Rectifying Potassium Channel Subunit Research Articles

Dynamic duo: Kir6 and SUR in KATP channel structure and function

ABSTRACT KATP channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit complex comprising four central pore-forming Kir6 inward rectifier potassium channel subunits surrounded by four regulatory subunits known as the sulfonylurea receptor, SUR, which confer homeostatic metabolic control of KATP gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for KATP expression and function. For more than four decades, understanding the structure-function relationship of Kir6 and SUR has remained a central objective of clinical significance. Here, we review progress in correlating the wealth of functional data in the literature with recent KATP cryoEM structures.

A critical review of ethanol effects on neuronal firing: A metabolic perspective.

Ethanol metabolism is relatively understudied in neurons, even though changes in neuronal metabolism are known to affect their activity. Recent work demonstrates that ethanol is preferentially metabolized over glucose as a source of carbon and energy, and it reprograms neurons to a state of reduced energy potential and diminished capacity to utilize glucose once ethanol is exhausted. Ethanol intake has been associated with changes in neuronal firing and specific brain activity (EEG) patterns have been linked with risk for alcohol use disorder (AUD). Furthermore, a haplotype of the inwardly rectifying potassium channel subunit, GIRK2, which plays a critical role in regulating excitability of neurons, has been linked with AUD and shown to be directly regulated by ethanol. At the same time, overexpression of GIRK2 prevents ethanol-induced metabolic changes. Based on the available evidence, we conclude thatthe mechanisms underlying the effects of ethanol on neuronal metabolism are a novel target for developing therapies for AUD.

Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons.

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2), have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants (“Affected: AF”) and four control subjects without variants (“Unaffected: UN”). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single-cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.

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.

Abstract P013: Role Of Kir4.1 ( Kcnj10 ) In The Regulation Of Salt-Induced Hypertension

In the kidney, K ir 4.1 ( Kcnj10 ) and K ir 5.1 ( Kcnj16 ) are highly expressed in the aldosterone sensitive distal nephron, a major target for hormones controlling blood pressure. These basolateral inwardly rectifying potassium channel subunits assemble to form both heteromeric K ir 4.1/K ir 5.1 and homomeric K ir 4.1 channels which control the transepithelial voltage, fine-tune renal electrolyte homeostasis, and contribute to long-term blood pressure control. To study the role of K ir 4.1 in the renal control of blood pressure, renin-angiotensin-aldosterone system (RAAS) balance, and salt-sensitive hypertension, we created a Kcnj10 knockout (SS Kcnj10-/- ) on the Dahl SS rat background using CRISPR/Cas9 mutagenesis. Homozygous SS Kcnj10-/- rats were hypokalemic, had reduced heart, kidney, and body weights, and did not survive more than 3 weeks from birth. Mass spectrometry-based quantification of RAAS peptides revealed increased Ang I, Ang 1-5, and Ang 1-7 levels in SS Kcnj10-/- rats compared to age-matched SS Kcnj10+/+ controls. There were no differences in Ang II, Ang III, Ang IV, or aldosterone levels. In addition, ACE activity was decreased in SS Kcnj10-/- rats. The observed increases in alternative RAS axis peptides and decrease in ACE activity in SS Kcnj10-/- rats are suggestive of a low blood pressure phenotype, however, limited survival precluded blood pressure measurement. Heterozygous (SS Kcnj10+/- ) rats developed normally and showed no changes in serum electrolytes. Survival rate of SS Kcnj10+/- rats was lower than wild-type rats but improved with dietary K + supplementation (1.41% K + ) allowing for telemetric blood pressure measurement. We found no differences in baseline blood pressure under control conditions, but salt-induced (4% NaCl) elevations in blood pressure were significantly attenuated in SS Kcnj10+/- rats. After 3 weeks on high salt, SS Kcnj10+/- rats showed decreased diuresis and improved albuminuria compared to wildtype. Our results demonstrate that renal K ir 4.1 containing channels mediate salt-sensitive increases in blood pressure through regulation of potassium homeostasis, modulation of alternative RAS axis hormones, and control of renal salt handling in the ASDN.

Impaired cytoplasmic domain interactions cause co-assembly defect and loss of function in the p.Glu293Lys KNCJ2 variant isolated from an Andersen-Tawil syndrome patient.

Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1. The p.Glu293Lys variant produced no current in homomeric form and showed dominant-negative effect over wild-type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoLuc® Binary Technology (NanoBiT) split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels. Loss of function and dominant-negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels.

Role of Kir4.1 (Kcnj10) in the Regulation of Salt‐Induced Hypertension

In the kidney, Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the aldosterone sensitive distal nephron (ASDN), a major target for multiple hormones controlling blood pressure. These basolateral inwardly rectifying potassium channel subunits assemble to form both heteromeric Kir4.1/Kir5.1 channels and homomeric Kir4.1 channels. These channels control the transepithelial voltage, fine‐tune renal electrolyte homeostasis and contribute to long‐term blood pressure control, correspondingly. To study the role of Kir4.1 containing channels in the renal control of blood pressure, renin‐angiotensin‐aldosterone system (RAAS) balance, and salt‐sensitive (SS) hypertension, we created a Kcnj10 knockout (SSKcnj10−/−) on the Dahl SS rat background. The SSKcnj10−/− rat was created using CRISPR/Cas9 mutagenesis, resulting in a single base pair insertion in exon 2 of the Kcnj10 gene and producing a frameshift in Kir4.1 protein translation after only 26 amino acids. Homozygous SSKcnj10−/− rats were hypokalemic, had reduced heart, kidney, and body weights, and did not survive more than 3 weeks after birth. Mass spectrometry based quantification of serum RAAS peptides revealed increased Ang I (1631 vs. 560 pM), Ang 1–5 (499 vs. 69 pM), and Ang 1–7 (598 vs. 42 pM) levels in SSKcnj10−/− rats compared to age‐matched SSKcnj10+/+ controls. No differences were observed in serum Ang II, Ang III, Ang IV, or aldosterone levels. In addition, ACE activity represented by the Ang II to Ang I ratio was decreased in SSKcnj10−/− rats (0.58 vs. 1.1). The increases in alternative RAS axis peptides (Ang 1–5 and Ang 1–7) and decrease in ACE activity in SSKcnj10−/− rats are suggestive of a low blood pressure phenotype, however, their limited survival precluded blood pressure measurement. Heterozygous (SSKcnj10+/−) developed normally and showed no changes in serum electrolytes. Although survival rate of both male and female SSKcnj10+/− rats was dramatically lower compared to SSKcnj10+/+ rats, they were able to survive much longer than SSKcnj10−/− rats (21 vs. 83 days on average, respectively) with dietary K+ supplementation (HK; 1.41% K+), allowing for telemetric blood pressure measurement. We found no differences in baseline blood pressure under control conditions (LS+HK; 123±2 vs 125±4, mmHg), but salt‐induced (HS, 4% NaCl) elevations in blood pressure were significantly attenuated in SSKcnj10+/− rats fed a HS diet (149±6 vs 183±9 mmHg for SSKcnj10+/− and SSKcnj10+/+ rats, respectively). Furthermore, after 3 weeks on HS, SSKcnj10+/− rats showed decreased diuresis (25±5 vs 38±2, ml/day) and improved albuminuria (6.0±4.0 vs 17.2±7.6, Alb/Cre ratio) compared to SSKcnj10+/+ rats. Our results demonstrate that renal Kir4.1 containing channels mediate salt‐sensitive increases in blood pressure through regulation of potassium homeostasis, modulation of alternative RAS axis hormones, and control of renal salt handling in the ASDN.Support or Funding InformationSupported by the National Institutes of Health grants R35 HL135749, R56 DK121750, R24 HL114474, and F31 DK122647

<p>G Protein-Gated Inwardly Rectifying Potassium Channel Subunit 3 is Upregulated in Rat DRGs and Spinal Cord After Peripheral Nerve Injury</p>

