Rectifying Potassium Channel Kir Research Articles

Engineering of an Artificial Light-Modulated Potassium Channel

Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1F137S (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.

Opening/blocking actions of pyruvate kinase antibodies on neuronal and muscular KATP channels

ATP-sensitive-K+ (KATP) channels couple metabolism to the electrical activity of the cells. This channel is associated with glycolytic enzymes to form complexes regulating the channel activity in various tissues. The pyruvate-kinase (PK) enzyme is an antigen in the Paediatric Autoimmune Neuropsychiatric Disorders Associated Streptococcal infection known as PANDAS which is characterized by an abnormal production of auto-antibodies against PK. Here, the effects of the anti-pyruvate kinase antibody (anti-PK-ab) on the muscle and neuronal KATP channels were investigated in native rat skeletal muscle fibres and human neuroblastoma cell-line (SH-SY5Y), respectively. Furthermore, the interaction of PK with the inwardly rectifier potassium channel (Kir6.1/Kir6.2) subunits of the KATP channels was investigated by co-immunoprecipitation experiments in mouse brain using the anti-PK-ab. Patch-clamp experiments showed that the short-term incubation (1h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 enhanced the KATP current of 19.6% and 33.5%, respectively. As opposite, the long-term incubation (24h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 reduced the KATP current of 16% and 24%, respectively, reducing the diameter with atrophy. The direct application of the anti-PK-ab to the excised patches in the absence of intracellular ATP caused channel block, while in the presence of nucleotide channel opened. In neuronal cell line, in the short-term the anti-PK-ab potentiated KATP currents without affecting survival, while in the long-term the anti-PK-ab reduced KATP currents inducing neuronal death. Opening/blocking actions of the anti-PK antibodies on the KATP channels were observed, the blocking action causes fibre atrophy and neuronal death. We demonstrated that PK and Kir subunits are physically/functionally coupled in neurons. The KATP/PK complex can be proposed a novel target in the autoimmune diseases associated with anti-PK production as in PANDAS.

Potassium Channel KIR4.1 as an Immune Target in Multiple Sclerosis

BackgroundMultiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system. Many findings suggest that the disease has an autoimmune pathogenesis; the target of the immune response is not yet known.MethodsWe screened serum IgG from persons with multiple sclerosis to identify antibodies that are capable of binding to brain tissue and observed specific binding of IgG to glial cells in a subgroup of patients. Using a proteomic approach focusing on membrane proteins, we identified the ATP-sensitive inward rectifying potassium channel KIR4.1 as the target of the IgG antibodies. We used a multifaceted validation strategy to confirm KIR4.1 as a target of the autoantibody response in multiple sclerosis and to show its potential pathogenicity in vivo.ResultsSerum levels of antibodies to KIR4.1 were higher in persons with multiple sclerosis than in persons with other neurologic diseases and healthy donors (P<0.001 for both comparisons). We replicated this finding in two independent groups of persons with multiple sclerosis or other neurologic diseases (P<0.001 for both comparisons). Analysis of the combined data sets indicated the presence of serum antibodies to KIR4.1 in 186 of 397 persons with multiple sclerosis (46.9%), in 3 of 329 persons with other neurologic diseases (0.9%), and in none of the 59 healthy donors. These antibodies bound to the first extracellular loop of KIR4.1. Injection of KIR4.1 serum IgG into the cisternae magnae of mice led to a profound loss of KIR4.1 expression, altered expression of glial fibrillary acidic protein in astrocytes, and activation of the complement cascade at sites of KIR4.1 expression in the cerebellum.ConclusionsKIR4.1 is a target of the autoantibody response in a subgroup of persons with multiple sclerosis. (Funded by the German Ministry for Education and Research and Deutsche Forschungsgemeinschaft.)

