Kir Channels Research Articles

Potassium channel‐mediated NO‐induced vasodilation during maturation: Dominance of Kv7 channels

AbstractMaturation represents a process characterized by adaptive changes, particularly in the circulatory system. However, it is not known whether, in neonates, potassium channels contribute to NO‐induced vasorelaxation at all and, if so, which potassium channels these are. Therefore, this study tested the hypothesis that potassium channels mediate NO‐induced vasorelaxation in newborn rats. Young (10‐ to 15‐day‐old) and adult (2‐ to 3‐month‐old) male rats were studied using real‐time PCR, isometric myography, and the sharp microelectrode technique on saphenous arteries. We observed prominent mRNA expression of several distinct isoforms of potassium channel families known to potentially mediate SNP‐induced vasodilation. Further, in both adult and young rats, SNP can relax vessels independently of potassium channels. A solely potassium channel‐independent anticontractile effect of SNP was observed also when either Kir6, or Kir2, or Kv2 channels, respectively, were available in both adult and young rats. However, when Kv1 channels were available, a Kv1 channel‐dependent component contributed to the anticontractile effect of SNP in young rats. When BKCa channels were available, a BKCa channel‐dependent component contributed to the anticontractile effect of SNP in adult rats. A considerable Kv7 channel‐dependent component contributed to the anticontractile effect of SNP in both adult and young rats. Thus, the data of the present study show for the first time that potassium channels, even multiple ones, contribute to SNP‐induced vasorelaxation in newborn rats and that the potassium channels involved in SNP‐induced vasorelaxation change from Kv1/Kv7 channels to BKCa/Kv7 channels during postnatal development.

Direct effects of antipsychotics on potassium channels.

Schizophrenia (SCZ) and bipolar disorder (BD) and are severe psychiatric conditions that contribute to disability and increased healthcare costs globally. Although first-, second-, and third-generation antipsychotics are available for treating BD and SCZ, most have various side effects unrelated to their unique functions. Many antipsychotics affect K+ channels (Kv, KCa, Kir, K2P, and other channels), which change the functions of various organs. This review summarizes the biological actions of antipsychotics, including off-target side effects involving K+ channels.

Involvement of Inwardly Rectifying Potassium (Kir) Channels in the Toxicity of Flonicamid to Drosophila melanogaster.

Inwardly rectifying potassium (Kir) channels regulate essential physiological processes in insects and have been identified as potential targets for developing new insecticides. Flonicamid has been reported to inhibit Kir channels, disrupting the functions of salivary glands and renal tubules. However, the precise molecular target of flonicamid remains debated. It is unclear whether flonicamid directly targets Kir channels or acts on other sites involved in the activation of transient receptor potential vanilloid (TRPV) channels. In this study, we observed that flonicamid is more toxic to flies than its metabolite, flumetnicam. This higher toxicity is difficult to reconcile if nicotinamidase is the active target, as flonicamid does not inhibit nicotinamidase. An alternative explanation is that flonicamid and flumetnicam may have distinct targets or act on multiple targets. Furthermore, reducing the expression of three individual Kir genes in the salivary glands of D. melanogaster significantly decreased the flies' susceptibility to both flonicamid and flumetnicam. The double knockdown of Kir1 with Kir3 or Kir2 with Kir3 further reduced the flies' sensitivity to both compounds. These findings confirm the involvement of Kir channels in mediating the toxic effects of flonicamid on flies. Overall, this study offers new insights into the physiological roles of insect Kir channels and flonicamid toxicity.

Structural Dynamics of the Slide Helix of Inactive/Closed Conformation of KirBac1.1 in Micelles and Membranes: A Fluorescence Approach.

