Rectifying Potassium Channel Research Articles

Hippocampal Vascular Dysfunction and Impaired Memory in a Neuroendocrine Model of Stress and Hypertension

The hippocampus is a deep brain structure critically involved in learning and memory that is highly susceptible to hypoxic/ischemic injury. Hypertension and psychological stress are major risk factors of dementia; however, their effects on the vasculature of the cognition-centric hippocampus remain unclear. This study investigated hippocampal vascular function and memory in a novel model of neuroendocrine-derived stress and hypertension. This model is induced via vector mediated brain-derived neurotrophic factor (BDNF) overexpression in the paraventricular nucleus of the hypothalamus (PVN) that causes chronic stimulation of the major hypothalamic stress pathways (e.g. hypothalamic-pituitary-adrenal axis), leading to significant long-term elevation of blood pressure. We hypothesized that function of the arterioles supplying the hippocampus and memory would be impaired in this BDNF-PVN model of chronic stress and hypertension. Potential sex differences in the susceptibility of the hippocampus to dysfunction were also investigated. Male and female Sprague Dawley rats received bilateral PVN injections of AAV2 vectors expressing BDNF or GFP for control (n=6-8/group) at 8 weeks of age. Four weeks later, an object recognition task was used to quantify the percent of time spent investigating a novel object as a measure of hippocampal-dependent long-term memory function. Hippocampal arterioles (HAs) from the same rats were then studied isolated and pressurized in an arteriograph chamber. Vasodilator responses of HAs to mediators of functional hyperemia were measured, including small- and intermediate-conductance calcium-activated potassium (SK/IK) channel activation via NS309, and activation of inward rectifier potassium (KIR) channels by increasing extracellular K+ from 3-15mM. Memory function was significantly impaired by PVN-BDNF treatment (two-way ANOVA, Tukey's post hoc test, F1,22=16.53; p=0.001), but sex had no effect (F1,22=1.16; p=0.29). Object recognition was lower in both male and female PVN-BDNF rats that spent 49±3% and 52±6% of the time with the novel object, compared to control male and female rats that spent 66±3% and 72±4% of the time investigating the novel object. HAs from male and female PVN-BDNF rats dilated significantly less to NS309 than controls (F1,20=12.35; p=0.002), suggesting endothelial damage. Surprisingly, activation of KIR channels with 15mM K+ caused a 3-5% vasoconstriction of HAs from both male and female PVN-BDNF rats that was in striking contrast to the 20-30% vasodilation that occurred in response to KIR channel activation in HAs from control rats (F1,19=24.90; p<0.0001). These data show that chronic neuroendocrine stress and hypertension caused hippocampal vascular dysfunction and impaired memory similarly in male and female rats. The blunted vasodilation of HAs from PVN-BDNF rats suggests functional hyperemia may be impaired in the hippocampus that could lead to neuronal dysfunction, memory loss and dementia.

Activation of the kidney sodium chloride cotransporter by the β2-adrenergic receptor agonist salbutamol increases blood pressure

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In exvivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

WIN55,212-2, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels.

The coupling of cannabinoid receptors, CB1 and CB2, to G protein-coupled inward rectifier potassium channels, GIRK1 and GIRK2, modulates neuronal excitability in the human brain. The present study established and validated the functional expression in a Xenopus laevis oocyte expression system of CB1 and CB2 receptors, interacting with heteromeric GIRK1/2 channels and a regulator of G protein signaling, RGS4. This ex vivo system enables the discovery of a wide range of ligands interacting orthosterically or allosterically with CB1 and/or CB2 receptors. WIN55,212-2, a non-selective agonist of CB1 and CB2, was used to explore the CB1- or CB2-GIRK1/2-RGS4 signaling cascade. We show that WIN55,212-2 activates CB1 and CB2 at low concentrations whereas at higher concentrations it exerts a direct block of GIRK1/2. This illustrates a dual modulatory function, a feature not described before, which helps to explain the adverse effects induced by WIN55,212-2 in vivo. When comparing the effects with other typical cannabinoids such as Δ9-THC, CBD, CP55,940, and rimonabant, only WIN55,212-2 can significantly block GIRK1/2. Interestingly, the inward rectifier potassium channel, IRK1, a non-G protein-coupled potassium channel important for setting the resting membrane voltage and highly similar to GIRK1 and GIRK2, is not sensitive to WIN55,212-2, Δ9-THC, CBD, CP55,940, or rimonabant. From this, it is concluded that WIN55,212-2 selectively blocks GIRK1/2.