Phenotype Variability in Patients Carrying KCNJ2 Mutations

Mutations of KCNJ2, the gene encoding the human inward rectifier potassium channel Kir2.1, cause Andersen-Tawil syndrome (ATS), a disease exhibiting ventricular arrhythmia, periodic paralysis, and dysmorphic features. However, some KCNJ2 mutation carriers lack the ATS triad and sometimes share the phenotype of catecholaminergic polymorphic ventricular tachycardia (CPVT). We investigated clinical and biophysical characteristics of KCNJ2 mutation carriers with "atypical ATS." Mutational analyses of KCNJ2 were performed in 57 unrelated probands showing typical (≥2 ATS features) and atypical (only 1 of the ATS features or CPVT) ATS. We identified 24 mutation carriers. Mutation-positive rates were 75% (15/20) in typical ATS, 71% (5/7) in cardiac phenotype alone, 100% (2/2) in periodic paralysis, and 7% (2/28) in CPVT. We divided all carriers (n=45, including family members) into 2 groups: typical ATS (A) (n=21, 47%) and atypical phenotype (B) (n=24, 53%). Patients in (A) had a longer QUc interval [(A): 695 ± 52 versus (B): 643 ± 35 ms] and higher U-wave amplitude (0.24 ± 0.07 versus 0.18 ± 0.08 mV). C-terminal mutations were more frequent in (A) (85% versus 38%, P<0.05). There were no significant differences in incidences of ventricular tachyarrhythmias. Functional analyses of 4 mutations found in (B) revealed that R82Q, R82W, and G144D exerted strong dominant negative suppression (current reduction by 95%, 97%, and 96%, respectively, versus WT at -50 mV) and T305S moderate suppression (reduction by 89%). KCNJ2 gene screening in atypical ATS phenotypes is of clinical importance because more than half of mutation carriers express atypical phenotypes, despite their arrhythmia severity.

Inwardly rectifying potassium channel Kir4.1 is localized at the calyx endings of vestibular afferents

Inwardly rectifying potassium (Kir) channel Kir4.1 (also called Kcnj10) is expressed in various cells such as satellite glial cells. It is suggested that these cells would absorb excess accumulated K+ from intercellular space which is surrounded by these cell membranes expressing Kir4.1. In the vestibular system, loss of Kir4.1 results in selective degeneration of type I hair cells despite normal development of type II hair cells. The mechanisms underlying this developmental disorder have been unclear, because it was thought that Kir4.1 is only expressed in glial cells throughout the entire nervous system. Here, we show that Kir4.1 is expressed not only in glial cells but also in neurons of the mouse vestibular system. In the vestibular ganglion, Kir4.1 mRNA is transcribed in both satellite cells and neuronal somata, whereas Kir4.1 protein is expressed only in satellite cells. On the other hand, in the vestibular sensory epithelia, Kir4.1 protein is localized at the calyx endings of vestibular afferents, which surround type I hair cells. Kir4.1 protein expression in the vestibular sensory epithelia is detected beginning after birth, and its localization gradually adopts a calyceal shape until type I hair cells are mature. Kir4.1 localized at the calyx endings may play a role in the K+-buffering action of vestibular afferents surrounding type I hair cells.

Mechanism of neuro‐vascular unit regulation by glutamate

Proper coupling between neuronal activity and cerebral blood flow is essential for brain function. The neurons, their surrounding glia and microvessels represent the neurovascular unit (NVU). A fundamental gap exists in our understanding of the mechanism(s) by which neuro-vascular regulation via glial astrocytes occurs and how disease impacts this vital function. Ischemic events including stroke may be associated with NVU dysregulation (increased vascular tone, decreased vasodilation). It is not completely understood if and how the main neurotransmitters (Glutamate, adenosine tri-phophate and D-serine) interact with the glia and/or microvessels to cause vessel dilation. Here we present a new ex vivo model of the NVU consisting of isolated penetrating arterioles placed on astrocytes and stimulated with the neurotransmitter glutamate as neuronal surrogate. Using this novel ex vivo model of the NVU, we determined that glutamate has no effect on arteriolar diameter but interacts with astrocytes to induce astrocyte-mediated arteriolar dilation via distinct mechanisms. Importantly, dilation to Glutamate is abolished in denuded vessels while smooth muscle - dependent dilation to ATP remains (see our companion poster). Further examination of the metabolic pathways involved indicates that glutamate releases a cytochrome P450-dependent mediator from the astrocyte with the large conductance calcium sensitive potassium channel (BKCa) as the main molecular target and, to a lesser degree, the inward rectifier Potassium channel KIR. Nitric oxide acts as a permissive factor necessary for potassium channel function. Involvement of other metabolic pathways and molecular targets on the vascular cells are discussed. These significant data indicate that astrocytes, exposed to different neurotransmitters, affect arteriolar tone via distinct molecular pathways.

