Tok1p activity following chemically-induced depolarization by measuring changes of plasma membrane potential (ΔΨ) using the diS-C3(3) fluorescence assay in a Tok1p-expressing and a Tok1p-deficient strain. We report that Tok1p channel activity in response to chemical stress does not depend solely on the extent of depolarization, as might have been expected, but may also be negatively influenced by accompanying effects of the used compound. The stressors may interact with the plasma membrane or the channel itself, or cause cytosolic acidification. All of these effects may negatively influence the Tok1p channel opening. While ODDC-induced depolarization exhibits the cleanest Tok1p activation, restoring an astonishing 75% of lost ΔΨ, higher BAC concentrations reduce Tok1p activity, probably because of direct interactions with the channel and/or its lipid microenvironment. This is not only the first study of the physiological role of Tok1p in ΔΨ maintenance under chemical stress, but also the first estimate of the extent of depolarization the channel is able to counterbalance.

The Segregated Expression of Voltage-Gated Potassium and Sodium Channels in Neuronal Membranes: Functional Implications and Regulatory Mechanisms.

Neurons are highly polarized cells with apparent functional and morphological differences between dendrites and axon. A critical determinant for the molecular and functional identity of axonal and dendritic segments is the restricted expression of voltage-gated ion channels (VGCs). Several studies show an uneven distribution of ion channels and their differential regulation within dendrites and axons, which is a prerequisite for an appropriate integration of synaptic inputs and the generation of adequate action potential (AP) firing patterns. This review article will focus on the signaling pathways leading to segmented expression of voltage-gated potassium and sodium ion channels at the neuronal plasma membrane and the regulatory mechanisms ensuring segregated functions. We will also discuss the relevance of proper ion channel targeting for neuronal physiology and how alterations in polarized distribution contribute to neuronal pathology.