Angiotensin II, ATP and high extracellular potassium induced intracellular calcium responses in primary rat brain endothelial cell cultures.

The meninges shield the nervous system from diverse, rather harmful stimuli and pathogens from the periphery. This tissue is composed of brain endothelial cells (BECs) that express diverse ion channels and chemical-transmitter receptors also expressed by neurons and glial cells to communicate with each other. However, information about the effects of ATP and angiotensin II on BECs is scarce, despite their essential roles in blood physiology. This work investigated in vitro if BECs from the meninges from rat forebrain respond to ATP, angiotensin II and high extracellular potassium, with intracellular calcium mobilizations and its second messenger-associated pathways. We found that in primary BEC cultures, both ATP and angiotensin II produced intracellular calcium responses linked to the activation of inositol trisphosphate receptors and ryanodine receptors, which led to calcium release from intracellular stores. We also used RT-PCR to explore what potassium channel subunits are expressed by primary BEC cultures and freshly isolated meningeal tissue, and which might be linked to the observed effects. We found that BECs mainly expressed the inward rectifier potassium channel subunits Kir1.1, Kir3.3, Kir 4.1 and Kir6.2. This study contributes to the understanding of the functions elicited by ATP and angiotensin II in BECs from rat meninges. SIGNIFICANCE OF THE STUDY: Brain endothelial cells (BECs) express diverse ion channels and membrane receptors, which they might use to communicate with neurons and glia. This work investigated in vitro, if BECs from the rat forebrain respond to angiotensin II and ATP with intracellular calcium mobilizations. We found that these cells did respond to said substances with intracellular calcium mobilizations linked to inositol trisphosphate and ryanodine receptor activation, which led to calcium release from intracellular stores. These findings are important because they might uncover routes of active communication between brain cells and endothelial cells.

Phylogenomics of Tick Inward Rectifier Potassium Channels and Their Potential as Targets to Innovate Control Technologies.

This study was conducted to enhance the identification of novel targets to develop acaricides that can be used to advance integrated tick-borne disease management. Drivers for the emergence and re-emergence of tick-borne diseases affecting humans, livestock, and other domestic animals in many parts of the world include the increased abundance and expanded geographic distribution of tick species that vector pathogens. The evolution of resistance to acaricides among some of the most important tick vector species highlights the vulnerability of relying on chemical treatments for tick control to mitigate the health burden of tick-borne diseases. The involvement of inward rectifier potassium (Kir) channels in homeostasis, diuresis, and salivary gland secretion in ticks and other pests identified them as attractive targets to develop novel acaricides. However, few studies exist on the molecular characteristics of Kir channels in ticks. This bioinformatic analysis described Kir channels in 20 species of hard and soft ticks. Summarizing relevant investigations on Kir channel function in invertebrate pests allowed the phylogenomic study of this class of ion channels in ticks. How this information can be adapted to innovate tick control technologies is discussed.

Cisplatin-induced activation and functional modulation of satellite glial cells lead to cytokine-mediated modulation of sensory neuron excitability

