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Sildenafil (Viagra), the first approved and widely used oral drug for the treatment of erectile dysfunction, was occasionally associated with life-threatening ventricular arrhythmias in patients. Since inward rectifier potassium current (IK1) may considerably contribute to this arrhythmogenesis, we investigated the effect of sildenafil on the human Kir2.1 and Kir2.2, the prevailing subunits forming the ventricular IK1 channels. Experiments were performed by the whole-cell patch clamp technique at 37°C using Chinese hamster ovary cells transiently expressing the human Kir2.1 and Kir2.2 channels. Changes of both the inward and outward current components (at -110 and -50 mV, respectively) were tested to be able to consider the physiological relevance of the sildenafil effect (changes at -110 and -50 mV did not significantly differ, results at -50 mV are listed below). A significant Kir2.1 inhibition was observed at all applied sildenafil concentrations (16.1% ± 3.7%, 20.0% ± 2.6%, and 15.0% ± 3.0% at 0.1, 1, and 10 µM, respectively). The inhibitory effect of 0.1 µM sildenafil was potentiated by the presence of a low concentration of Ba2+ (0.1 µM) which induced only a slight Kir2.1 inhibition by 5.95% ± 0.75% alone (the combined effect was 35.5% ± 3.4%). The subtherapeutic and therapeutic sildenafil concentrations (0.1 and 1 µM) caused a dual effect on Kir2.2 channels whereas a significant Kir2.2 activation was observed at the supratherapeutic sildenafil concentration (10 µM: 34.1% ± 5.6%). All effects were fully reversible. This is the first study demonstrating that sildenafil at clinically relevant concentrations inhibits both the inward and outward current components of the main human ventricular IK1 subunit Kir2.1. This inhibitory effect was significantly potentiated by a low concentration of environmental contaminant Ba2+ in agreement with recently reported data on rat ventricular IK1 which additionally showed a significant repolarization delay. Considering the similar subunit composition of the human and rat ventricular IK1 channels, the observed effects might contribute to sildenafil-associated arrhythmogenesis in clinical practice. [ABSTRACT FROM AUTHOR]

Inward rectifier K+(Kir) channels regulate cell membrane potential. Different Kir channels respond to unique ligands, but all are regulated by phosphatidylinositol 4,5bisphosphate (PI(4,5)P2). Using planar lipid bilayers, we show that Kir2.2 exhibits bursts of openings separated by long quiescent interburst periods. Increasing PI(4,5)P2 concentration shortens the Kir2.2 interburst duration and lengthens the burst duration without affecting dwell times within a burst. From this, we propose that burst and interburst durations correspond to the cytoplasmic domain (CTD)–docked and CTDundocked conformations observed in the presence and absence of PI(4,5)P2 in atomic structures. We also studied the effect of different phosphatidylinositol lipids on Kir2.2 activation and conclude that the 50 phosphate is essential to Kir2.2 pore opening. Other phosphatidylinositollipidscan compete with PI(4,5)P2 but cannot activate Kir2.2without the 50 phosphate. PI(4)P, which is directly interconvertible to and from PI(4,5)P2, might thus be a regulator of Kir channels in the plasma membrane. [ABSTRACT FROM AUTHOR]

Bronchodilator aminophylline may induce atrial or less often ventricular arrhythmias. The mechanism of this proarrhythmic side effect has not been fully explained. Modifications of inward rectifier potassium (Kir) currents including IK1 are known to play an important role in arrhythmogenesis; however, no data on the aminophylline effect on these currents have been published. Hence, we tested the effect of aminophylline (3–100 µM) on IK1 in enzymatically isolated rat ventricular myocytes using the whole-cell patch-clamp technique. A dual steady-state effect of aminophylline was observed; either inhibition or activation was apparent in individual cells during the application of aminophylline at a given concentration. The smaller the magnitude of the control IK1, the more likely the activation of the current by aminophylline and vice versa. The effect was reversible; the relative changes at −50 and −110 mV did not differ. Using IK1 channel population model, the dual effect was explained by the interaction of aminophylline with two different channel populations, the first one being inhibited and the second one being activated. Considering various fractions of these two channel populations in individual cells, varying effects observed in the measured cells could be simulated. We propose that the dual aminophylline effect may be related to the direct and indirect effect of the drug on various Kir2.x subunits forming the homo- and heterotetrameric IK1 channels in a single cell. The observed IK1 changes induced by clinically relevant concentrations of aminophylline might contribute to arrhythmogenesis related to the use of this bronchodilator in clinical medicine. [ABSTRACT FROM AUTHOR]