Inward rectifying potassium (Kir) channels play a critical role in maintaining the resting membrane potential and cellular homeostasis. The high-resolution crystal structure of homotetrameric KirBac1.1 in detergent micelles provides a snapshot of the closed state. Similar to micelles, KirBac1.1 is reported to be in the inactive/closed conformation in POPC membranes. The slide helix of KirBac1.1 is an important structural motif that regulates channel gating. Despite the importance of slide helix in lipid-dependent gating, conflicting models have emerged for the location of slide helix and its structural dynamics in membrane mimetics is poorly understood. Here, we monitored the structural dynamics of the slide helix (residues 46-57) of KirBac1.1 in both DM micelles and POPC membranes utilizing various site-directed fluorescence approaches. We show, using ACMA-based liposome-flux assay, the cysteine mutants of the slide helix are not functional, ensuring the inactive/closed conformation in POPC membranes similar to wild-type channel. Time-resolved fluorescence and water accessibility measurements of NBD-labeled single-cysteine mutants of slide-helix residues suggest that the location of the slide helix at the interfacial region might be shallower in membranes compared to micelles. Interestingly, the slide helix of KirBac1.1 is more dynamic in the physiologically relevant membrane environment, which is accompanied by a differential hydration dynamics throughout the slide helix. Further, REES and lifetime distribution analyses suggest significant changes in conformational heterogeneity of the slide helix in membrane mimetics. Overall, our results give an insight into how membrane mimetics affect the organization and dynamics of slide helix of the closed state of KirBac1.1, and highlight the importance of lipid-protein interactions in membranes.

RESPIRATORY SURVEILLANCE AND INWARD RECTIFIER POTASSIUM CHANNEL EXPRESSION IN LUNG TISSUE WITHIN AN EXPERIMENTAL EPILEPSY MODEL.

Epilepsy is characterized by neuronal discharges that occur as a result of disruption of the excitatory and inhibitory balance of the brain due to functional and structural changes. It has been shown in the literature that this neurological disorder may be related to the expression of ion channels. Any defect in the function or expression mechanism of these channels can lead to various neuronal disorders such as epilepsy. Epileptic seizures occur as a result of the accumulation of biological disorders in the circulatory, respiratory and nervous systems. In this study, we aimed to examine the changes in the expression of inward-directing potassium channels (Kir 3.1 and 6.2) in lung tissue and respiratory functions, considering that it will contribute to the elucidation of the mechanisms of sudden deaths thought to be caused by cardiorespiratory complications in epilepsy. In the study, 48 adult male Wistar albino rats weighing 250-300 g were used in the study. During the research process, respiratory function tests were performed on epileptic rats induced with pentylenetetrazol (PTZ) firing model, and then histopathological changes in lung and hippocampus tissues, and expression levels of the Kir (3.1 and 6.2) channels were evaluated by immunohistochemistry, qRT-PCR and Western blot analysis. Memantine and tertiapin-Q have been shown to protect epileptic groups from histopathological harm induced by PTZ application and also reduce HIF-1α, Kir 3.1 and Kir 6.2 expression. The findings imply that memantine and tertiapin-Q would be suitable options for treating epilepsy patients.

Pore blocking mechanisms of centipede toxin SsTx-4 on the inwardly rectifying potassium channels.

The peptide toxin SsTx-4 derived from venom of centipede Scolopendra subspinipes mutilans was characterized as a potent antagonist of the inwardly rectifying potassium (Kir) channel subtypes Kir1.1, Kir4.1, and Kir6.2 in our previous study. Alanine-scanning mutagenesis analysis identified key molecular determinants on the SsTx-4 toxin interacting with these Kir channels, as well as those on the Kir6.2 channel interacting with the toxin. However, the key residues on Kir1.1 and Kir4.1 channels responsible for binding SsTx-4 remain unclear. Here, using a combination of site-directed mutagenesis, patch-clamp analysis, molecular docking with AlphaFold 3, and molecular dynamic simulations, we revealed that SsTx-4 acted on the Kir channels as a pore blocker, with K13 on toxin serving as the functional pore-blocking residue and other residues on it contributing to stabilize the toxin-channel complex by binding to multiple residues on the wall of the channels' outer vestibule, involving E104 on Kir1.1; D100, L115, and F133 on Kir4.1; and E108, S113, H115, and M137 on Kir6.2. Collectively, these findings advanced our understanding on the interaction between Kir channels and this prototype Kir antagonist, providing insights that could inspire the development of more potent and specific Kir subtype blockers in the future.

Toxicity of Flonicamid to Diaphorina citri (Hemiptera: Liviidae) and Its Identification and Expression of Kir Channel Genes.