Cisplatin plays an essential role in the treatment of various cancers. Cisplatin exhibits high efficacy, but it often leads to severe neurotoxic side effects, such as chemotherapy-induced polyneuropathy (CIPN). The pathophysiology of CIPN is not fully understood. There is increasing evidence for damage to satellite glial cells (SGC) and dorsal root ganglion (DRG) neurons. We investigated the influence of cisplatin on the function of SGCs and the direct influence on DRGs. Satellite glial cells were isolated from DRG and exposed to 0.1, 1, 10, or 100 μM cisplatin for 2 h, 4 h, and 24 h. Using immunocytochemical staining and Western blot analysis, the expression of the glial fibrillary acid protein (GFAP), reactive oxygen species (ROS), and inward rectifier potassium channel 4.1 (Kir4.1) was determined. An increase in the immune reactivity (IR) and protein levels of GFAP and ROS was measured, and a reduction of IR and protein level of Kir4.1 was detected. A decrease in these channels' current density was observed using the whole-cell patch-clamp recording. The interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) release of SGCs increased after cisplatin exposure as measured using ELISA, and interleukin-1β (IL-1β) decreased. The SGC-secreted factors in the supernatant after cisplatin treatment led to a modulation of cultured DRG neurons' excitability.Taken together, the modulation and function of different SGC proteins could be linked to a direct impact of cisplatin. Further, SGC-secreted factors influenced the excitability of sensory neurons. Overall, SGCs could be a potential target in preventing and treating chemotherapy-induced neuropathic pain.

Reciprocal interaction between IK1 and If in biological pacemakers: A simulation study.

Pacemaking dysfunction (PD) may result in heart rhythm disorders, syncope or even death. Current treatment of PD using implanted electronic pacemakers has some limitations, such as finite battery life and the risk of repeated surgery. As such, the biological pacemaker has been proposed as a potential alternative to the electronic pacemaker for PD treatment. Experimentally and computationally, it has been shown that bio-engineered pacemaker cells can be generated from non-rhythmic ventricular myocytes (VMs) by knocking out genes related to the inward rectifier potassium channel current (IK1) or by overexpressing hyperpolarization-activated cyclic nucleotide gated channel genes responsible for the “funny” current (If). However, it is unclear if a bio-engineered pacemaker based on the modification of IK1- and If-related channels simultaneously would enhance the ability and stability of bio-engineered pacemaking action potentials. In this study, the possible mechanism(s) responsible for VMs to generate spontaneous pacemaking activity by regulating IK1 and If density were investigated by a computational approach. Our results showed that there was a reciprocal interaction between IK1 and If in ventricular pacemaker model. The effect of IK1 depression on generating ventricular pacemaker was mono-phasic while that of If augmentation was bi-phasic. A moderate increase of If promoted pacemaking activity but excessive increase of If resulted in a slowdown in the pacemaking rate and even an unstable pacemaking state. The dedicated interplay between IK1 and If in generating stable pacemaking and dysrhythmias was evaluated. Finally, a theoretical analysis in the IK1/If parameter space for generating pacemaking action potentials in different states was provided. In conclusion, to the best of our knowledge, this study provides a wide theoretical insight into understandings for generating stable and robust pacemaker cells from non-pacemaking VMs by the interplay of IK1 and If, which may be helpful in designing engineered biological pacemakers for application purposes.

Effects of modulation on sodium and potassium channel currents by extremely low frequency electromagnetic fields stimulation on hippocampal CA1 pyramidal cells

ABSTRACT To investigate the effects of extremely low-frequency electromagnetic fields (ELF-EMFs) stimulation on sodium channel currents (INa), transient outward potassium channel currents (IA) and delayed rectifier potassium channel currents (IK) on hippocampal CA1 pyramidal neurons of young Sprague–Dawley rats. CA1 pyramidal neurons of rat hippocampal slices were subjected to ELF-EMFs stimulation with different frequencies (15 and 50 Hz), intensities (0.5, 1 and 2 mT) and durations (10, 20 and 30 min). The INa, IA and IK of neurons were recorded by a whole-cell patch-clamp method. ELF-EMFs stimulation enhanced INa densities, and depressed IA and IK densities. In detail, INa was more sensitive to the variation of intensities and frequencies of ELF-EMFs, whereas IA and IK were mainly affected by the variation of the duration of ELF-EMFs. ELF-EMFs stimulation altered activation and deactivation properties of INa, IA and IK. ELF-EMFs stimulation plays a role as a regulator rather than an inducer for ion channels. It might change the transition probability of ion channel opening or closing, and might also change the structure and function of the ion channel which need to be proved by the further technical method.

