Outward Rectification Research Articles

Neurocardiac pathologies associated with potassium channelopathies.

Voltage-gated potassium channels are expressed throughout the human body and are essential for physiological functions. These include delayed rectifiers, A-type channels, outward rectifiers, and inward rectifiers. They impact electrical function in the heart (repolarization) and brain (repolarization and stabilization of the resting membrane potential). KCNQx and KCNHx encode Kv7.x and Kv11.x proteins, which form delayed rectifier potassium channels. KCNQx and KCNHx channelopathies are associated with both cardiac and neuronal pathologies. These include electrocardiographic abnormalities, cardiac arrhythmias, sudden cardiac death (SCD), epileptiform discharges, seizures, bipolar disorder, and sudden unexpected death in epilepsy (SUDEP). Due to the ubiquitous expression of KCNQx and KCNHx channels, abnormalities in their function can be particularly harmful, increasing the risk of sudden death. For example, KCNH2 variants have a dual role in both cardiac and neuronal pathologies, whereas KCNQ2 and KCNQ3 variants are associated with severe and refractory epilepsy. Recurrent and uncontrolled seizures lead to secondary abnormalities, which include autonomics, cardiac electrical function, respiratory drive, and neuronal electrical activity. Even with a wide array of anti-seizure therapies available on the market, one-third of the more than 70 million people worldwide with epilepsy have uncontrolled seizures (i.e., intractable/drug-resistant epilepsy), which negatively impact neurodevelopment and quality of life. To capture the current state of the field, this review examines KCNQx and KCNHx expression patterns and electrical function in the brain and heart. In addition, it discusses several KCNQx and KCNHx variants that have been clinically and electrophysiologically characterized. Because these channel variants are associated with multi-system pathologies, such as epileptogenesis, Kv7 channel modulators provide a potential anti-seizure therapy, particularly for people with intractable epilepsy. Ultimately an increased understanding of the role of Kv channels throughout the body will fuel the development of innovative, safe, and effective therapies for people at a high risk of sudden death (SCD and SUDEP).

The 14-3-3 protein nt GF14e interacts with CIPK2 and increases low potassium stress in tobacco

ABSTRACT Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.

Acid-Sensitive Outwardly Rectifying Cl- Current in OV2944 Mouse Ovarian Cancer Cells.

Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3)but was slightly restored upon PS administration. These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.

Роль К+-провідного каналу TREK-1 у механочутливості гладеньком’язових клітин детрузора сечового міхура щура

Currently, TREK-1 is considered to be the main mechanosensitive channel in detrusor smooth muscle (DSM) cells. The aim of our study was to detect the functioning of the K+-conducting mechanosensitive TREK-1 channel in rat DSM cells using the patch-clamp technique in response to hydrodynamic stimulation (shear stress) and to determine the effects of a TREK-1 agonist – arachidonic acid (AA) and an antagonist – L-methionine. Mechanical stimulation of DSM cells using hydrodynamic stress led to the appearance of a membrane current with signs of pronounced outward rectification at positive membrane potentials, which is typical of TREK-1 activation. The application of AA (50 mcmol/l) activated a current with similar characteristics of the outward rectification to the shear stress-activated one. L-methionine (10 mcmol/l) almost completely prevented the generation of an outwardly rectifying current in response to shear stress stimulation. DSM cells also retained the ability to generate a mechanoactivated current with a more pronounced inward component when extracellular and intracellular K+ were replaced by Cs+. It was concluded that the dominant mechanoactivated current in rat DSM cells is carried by K+-selective TREK-1 channels, but a small portion of this current can also be carried by other nonselective mechanosensitive cation channels.