BackgroundG protein-gated inwardly rectifying potassium (GIRK) channels are involved in the regulation of neuronal excitability. Four GIRK subunits (GIRK1-4) are expressed in rat dorsal root ganglia (DRGs). Recently, we have characterized the expression of GIRK1 and −2, and both are downregulated in rat DRGs and spinal cord after a complete sciatic nerve transection (axotomy). Here, we aimed to study the neurochemical characteristics of GIRK3, and its regulation in rat DRGs and spinal cord induced by nerve injury.MethodsA sciatic nerve axotomy was performed to study the influences of injury on GIRK3 expression in DRGs and spinal cord. A dorsal root rhizotomy and a sciatic nerve crush were employed to study the axonal transport of GIRK3 protein, respectively. Immunohistochemistry analysis was employed for investigating the neurochemical characteristics of GIRK3.ResultsIn control DRGs, ~18% of neuron profiles (NPs) were GIRK3-positive (+), and ~41%, ~48% and ~45% of GIRK3+ NPs were CGRP+, IB4+ and NF200+, respectively. GIRK3-like immunoreactivity was observed in glabrous skin of hind paws and axons originating from DRG neurons. Fourteen days after axotomy, more than one-third of DRG NPs were GIRK3+, and among these ~51% and ~56% coexpressed galanin and neuropeptide Y, respectively. In control animals, a small group of interneurons found in the dorsal horn was GIRK3+. In addition, GIRK3+ processes could be observed in superficial laminae of spinal dorsal horn. After nerve injury, the intensity of GIRK3-like immunoreactivity in the superficial layers was increased. Evidence based on rhizotomy and sciatic nerve crush indicated both anterograde and retrograde transport of GIRK3.ConclusionOur study demonstrates that GIRK3 is expressed in sensory neurons and spinal cord. GIRK3 has both anterograde and retrograde axonal transport. GIRK3 expression can be regulated by peripheral nerve injury.

Defective bicarbonate reabsorption in Kir4.2 potassium channel deficient mice impairs acid-base balance and ammonia excretion

The kidneys excrete the daily acid load mainly by generating and excreting ammonia but the underlying molecular mechanisms are not fully understood. Here we evaluated the role of the inwardly rectifying potassium channel subunit Kir4.2 (Kcnj15 gene product) in this process. In mice, Kir4.2 was present exclusively at the basolateral membrane of proximal tubular cells and disruption of Kcnj15 caused a hyperchloremic metabolic acidosis associated with a reduced threshold for bicarbonate in the absence of a generalized proximal tubule dysfunction. Urinary ammonium excretion rates in Kcnj15- deleted mice were inappropriate to acidosis under basal and acid-loading conditions, and not related to a failure to acidify urine or a reduced expression of ammonia transporters in the collecting duct. In contrast, the expression of key proteins involved in ammonia metabolism and secretion by proximal cells, namely the glutamine transporter SNAT3, the phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase enzymes, and the sodium-proton exchanger NHE-3 was inappropriate in Kcnj15-deleted mice. Additionally, Kcnj15 deletion depolarized the proximal cell membrane by decreasing the barium-sensitive component of the potassium conductance and caused an intracellular alkalinization. Thus, the Kir4.2 potassium channel subunit is a newly recognized regulator of proximal ammonia metabolism. The kidney consequences of its loss of function in mice support the proposal for KCNJ15 as a molecular basis for human isolated proximal renal tubular acidosis.

Altered mRNA Expression of ATP-Sensitive and Inward Rectifier Potassium Channel Subunits in Streptozotocin-Induced Diabetic Rat Heart and Aorta

Altered mRNA Expression of ATP-Sensitive and Inward Rectifier Potassium Channel Subunits in Streptozotocin-Induced Diabetic Rat Heart and Aorta

Proestrus Differentially Regulates Expression of Ion Channel and Calcium Homeostasis Genes in GnRH Neurons of Mice.