An Inhibitory Circuit From Brainstem to GnRH Neurons in Male Mice: A New Role for the RFRP Receptor

RFamide-related peptides (RFRPs, mammalian orthologs of gonadotropin-inhibitory hormone) convey circadian, seasonal and social cues to the reproductive system. They regulate gonadotropin secretion by modulating gonadotropin-releasing hormone (GnRH) neurons via the RFRP receptor. Mice lacking this receptor are fertile but exhibit abnormal gonadotropin responses during metabolic challenges such as acute fasting, when the normal drop in gonadotropin levels is delayed. Although it is known that these food intake signals to the reproductive circuit originate in the nucleus tractus solitarius (NTS) in the brainstem, the phenotype of the neurons conveying the signal remains unknown. Given that neuropeptide FF (NPFF), another RFamide peptide, resides in the NTS and can bind to the RFRP receptor, we hypothesized that NPFF may regulate GnRH neurons. To address this question, we used a combination of techniques: cell-attached electrophysiology on GnRH-driven green fluorescent protein-tagged neurons in acute brain slices; calcium imaging on cultured GnRH neurons; and immunostaining on adult brain tissue. We found 1) NPFF inhibits GnRH neuron excitability via the RFRP receptor and its canonical signaling pathway (Gi/o protein and G protein-coupled inwardly-rectifying potassium channels), 2) NPFF-like fibers in the vicinity of GnRH neurons coexpress neuropeptide Y, 3) the majority of NPFF-like cell bodies in the NTS also coexpress neuropeptide Y, and 4) acute fasting increased NPFF-like immunoreactivity in the NTS. Together these data indicate that NPFF neurons within the NTS inhibit GnRH neurons, and thus reproduction, during fasting but prior to the energy deficit.

Kir6.1 improves cardiac dysfunction in diabetic cardiomyopathy via the AKT-FoxO1 signalling pathway.

Previous studies have shown that the expression of inwardly rectifying potassium channel 6.1 (Kir6.1) in heart mitochondria is significantly reduced in type 1 diabetes. However, whether its expression and function are changed and what role it plays in type 2 diabetic cardiomyopathy (DCM) have not been reported. This study investigated the role and mechanism of Kir6.1 in DCM. We found that the cardiac function and the Kir6.1 expression in DCM mice were decreased. We generated mice overexpressing or lacking Kir6.1 gene specifically in the heart. Kir6.1 overexpression improved cardiac dysfunction in DCM. Cardiac‐specific Kir6.1 knockout aggravated cardiac dysfunction. Kir6.1 regulated the phosphorylation of AKT and Foxo1 in DCM. We further found that Kir6.1 overexpression also improved cardiomyocyte dysfunction and up‐regulated the phosphorylation of AKT and FoxO1 in neonatal rat ventricular cardiomyocytes with insulin resistance. Furthermore, FoxO1 activation down‐regulated the expression of Kir6.1 and decreased the mitochondrial membrane potential (ΔΨm) in cardiomyocytes. FoxO1 inactivation up‐regulated the expression of Kir6.1 and increased the ΔΨm in cardiomyocytes. Chromatin immunoprecipitation assay demonstrated that the Kir6.1 promoter region contains a functional FoxO1‐binding site. In conclusion, Kir6.1 improves cardiac dysfunction in DCM, probably through the AKT‐FoxO1 signalling pathway.

Orexin-A Intensifies Mouse Pupillary Light Response by Modulating Intrinsically Photosensitive Retinal Ganglion Cells.