Rectification of ATP-gated current of rat P2X2 and P2X7 receptors depends on the cytoplasmic N-terminus

The phenotypes of ATP-gated currents thought ionotropic P2X channels depend on the composition of the oligomeric receptor. We constructed chimeric P2X2/P2X7 receptors to study the effect of cytoplasmic domains on rectification of current flow through the open channel. We found that the identity of the N-terminus determines the pattern of rectification, with chimeric receptors containing the N-terminus of the P2X2 receptor displaying inward rectification, and chimeric receptors containing the N-terminus of the P2X7 receptor displaying slightly outward rectification. In contrast, rectification of current through chimeric receptors with swapped C-termini always mimicked the wild-type receptor. Thus, our findings suggest that the N-terminus of P2X receptors regulate ion flow through the channel pore and are responsible in part for determining current rectification.

Osmotically Activated Anion Current of Phycomyces Blakesleeanus-Filamentous Fungi Counterpart to Vertebrate Volume Regulated Anion Current.

Studies of ion currents in filamentous fungi are a prerequisite for forming a complete understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in the native membrane, including the currents mediated by the channels not yet molecularly identified. Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity sequence, and time and voltage dependent profile of the current. Using the patch clamp method on the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation.

Optogenetic Activation of PVN-projecting MnPO Neurons Induces Changes in Type I PVN Neurons

The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center that receives afferent inputs from the median preoptic nucleus (MnPO). The link between the MnPO and the PVN is essential in generating chronic intermittent hypoxia hypertension. An optogenetic intersectional viral approach was used to gain more insight into the contribution of MnPO inputs to changes in PVN function that could contribute to hypertension. Adult male Sprague-Dawley rats were anesthetized with isoflurane and injected with 100 nL of AAV9.hSyn.HI.eGFP-cre.WPRE.SV40 in the PVN bilaterally and AAV1-Ef1a-DIO-hChR2(H134R)-mCherry-WPRE-HGHpA in the MnPO. This method induced a CRE-dependent expression of channel rhodopsin in PVN-projecting MnPO neurons. Three weeks after the injections, the rats were sacrificed as oblique brain slices of PVN were made. Postsynaptic currents (PSCs) were recorded from PVN neurons (VHold = -70 mV) with an aCSF (2-3 ml/min) bath solution. MnPO axon terminals in the PVN were stimulated with 15 Hz LED-generated blue light (470nM) pulses of 20-ms duration for a total of 1 min with an optical fiber directed at PVN. The stimulation train was repeated 5 times at 5 minutes intervals. PSCs were recorded for 40 minutes, including 5 min baseline periods before stimuli and 10 min post-stimulation period. Cells were characterized as type I, type II, or type III PVN neurons based on the presence of transient outward rectification. Amplitude and frequency data were analyzed offline using Easy Electrophysiology v2.5.0 software from a total of 17 Type I PVN neurons. Optogenetic stimulation evoked EPSC or mixed EPSC/IPSCs in 13 Type I neurons. From the 13 photo-evoked neurons, 10 neurons showed a significant increase in EPSC frequency following stimulation train as compared to the preceding baseline (1st: 6.149±1.438 Hz vs 27.377±5.474 Hz, p=0.0187; 2nd 7.532±1.670 Hz vs 27.562±5.514 Hz, p=0.0161; 3rd 7.395±1.659 Hz vs 25.415±5.028 Hz, p=0.0170; 4th 7.343±1.466 Hz vs 24.225±4.750 Hz, p=0.0183; 5th 6.709±1.375 Hz vs 21.837±4.464 Hz, p=0.0220). In the same neurons, spontaneous EPSC frequency in the first minute after stimulation trains 1 to 4 also was significantly higher than the first pre-stimulus baseline but the effect gradually decreased over time (baseline 6.149±1.438 Hz vs 14.615±3.260 Hz, p=0.0236; vs 12.055±2.519 Hz, p=0.0213; vs 10.904±2.340 Hz, p=0.0247; vs 9.801±2.027 Hz, p=0.0331). There were no changes in EPSC amplitude. Repetitive optogenetic stimulation of MnPO inputs to Type I PVN neurons increased EPSC frequency in a time-dependent manner. Additional experiments will be needed to specify the mechanism behind the increase in frequency and whether this phenomenon occurs in other PVN cell types. P01 HL088052; R01 HL155977 This is the full abstract presented at the American Physiology Summit 2023 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.