In the present study, the functional role of the inwardly rectifying K+ channel, Kir4.1, and large-conductance Ca2+-activated K+ (BK) channel during cell migration in U251 cell line was investigated. We focused on polarised cells which are positive for the active-Cdc42 migration marker. The perforated patch technique was used to avoid intracellular dialysis and to maintain physiological changes in intracellular calcium. Wound healing was employed to assay migration after 24 h. Polarised cells recorded displayed different hallmarks of undifferentiated glial cells: depolarised resting membrane potential and high membrane resistance. Cells recorded outside wounded area did not display either constitutive inward or outward rectification. After migration, U251 cells were characterised by a constitutively smaller Kir4.1 and larger BK currents with a linearly related amplitude. Menthol modulation increased both currents in a linearly dependent manner, indicating a common mechanism triggered by activation of transient receptor potential melastatin 8 (TRPM8), a Ca2+-permeable non-selective cation channel. We hypothesised that both migration and menthol modulation would share an increase of intracellular calcium triggering the increase in Kir4.1 and BK channels. Immunocytochemistry demonstrated the cytoplasmic expression of both Kir4.1 and BK channels and a mislocation in the nucleus under basal conditions. Before and after migration, polarised cells increased the expression of Kir4.1 and BK channels both in the cytoplasm and nucleus. TEM ultrastructural analysis displayed a different nuclear distribution of Kir4.1 and BK channels. In the present study, the physiological role of Kir4.1 and BK currents at membrane potential, their involvement in migration, and the functional role of nuclear channels were discussed. [ABSTRACT FROM AUTHOR]

KIR2.1 potassium channels, producing inward rectifier potassium current (I K1 ), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the "epestfind" webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1 , however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels. [ABSTRACT FROM AUTHOR]

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current ( IK1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK- KWGF cells, both drugs dose- and time-dependently increased KIR2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co-expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK1 by inhibiting KIR2.1 degradation. [ABSTRACT FROM AUTHOR]

Nicotine abuse is associated with variety of diseases including arrhythmias, most often atrial fibrillation (AF). Altered inward rectifier potassium currents including acetylcholine-sensitive current I are known to be related to AF pathogenesis. Since relevant data are missing, we aimed to investigate I changes at clinically relevant concentrations of nicotine. Experiments were performed by the whole cell patch clamp technique at 23 ± 1 °C on isolated rat atrial myocytes. Nicotine was applied at following concentrations: 4, 40 and 400 nM; ethanol at 20 mM (∼0.09%). Nicotine at 40 and 400 nM significantly activated constitutively active component of I with the maximum effect at 40 nM (an increase by ∼100%); similar effect was observed at −110 and −50 mV. Changes at 4 nM nicotine were negligible on average. Coapplication of 40 nM nicotine and 20 mM ethanol (which is also known to activate this current) did not show cumulative effect. In the case of acetylcholine-induced component of I , a dual effect of nicotine and its correlation with the current magnitude in control were apparent: the current was increased by nicotine in the cells showing small current in control and vice versa . The effect of 40 and 400 nM nicotine on acetylcholine-induced component of I was significantly different at −110 and −50 mV. We conclude that nicotine at clinically relevant concentrations significantly increased constitutively active component of I and showed a dual effect on its acetylcholine-induced component, similarly as ethanol. Synchronous application of nicotine and ethanol did not cause additive effect. [ABSTRACT FROM AUTHOR]

In many addictive drugs including alcohol and nicotine, proarrhythmic effects were reported. This review provides an overview of the current knowledge in this field (with a focus on the inward rectifier potassium currents) to promote the lacking data and appeal for their completion, thus, to improve understanding of the proarrhythmic potential of addictive drugs. [ABSTRACT FROM AUTHOR]

Alcohol intoxication tends to induce arrhythmias, most often the atrial fibrillation. To elucidate arrhythmogenic mechanisms related to alcohol consumption, the effect of ethanol on main components of the ionic membrane current is investigated step by step. Considering limited knowledge, we aimed to examine the effect of clinically relevant concentrations of ethanol (0.8-80 mM) on acetylcholine-sensitive inward rectifier potassium current I . Experiments were performed by the whole-cell patch clamp technique at 23 ± 1 °C on isolated rat and guinea-pig atrial myocytes, and on expressed human Kir3.1/3.4 channels. Ethanol induced changes of I in the whole range of concentrations applied; the effect was not voltage dependent. The constitutively active component of I was significantly increased by ethanol with the maximum effect (an increase by ∼100 %) between 8 and 20 mM. The changes were comparable in rat and guinea-pig atrial myocytes and also in expressed human Kir3.1/3.4 channels (i.e., structural correlate of I ). In the case of the acetylcholine-induced component of I , a dual ethanol effect was apparent with a striking heterogeneity of changes in individual cells. The effect correlated with the current magnitude in control: the current was increased by eth-anol in the cells showing small current in control and vice versa. The average effect peaked at 20 mM ethanol (an increase of the current by ∼20 %). Observed changes of action potential duration agreed well with the voltage clamp data. Ethanol significantly affected both components of I even in concentrations corresponding to light alcohol consumption. [ABSTRACT FROM AUTHOR]

Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn2+-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K+ current. We identify KIR2.1 as the acid-sensitive K+ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissuespecific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of KIR2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K+ current in amplifying the sensory response, entry of protons through the Zn2+-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K+ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium channels play key roles in many tissues by coupling metabolic status to membrane potential. In contrast with other potassium channels, the pore-forming Kir6 subunits must co-assemble in hetero-octameric complexes with ATP-binding cassette (ABC) family sulfonylurea receptor (SUR) subunits to facilitate cell surface expression. Binding of nucleotides and drugs to SUR regulates channel gating but how these responses are communicated within the complex has remained elusive to date. We have now identified an electrostatic interaction, forming part of a functional interface between the cytoplasmic nucleotide-binding domain-2 of SUR2 subunits and the distal C-terminus of Kir6 polypeptides that determines channel response to nucleotide, potassium channel opener and antagonist. Mutation of participating residues disrupted physical interaction andregulation of expressed channels, properties that were restored in paired charge-swap mutants. Equivalent interactions were identified in Kir6.1- and Kir6.2-containing channels suggesting a conserved mechanism of allosteric regulation. [ABSTRACT FROM AUTHOR]

Inwardly-rectifying potassium (Kir) channels contribute to maintenance of the resting membrane potential and regulation of electrical excitation in many cell types. Strongly rectifying Kir channels exhibit a very steep voltage dependence resulting in silencing of their activity at depolarized membrane voltages. The mechanism underlying this steep voltage dependence is blockade by endogenous polyamines. These small multifunctional, polyvalent metabolites enter the long Kir channel pore from the intracellular side, displacing multiple occupant ions as they migrate to a stable binding site in the transmembrane region of the channel. Numerous structure-function studies have revealed structural elements of Kir channels that determine their susceptibility to polyamine block, and enable the steep voltage dependence of this process. In addition, various channelopathies have been described that result from alteration of the polyamine sensitivity or activity of strongly rectifying channels. The primary focus of this article is to summarize current knowledge of the molecular mechanisms of polyamine block, and provide some perspective on lingering uncertainties related to this physiologically important mechanism of ion channel blockade. We also briefly review some of the important and well understood physiological roles of polyamine sensitive, strongly rectifying Kir channels, primarily of the Kir2 family. [ABSTRACT FROM AUTHOR]

Background/Aims: Mutations in the inwardly-rectifying K+-channel KCNJ10/Kir4.1 cause autosomal recessive EAST syndrome (epilepsy, ataxia, sensorineural deafness and tubulopathy). KCNJ10 is expressed in the distal convoluted tubule of the kidney, stria vascularis of the inner ear and brain glial cells. Patients diagnosed clinically with EAST syndrome were genotyped and mutations in KCNJ10 were studied functionally. Methods: Patient DNA was amplified and sequenced, and new mutations were identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K+ currents were measured by 2-electrode voltage clamping and channel expression was analysed by Western blotting. Results: We identified 3 homozygous mutations in KCNJ10 (p.F75C, p.A167V and p.V91fs197X), with mutation p.A167V previously reported in a compound heterozygous state. Oocytes expressing wild-type human KCNJ10 showed inwardly rectified currents, which were significantly reduced in all of the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba2+ demonstrated a large residual function in p.A167V only, which was not compatible with causing disease. However, co-expression with KCNJ16 abolished function in these heteromeric channels almost completely. Conclusion: This study provides an explanation for the pathophysiology of the p.A167V KCNJ10 mutation, which had previously not been considered pathogenic on its own. These findings provide evidence for the functional cooperation of KCNJ10 and KCNJ16. Thus, in vitro ascertainment of KCNJ10 function may necessitate co-expression with KCNJ16. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aim Hematopoietic stem cells, especially CD117pos cells, have been found to possess a regenerative potential in various tissues, in particular cardiac muscle. However, the characterization of the relevant ion currents of stem cells prior to implantation lacks documentation. Activation of angiotensin II type 2 receptor ( AT2R) can lead to further cell differentiation and receptor auto-expression and might thus influence electrophysiological properties of CD117pos stem cells. This study was designed to functionally characterize membrane currents of CD117pos cells under normal and AT2R-stimulated conditions. Methods CD117pos murine bone marrow stem cells were isolated with MACS technique and stimulated for the AT2R with angiotensin II and losartan for 3-5 days prior to patch-clamp measurements. RT- PCR was used to determine channel expression. Endothelial properties were analysed with immunocytochemistry and ac LDL uptake assay. Results A well-expressed inward rectifying current ( IKir) was identified in cultured CD117pos cells. Furthermore, a ZD 7288 ( HCN channel blocker)-sensitive current component was isolated. Voltage-dependent potassium currents and chloride currents were less expressed. A small fraction of cells demonstrated voltage- and time-dependent inward currents. In AT2R-stimulated cells inward rectifying the hyperpolarization-induced inward currents were slightly attenuated on the translational level but showed increased mRNA expression. Cultured CD117pos cells express CD31 and VEGFR-2 and significantly increased the uptake of ac LDL. Conclusions CD117pos cells do not have properties of action potential-generating cells and moderately change their excitability during AT2R stimulation. Electrophysiological and molecular properties of control and AT2R-stimulated cells point to a differentiation to vascular endothelial cells. This could increase beneficial vascularization in injured tissues. [ABSTRACT FROM AUTHOR]