We show for the first time that the neuropeptide orexin modulates pupillary light response, a non-image-forming visual function, in mice of either sex. Intravitreal injection of the orexin receptor (OXR) antagonist TCS1102 and orexin-A reduced and enhanced pupillary constriction in response to light, respectively. Orexin-A activated OX1Rs on M2-type intrinsically photosensitive retinal ganglion cells (M2 cells), and caused membrane depolarization of these cells by modulating inward rectifier potassium channels and nonselective cation channels, thus resulting in an increase in intrinsic excitability. The increased intrinsic excitability could account for the orexin-A-evoked increase in spontaneous discharges and light-induced spiking rates of M2 cells, leading to an intensification of pupillary constriction. Orexin-A did not alter the light response of M1 cells, which could be because of no or weak expression of OX1Rs on them, as revealed by RNAscope in situ hybridization. In sum, orexin-A is likely to decrease the pupil size of mice by influencing M2 cells, thereby improving visual performance in awake mice via enhancing the focal depth of the eye's refractive system.SIGNIFICANCE STATEMENT This study reveals the role of the neuropeptide orexin in mouse pupillary light response, a non-image-forming visual function. Intravitreal orexin-A administration intensifies light-induced pupillary constriction via increasing the excitability of M2 intrinsically photosensitive retinal ganglion cells by activating the orexin receptor subtype OX1R. Modulation of inward rectifier potassium channels and nonselective cation channels were both involved in the ionic mechanisms underlying such intensification. Orexin could improve visual performance in awake mice by reducing the pupil size and thereby enhancing the focal depth of the eye's refractive system.

Inositol Phospholipid Interactions with Inward Rectifier Potassium Channels and Their Role in Disease

Ion channels are frequently modulated by specific protein-lipid interactions in the phospholipid bilayer. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised in biophysical studies. Here, we provide an extensive application of a coarse-grained molecular dynamics free-energy perturbation (CG-MD-FEP) methodology to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step protocol, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid and in the presence of anionic phospholipid such as phosphatidylserine (PS). By perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K has increased PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. This is in good agreement with electrophysiological studies showing the E179K mutant exhibits a reduction in neomycin sensitivity, which implies that PIP2 binds more tightly to E179K mutant channels. We conclude that our application provides a valuable method to compare affinities between lipid species, and for annotating amino acid side chains within lipid-binding pockets.

Jostling for Position: Defining How Multiple Lipid Species Interact with Inward Rectifier Potassium (Kir) Channels

Protein-lipid interactions are a key element of the function of many integral membrane proteins. These potential interactions should be considered alongside the complexity and diversity of membrane lipid composition. inward rectifier potassium channel (Kir) Kir2.2 has multiple interactions with plasma membrane lipids: Phosphatidylinositol (4,5)-bisphosphate (PIP2) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP2; and cholesterol inhibits the channel.

Investigation of the Inactivation of HERG using Molecular Dynamics Simulations of New cryo-EM Structures

The human ether á-go-go related gene (hERG) encodes the delayed rectifier potassium channel (Kv11.1), best known for mediation of the repolarising current in the cardiac action potential. Loss of function of hERG can lead to long QT-syndrome which causes cardiac arrhythmias and sudden cardiac arrest. hERG is susceptible to a wide range of drugs which can lead to drug induced long QT-syndrome. Here, the recent cryo-EM structure of hERG in an activated state, together with newly-solved structures that are open or inactivated serve as the basis to investigate the structural determinants of hERG inactivation. We used molecular dynamics flexible fitting to refine and relax these new structures in micelles consisting of detergents, lipids and cholesterol. A combination of long simulations and libraries of short simulations were performed with a variety of ion concentrations to study different conformational states. These simulations highlighted the amino acids important for maintaining each state of the selectivity filter and therefore may be involved in hERG inactivation. Furthermore, we have used replica exchange with solute tempering to enhance sampling of the exchange of ions in the hERG selectivity filter. We observe correlations between channel conformation and ion occupancy that shed light on the coupling of ions to inactivation. Together with mutagenesis experiments, these simulations provide molecular-level understanding of hERG inactivation, with considerable clinical and pharmacological potential.

Genetic Ablation of G Protein-Gated Inwardly Rectifying K+ Channels Prevents Training-Induced Sinus Bradycardia.