Neuron-oligodendrocyte potassium shuttling at nodes of Ranvier protects against inflammatory demyelination.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward-rectifying) and oligodendroglial Kir4.1 (inward-rectifying) potassium channels have important roles in regulating neuronal excitability at and around the nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE), with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs, and rescued neuronal pathology in oligodendrocyte-Kir4.1-deficient (OL-Kir4.1-deficient) mice. In summary, our findings indicate that neuron-OL compensatory interactions promoted resilience through Kv7 and Kir4.1 channels and identify pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination.

The role of potassium current in the pulmonary response to environmental oxidative stress

Exposure of human lung epithelial cells (A549 cell line) to the oxidant pollutant ozone (O3) alters cell membrane currents inducing its decrease, when the cell undergoes to a voltage-clamp protocol ranging from −90 to +70mV. The membrane potential of these cells is mainly maintained by the interplay of potassium and chloride currents. Our previous studies indicated the ability of O3 to activate ORCC (Outward Rectifier Chloride Channel) and consequently increases the chloride current.In this paper our aim was to understand the response of potassium current to oxidative stress challenge and to identify the kind potassium channel involved in O3 induced current changes.After measuring the total membrane current using an intracellular solution with or without potassium ions, we obtained the contribution of potassium to the overall membrane current in control condition by a mathematical approach. Repeating these experiments after O3 treatment we observed a significant decrease of Ipotassium. Treatment of the cells with Iberiotoxin (IbTx), a specific inhibitor of BK channel, we were able to verify the presence and the functionality of BK channels. In addition, the administration of 4-Aminopyridine (an inhibitor of voltage dependent K channels but not BK channels) and Tetraethylammonium (TEA) before and after O3 treatment we observed the formation of BK oxidative post-translation modifications.Our data suggest that O3 is able to inhibit potassium current by targeting BK channel. Further studies are needed to better clarify the role of this BK channel and its interplay with the other membrane channels under oxidative stress conditions. These findings can contribute to identify the biomolecular pathway induced by O3 allowing a possible pharmacological intervention against oxidative stress damage in lung tissue.

The anion channel SLAH3 interacts with potassium channels to regulate nitrogen-potassium homeostasis and the membrane potential in Arabidopsis.

Nitrogen (N) and potassium (K) are essential macronutrients for plants. Sufficient N and K uptake from the environment is required for successful growth and development. However, how N and K influence each other at the molecular level in plants is largely unknown. In this study, we found loss-of-function mutation in SLAH3 (SLAC1 HOMOLOGUE 3), encoding a NO3- efflux channel in Arabidopsis thaliana, enhanced tolerance to high KNO3 concentrations. Surprisingly, slah3 mutants were less sensitive to high K+ but not NO3-. Addition of NO3- led to reduced phenotypic difference between wild-type and slah3 plants, suggesting SLAH3 orchestrates NO3--K+ balance. Non-invasive Micro-test Technology analysis revealed reduced NO3- efflux and enhanced K+ efflux in slah3 mutants, demonstrating that SLAH3-mediated NO3- transport and SLAH3-affected K+ flux are critical in response to high K +. Further investigation showed that two K+ efflux channels, GORK (GATED OUTWARDLY-RECTIFYING K+ CHANNEL) and SKOR (STELAR K+ OUTWARD RECTIFIER), interacted with SLAH3 and played key roles in high K+ response. The gork and skor mutants were slightly more sensitive to high K+ conditions. Less depolarization occurred in slah3 mutants and enhanced depolarization was observed in gork and skor mutants upon K+ treatment, suggesting NO3-/K+ efflux-mediated membrane potential regulation is involved in high K+ response. Electrophysiological results showed that SLAH3 partially inhibited the activities of GORK and SKOR in Xenopus laevis oocytes. This study revealed that the anion channel SLAH3 interacts with the potassium channels GORK and SKOR to modulate membrane potential by coordinating N-K balance.