Striatal D2 Receptors Regulate Dendritic Morphology of Medium Spiny Neurons via Kir2 Channels

Structural plasticity in the adult brain is essential for adaptive behaviors and is thought to contribute to a variety of neurological and psychiatric disorders. Medium spiny neurons of the striatum show a high degree of structural plasticity that is modulated by dopamine through unknown signaling mechanisms. Here, we demonstrate that overexpression of dopamine D2 receptors in medium spiny neurons increases their membrane excitability and decreases the complexity and length of their dendritic arbors. These changes can be reversed in the adult animal after restoring D2 receptors to wild-type levels, demonstrating a remarkable degree of structural plasticity in the adult striatum. Increased excitability and decreased dendritic arborization are associated with downregulation of inward rectifier potassium channels (Kir2.1/2.3). Downregulation of Kir2 function is critical for the neurophysiological and morphological changes in vivo because virally mediated expression of a dominant-negative Kir2 channel is sufficient to recapitulate the changes in D2 transgenic mice. These findings may have important implications for the understanding of basal ganglia disorders, and more specifically schizophrenia, in which excessive activation of striatal D2 receptors has long been hypothesized to be of pathophysiologic significance.

Functional adaptation of cortical interneurons to attenuated activity is subtype-specific

Functional neuronal homeostasis has been studied in a variety of model systems and contexts. Many studies have shown that there are a number of changes that can be activated within individual cells or networks in order to compensate for perturbations or changes in levels of activity. Dissociating the cell autonomous from the network-mediated events has been complicated due to the difficulty of sparsely targeting specific populations of neurons in vivo. Here, we make use of a recent in vivo approach we developed that allows for the sparse labeling and manipulation of activity within superficial caudal ganglionic eminence (CGE)-derived GABAergic interneurons. Expression of the inward rectifying potassium channel Kir2.1 cell-autonomously reduced neuronal activity and lead to specific developmental changes in their intrinsic electrophysiological properties and the synaptic input they received. In contrast to previous studies on homeostatic scaling of pyramidal cells, we did not detect any of the typically observed compensatory mechanisms in these interneurons. Rather, we instead saw a specific alteration of the kinetics of excitatory synaptic events within the reelin-expressing subpopulation of interneurons. These results provide the first in vivo observations for the capacity of interneurons to cell-autonomously regulate their excitability.

The Potassium Channel Kir2.1 Activity is Required for Osteoblastogenesis

Andersen's syndrome (AS) is a rare and dominant pathology linked to the inward rectifying potassium channel Kir 2.1. Patients exhibit a triad of symptoms: periodic paralysis, cardiac dysrhythmia and bone malformations. We have made progress in understanding the contribution of the Kir2.1 channel in skeletal and cardiac muscle dysfunctions but we are still missing the link to bone morphogenesis.We isolated myoblast precursors from muscle biopsies from three healthy individuals and three typical AS patients that exhibit dysmorphic features. Myoblast cultures underwent either myogenic or osteogenic differentiation using the appropriate medium. We used Alizarin, Von Kossa, and alkaline phosphatase staining techniques to monitor osteobalst differentiation mineralization. While myogenic differentiation was not affected, we found that only cells from control patients underwent osteoblastic differentiation as they were positively stained.To further demonstrate the role of the Kir2.1 channels in the osteogenic differentiation of myoblasts, we blocked Kir2.1 channel activity in control patient's cells either by treatment with 0.5 mM BaCl2 or by inhibiting its expression by overexpression of a dominant negative form of the channel. In both cases, cells lost the capacity to mineralize their extracellular matrix.We also carried out rescue experiments on AS patient's cells by infection with adenovirus carrying the wild type form of the channel. These cells become able to express alkaline phosphatase and mineralize their extracellular matrix. Thus, our study strongly suggests that the potassium channel Kir 2.1 is required for the differentiation of myoblast precursors into osteoblast in vitro. Further work will shed light on the molecular mechanisms leading to bone dysmorphology associated with Kir2.1 dysfunction.