Flonicamid is a selective insecticide effective against piercing-sucking insects. Although its molecular target has been identified in other species, the specific effects and detailed mechanism of action in Diaphorina citri Kuwayama remain poorly understood. In this study, we determined that the LC50 of flonicamid for D. citri adults was 16.6 mg AI L-1 after 4 days of exposure. To explore the relevant mechanisms, the treatments with acetone and with 20 mg AI L-1 flonicamid for 96 h were collected as samples for RNA-Seq. The analysis of the transcriptomes revealed 345 differentially expressed genes (DEGs) in D. citri adults subjected to different treatments. Among these DEGs, we focused on the inward-rectifying potassium (Kir) channel genes, which have been extensively studied as potential targets of flonicamid. Three Kir subunit genes (Dckir1, Dckir2, Dckir3) in D. citri were successfully cloned and identified. Furthermore, the expression profiles of these DcKirs were investigated using RT-qPCR and showed that their expression significantly increased after D. citri eclosion to adulthood, particularly for DcKir3. The DcKirs were predominantly expressed in gut tissues, with DcKir1 and DcKir2 exhibiting high expression levels in the hindgut and midgut, respectively, while DcKir3 showing high expression in the midgut and Malpighian tubules. This study provides insights into the potential roles of Kir subunits in D. citri and enhances our understanding of the physiological effects of flonicamid in this pest.

Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells, which is involved in retinal ganglion cell apoptosis in glaucoma. Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma. In this study, we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation. Following this, we treated Müller glial cells in vitro and in vivo with the mGluR I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr9Asp overexpression. We found that both Kir4.1 and Kir4.1 Tyr9Asp overexpression inhibited activation of Müller glial cells. Subsequently, we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr9Asp in the eye, and observed similar results in Müller cells in vivo as those seen in vitro. Both Kir4.1 and Kir4.1 Tyr9Asp overexpression inhibited Müller cell activation, regulated the balance of Bax/Bcl-2, and reduced the mRNA and protein levels of pro-inflammatory factors, including interleukin-1β and tumor necrosis factor-α. Furthermore, we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr9Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia. In this co-culture system, we observed elevated adenosine triphosphate concentrations in activated Müller cells, increased levels of translocator protein (a marker of microglial activation), and elevated interleukin-1β mRNA and protein levels in microglia induced by activated Müller cells. These changes could be reversed by Kir4.1 and Kir4.1 Tyr9Asp overexpression in Müller cells. Kir4.1 overexpression, but not Kir4.1 Tyr9Asp overexpression, reduced the number of proliferative and migratory microglia induced by activated Müller cells. Collectively, these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma. Kir4.1 and Kir4.1 Tyr9Asp overexpression attenuated Müller cell activation, reduced ATP/P2X receptor-mediated interactions between glial cells, inhibited microglial activation, and decreased the synthesis and release of pro-inflammatory factors, consequently ameliorating retinal ganglion cell apoptosis in glaucoma.

Biochemical, biophysical, and structural investigations of two mutants (C154Y and R312H) of the human Kir2.1 channel involved in the Andersen-Tawil syndrome.

Inwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to the cellular repolarization of many excitable cells. Dysfunction in Kir2.1 channels is implicated in several chronic and debilitating human diseases for which there are currently no effective treatments. Specifically, Kir2.1-R312H and Kir2.1-C154Y mutations are associated with Andersen-Tawil syndrome (ATS) in humans. We have investigated the impact of these two mutants in the trafficking of the channel to the cell membrane and function in Xenopus laevis oocytes. Despite both mutations being trafficked to the cell membrane at different extents and capable of binding PIP2 (phosphatidylinositol-4,5-bisphosphate), the main modulator for channel activity, they resulted in defective channels that do not display K+ current, albeit through different molecular mechanisms. Coexpression studies showed that R312H and C154Y are expressed and associated with the WT subunits. While WT subunits could rescue R312H dysfunction, the presence of a unique C154Y subunit disrupts the function of the entire complex, which is a typical feature of mutations with a dominant-negative effect. Molecular dynamics simulations showed that Kir2.1-C154Y mutation induces a loss in the structural plasticity of the selectivity filter, impairing the K+ flow. In addition, the cryo-EM structure of the Kir2.1-R312H mutant has been reconstructed. This study identified the molecular mechanisms by which two ATS-causing mutations impact Kir2.1 channel function and provide valuable insights that can guide potential strategies for the development of future therapeutic interventions for ATS.