Inhibitory Effects of 2-Aminoethoxydiphenyl Borate (2-APB) on Three KV1 Channel Currents.

2-Aminoethoxydiphenyl borate (2-APB), a boron-containing compound, is a multitarget compound with potential as a drug precursor and exerts various effects in systems of the human body. Ion channels are among the reported targets of 2-APB. The effects of 2-APB on voltage-gated potassium channels (KV) have been reported, but the types of KV channels that 2-APB inhibits and the inhibitory mechanism remain unknown. In this paper, we discovered that 2-APB acted as an inhibitor of three representative human KV1 channels. 2-APB significantly blocked A-type Kv channel KV1.4 in a concentration-dependent manner, with an IC50 of 67.3 μM, while it inhibited the delayed outward rectifier channels KV1.2 and KV1.3, with IC50s of 310.4 μM and 454.9 μM, respectively. Further studies on KV1.4 showed that V549, T551, A553, and L554 at the cavity region and N-terminal played significant roles in 2-APB's effects on the KV1.4 channel. The results also indicated the importance of fast inactivation gating in determining the different effects of 2-APB on three channels. Interestingly, a current facilitation phenomenon by a short prepulse after 2-APB application was discovered for the first time. The docked modeling revealed that 2-APB could form hydrogen bonds with different sites in the cavity region of three channels, and the inhibition constants showed a similar trend to the experimental results. These findings revealed new molecular targets of 2-APB and demonstrated that 2-APB's effects on KV1 channels might be part of the reason for the diverse bioactivities of 2-APB in the human body and in animal models of human disease.

Mapping the functional expression of auxiliary subunits of KCa1.1 in glioblastoma

Glioblastoma (GBM) is the most aggressive glial tumor, where ion channels, including KCa1.1, are candidates for new therapeutic options. Since the auxiliary subunits linked to KCa1.1 in GBM are largely unknown we used electrophysiology combined with pharmacology and gene silencing to address the functional expression of KCa1.1/β subunits complexes in both primary tumor cells and in the glioblastoma cell line U-87 MG. The pattern of the sensitivity (activation/inhibition) of the whole-cell currents to paxilline, lithocholic acid, arachidonic acid, and iberiotoxin; the presence of inactivation of the whole-cell current along with the loss of the outward rectification upon exposure to the reducing agent DTT collectively argue that KCa1.1/β3 complex is expressed in U-87 MG. Similar results were found using human primary glioblastoma cells isolated from patient samples. Silencing the β3 subunit expression inhibited carbachol-induced Ca2+ transients in U-87 MG thereby indicating the role of the KCa1.1/β3 in the Ca2+ signaling of glioblastoma cells. Functional expression of the KCa1.1/β3 complex, on the other hand, lacks cell cycle dependence. We suggest that the KCa1.1/β3 complex may have diagnostic and therapeutic potential in glioblastoma in the future.

Биоэлектрические эффекты отмывания клеток Сhara fragilis от растворов фитокомпозиции одуванчик-зверобой-календулы

By using of microelectrode techniques, patterns of change in the potential (φm) and resistance (Rm) of the plasma membrane (PM) of Chara fragilis cells under the influence of Dandelion–Hypericum-Calendula (DHC) phytocomposition solutions were studied. The values of φm and Rm are widely scattered: -90 ÷ -300 mV and 1 ÷ 32.6 Ohm·m2, respectively under the standard conditions. The average value of φm was -183 ± 4.9 mV, Rm = 9.2 ± 1.2 Ohm·m2. Using the dependences of φm, Rm on the concentration of K+ in the external environment, the ranges of activation of the K+ - channels to the inward (KCİR) and outward rectification (KCOR) were determined. Under the action of the DHC phytocomposition, while the cell was in the medium with the concentration of the composite 10-1 or 1 mg/ml, we did not detect changes in φm and Rm for 25 min. Stimulation or suppression of transport processes under the influence of composites occurred only with the exclusion of DHC from the composition of the nutrient medium and with a stepwise increase in the concentration of the composition in the medium. The electrophysiological effects of the action of the DHC composition are discussed from the point of view of its action on the lipid phase of the plasma membrane of the experimental cells.