Experimental study on inhibition of rat ventricular Ik1 by RNA interference targeting the KCNJ2 gene

The dominant-negative inhibition of KCNJ2-encoded inward rectifier potassium channels (Kir2) is currently considered the best approach to biological pacemakers. We hypothesized that inhibition of the inward rectifier potassium current (IK1) in ventricular myocytes by RNA interference (RNAi) would convert ventricular myocytes into pacemaker cells. Five pieces of short hairpin RNA (shRNA) were designed to target the KCNJ2 gene and then plasmids incorporating shRNA and green fluorescent protein (GFP) as a marker were constructed for transfection into rat ventricular myocytes. The levels of KCNJ2 mRNA were analyzed with real-time quantitative RT-PCR to screen for pieces of shRNA that were effective at inhibiting the expression of the KCNJ2 gene. The activity of potassium ionic channels was then studied in the transfected ventricular myocytes. In the recombinant plasmids, LYS2 transfection significantly inhibited the mRNA of the KCNJ2 gene in comparison to other groups (p < 0.05), and the beating frequency of ventricular myocytes increased after LYS2 transfection. The open probability of IK1 potassium ion channels of cardiac myocytes transfected with the LYS2 plasmid was significantly downregulated (p < 0.05) and the IK1 of ventricular myocytes was also significantly suppressed compared to the negative group (p < 0.05). Our study demonstrated that IK1 was clearly inhibited after the inhibition of KCNJ2 gene expression by RNAi, and this may represent a new approach to the study of biological pacemakers.

Potassium‐dependent activation of Kir4.2 K+ channels

The inwardly rectifying potassium channel Kir4.2 is sensitive to changes in the extracellular potassium concentration ([K(+)](o)). This form of regulation is manifest as a slow (tens of minutes) increase in the whole-cell currents when [K(+)](o) is increased. Here we have investigated the mechanism of K(o)(+) sensitivity of Kir4.2 expressed in Xenopus oocytes. Using two-electrode voltage clamp we found that the sensitivity is specific for the homomeric form of the channel and is completely abolished when coexpressed with Kir5.1. Furthermore, unlike Kir1.1, there is no coupling between the intracellular pH sensitivity and K(o)(+) sensitivity, as is evident by introducing a mutation (K66M), which greatly decreases the pH(i) sensitivity while the K(o)(+) sensitivity remains unchanged. K(o)(+)-dependent activation of Kir4.2 does not involve an increase in the surface expression of the channel, nor is there a difference in the open probability between high and low [K(+)] as determined through patch-clamp measurements. We also found that there is an inverse relationship between the rates of both activation and deactivation and [K(+)](o). Using a kinetic model we argue that Kir4.2 exists in at least three states at the plasma membrane: a deactivated state, an intermediate unstable state and an active state, and that [K(+)](o) affects the rate of transition from the intermediate state to the active state.

Expression of ATP-Sensitive Potassium (K ATP) Channel Subunits in Mammalian Liver