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K2.x carried I current and that inhibiting protein degradation in the lysosome increased intracellular K2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of K2.1 to further restore normal levels of functional K2.1 channels. K2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). K2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved K2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased I (at −60 mV) from 31 ± 6 to 55 ± 7 pA/pF ( N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute I block. Baf and CQ did not significantly enhance I densities. Pentamidine (10 μM, 48 h) reduced K2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in K2.1 degradation. Pentamidine-induced downregulation of K2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued. [ABSTRACT FROM AUTHOR]

Aims One form of the short QT syndrome (SQT3) has been linked to the D172N gain-in-function mutation to Kir2.1, which preferentially increases outward current through channels responsible for inward rectifier K+ current (IK1). This study investigated mechanisms by which the Kir2.1 D172N mutation facilitates and perpetuates ventricular arrhythmias. Methods and results The ten Tusscher et al. model for human ventricular action potentials (APs) was modified to incorporate changes to IK1 based on experimentally observed changes to Kir2.1 function: both heterozygous (WT-D172N) and homozygous (D172N) mutant scenarios were studied. Cell models were incorporated into heterogeneous one-dimensional (1D), 2D tissue, and 3D models to compute the restitution curves of AP duration (APD-R), effective refractory period (ERP-R), and conduction velocity (CV). Temporal and spatial vulnerability of ventricular tissue to re-entry was measured and dynamic behaviour of re-entrant excitation waves (lifespan and dominant frequency) in 2D and 3D models of the human ventricle was characterized. D172N ‘mutant’ IK1 led to abbreviated APD and ERP, as well as steeper APD-R and ERP-R curves. It reduced tissue excitability at low excitation rates but increased it at high rates. It increased tissue temporal vulnerability for initiating re-entry, but reduced the minimal substrate size necessary to sustain re-entry. SQT3 ‘mutant’ IK1 also stabilized and accelerated re-entrant excitation waves, leading to sustained rapid re-entry. Conclusion Increased IK1 due to the Kir2.1 D172N mutation increases arrhythmia risk due to increased tissue vulnerability, shortened ERP, and altered excitability, which in combination facilitate initiation and maintenance of re-entrant circuits. [ABSTRACT FROM PUBLISHER]