In proestrus, the changing gonadal hormone milieu alters the physiological properties of GnRH neurons and contributes to the development of the GnRH surge. We hypothesized that proestrus also influences the expression of different ion channel genes in mouse GnRH neurons. Therefore, we performed gene expression profiling of GnRH neurons collected from intact, proestrous and metestrous GnRH-GFP transgenic mice, respectively. Proestrus changed the expression of 37 ion channel and 8 calcium homeostasis-regulating genes. Voltage-gated sodium channels responded with upregulation of three alpha subunits (Scn2a1, Scn3a, and Scn9a). Within the voltage-gated potassium channel class, Kcna1, Kcnd3, Kcnh3, and Kcnq2 were upregulated, while others (Kcna4, Kcnc3, Kcnd2, and Kcng1) underwent downregulation. Proestrus also had impact on inwardly rectifying potassium channel subunits manifested in enhanced expression of Kcnj9 and Kcnj10 genes, whereas Kcnj1, Kcnj11, and Kcnj12 subunit genes were downregulated. The two-pore domain potassium channels also showed differential expression with upregulation of Kcnk1 and reduced expression of three subunit genes (Kcnk7, Kcnk12, and Kcnk16). Changes in expression of chloride channels involved both the voltage-gated (Clcn3 and Clcn6) and the intracellular (Clic1) subtypes. Regarding the pore-forming alpha-1 subunits of voltage-gated calcium channels, two (Cacna1b and Cacna1h) were upregulated, while Cacna1g showed downregulation. The ancillary subunits were also differentially regulated (Cacna2d1, Cacna2d2, Cacnb1, Cacnb3, Cacnb4, Cacng5, Cacng6, and Cacng8). In addition, ryanodine receptor 1 (Ryr1) gene was downregulated, while a transient receptor potential cation channel (Trpm3) gene showed enhanced expression. Genes encoding proteins regulating the intracellular calcium homeostasis were also influenced (Calb1, Hpca, Hpcal1, Hpcal4, Cabp7, Cab 39l, and Cib2). The differential expression of genes coding for ion channel proteins in GnRH neurons at late proestrus indicates that the altering hormone milieu contributes to remodeling of different kinds of ion channels of GnRH neurons, which might be a prerequisite of enhanced cellular activity of GnRH neurons and the subsequent surge release of the neurohormone.

Inwardly Rectifying Potassium Channel Kir4.1 as a Novel Modulator of BDNF Expression in Astrocytes.

Brain-derived neurotrophic factor (BDNF) is a key molecule essential for neural plasticity and development, and is implicated in the pathophysiology of various central nervous system (CNS) disorders. It is now documented that BDNF is synthesized not only in neurons, but also in astrocytes which actively regulate neuronal activities by forming tripartite synapses. Inwardly rectifying potassium (Kir) channel subunit Kir4.1, which is specifically expressed in astrocytes, constructs Kir4.1 and Kir4.1/5.1 channels, and mediates the spatial potassium (K+) buffering action of astrocytes. Recent evidence illustrates that Kir4.1 channels play important roles in bringing about the actions of antidepressant drugs and modulating BDNF expression in astrocytes. Although the precise mechanisms remain to be clarified, it seems likely that inhibition (down-regulation or blockade) of astrocytic Kir4.1 channels attenuates K+ buffering, increases neuronal excitability by elevating extracellular K+ and glutamate, and facilitates BDNF expression. Conversely, activation (up-regulation or opening) of Kir4.1 channels reduces neuronal excitability by lowering extracellular K+ and glutamate, and attenuates BDNF expression. Particularly, the former pathophysiological alterations seem to be important in epileptogenesis and pain sensitization, and the latter in the pathogenesis of depressive disorders. In this article, we review the functions of Kir4.1 channels, with a focus on their regulation of spatial K+ buffering and BDNF expression in astrocytes, and discuss the role of the astrocytic Kir4.1-BDNF system in modulating CNS disorders.

Erratum.

Alcohol: Clinical and Experimental ResearchVolume 40, Issue 10 p. 2253-2253 ErratumFree Access Erratum This article corrects the following: G Protein-Gated Inwardly Rectifying Potassium Channel Subunit 3 Knock-Out Mice Show Enhanced Ethanol Reward Megan E. Tipps, Jonathan D. Raybuck, Laura B. Kozell, K. Matthew Lattal, Kari J. Buck, Volume 40Issue 4Alcoholism: Clinical and Experimental Research pages: 857-864 First Published online: March 25, 2016 First published: 20 September 2016 https://doi.org/10.1111/acer.13236AboutSectionsPDF 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 onFacebookTwitterLinkedInRedditWechat In the article by Tipps et al., “G protein-gated inwardly rectifying potassium channel subunit 3 knock-out mice show enhanced ethanol reward,” 2016, 40:857–864, author C. Contet was omitted from the Herman et al. (2015) reference. The corrected reference is listed below: Herman MA, Sidhu H, Stouffer DG, Kreifeld M, Le D, Gates-Gatto C, Munoz MB, Roberts AJ, Parsons LH, Roberto M, Wickman K, Slesinger PA, Contet C (2015) GIRK3 gates activation of the mesolimbic dopaminergic pathway by ethanol. Proc Natl Acad Sci USA 112:7091–7096. We apologize for this error. Reference Tipps ME, Raybuck JD, Kozell LB, Lattal KM, Buck KJ (2016) G protein-gated inwardly rectifying potassium channel subunit 3 knock-out mice show enhanced ethanol reward. Alcohol Clin Exp Res 40: 857– 864; doi: 10.1111/acer.13012. Volume40, Issue10October 2016Pages 2253-2253 ReferencesRelatedInformation

Infantile spasms in down syndrome: Rescue by knockdown of the GIRK2 channel.