ATP-sensitive potassium (KATP) channels are expressed in multiple tissues, where they provide a link between cellular metabolism and membrane excitability. They consist of two subunits: the inward rectifier potassium channel Kir6, which forms the pore, and the sulfonylurea receptor SUR, an ATP-Binding Cassette (ABC) transporter that functions as the regulatory subunit. Differential expression of Kir6 and SUR isotypes leads to formation of KATP channels with distinct electrophysiological and pharmacological properties, and a number of tissue-specific subunit combinations have been described. Although KATP channels in the liver remain largely unexplored, there is some evidence that KATP channels are expressed in hepatocytes. However, a rigorous characterization of the subunit composition of hepatocytic KATP channels is still lacking. In order to address this, we performed end-point RT-PCR for KATP channel subunit mRNA in extracts from mouse and rat liver, as well as from the human-derived hepatocyte cell line HepG2. Interestingly, a range of expression patterns was observed. In mouse liver, Kir6.1 and SUR2 were detected, while no signal was seen for Kir6.2 and SUR1 (n=3-5). In rat liver, signal was detected for all major subunits: Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B (n=3). In HepG2 cells, signal was detected for Kir6.1, Kir6.2, and SUR2B, but not for SUR1 or SUR2A (n=3). While detection of Kir6.1 and SUR2/SUR2B in mouse and rat liver may reflect vascular smooth muscle channels, the presence of these subunits in HepG2 cells indicates that they may in fact represent KATP channels expressed in hepatocytes. Additionally, expression of Kir6.2 in both rat liver and HepG2 cells is suggestive of a Kir6.2-containing hepatocytic KATP channel. Ultimately, correlation with electrophysiological and pharmacological experiments will be necessary to elucidate the subunit composition of hepatocytic KATP channels, as well as their physiological role.

Structural bases for the different anti-fibrillatory effects of chloroquine and quinidine

Chloroquine, an anti-malarial quinoline, is structurally similar to quinidine. Both drugs have been shown to block ion channels. We tested the hypothesis that chloroquine's mode of interaction with the vestibule of the cytoplasmic domain of the inward rectifier potassium channel Kir2.1 makes it a more effective I(K1) blocker and anti-fibrillatory agent than quinidine. We used comparative molecular modelling and ligand docking of the three-dimensional structures of quinidine and chloroquine in the intracellular domain of Kir2.1. Simulations predicted that chloroquine effectively blocks potassium flow by binding at the centre of the ion permeation vestibule of Kir2.1. In contrast, quinidine binds the vestibular side, only partially blocking ion movement. We tested the modelling predictions in Kir2.1-expressing human embryonic kidney (HEK)-293 cells. The half-maximal inhibitory concentration for chloroquine block of I(K1) was 1.2 µM, while that of quinidine was 57 µM. Finally, we used optical mapping of Langendorff-perfused mouse hearts with cardiac-specific Kir2.1 up-regulation to compare the anti-fibrillatory effects of the drugs. In five of six hearts, 10 μM quinidine slowed the frequency but did not terminate the tachyarrhythmia. In five of five hearts, 10 μM chloroquine terminated the arrhythmia, restoring sinus rhythm. Quinidine only partially blocks I(K1). Chloroquine binds at the centre of the ion permeation vestibule of Kir2.1, which makes it a more effective I(K1) blocker and anti-fibrillatory agent than quinidine. Integrating the structural biology of drug-ion channel interactions with cellular electrophysiology and optical mapping is an excellent approach to understand the molecular mechanisms of anti-arrhythmic drug action and for drug discovery.

Biophysical and Molecular Characterization of a Novel De Novo KCNJ2 Mutation Associated With Andersen-Tawil Syndrome and Catecholaminergic Polymorphic Ventricular Tachycardia Mimicry

Mutations in KCNJ2, the gene encoding the human inward rectifier potassium channel Kir2.1 (IK1 or IKir2.1), have been identified in Andersen-Tawil syndrome. Andersen-Tawil syndrome is a multisystem inherited disease exhibiting periodic paralysis, cardiac arrhythmias, and dysmorphic features at times mimicking catecholaminergic polymorphic ventricular tachycardia. Our proband displayed dysmorphic features including micrognathia, clinodactyly, and syndactyly and exhibited multiform extrasystoles and bidirectional ventricular tachycardia both at rest and during exercise testing. The patient's symptoms continued after administration of nadolol but subsided after treatment with flecainide. Molecular genetic screening revealed a novel heterozygous mutation (c.779G>C/p.R260P) in KCNJ2. Whole-cell patch-clamp studies conducted in TSA201 cells transfected with wild-type human KCNJ2 cDNA (WT-KCNJ2) yielded robust IKir2.1 but no measurable current in cells expressing the R260P mutant. Coexpression of WT and R260P-KCNJ2 (heterozygous expression) yielded a markedly reduced inward IKir2.1 compared with WT alone (-36.5±9.8 pA/pF versus -143.5±11.4 pA/pF, n=8 for both, P<0.001, respectively, at -90 mV), indicating a strong dominant negative effect of the mutant. The outward component of IKir2.1 measured at -50 mV was also markedly reduced with the heterozygous expression versus WT (0.52±5.5 pA/pF versus 23.4±6.7 pA/pF, n=8 for both, P<0.001, respectively). Immunocytochemical analysis indicates that impaired trafficking of R260P-KCNJ2 channels. We report a novel de novo KCNJ2 mutation associated with classic phenotypic features of Andersen-Tawil syndrome and catecholaminergic polymorphic ventricular tachycardia mimicry. The R260P mutation produces a strong dominant negative effect leading to marked suppression of IK1 secondary to a trafficking defect.