Danggui Buxue Decoction and its components dilate coronary artery through activating the inward rectification K+ channels pathway

Ethnopharmacological relevanceDanggui Buxue Decoction (DBD), a classic representative prescription of invigorating Qi and producing blood, is used to treat coronary heart disease angina pectoris and vascular injury diseases. Abnormal coronary artery is an important cause of cardiovascular disease. However, the mechanism of DBD dilates coronary arteries is still unclear. Aim of the studyThis study aimed to elucidate the impacts and distinctions among DBD, Astragalus, Angelica sinensis, and identified active components on pre-constricted coronary arteries, as well as to delve deeper into their respective mechanisms. Materials and methodsAfter the preconstriction of a rat isolated coronary artery ring with either 30 mM KCl or 200 nM U46619, the vascular tension was observed following the addition of DBD, and other components. Subsequently, the impact of these active components on coronary blood flow (CBF) was confirmed through in vivo testing. Further investigation into the underlying mechanism was carried out using a combination of blockers, molecular docking, surface plasmon resonance (SPR), cell heat transfer analysis (CETSA), and patch-clamp techniques. ResultsIn vitro experiments showed that DBD and its components butylidenephthalide, ligustilide, calycosin, and quercetin could dilate coronary artery preconstricted with either 30 mM KCl or 200 nM U46619. In addition, the active ingredient was found to significantly increase CBF. Mechanistically, BaCl2 was found to reduce the relaxation effect of the drug by adding a blocker. Molecular docking, SPR and CETSA results showed that the active ingredients had a strong binding potential with inward rectification K+ channels (KIR) channel protein. Patch clamp studies demonstrate that quercetin can increase KIR current, and BaCl2 can significantly reduce its current. ConclusionsThe active components of DBD, butylidenephthalide, ligustilide, calycosin, and quercetin, activate KIR channels to relax coronary artery and increase CBF.

Development of new Kir2.1 channel openers from propafenone analogues.

Reduced inward rectifier potassium channel (Kir2.1) functioning is associated with heart failure and may cause Andersen-Tawil Syndrome, among others characterized by ventricular arrhythmias. Most heart failure or Andersen-Tawil Syndrome patients are treated with β-adrenoceptor antagonists (β-blockers) or sodium channel blockers; however, these do not specifically address the inward rectifier current (IK1) nor aim to improve resting membrane potential stability. Consequently, additional pharmacotherapy for heart failure and Andersen-Tawil Syndrome treatment would be highly desirable. Acute propafenone treatment at low concentrations enhances IK1 current, but it also exerts many off-target effects. Therefore, discovering and exploring new IK1-channel openers is necessary. Effects of propafenone and 10 additional propafenone analogues were analysed. Currents were measured by single-cell patch-clamp electrophysiology. Kir2.1 protein expression levels were determined by western blot analysis and action potential characteristics were further validated in human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMCs). Molecular docking was performed to obtain detailed information on drug-channel interactions. Analogues GPV0019, GPV0057 and GPV0576 strongly increased the outward component of IK1 while not affecting the Kir2.1 channel expression levels. GPV0057 did not block IKr at concentrations below 0.5μmol L-1 nor NaV1.5 current below 1μmol L-1. Moreover, hiPSC-CMC action potential duration was also not affected by GPV0057 at 0.5 and 1μmol L-1. Structure analysis indicates a mechanism by which GPV0057 might enhance Kir2.1 channel activation. GPV0057 has a strong efficiency towards increasing IK1, which makes it a good candidate to address IK1 deficiency-associated diseases.

Chronic ethanol exposure in mice evokes pre- and postsynaptic deficits in GABAergic transmission in ventral tegmental area GABA neurons.