The Ts65Dn (Ts) mouse model of Down syndrome (DS) is exquisitely sensitive to an infantile spasms phenotype induced by γ-aminobutyric acidB receptor (GABAB R) agonists. The Ts mouse contains the core genomic triplication of the DS critical region, which includes 3 copies of the Kcnj6 gene that encodes the GABAB R-coupled G protein-coupled inward rectifying potassium channel subunit 2 (GIRK2) channel. We test the hypothesis that GIRK2 is necessary for the GABAB R agonist-induced infantile spasms phenotype in Ts. We assessed the result of either genetic or pharmacological knockdown of the GIRK2 channel in Ts brain upon the GABAB R agonist-induced infantile spasms phenotype in the Ts mouse model of DS. As well, we examined GABAB R currents in hippocampal neurons prepared from GIRK2-trisomic Ts control mice and GIRK2-disomic Ts mice in which Kcnj6 had been genetically knocked down from 3 to 2 copies. The reduction of the copy number of Kcnj6 in Ts mice rescued the GABAB R agonist-induced infantile spasms phenotype. There was an increase in GABAB R-mediated GIRK2 currents in GIRK2-trisomic Ts mouse hippocampal neurons, which were normalized in the GIRK2-disomic Ts mice. Similarly, pharmacological knockdown of the GIRK2 channel in Ts brain using the GIRK antagonist tertiapin-Q also rescued the GABAB R agonist-induced infantile spasms phenotype in Ts mutants. The GABAB R-coupled GIRK2 channel is necessary for the GABAB R agonist-induced infantile spasms phenotype in the Ts mouse and may represent a novel therapeutic target for the treatment of infantile spasms in DS. Ann Neurol 2016;80:511-521.

Nogo-A Neutralization Improves Graft Function in a Rat Model of Parkinson's Disease.

Transplantation of fetal human ventral mesencephalic (VM) dopaminergic neurons into the striatum is a promising strategy to compensate for the characteristic dopamine deficit observed in Parkinson’s disease (PD). This therapeutic approach, however, is currently limited by the high number of fetuses needed for transplantation and the poor survival and functional integration of grafted dopaminergic neurons into the host brain. Accumulating evidence indicates that contrasting inhibitory signals endowed in the central nervous system (CNS) might support neuronal regeneration. Hence, in the present study we aimed at improving survival and integration of grafted cells in the host brain by neutralizing Nogo-A, one of the most potent neurite growth inhibitors in the CNS. For that purpose, VM tissue cultures were transplanted into rats with a partial 6-hydroxydopamine (6-OHDA) lesion causing a hemi-PD model and concomitantly treated for 2 weeks with intra-ventricular infusion of neutralizing anti-Nogo-A antibodies. Motor behavior using the cylinder test was assessed prior to and after transplantation as functional outcome. At the end of the experimental period the number of dopaminergic fibers growing into the host brain, the number of surviving dopaminergic neurons in the grafts as well as graft size was examined. We found that anti-Nogo-A antibody infusion significantly improved the asymmetrical forelimb use observed after lesions as compared to controls. Importantly, a significantly three-fold higher dopaminergic fiber outgrowth from the transplants was detected in the Nogo-A antibody treated group as compared to controls. Furthermore, Nogo-A neutralization showed a tendency for increased survival of dopaminergic neurons (by two-fold) in the grafts. No significant differences were observed for graft volume and the number of dopaminergic neurons co-expressing G-protein-coupled inward rectifier potassium channel subunit two between groups. In sum, our findings support the view that neutralization of Nogo-A in the host brain may offer a novel and therapeutically meaningful intervention for cell transplantation approaches in PD.

G Protein-Gated Inwardly Rectifying Potassium Channel Subunit 3 Knock-Out Mice Show Enhanced Ethanol Reward.