Postsynaptic Spiking Homeostatically Induces Cell-Autonomous Regulation of Inhibitory Inputs via Retrograde Signaling

Developing neural circuits face the dual challenge of growing in an activity-induced fashion and maintaining stability through homeostatic mechanisms. Compared to our understanding of homeostatic regulation of excitatory synapses, relatively little is known about the mechanism mediating homeostatic plasticity of inhibitory synapses, especially that following activity elevation. Here, we found that elevating neuronal activity in cultured hippocampal neurons for 4 h significantly increased the frequency and amplitude of mIPSCs, before detectable change at excitatory synapses. Consistently, we observed increases in presynaptic and postsynaptic proteins of GABAergic synapses, including GAD65, vGAT, and GABA(A)Rα1. By suppressing activity-induced increase of neuronal firing with expression of the inward rectifier potassium channel Kir2.1 in individual neurons, we showed that elevation in postsynaptic spiking activity is required for activity-dependent increase in the frequency and amplitude of mIPSCs. Importantly, directly elevating spiking in individual postsynaptic neurons, by capsaicin activation of overexpressed TRPV1 channels, was sufficient to induce increased mIPSC amplitude and frequency, mimicking the effect of elevated neuronal activity. Downregulating BDNF expression in the postsynaptic neuron or its extracellular scavenging prevented activity-induced increase in mIPSC frequency, consistent with a role of BDNF-dependent retrograde signaling in this process. Finally, elevating activity in vivo by kainate injection increased both mIPSC amplitude and frequency in CA1 pyramidal neurons. Thus, spiking-induced, cell-autonomous upregulation of GABAergic synaptic inputs, through retrograde BDNF signaling, represents an early adaptive response of neural circuits to elevated network activity.

Knockdown of Inwardly Rectifying Potassium Channel Kir2.2 Suppresses Tumorigenesis by Inducing Reactive Oxygen Species–Mediated Cellular Senescence

Senescence is an important determinant of treatment outcome in cancer therapy. In the present study, we show that knockdown of the inwardly rectifying K(+) channel Kir2.2 induced growth arrest without additional cellular stress in cancer cells lacking functional p53, p16, and/or Rb. Kir2.2 knockdown also induced senescence-associated β-galactosidase activity and upregulated senescence marker proteins in multiple cancer cell lines derived from different tissues, including prostate, stomach, and breast. Interestingly, knockdown of Kir2.2 induced a significant increase in reactive oxygen species (ROS) that was accompanied by cell cycle arrest, characterized by significant upregulation of p27, with concomitant downregulation of cyclinA, cdc2, and E2F1. Kir2.2 knockdown cells displayed increased levels of PML bodies, DNA damage (γH2AX) foci, senescence-associated heterochromatin foci, mitochondrial dysfunction, secretory phenotype, and phosphatase inactivation. Conversely, overexpression of Kir2.2 decreased doxorubicin-induced ROS accumulation and cell growth inhibition. Kir2.2 knockdown-induced cellular senescence was blocked by N-acetylcysteine, indicating that ROS is a critical mediator of this pathway. In vivo tumorigenesis analyses revealed that tumors derived from Kir2.2 knockdown cells were significantly smaller than those derived from control cells (P < 0.0001) and showed a remarkable increase in senescence-associated proteins, including senescence-associated β-galactosidase, p27, and plasminogen activator inhibitor-1. Moreover, the preestablished tumors are reduced in size after the injection of siKir2.2 (P = 0.0095). Therefore, we propose for the first time that Kir2.2 knockdown induces senescence of cancer cells by a mechanism involving ROS accumulation that requires p27, but not Rb, p53, or p16.