Background: Endurance athletes are prone to bradyarrhythmias, which in the long-term may underscore the increased incidence of pacemaker implantation reported in this population. Our previous work in rodent models has shown training-induced sinus bradycardia to be due to microRNA (miR)-mediated transcriptional remodeling of the HCN4 channel, leading to a reduction of the “funny” (If) current in the sinoatrial node (SAN).Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as IKACh channels prevents sinus bradycardia induced by intensive exercise training in mice.Methods: Control wild-type (WT) and mice lacking GIRK4 (Girk4–/–), an integral subunit of IKACh were assigned to trained or sedentary groups. Mice in the trained group underwent 1-h exercise swimming twice a day for 28 days, 7 days per week. We performed electrocardiogram recordings and echocardiography in both groups at baseline, during and after the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and molecular profiling by quantitative PCR (qPCR) and western blotting.Results: At swimming cessation trained WT mice presented with a significantly lower resting HR that was reversible by acute IKACh block whereas Girk4–/– mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of If, as well as of T- and L-type Ca2+ currents (ICaT and ICaL) were significantly reduced only in SAN cells obtained from WT-trained mice. If reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced ICaL in WT mice was associated with reduced Cav1.3 protein levels. Strikingly, IKACh ablation suppressed all training-induced molecular remodeling observed in WT mice.Conclusion: Genetic ablation of cardiac IKACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents If, ICaT and ICaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Cav1.3. Strategies targeting cardiac IKACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes.

A Novel Medicago truncatule AKT1-null Mutant Impairs Potassium and Sodium Ions Uptake and Affects Root Nodulation

REVERSE genetic approach was used to isolate and characterize Medicago truncatula containing “knockout” mutations in gene involved in nodulation process and in many other physiological processes. More than 60 Tnt1-Flanking sequence tags (FSTs) ranging from ~70bp to ~600bp were isolated, sequenced and submitted to Genebank referring for akt1 mutant characterization. The proper Tnt1-insertion was mapped in chromosome number 8 of Medicago truncatula genome. It was precisely identified upstream the base number 141 and downstream of the ATG start codon of Medicago truncatula potassium channel AKT1 gene (MtAKT1). MtAKT1 gene encodes inwardly rectifying potassium channel was isolated, sequenced, and submitted to Genebank with accession number MN649185.1 .M. truncatula akt1 mutant is achieving higher number of deformed root nodules and shorter root length compared to wild type. akt1 nodules exhibited reduced size occupied with un-differentiated cells and abnormalities in symbiotic nodule zones. Non-functional nitrogen fixation zone and compacted infection zone were observed as well. AKT1 null mutant exhibits attenuation in its ability to maintain the proper K+ and Na+ ions content in akt1 seedlings which is 2-3 fold less than wild type seedlings,. In contrast, akt1 seedlings showed three-fold increase in Ca++ ion concentration compared to wild type.In conclusion Medicago truncatula mutant, akt1 is Tnt1-retrotransposon mutant impaired in inwardly rectifying potassium channel AKT1. As the first reported AKT1 null mutant was isolated from legume plants, this mutant is displaying abnormalities in nitrogen fixation organ and affecting ions uptake.

Flonicamid and knockdown of inward rectifier potassium channel gene CsKir2B adversely affect the feeding and development of Chilo suppressalis.

The selective insecticide flonicamid shows highly insecticidal activities against piercing-sucking insects and has been widely used for the control of Hemipteran insect pests, whereas its effects on Lepidopteran insect pests remain largely unknown. Recently, inward rectifier potassium (Kir) channel has been verified to be a target of flonicamid, however, functional characterization of Lepidopteran Kir genes is still lacking. Flonicamid shows no insecticidal toxicity against Chilo suppressalis larvae. However, the feeding and growth of larvae were reversibly inhibited by flonicamid (50-1200 mg L-1 ). Flonicamid treatment also remarkably reduced and delayed the pupation and eclosion of Chilo suppressalis. Additionally, five distinct Kir channel genes (CsKir1, CsKir2A, CsKir2B, CsKir3A and CsKir3B) were cloned from Chilo suppressalis. Expression profiles analysis revealed that CsKir2A was predominately expressed in the hindgut of larvae, whereas CsKir2B had high expressions in the Malpighian tubules and hindgut. RNA interference (RNAi)-mediated knockdown of CsKir2B significantly reduced the growth and increased the mortalities of larvae, whereas silencing of CsKir2A had no obvious effects on Chilo suppressalis. Flonicamid exhibits adverse effects on the growth and development of Chilo suppressalis. CsKir2B might be involved in the feeding behavior of Chilo suppressalis. These results provide valuable information on the effects of flonicamid on non-target insects as well as the function of insect Kir channels, and are helpful in developing new insecticide targeting insect Kir channels. © 2020 Society of Chemical Industry.