Background/Aims: Mutations in the inwardly-rectifying K+ channel KCNJ10/Kir4.1 cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (EAST syndrome). KCNJ10 is expressed in the kidney distal convoluted tubule, cochlear stria vascularis and brain glial cells. Patients clinically diagnosed with EAST syndrome were genotyped to identify and study mutations in KCNJ10. Methods: Patient DNA was sequenced and new mutations identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K+ currents were measured by two-electrode voltage clamping. Results: Three new mutations in KCNJ10 (p.R65C, p.F75L and p.V259fs259X) were identified, and mutation p.R297C, previously only seen in a compound heterozygous patient, was found in a homozygous state. Wild-type human KCNJ10-expressing oocytes showed strongly inwardly-rectified currents, which by comparison were significantly reduced in all the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba2+ demonstrated residual function in all mutant channels (p < 0.05) but V259X. Conclusion: This study confirms that EAST syndrome can be caused by many different mutations in KCNJ10 that significantly reduce K+ conductance. EAST syndrome should be considered in any patient with a renal Gitelman-like phenotype with additional neurological signs and symptoms like ataxia, epilepsy or sensorineural deafness. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Patch clamp studies of the potassium-transport proteins TRK1,2 in Saccharomyces cerevisiae have revealed large chloride efflux currents: at clamp voltages negative to −100 mV, and intracellular chloride concentrations >10 mM (J. Membr. Biol. 198:177, 2004). Stationary-state current-voltage analysis led to an in-series two-barrier model for chloride activation: the lower barrier (α) being 10-13 kcal/mol located ∼30% into the membrane from the cytoplasmic surface; and the higher one (β) being 12-16 kcal/mol located at the outer surface. Measurements carried out with lyotrophic anions and osmoprotective solutes have now demonstrated the following new properties: (1) selectivity for highly permeant anions changes with extracellular pH; at pH = 5.5: I ≈ Br > Cl > SCN > NO, and at pH 7.5: I ≈ Br > SCN > NO > Cl. (2) NO acts like 'superchoride', possibly enhancing the channel's intrinsic permeability to Cl. (3) SCN and NO block chloride permeability. (4) The order of selectivity for several slightly permeant anions (at pH = 5.5 only) is formate > gluconate > acetate >> phosphate. (5) All anion conductances are modulated (choked) by osmoprotective solutes. (6) The data and descriptive two-barrier model evoke a hypothetical structure (Biophys. J. 77:789, 1999) consisting of an intramembrane homotetramer of fungal TRK molecules, arrayed radially around a central cluster of four single helices (TM7) from each monomer. (7) That tetrameric cluster would resemble the hydrophobic core of (pentameric) ligand-gated ion channels, and would suggest voltage-modulated hydrophobic gating to underlie anion permeation. [ABSTRACT FROM AUTHOR]

CO chemosensing is a vital function for the maintenance of life that helps to control acid-base balance. Most studies have reported that CO is measured via its proxy, pH. Here we report an inwardly rectifying channel, in outside-out excised patches from HeLa cells that was sensitive to modest changes in PCO under conditions of constant extracellular pH. As PCO increased, the open probability of the channel increased. The single-channel currents had a conductance of 6.7 pS and a reversal potential of -70 mV, which lay between the K and Cl equilibrium potentials. This reversal potential was shifted by +61 mV following a tenfold increase in extracellular [K] but was insensitive to variations of extracellular [Cl]. The single-channel conductance increased with extracellular [K]. We propose that this channel is a member of the Kir family. In addition to this K channel, we found that many of the excised patches also contained a conductance carried via a Cl-selective channel. This CO-sensitive Kir channel may hyperpolarize excitable cells and provides a potential mechanism for CO-dependent inhibition during hypercapnia. [ABSTRACT FROM AUTHOR]

The renal outer medullary K+ (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592-599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP2) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP2 depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP2 hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure. [ABSTRACT FROM AUTHOR]

Auditory transduction, amplification, and hair cell survival depend on the regulation of extracellular [K(+)] in the cochlea. K(+) is removed from the vicinity of sensory hair cells by epithelial cells, and may be distributed through the epithelial cell syncytium, reminiscent of "spatial buffering" in glia. Hypothetically, K(+) is then transferred from the epithelial syncytium into the connective tissue syncytium within the cochlear lateral wall, enabling recirculation of K(+) back into endolymph. This may involve secretion of K(+) from epithelial root cells, and its re-uptake via transporters into spiral ligament fibrocytes. The molecular basis of this secretion is not known. Using a combination of approaches we demonstrated that the resting conductance in guinea pig root cells was dominated by K(+) channels, most likely composed of the Kir4.1 subunit. Dye injections revealed extensive intercellular gap junctional coupling, and delineated the root cell processes that penetrated the spiral ligament. Following uncoupling using 1-octanol, individual cells had Ba(2+)-sensitive weakly rectifying currents. In the basal (high-frequency encoding) cochlear region K(+) loads are predicted to be the highest, and root cells in this region had the largest surface area and the highest current density, consistent with their role in K(+) secretion. Kir4.1 was localized within root cells by immunofluorescence, and specifically to root cell process membranes by immunogold labeling. These results support a role for root cells in cochlear K(+) regulation, and suggest that channels composed of Kir4.1 subunits may mediate K(+) secretion from the epithelial gap junction network. [ABSTRACT FROM AUTHOR]