New Insights into Ion Channels: Predicting hERG-Drug Interactions.

Drug-induced long QT syndrome can be a very dangerous side effect of existing and developmental drugs. In this work, a model proposed two decades ago addressing the ion specificity of potassium channels is extended to the human ether-à-gogo gene (hERG). hERG encodes the protein that assembles into the potassium channel responsible for the delayed rectifier current in ventricular cardiac myocytes that is often targeted by drugs associated with QT prolongation. The predictive value of this model can guide a rational drug design decision early in the drug development process and enhance NCE (New Chemical Entity) retention. Small molecule drugs containing a nitrogen that can be protonated to afford a formal +1 charge can interact with hERG to prevent the repolarization of outward rectifier currents. Low-level ab initio calculations are employed to generate electronic features of the drug molecules that are known to interact with hERG. These calculations were employed to generate structure–activity relationships (SAR) that predict whether a small molecule drug containing a protonated nitrogen has the potential to interact with and inhibit the activity of the hERG potassium channels of the heart. The model of the mechanism underlying the ion specificity of potassium channels offers predictive value toward optimizing drug design and, therefore, minimizes the effort and expense invested in compounds with the potential for life-threatening inhibitory activity of the hERG potassium channel.

Structure-function relationships of the LRRC8 subunits and subdomains of the volume-regulated anion channel (VRAC).

Volume Regulated Anion Channels (VRAC) are critical contributors to cell volume homeostasis and are expressed ubiquitously in all vertebrate cells. VRAC sense increases in cell volume, and act to return cells to baseline volume in a process known as regulatory volume decrease (RVD) through the efflux of anions and organic osmolytes. This review will highlight seminal studies that elucidated the role of VRAC in RVD, their characteristics as a function of subunit specificity, and their clinical relevance in physiology and pathology. VRAC are also known as volume-sensitive outward rectifiers (VSOR) and volume-sensitive organic osmolyte/anion channels (VSOAC). In this review, the term VRAC will be used to refer to this family of channels.

An extracellular scaffolding complex confers unusual rectification upon an ionotropic acetylcholine receptor in C. elegans

Biophysical properties of ligand-gated receptors can be profoundly modified by auxiliary subunits or by the lipid microenvironment of the membrane. Hence, it is sometimes challenging to relate the properties of receptors reconstituted in heterologous expression systems to those of their native counterparts. Here we show that the properties of Caenorhabditis elegans levamisole-sensitive acetylcholine receptors (L-AChRs), the ionotropic acetylcholine receptors targeted by the cholinergic anthelmintic levamisole at neuromuscular junctions, can be profoundly modified by their clustering machinery. We uncovered that L-AChRs exhibit a strong outward rectification invivo, which was not previously described in heterologous systems. This unusual feature for an ionotropic AChR is abolished by disrupting the interaction of the receptors with the extracellular complex required for their synaptic clustering. When recorded at -60 mV, levamisole-induced currents are similar in the wild type and in L-AChR-clustering-defective mutants, while they are halved in these mutants at more depolarized physiological membrane potentials. Consequently, levamisole causes a strong muscle depolarization in the wild type, which leads to complete inactivation of the voltage-gated calcium channels and to an irreversible flaccid paralysis. In mutants defective for L-AChR clustering, the levamisole-induced depolarization is weaker, allowing voltage-gated calcium channels to remain partially active, which eventually leads to adaptation and survival of the worms. This explains why historical screens for C. elegans mutants resistant to levamisole identified the components of the L-AChR clustering machinery, in addition to proteins required for receptor biosynthesis or efficacy. This work further emphasizes the importance of pursuing ligand-gated channel characterization in their native environment.

Identification of the chloride channel, leucine-rich repeat-containing protein 8, subfamily a (LRRC8A), in mouse cholangiocytes.