Reduced motor cortex GABABR function following chronic alcohol exposure.

The GABAB receptor (GABABR) agonist baclofen has been used to treat alcohol and several other substance use disorders (AUD/SUD), yet its underlying neural mechanism remains unclear. The present study aimed to investigate cortical GABABR dynamics following chronic alcohol exposure. Ex vivo brain slice recordings from mice chronically exposed to alcohol revealed a reduction in GABABR-mediated currents, as well as a decrease of GABAB1/2R and G-protein-coupled inwardly rectifying potassium channel 2 (GIRK2) activities in the motor cortex. Moreover, our data indicated that these alterations could be attributed to dephosphorylation at the site of serine 783 (ser-783) in GABAB2 subunit, which regulates the surface expression of GABABR. Furthermore, a human study using paired-pulse-transcranial magnetic stimulation (TMS) analysis further demonstrated a reduced cortical inhibition mediated by GABABR in patients with AUD. Our findings provide the first evidence that chronic alcohol exposure is associated with significantly impaired cortical GABABR function. The ability to promote GABABR signaling may account for the therapeutic efficacy of baclofen in AUD.

Physiological roles of heteromerization: focus on the two-pore domain potassium channels.

Potassium channels form the largest family of ion channels with more than 80 members involved in cell excitability and signalling. Most of them exist as homomeric channels, whereas specific conditions are required to obtain heteromeric channels. It is well established that heteromerization of voltage-gated and inward rectifier potassium channels affects their function, increasing the diversity of the native potassium currents. For potassium channels with two pore domains (K2P ), homomerization has long been considered the rule, their polymodal regulation by a wide diversity of physical and chemical stimuli being responsible for the adaptation of the leak potassium currents to cellular needs. This view has recently evolved with the accumulation of evidence of heteromerization between different K2P subunits. Several functional intragroup and intergroup heteromers have recently been identified, which contribute to the functional heterogeneity of this family. K2P heteromerization is involved in the modulation of channel expression and trafficking, promoting functional and signalling diversity. As illustrated in the Abstract Figure, heteromerization of TREK1 and TRAAK provides the cell with more possibilities of regulation. It is becoming increasingly evident that K2P heteromers contribute to important physiological functions including neuronal and cardiac excitability. Since heteromerization also affects the pharmacology of K2P channels, this understanding helps to establish K2P heteromers as new therapeutic targets for physiopathological conditions.

Production and purification of ATP-sensitive potassium channel particles for cryo-electron microscopy.

ATP-sensitive potassium (KATP) channels are multimeric protein complexes made of four inward rectifying potassium channel (Kir6.x) subunits and four ABC protein sulfonylurea receptor (SURx) subunits. Kir6.x subunits form the potassium ion conducting pore of the channel, and SURx functions to regulate Kir6.x. Kir6.x and SURx are uniquely dependent on each other for expression and function. In pancreatic β-cells, channels comprising SUR1 and Kir6.2 mediate glucose-stimulated insulin secretion and are the targets of antidiabetic sulfonylureas. Mutations in genes encoding SUR1 or Kir6.2 are linked to insulin secretion disorders, with loss- or gain-of-function mutations causing congenital hyperinsulinism or neonatal diabetes mellitus, respectively. Defects in the KATP channel in other tissues underlie human diseases of the cardiovascular and nervous systems. Key to understanding how channels are regulated by physiological and pharmacological ligands and how mutations disrupt channel assembly or gating to cause disease is the ability to observe structural changes associated with subunit interactions and ligand binding. While recent advances in the structural method of single-particle cryo-electron microscopy (cryoEM) offers direct visualization of channel structures, success of obtaining high-resolution structures is dependent on highly concentrated, homogeneous KATP channel particles. In this chapter, we describe a method for expressing KATP channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying KATP channels using an affinity tag to the SURx subunit for cryoEM structural studies.