Molecular Mechanisms of EAST/SeSAME Syndrome Mutations in Kir4.1 (KCNJ10)

Inwardly rectifying potassium channel Kir4.1 is critical for glial function, control of neuronal excitability, and systemic K(+) homeostasis. Novel mutations in Kir4.1 have been associated with EAST/SeSAME syndrome, characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt waste. Patients are homozygous for R65P, G77R, C140R or T164I; or compound heterozygous for A167V/R297C or R65P/R199Stop, a deletion of the C-terminal half of the protein. We investigated the functional significance of these mutations by radiotracer efflux and inside-out membrane patch clamping in COSm6 cells expressing homomeric Kir4.1 or heteromeric Kir4.1/Kir5.1 channels. All of the mutations compromised channel function, but the underlying mechanisms were different. R65P, T164I, and R297C caused an alkaline shift in pH sensitivity, indicating that these positions are crucial for pH sensing and pore gating. In R297C, this was due to disruption of intersubunit salt bridge Glu(288)-Arg(297). C140R breaks the Cys(108)-Cys(140) disulfide bond essential for protein folding and function. A167V did not affect channel properties but may contribute to decreased surface expression in A167V/R297C. In G77R, introduction of a positive charge within the bilayer may affect channel structure or gating. R199Stop led to a dramatic decrease in surface expression, but channel activity was restored by co-expression with intact subunits, suggesting remarkable tolerance for truncation of the cytoplasmic domain. These results provide an explanation for the molecular defects that underlie the EAST/SeSAME syndrome.

Variable loss of Kir4.1 channel function in SeSAME syndrome mutations

SeSAME syndrome is a complex disease characterized by seizures, sensorineural deafness, ataxia, mental retardation and electrolyte imbalance. Mutations in the inwardly rectifying potassium channel Kir4.1 ( KCNJ10 gene) have been linked to this condition. Kir4.1 channels are weakly rectifying channels expressed in glia, kidney, cochlea and possibly other tissues. We determined the electrophysiological properties of SeSAME mutant channels after expression in transfected mammalian cells. We found that a majority of mutations (R297C, C140R, R199X, T164I) resulted in complete loss of Kir4.1 channel function while two mutations (R65P and A167V) produced partial loss of function. All mutant channels were rescued upon co-transfection of wild-type Kir4.1 but not Kir5.1 channels. Cell-surface biotinylation assays indicate significant plasma membrane expression of all mutant channels with exception of the non-sense mutant R199X. These results indicate the differential loss of Kir channel function among SeSAME syndrome mutations.

Andersen Cardiodysrhythmic Periodic Paralysis With KCNJ2 Mutations: A Novel Mutation in the Pore Selectivity Filter Residue

Andersen cardiodysrhythmic periodic paralysis or Andersen-Tawil syndrome includes the distinct clinical features of periodic paralysis, cardiac arrhythmia, and facial and skeletal dysmorphisms and exhibits autosomal dominant inheritance. Mutations in the KCNJ2 gene, which encodes the human inward rectifier potassium channel Kir2.1, have been identified in the majority of cases. Despite well-established clinical and molecular characteristics, treatment is still case oriented, and timely diagnosis could be delayed because of the low incidence and phenotypic heterogeneity of this disease. This article describes the clinical and molecular features of 3 cases of Andersen-Tawil syndrome in 2 families. One of the mutations (G144D) was located in the pore selectivity filter residue (which is mutated recurrently) and was considered novel. Intermittent muscle weakness in childhood warrants careful evaluation of cardiac dysrhythmia and skeletal anomalies.

Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K(ATP)) channels that regulate insulin secretion in pancreatic beta-cells. Mutations in K(ATP) channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K(ATP) channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.