Chloride (Cl- ) channels in the apical membrane of biliary epithelial cells (BECs), also known as cholangiocytes, provide the driving force for biliary secretion. Although two Cl- channels have been identified on a molecular basis, the Cystic Fibrosis Transmembrane Conductance Regulator and Transmembrane Member 16A, a third Cl- channel with unique biophysical properties has been described. Leucine-Rich Repeat-Containing Protein 8, subfamily A (LRRC8A) is a newly identified protein capable of transporting Cl- in other epithelium in response to cell swelling. The aim of the present study was to determine if LRRC8A represents the volume-regulated anion channel in mouse BECs. Studies were performed in mouse small (MSC) and large (MLC) cholangiocytes. Membrane Cl- currents were measured by whole-cell patch-clamp techniques and cell volume measurements were performed by calcein-AM fluorescence. Exposure of either MSC or MLC to hypotonicity (190 mOsm) rapidly increased cell volume and activated Cl- currents. Currents exhibited outward rectification, time-dependent inactivation at positive membrane potentials, and reversal potential at 0 mV (ECl ). Removal of extracellular Cl- or specific pharmacological inhibition of LRRC8A abolished currents. LRRC8A was detected in both MSC and MLC by reverse transcription polymerase chain reaction and confirmed by western blot. Transfection with LRRC8A small interfering RNA decreased protein levels by >70% and abolished volume-stimulated Cl- currents. These results demonstrate that LRRC8A is functionally present in mouse BECs, contributes to volume-activated Cl- secretion, and, therefore, may be a target to modulate bile formation in the treatment of cholestatic liver disorders.

Changes in PVN Neurons after Low‐Frequency Acute Optogenetic Stimulation

The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center. It receives afferent inputs from many brain regions, including the median preoptic nucleus (MnPO). The connection between the MnPO and the PVN is particularly important in generating chronic intermittent hypoxia phenotypes such as increased sympathetic activity leading to the development of hypertension. PVN‐projecting MnPO neurons discharge frequency is <5Hz. To gain more insight into the acute change in PVN neuron properties after low‐frequency stimulation, whole‐cell patch‐clamp recordings were performed on PVN neurons. Adult 250‐350g male rats were injected bilaterally in the PVN with 100 nL retrogradely transported adeno‐associated virus encoding Channelrhodopsin (AAVrg‐CaMKIIa‐hChR2(H134R)‐mCherry) or with AAVrg‐CAG‐tdTomato as control. Three weeks after the injections, the rats were anesthetized with isoflurane (2‐3%) and sacrificed to prepare horizontal brain slices containing the PVN and MnPO. Postsynaptic currents were recorded from PVN neurons in whole‐cell voltage clamp configuration (Vhold=‐60 mV) under constant bath perfusion of normal aCSF (2‐3 ml/min). Axon terminals in the PVN were stimulated with 5Hz LED‐generated blue light (470nM) pulses of 50‐ms duration, 5 seconds off/on for a total of 1 min, delivered using optical fiber directed to the MnPO. Photo‐evoked currents were measured during 5 min before and after the photo‐stimulation train. Amplitude and frequency of spontaneous currents were compared before and after stimulation as well. At the end of each recording, cells were characterized as type I (magnocellular) or type II (parvocellular) PVN neurons based on the presence of transient outward rectification. Data were analyzed offline using Easy Electrophysiology v2.3.3 software. For both cell types, 5 Hz optogenetic stimulation caused a significant reduction in the optogenetic‐evoked current amplitude during the first 15 minutes after stimulation (before stimulation 94.88±2.85 %, n=9 cells vs. 15 minutes after stimulation 67.52±6.15 %, n=9. P=0.0027) before returning to baseline values. Post‐stimulation spontaneous postsynaptic current amplitude and frequency decreased in type II PVN neurons from retrograde ChR2 animals (pre‐stimulation amplitude 100±0.62 % vs. post‐stimulation amplitude 86.50±0.54 %, P=0.0371; pre‐stimulation frequency 3.82±1.7 Hz, post‐stimulation frequency 2.396±1.2 Hz, P= 0.0385) but not in type I PVN neurons from retrograde ChR2 animals or cells from control animals. Kinetic profiles of spontaneous current in type I and II PVN neurons did not change after 5 Hz optogenetic stimulation. The results suggest that low‐frequency stimulation discriminately alters spontaneous presynaptic neurotransmitter release and possibly postsynaptic responsiveness in PVN neurons. Additional experiments will be needed to specifically stimulate PVN afferents from the MnPO.