As a multisystem disorder, Andersen–Tawil syndrome (ATS) is rather unique in the family of channelopathies. The full spectrum of the disease is characterized by ventricular arrhythmias, dysmorphic features, and periodic paralysis. Most ATS patients have a mutation in the ion channel gene, KCNJ2, which encodes the inward rectifier K+ channel Kir2.1, a component of the inward rectifier IK1. IK1 provides repolarizing current during the most terminal phase of repolarization and is the primary conductance controlling the diastolic membrane potential. Thus, ATS is a disorder of cardiac repolarization. The chapter will discuss the most recent data concerning the genetic, cellular, and clinical data underlying this unique disorder. [ABSTRACT FROM AUTHOR]

The family of voltage-gated (Shaker-like) potassium channels in plants includes both inward-rectifying (Kin) channels that allow plant cells to accumulate K+ and outward-rectifying (Kout) channels that mediate K+ efflux. Despite their close structural similarities, Kin and Kout channels differ in their gating sensitivity towards voltage and the extracellular K+ concentration. We have carried out a systematic program of domain swapping between the Kout channel SKOR and the Kin channel KAT1 to examine the impacts on gating of the pore regions, the S4, S5, and the S6 helices. We found that, in particular, the N-terminal part of the S5 played a critical role in KAT1 and SKOR gating. Our findings were supported by molecular dynamics of KAT1 and SKOR homology models. In silico analysis revealed that during channel opening and closing, displacement of certain residues, especially in the S5 and S6 segments, is more pronounced in KAT1 than in SKOR. From our analysis of the S4–S6 region, we conclude that gating (and K+-sensing in SKOR) depend on a number of structural elements that are dispersed over this ∼145-residue sequence and that these place additional constraints on configurational rearrangement of the channels during gating. [ABSTRACT FROM PUBLISHER]

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channeI conductance, or channel open probability. In the absence of:SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicuJar compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was ∼13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation. [ABSTRACT FROM AUTHOR]

Short QT Syndrome (SQTS) is a novel clinical entity characterized by markedly rapid cardiac repolarization and lethal arrhythmias. A mutation in the Kir2.1 inward rectifier K+ channel (D172N) causes one form of SQTS (SQT3). Pharmacologic block of Kir2.1 channels may hold promise as potential therapy for SQT3. We recently reported that the anti-malarial drug chloroquine blocks Kir2.1 channels by plugging the cytoplasmic pore domain. In this study, we tested whether chloroquine blocks D172N Kir2.1 channels in a heterologous expression system and if chloroquine normalizes repolarization properties using a mathematical model of a human ventricular myocyte. Chloroquine caused a dose- and voltage-dependent reduction in wild-type (WT), D172N and WT-D172N heteromeric Kir2.1 current. The potency and kinetics of chloroquine block of D172N and WT-D172N Kir2.1 current were similar to WT. In silico modeling of the heterozygous WT-D172N Kir2.1 condition predicted that 3 μM chloroquine normalized inward rectifier K+ current magnitude, action potential duration and effective refractory period. Our results suggest that therapeutic concentrations of chloroquine might lengthen cardiac repolarization in SQT3. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at +90 mV, rising from control densities of 20.9 ± 2.8 to 34.0 ± 5.4 pAIpF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 ± 61.0 pA/pF in control cells and 658 ± 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels. [ABSTRACT FROM AUTHOR]

Inwardly-rectifying K+ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by IK1, carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M2 receptors (IK(ACh)), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (IK(ATP), IK(ACh), IK1) in both, AM and VM is limited, due to K+ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings IK(ACh) in VM is underestimated as a consequence of constitutive IK1. In that case a reduction in density of IK1 should be paralleled by an increase in density of IK(ACh). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form IK1 channels, in VM caused a reduction in IK1-density by about 80 %. In those cells IK(ACh) was increased about 4 fold. (ii) A comparable increase in IK(ACh) was observed upon reduction of IK1 caused by adenovirus-mediated RNA interference.(iii) Ba2+ in a concentration of 2 μM blocks IK1 in VM by about 60 % without affecting atrial IK(ACh). The reduction in IK1 by 2 μM Ba2+ is paralleled by a reversible increase in IK(ACh) by about 100%. These data demonstrate that the increase in K+ conductance underlying ventricular IK(ACh) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The familial periodic paralyses (PP) were commonly considered to be benign diseases since frequency and severity of the paralytic attacks decrease in adulthood. However, with increasing age, a third of the patients develop permanent weakness and muscle degeneration with fatty replacement. Another complication, cardiac arrhythmia, can result from the dyskalemia during paralytic attacks. The familial PP are typical dominant ion channelopathies: the function of the mutant muscular channel is compensated in the interictal state but defective under certain conditions which then cause flaccid weakness. A triggering factor is the level of serum potassium, the extracellular ion decisive for membrane excitability. In hyper- and hypokalemic periodic paralysis, the mutations are specifically located in the voltage-gated sodium and calcium channels which are essential for action potential generation or excitation-contraction coupling. The common mechanism for the membrane inexcitability during paralytic attacks is a transient membrane depolarization that inactivates the sodium channels which are then no longer available for action potential generation. For the third PP type, the Andersen syndrome, the responsible gene is also expressed in cardiac muscle, and, independently of paralytic attacks, the hazard of ventricular arrhythmias is inherent. The gene product, an inwardly rectifying potassium channel, is responsible for maintaining the resting membrane potential, and all known mutations cause dominant–negative effects on the tetrameric channel complexes. In this article the clinical consequences of the mutations and the therapeutic strategies for all three types of PP are reported. [ABSTRACT FROM AUTHOR]