GABAergic neurons in mouse ventral tegmental area (VTA) exhibit elevated activity during withdrawal following chronic ethanol exposure. While increased glutamatergic input and decreased GABAA receptor sensitivity have been implicated, the impact of inhibitory signaling in VTA GABA neurons has not been fully addressed. We used electrophysiological and ultrastructural approaches to assess the impact of chronic intermittent ethanol vapour exposure in mice on GABAergic transmission in VTA GABA neurons during withdrawal. We used CRISPR/Cas9 ablation to mimic a somatodendritic adaptation involving the GABAB receptor (GABABR) in ethanol-naïve mice to investigate its impact on anxiety-related behaviour. The frequency of spontaneous inhibitory postsynaptic currents was reduced in VTA GABA neurons following chronic ethanol treatment and this was reversed by GABABR inhibition, suggesting chronic ethanol strengthens the GABABR-dependent suppression of GABAergic input to VTA GABA neurons. Similarly, paired-pulse depression of GABAA receptor-dependent responses evoked by optogenetic stimulation of nucleus accumbens inputs from ethanol-treated mice was reversed by GABABR inhibition. Somatodendritic currents evoked in VTA GABA neurons by GABABR activation were reduced following ethanol exposure, attributable to the suppression of GIRK (Kir3) channel activity. Mimicking this adaptation enhanced anxiety-related behaviour in ethanol-naïve mice. Chronic ethanol weakens the GABAergic regulation of VTA GABA neurons in mice via pre- and postsynaptic mechanisms, likely contributing to their elevated activity during withdrawal and expression of anxiety-related behaviour. As anxiety can promote relapse during abstinence, interventions targeting VTA GABA neuron excitability could represent new therapeutic strategies for treatment of alcohol use disorder.

The Kir channel in the nucleus tractus solitarius integrates the chemosensory system with REM sleep executive machinery for homeostatic balance

The locus coeruleus (LC), nucleus tractus solitarius (NTS), and retrotrapezoid nucleus (RTN) are critical chemosensory regions in the brainstem. In the LC, acid-sensing ion channels and proton pumps serve as H+ sensors and facilitate the transition from non-rapid eye movement (NREM) to rapid eye movement (REM) sleep. Interestingly, the potassium inward rectifier (KIR) channels in the LC, NTS, and RTN also act as H+-sensors and are a primary target for improving sleep in obstructive sleep apnea and Rett syndrome patients. However, the role of Kir channels in NREM to REM sleep transition for H+ homeostasis is not known. Male Wistar rats were surgically prepared for chronic sleep–wake recording and drug delivery into the LC, NTS, and RTN. In different animal cohorts, microinjections of the Kir channel inhibitor, barium chloride (BaCl2), at concentrations of 1 mM (low dose) and 2 mM (high dose) in the LC and RTN significantly increased wakefulness and decreased NREM sleep. However, BaCl2 microinjection into the LC notably reduced REM sleep, whereas it didn’t change in the RTN-injected group. Interestingly, BaCl2 microinjections into the NTS significantly decreased wakefulness and increased the percent amount of NREM and REM sleep. Additionally, with the infusion of BaCl2 into the NTS, the mean REM sleep episode numbers significantly increased, but the length of the REM sleep episode didn’t change. These findings suggest that the Kir channels in the NTS, but not in the LC and RTN, modulate state transition from NREM to REM sleep.

Endocannabinoid regulation of inward rectifier potassium (Kir) channels.

The inward rectifier potassium channel Kir2.1 (KCNJ2) is an important regulator of resting membrane potential in both excitable and non-excitable cells. The functions of Kir2.1 channels are dependent on their lipid environment, including the availability of PI(4,5)P2, secondary anionic lipids, cholesterol and long-chain fatty acids acyl coenzyme A (LC-CoA). Endocannabinoids are a class of lipids that are naturally expressed in a variety of cells, including cardiac, neuronal, and immune cells. While these lipids are identified as ligands for cannabinoid receptors there is a growing body of evidence that they can directly regulate the function of numerous ion channels independently of CBRs. Here we examine the effects of a panel of endocannabinoids on Kir2.1 function and demonstrate that a subset of endocannabinoids can alter Kir2.1 conductance to varying degrees independently of CBRs. Using computational and Surface plasmon resonance analysis, endocannabinoid regulation of Kir2.1 channels appears to be the result of altered membrane properties, rather than through direct protein-lipid interactions. Furthermore, differences in endocannabinoid effects on Kir4.1 and Kir7.1 channels, indicating that endocannabinoid regulation is not conserved among Kir family members. These findings may have broader implications on the function of cardiac, neuronal and/or immune cells.

Vasorelaxant mechanisms of the antidiabetic anagliptin in rabbit aorta: roles of Kv channels and SERCA pump.