Elevated 5‐hydroxytryptamine in COVID‐19 Stimulates ANO1 Mediated Cl Secretion in Lung & Intestinal Epithelial Cells

Earlier studies demonstrated that blood 5‐hydroxytryptamine (5‐HT) is elevated in patients with COVID‐19‐associated diarrhea with higher severity of symptoms. We hypothesize that disruption of vectorial Cl transport by 5‐HT may be critical in determining the alveolar flooding and abnormalities in intestinal Cl secretion through stimulation of anoctamin 1(ANO1) Cl channel. Using western blot, immunostaining, and electrophysiology, we characterized the localization of human ANO1 and its stimulation by 5‐HT on Cl secretion in lung and intestinal epithelium. Because calcium‐activated chloride current in human intestinal epithelia remains controversial, we examined the localization of ANO1 in the human terminal ileum and colonic tissue using confocal microscopy. Our results indicated that ANO1 was localized predominantly at the brush‐border membrane and co‐localized with the brush‐border membrane marker villin. ANO1 is not present in goblet cells. The anti‐ANO1 antibody recognized a protein of appropriate size in human colonic tissue. The specificity of the ANO1 antibody was tested by immunoblot analysis of lysates from human colorectal cancer tissues, where it displayed amplified ANO1 protein expression. We next confirmed ANO1‐currents activated by 5‐HT in the Caco‐2 cells by patch‐clamp measurements of whole‐cell current. The application of 100 nM 5‐HT produced a typical outward rectification. CaCCinh‐A01, a specific ANO1 blocker, inhibited the currents. The half‐maximal effective concentration value for the effects of 5‐HT was estimated at 21.8 ± 13.7 nM with a Hill coefficient of 0.89 ± 0.16. These results indicated that 5‐HT evoked calcium‐activated Cl currents through ANO1 channels. ANO1 is expressed in Calu 3 cells. We next confirmed the presence of ANO1 currents activated by 5‐HT in Calu‐3 cells by the Ussing chamber experiments. Serosal addition of 5‐HT produced an immediate and significant increase in Isc in Calu‐3 cells that was inhibited by the ANO1 selective inhibitor T16Ainh‐A01. Finally, we demonstrate that SARS‐CoV2 infection led to enterochromaffin cell hyperplasia in the intestinal epithelium of Syrian Hamster with a possible elevation of 5‐HT, which could explain the severity of symptoms in COVID‐19 associated diarrheal patients.In conclusion, SARS‐CoV2 infection resulted in intestinal enterochromaffin cells hyperplasia that could elevate 5‐HT. Elevated 5‐HT activates luminal ANO1 CaCC in the intestinal and lung epithelium by a mechanism that appears to involve the rise of [Ca2+]i. Our data suggest that 5‐HT may be a critical determinant of the COVID‐19 associated diarrhea and flooding of alveoli that have considerable implications for COVID‐19 therapy.