Recent studies implicated involvement of the 5-hydroxytryptamine4 (5-HT4) receptor in cognitive and emotional processes. The highest 5-HT4 receptor densities in the brain are found in the limbic system including the hippocampus. Here we used the selective 5-HT4 receptor full agonist, N-pentyl- N′-aminoguanidine carbazimidamide (SDZ-216454) to characterize effects of 5-HT4 receptor activation in whole-cell and field recordings in the area CA1 in hippocampal slices prepared from 3 to 4- and 6 to 9-week-old rats, respectively. Extracellular recordings showed that transient 5-HT4 receptor activation by 10–20 min application of SDZ-216454 induces field excitatory postsynaptic potential (fEPSP)-population spike potentiation (ESP5-HT4), which persisted for as long as we held the recordings (> 2 h). ESP5-HT4 displayed characteristics different from EPSP-spike potentiation that accompanies long-term potentiation; it developed without an associated increase in synaptic transmission, was independent on afferent input, activity of postsynaptic neurons and N-methyl-d-aspartate receptor activation; and was expressed in the presence of GABA receptor antagonists. ESP5-HT4 was also induced by transient application of the natural neurotransmitter, 5-HT. The increase in the evoked population spike (PS) induced by SDZ-216454 was not prevented by blockers of hyperpolarization-activated cation current (Ih), Cs+ and ZD-7288, but was mimicked and occluded by 150 µm Ba2+. Whole-cell voltage-clamp recordings from pyramidal neurons demonstrated that SDZ-216454 application increases membrane resistance with a concomitant decrease in a Ba2+-sensitive inwardly rectifying K+ current and the Ba2+-insensitive K+ current underlying slow afterhyperpolarization (IsAHP). We conclude that 5-HT4 receptor activation may cause a long-lasting excitability increase in CA1 pyramidal neurons by inhibition of a Ba2+-sensitive inwardly rectifying K+ current. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA)B receptors are known to enhance activation of Kir3 channels generating G-protein-dependent inward rectifier K+-currents (GIRK). In some neurons, GABAB receptors either cause a tonic GIRK activation or generate a late K+-dependent inhibitory postsynaptic current component. However, other neurons express Kir2 channels, which generate a constitutive inward rectifier K+-current (CIRK) without requiring G-protein activation. The functional coupling of CIRK with GABAB receptors remained unexplored so far. About 50% of rat cerebellar granule cells in the internal granular layer of P19–26 rats showed a sizeable CIRK current. Here, we have investigated CIRK current regulation by GABAB receptors in cerebellar granule cells, which undergo GABAergic inhibition through Golgi cells. By using patch-clamp recording techniques and single-cell reverse transcriptase-polymerase chain reaction in acute cerebellar slices, we show that granule cells co-express Kir2 channels and GABAB receptors. CIRK current biophysical properties were compatible with Kir2 but not Kir3 channels, and could be inhibited by the GABAB receptor agonist baclofen. The action of baclofen was prevented by the GABAB receptor blocker CGP35348, involved a pertussis toxin-insensitive G-protein-mediated pathway, and required protein phosphatases inhibited by okadaic acid. GABAB receptor-dependent CIRK current inhibition could also be induced by repetitive GABAergic transmission at frequencies higher than the basal autorhythmic discharge of Golgi cells. These results suggest therefore that GABAB receptors can exert an inhibitory control over CIRK currents mediated by Kir2 channels. CIRK inhibition was associated with an increased input resistance around rest and caused a ∼ 5 mV membrane depolarization. The pro-excitatory action of these effects at an inhibitory synapse may have an homeostatic role re-establishing granule cell readiness under conditions of strong inhibition. [ABSTRACT FROM AUTHOR]