The present study investigated the vasorelaxant mechanisms of an oral antidiabetic drug, anagliptin, using phenylephrine (Phe)-induced pre-contracted rabbit aortic rings. Arterial tone measurement was performed in rabbit thoracic aortic rings. Anagliptin induced vasorelaxation in a dose-dependent manner. Pre-treatment with the classical voltagedependent K+ (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly decreased the vasorelaxant effect of anagliptin, whereas pre-treatment with the inwardly rectifying K+ (Kir) channel inhibitor Ba2+, the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, and the large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline did not attenuate the vasorelaxant effect. Furthermore, the vasorelaxant response of anagliptin was effectively inhibited by pre-treatment with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid. Neither cAMP/protein kinase A (PKA)-related signaling pathway inhibitors (adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor KT 5720) nor cGMP/protein kinase G (PKG)-related signaling pathway inhibitors (guanylyl cyclase inhibitor ODQ and PKG inhibitor KT 5823) reduced the vasorelaxant effect of anagliptin. Similarly, the anagliptin-induced vasorelaxation was independent of the endothelium. Based on these results, we suggest that anagliptin-induced vasorelaxation in rabbit aortic smooth muscle occurs by activating Kv channels and the SERCA pump, independent of other vascular K+ channels, cAMP/PKA- or cGMP/PKG-related signaling pathways, and the endothelium.

Ionic mechanisms involved in arginine vasopressin-mediated excitation of auditory cortical and thalamic neurons

The axons containing arginine vasopressin (AVP) from the hypothalamus innervate a variety of structures including the cerebral cortex, thalamus, hippocampus and amygdala. A plethora amount of evidence indicates that activation of the V1a subtype of the vasopressin receptors facilitates anxiety-like and fear responses. As an essential structure involved in fear and anxiety responses, the amygdala, especially the lateral nucleus of amygdala (LA), receives glutamatergic innervations from the auditory cortex and auditory thalamus where high density of V1a receptors have been detected. However, the roles and mechanisms of AVP in these two important areas have not been determined, which prevents the understanding of the mechanisms whereby V1a activation augments anxiety and fear responses. Here, we used coronal brain slices and studied the effects of AVP on neuronal activities of the auditory cortical and thalamic neurons. Our results indicate that activation of V1a receptors excited both auditory cortical and thalamic neurons. In the auditory cortical neurons, AVP increased neuronal excitability by depressing multiple subtypes of inwardly rectifying K+ (Kir) channels including the Kir2 subfamily, the ATP-sensitive K+ channels and the G protein-gated inwardly rectifying K+ (GIRK) channels, whereas activation of V1a receptors excited the auditory thalamic neurons by depressing the Kir2 subfamily of the Kir channels as well as activating the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and a persistent Na+ channel. Our results may help explain the roles of V1a receptors in facilitating fear and anxiety responses.Categories: Cell Physiology.

Angiotensin II-Type-1a Receptor and Renal K + Wasting during Overnight Low-Na + Intake.

Chronic Angiotensin-II (Ang-II) perfusion stimulates Kir4.1/Kir5.1 of the DCT via angiotensin-II-type-1a-receptor (AT1aR) and low-sodium-intake also stimulates Kir4.1/Kir5.1. However, it is not explored the role of AT1aR in mediating the effect of LS on Kir4.1/Kir5.1. We used patch-clamp-technique to examine Kir4.1/Kir5.1 activity of the DCT, employed immunoblotting to examine NCC expression/activity, and used in vivo perfusion-technique to measure renal-Na+ and renal-K+-excretion in control, kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) and DCT-specific-AT1aR-knockout mice (DCT-AT1aR- KO). Ang-II acutely stimulated 40-pS-K+ channel (Kir4.1/Kir5.1-heterotetramer), increased whole-cell Kir4.1/Kir5.1-mediated K+-currents and the negativity of DCT-membrane-potential only in late-DCT2 but not in early-DCT. Acute Ang-II increased thiazide-induced renal Na+-excretion (ENa). The effect of Ang-II on Kir4.1/Kir5.1 and HCTZ-induced-ENa was absent in Ks-AT1aR-KO mice. Overnight-low-salt stimulated the expression of Agtr1a mRNA in DCT, increased whole-cell Kir4.1/Kir5.1-mediated K+-currents in late-DCT, hyperpolarized late-DCT membrane, augmented the expression of phosphor-Na-Cl-cotransporter (pNCC) and enhanced thiazide-induced renal-ENa in the control mice. However, the effect of overnight-low-salt on Kir4.1/Kir5.1-activity, DCT membrane potential and NCC activity/expression was abolished in DCT-AT1aR-KO or Ks-AT1aR-KO mice. Overnight-low-salt had no effect on baseline renal K+-excretion (EK) and plasma K+-concentrations in the control mice but it increased baseline renal-EK and decreased plasma K+-concentrations in DCT-AT1aR-KO or in Ks-AT1aR-KO mice. Acute Ang-II or overnight-LS stimulated Kir4.1/Kir5.1 in late-DCT and that AT1aR was responsible for acute Ang-II or overnight-low-salt-induced stimulation of Kir4.1/Kir5.1 and NCC. AT1aR of the DCT plays a role in maintaining adequate baseline renal-EK and plasma K+ concentrations during overnight-LS.