G protein-gated inwardly rectifying potassium (GIRK) channels contribute to the effects of a number of drugs of abuse, including ethanol. However, the roles of individual subunits in the rewarding effects of ethanol are poorly understood. We compare conditioned place preference (CPP) in GIRK3 subunit knock-out (GIRK3(-/-)), heterozygote (GIRK3(+/-)), and wild-type (WT) mice. In addition, the development of locomotor tolerance/sensitization and the effects of EtOH intoxication on associative learning (fear conditioning) are also assessed. Our data show significant EtOH CPP in GIRK3(-/-) and GIRK3(+/-) mice, but not in the WT littermates. In addition, we demonstrate that these effects are not due to differences in EtOH metabolism, the development of EtOH tolerance/sensitivity, or associative learning abilities. While there were no consistent genotype differences in the fear conditioning assay, our data do show a selective sensitization of the impairing effects of EtOH intoxication on contextual learning, but no effect on cued learning. These findings suggest that GIRK3 plays a role in EtOH reward. Furthermore, the selectivity of this effect suggests that GIRK channels could be an effective therapeutic target for the prevention and/or treatment of alcoholism.

G protein-gated inwardly rectifying potassium channel subunits 1 and 2 are down-regulated in rat dorsal root ganglion neurons and spinal cord after peripheral axotomy.

BackgroundIncreased nociceptive neuronal excitability underlies chronic pain conditions. Various ion channels, including sodium, calcium and potassium channels have pivotal roles in the control of neuronal excitability. The members of the family of G protein-gated inwardly rectifying potassium (GIRK) channels, GIRK1–4, have been implicated in modulating excitability. Here, we investigated the expression and distribution of GIRK1 and GIRK2 in normal and injured dorsal root ganglia (DRGs) and spinal cord of rats.ResultsWe found that ~70% of the DRG neurons expressed GIRK1, while only <10% expressed GIRK2. The neurochemical profiles of GIRK1- and GIRK2-immunoreactive neurons were characterized using the neuronal markers calcitonin gene-related peptide, isolectin-B4 and neurofilament-200, and the calcium-binding proteins calbindin D28k, calretinin, parvalbumin and secretagogin. Both GIRK subunits were expressed in DRG neurons with nociceptive characteristics. However, while GIRK1 was widely expressed in several sensory neuronal subtypes, GIRK2 was detected mainly in a group of small C-fiber neurons. In the spinal dorsal horn, GIRK1- and -2-positive cell bodies and processes were mainly observed in lamina II, but also in superficial and deeper layers. Abundant GIRK1-, but not GIRK2-like immunoreactivity, was found in the ventral horn (laminae VI–X). Fourteen days after axotomy, GIRK1 and GIRK2 were down-regulated in DRG neurons at the mRNA and protein levels. Both after axotomy and rhizotomy there was a reduction of GIRK1- and -2-positive processes in the dorsal horn, suggesting a presynaptic localization of these potassium channels. Furthermore, nerve ligation caused accumulation of both subunits on both sides of the lesion, providing evidence for anterograde and retrograde fast axonal transport.ConclusionsOur data support the hypothesis that reduced GIRK function is associated with increased neuronal excitability and causes sensory disturbances in post-injury conditions, including neuropathic pain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12990-015-0044-z) contains supplementary material, which is available to authorized users.

The GIRK2 subunit is involved in IS-like seizures induced by GABA(B) receptor agonists.