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European Journal of NeuroscienceVolume 55, Issue 7 p. 1887-1889 ERRATUMFree Access ERRATUM This article corrects the following: Cyclic nucleotide-dependent ionic currents in olfactory receptor neurons of the hawkmoth Manduca sexta suggest pull–push sensitivity modulation Jan Dolzer, Katrin Schröder, Monika Stengl, Volume 54Issue 3European Journal of Neuroscience pages: 4804-4826 First Published online: July 8, 2021 First published: 28 March 2022 https://doi.org/10.1111/ejn.15638AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Dolzer et al. (2021), some reference citations in Table 3 were missing. Table 3 should be presented as below: The Publisher apologises for this error. TABLE 3. Currents and current components in ORNs of Manduca sexta Name Abbreviation I-V curve Ion channel conductance/reversal potential (RP) Characteristics Activation Inactivation/block No effects References Potassium currents delayed rectifier IK ~ 30 pS outward rectification delayed onset depol. TEA, Cs+ mM Ni2+ Dolzer 2002 Zufall et al. 1991b cGMP-inactivted outward rectifier IK(cGMP-) 30 pS cNMP block depol. TEA, cAMP, cGMP, ATP Dolzer 2002 Zufall et al. 1991b Stengl et al. 1992 non-TEA-blockable delayed rectifier IK(noTEA) 40 pS? delayed onset no TEA block ~10−6 Ca2+ active? depol. PKC? cGMP?, Zn3+? TEA, cAMP Dolzer 2002 Dolzer et al. 2008 rapidly inactivating outward rectifier IK(A) 12–18 pA not tested depol. 4-AP 5 mM Zufall et al. 1991b slowly inactivating delayed rectifier IK(cGMP+) 70 pS? slow inactivation after depol. cGMP cAMP TEA (slow, incomplete), Ni2+, Cs+? Dolzer 2002 Dolzer et al. 2008 calcium-activated outward rectifier IK(Ca2+) 66 pS I-V flattened in positive Vm >10−6 M Ca2+ cGMP TEA, Cs+, CTX cAMP Dolzer 2002 Zufall et al. 1991b Nonspecific cation currents calcium-dependent Icat(Ca2+) RP = 0 I-V steeper in positive and negative Vm >10−6 M Ca2+ mM Ca2+, TEA, Ni2+, cGMP? Dolzer 2002 Stengl 1993, 1994 fast, steady, non-inactivating Icat(nRP) RP < 0 g (K) > g (Ca2+) clearly negative reversal potential TEA, Ni2+ cGMP? cAMP Dolzer 2002 late, large, long-lasting ILL RP = 0 slow activation after depol., slow inactivation after hyperpol. long, large depol. cAMP long, large depol. Ca2+?? TEA (partly) cGMP? Dolzer 2002 fast Unknown cation current Icat(PKC?) RP = 0 linear I-V TEA block Ca2+? PKC? TEA cAMP cGMP? Dolzer 2002 PKC-dependent Icat+(PKC) not tested PKC TEA Stengl 1993 Unknown currents non-specific cat- or anion I? RP = 0 linear I-V no TEA block TEA, Ni2+, cAMP?, cGMP?, Ca2+?? TEA, SIST, (DIDS), 9 AC, PTX, ZN2+, niflumic acid Dolzer 2002 inward rectifier Iir RP ~ 0 I-V steeper in negative Vm TEA Ni2+ Dolzer 2002 Abbreviations: cNMP, cyclic nucleotides; CTX, charybdotoxin; depol, depolarization; DIDS, 4,4′-diisothiocyanostilbene-2,20-disulfonic acid; hyperpol, hyperpolarization; I–V, current–voltage relation; PKC, protein kinase C; PTX, picrotoxin; SITS, 4-acetamido-4-thiocyanostilbene-2,2′-disulfonic acid; Vm, membrane potential; 4-AP, 4-aminopyridine; 9 AC, anthracene-9-carboxylic acid. Note: Currents that were not tested in this study, (IK(A) and Icat + (PKC)), are on grey background. Newly observed properties are underlined, and findings that are only described in the literature are in bold. REFERENCE Dolzer, J., Schröder, K., & Stengl, M. (2021). Cyclic nucleotide-dependent ionic currents in olfactory receptor neurons of the hawkmoth Manduca sexta suggest pull–push sensitivity modulation. European Journal of Neuroscience, 54(3), 4804– 4826. https://doi.org/10.1111/ejn.15346Wiley Online LibraryPubMedWeb of Science®Google Scholar Volume55, Issue7April 2022Pages 1887-1889 ReferencesRelatedInformation