Structure of the Ion Channel Kir7.1 and Implications for its Function in Normal and Pathophysiologic States.

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

Direct modulation of G protein-gated inwardly rectifying potassium (GIRK) channels.

Ion channels play a pivotal role in regulating cellular excitability and signal transduction processes. Among the various ion channels, G-protein-coupled inwardly rectifying potassium (GIRK) channels serve as key mediators of neurotransmission and cellular responses to extracellular signals. GIRK channels are members of the larger family of inwardly-rectifying potassium (Kir) channels. Typically, GIRK channels are activated via the direct binding of G-protein βγ subunits upon the activation of G-protein-coupled receptors (GPCRs). GIRK channel activation requires the presence of the lipid signaling molecule, phosphatidylinositol 4,5-bisphosphate (PIP2). GIRK channels are also modulated by endogenous proteins and other molecules, including RGS proteins, cholesterol, and SNX27 as well as exogenous compounds, such as alcohol. In the last decade or so, several groups have developed novel drugs and small molecules, such as ML297, GAT1508 and GiGA1, that activate GIRK channels in a G-protein independent manner. Here, we aim to provide a comprehensive overview focusing on the direct modulation of GIRK channels by G-proteins, PIP2, cholesterol, and novel modulatory compounds. These studies offer valuable insights into the underlying molecular mechanisms of channel function, and have potential implications for both basic research and therapeutic development.

MTORc2 in Distal Convoluted Tubule and Renal K + Excretion during High Dietary K + Intake.

Renal mTORc2 plays a role in regulating renal K+-excretion (renal-EK) and K+-homeostasis. Inhibition of renal mTORc2 caused hyperkalemia due to suppressing epithelial-Na+-channel (ENaC) and ROMK (Kir1.1) in the collecting duct. We now explore whether mTORc2 of DCT regulates basolateral Kir4.1/Kir5.1, NCC and renal-EK. We used patch-clamp-technique to examine basolateral Kir4.1/Kir5.1 in early-DCT, immunoblotting and immunofluorescence to examine NCC expression and in vivo measurement of urinary K+-excretion to determine baseline renal-EK in the mice treated with mTORc2-inhibitor and in DCT-specific Rapamycin-Insensitive-Companion- of-mTOR knockout (DCT-RICTOR-KO) mice. Inhibition of mTORc2 with AZD8055 abolished high-K+-induced inhibition of Kir4.1/Kir5.1 in DCT, high-K+-induced depolarization of DCT membrane and high-K+-induced suppression of pNCC expression. AZD8055 stimulated the 40-pS-inwardly-rectifying-K+ channel (Kir4.1/Kir5.1-heterotetramer) in early-DCT in the mice on overnight-high-K+, this effect was absent in the presence of PKC-inhibitor which also stimulated Kir4.1/Kir5.1. AZD8055-treatment decreased renal-EK in animals on overnight-high-K+. Deletion of RICTOR in the DCT increased the Kir4.1/Kir5.1-mediated K+-currents, hyperpolarized DCT membrane and increased the expression of pWNK4 and pNCC. Renal-EK was lower and plasma-K+ was higher in DCT-RICTOR-KO mice than corresponding control mice. Also, overnight-high-K+ did not inhibit Kir4.1/Kir5.1 activity in the DCT and failed to inhibit the expression of pNCC in DCT-RICTOR-KO mice. Overnight-high-K+ stimulated renal-EK in control mice, but this effect was attenuated in DCT-RICTOR-KO mice. Thus, overnight-high-K+ induced hyperkalemia in DCT-RICTOR-KO mice but not in control mice. mTORc2 of the DCT inhibits Kir4.1/Kir5.1 activity and NCC expression, and stimulates renal-EK during high-K+-intake.