Infantile spasms (or IS) is a catastrophic childhood epilepsy that is particularly prevalent in children with Down syndrome. Previously, we have shown that the Ts65Dn (Ts) mouse model of Down syndrome is a useful substrate upon which to develop an animal model of infantile spasms. Specifically, the Ts mouse is exquisitely sensitive to the electroencephalography (EEG) and behavioral effects of γ-aminobutyric acid (GABA) B receptor (GABA(B)R) agonists with a resultant phenotype that bears behavioral, EEG, and pharmacologic semblance to infantile spasms in humans. The G protein-coupled inward rectifying potassium channel subunit 2 (GIRK2) gene, KCNJ6, is overexpressed in Ts mice, and the GABA(B)R-mediated GIRK2 current is significantly increased in these mutant animals as well. Therefore, we formulated the hypothesis that the GIRK2 channel plays a significant role in the behavioral (measured by acute extensor spasms quantification) and EEG (measured by the electrodecremental response duration) phenotype induced in the Ts mice by GABA(B)R agonists. GIRK2(-/-), (+/-), and (+/+) mice were treated with γ-butyrolactone (GBL), a pro-drug of the GABA(B)R agonist γ-hydroxybutyric acid, and the specific GABA(B)R agonist baclofen (BAC) under continuous EEG monitoring. These drugs induce epileptiform bursts, extensor spasms, and an electrodecremental response (EDR) in Ts mice at low doses, and in wild-type mice at high doses. A dose-response curve was ascertained with two treatment groups: GBL (100, 200, and 400 mg/kg) and BAC (4, 8, 12, and 16 mg/kg). We determined the baseline, the presence and duration of electrodecremental epochs (EDEs), and quantified acute epileptic extensor spasms. Analysis of EEG and behavior of GIRK2(-/-), (+/-), and (+/+) mice after treatment with GABA(B)R agonists and antagonists, indicate that GIRK2(-/-) mice are highly resistant to GABA(B)R agonist-induced EEG and behavioral changes. These data increase the possibility that GIRK2 channel function plays a major role in the genesis of infantile spasms.

Expressional Analysis of Inwardly Rectifying Kir4.1 Channels in Groggy Rats, a Rat Model of Absence Seizures

Background: The inwardly rectifying potassium channel subunit Kir4.1 is specifically expressed in astrocytes, which mediates spatial K+ buffering and is implicated in the pathogenesis of convulsive epileptic disorders (ie, generalized tonic-clonic (GTC) and temporal lobe seizures). Objectives: This study aimed to explore the pathophysiological role of Kir4.1 channels in modulating absence seizure incidence, using a spontaneously epileptic animal model. Materials and Methods: Groggy rats, a rat model of human absence seizures, and Slc:Wistar (control) rats, were used in this study. Cortical and hippocampal EEG were recorded to confirm the seizure incidence in Groggy rats. The expression levels of Kir subunits (ie, Kir4.1, Kir5.1 and Kir2.1) in ten brain regions were analyzed by Western blotting. Results: Groggy rats showed a high incidence (ca. 350 seconds total duration/15 minutes observation period) of absence-like seizures, which were characterized by a sudden immobile posture and synchronously-associated spike and wave discharges. However, Western blot analysis revealed that Kir4.1 expression in Groggy rats was not significantly different from that of control rats in any of the brain regions examined (eg, cerebral cortex, striatum, hippocampus, diencephalon, midbrain, pons/medulla oblongata and cerebellum). In addition, expressional levels of Kir5.1 and Kir2.1, which are also expressed in astrocytes, were unaltered in Groggy rats. Conclusions: The present results suggest that unlike GTC and temporal lobe seizures, pathophysiological alterations (eg, dysfunction and/or expressional changes) of Kir4.1 are not linked to non-convulsive absence seizures.

Expressional analysis of inwardly rectifying Kir4.1 channels in Noda epileptic rat (NER)

The inwardly rectifying potassium channel subunit Kir4.1 is expressed in brain astrocytes and involved in spatial K+ buffering, regulating neural activity. To explore the pathophysiological alterations of Kir4.1 channels in epileptic disorders, we analyzed interictal expressional levels of Kir4.1 in the Noda epileptic rat (NER), a hereditary animal model for generalized tonic-clonic (GTC) seizures. Western blot analysis showed that Kir4.1 expression in NERs was significantly reduced in the occipito-temporal cortical region and thalamus. However, the expression of Kir5.1, another Kir subunit mediating spatial K+ buffering, remained unaltered in any brain regions examined. Immunohistochemical analysis revealed that Kir4.1 was primarily expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes (somata) and foot processes clustered around neurons proved with anti-neuronal nuclear antigen (NeuN) antibody. In NERs, Kir4.1 expression in astrocytic processes was region-selectively diminished in the amygdaloid nuclei (i.e., medial amygdaloid nucleus and basomedial amygdaloid nucleus) while Kir4.1 expression in astrocytic somata was unchanged. Furthermore, the amygdala regions with reduced Kir4.1 expression showed a marked elevation of Fos protein expression following GTC seizures. The present results suggest that reduced activity of astrocytic Kir4.1 channels in the amygdala is involved in limbic hyperexcitability in NERs.