A biological pacemaker might be created by generation of a cellular construct consisting of cardiac cells that display spontaneous membrane depolarization, and that are electrotonically coupled to surrounding myocardial cells by means of gap junctions. Depending on the frequency of the spontaneously beating cells, frequency regulation might be required. We hypothesized that application of Kir2.1 expressing non-cardiac cells, which provide I (K1) to spontaneously active neonatal cardiomyocytes (NCMs) by electrotonic coupling in such a cellular construct, would generate an opportunity for pacemaker frequency control. Non-cardiac Kir2.1 expressing cells were co-cultured with spontaneously active rat NCMs. Electrotonic coupling between the two cell types resulted in hyperpolarization of the cardiomyocyte membrane potential and silencing of spontaneous activity. Either blocking of gap-junctional communication by halothane or inhibition of I (K1) by BaCl(2) restored the original membrane potential and spontaneous activity of the NCMs. Our results demonstrate the power of electrotonic coupling for the application of specific ion currents into an engineered cellular construct such as a biological pacemaker. [ABSTRACT FROM AUTHOR]

Inwardly rectifying potassium channels Kir2.1–Kir2.3 are important regulators of membrane potential and, thus, control cellular excitability. However, little is known about the regulation of these channels. Therefore, we studied the mechanisms mediating the regulation of Kir2.1–Kir2.3 by the G-protein-coupled m1 muscarinic receptor using the whole-cell patch-clamp technique and recombinant expression in the tsA201 mammalian cell line. Stimulation of the m1 muscarinic receptor inhibited all subtypes of inward rectifier tested, Kir2.1–Kir2.3. The inhibition of each channel subtype was reversible and was attenuated by the muscarinic receptor antagonist, atropine. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) mimicked the effects of m1 receptor activation by inhibiting Kir2.1 currents. However, PMA had no effect on Kir2.2 or Kir2.3. Inclusion of 200-μM guanosine 5′- O-(2-thiodiphosphate) (GDPβS) in the patch pipette solution prevented the effects of m1 muscarinic receptor stimulation on all three of the channel subtypes tested, confirming the mediation of the responses by G-proteins. Cotransfection with the activated mutant of the small GTPase Rho reduced current density, while C3 exoenzyme, a selective inhibitor of Rho, attenuated the m1 muscarinic receptor-induced inhibition of Kir2.1–Kir2.3. Also, buffering the intracellular calcium concentration with a high concentration of EGTA abolished the m1 receptor-induced inhibition of Kir2.1–Kir2.3, implicating a role for calcium in these responses. These results indicate that all three of the Kir2 channels are similarly inhibited by m1 muscarinic receptor stimulation through calcium-dependent activation of the small GTPase Rho. [ABSTRACT FROM AUTHOR]

The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K(+) channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not. [ABSTRACT FROM AUTHOR]

The tetrameric K channel ROMK provides an important pathway for K secretion by the mammalian kidney, and the gating of this channel is highly sensitive to changes in cytosolic pH. Although charge-charge interactions have been implicated in pH sensing by this K channel tetramer, the molecular mechanism linking pH sensing and the gating of ion channels is poorly understood. The x-ray crystal structure KirBac1.1, a prokaryotic ortholog of ROMK, has suggested that channel gating involves intermolecular inter- actions of the N- and C-terminal domains of adjacent subunits. Here we studied channel gating behavior to changes in pH using giant patch clamping of Xenopus laevis oocytes expressing WI or mutant ROMK, and we present evidence that no single charged residue provides the pH sensor. Instead, we show that N-C- and C-C- terminal subunit-subunit interactions form salt bridges, which function to stabilize ROMK in the open state and which are modified by protons. We identify a highly conserved C-C-terminal arginine-glutamate (R-E) ion pair that forms an intermolecular salt bridge and responds to changes in proton concentration. Our results support the intermolecular model for pH gating of inward rectifier K channels. [ABSTRACT FROM AUTHOR]

The weak inward rectifier potassium channel ROMK is important for water and salt reabsorption in the kidney. Here we identified Golgin-160 as a novel interacting partner of the ROMK channel. By using yeast two-hybrid assays and co-immunoprecipitations from transfected cells, we demonstrate that Golgin-160 associates with the ROMK C-terminus. Immunofluorescence microscopy confirmed that both proteins are co-localized in the Golgi region. The interaction was further confirmed by the enhancement of ROMK currents by the co-expressed Golgin-160 in Xenopus oocytes. The increase in ROMK current amplitude was due to an increase in cell surface density of ROMK protein. Golgin-160 also stimulated current amplitudes of the related Kir2.1, and of voltage-gated Kv1.5 and Kv4.3 channels, but not the current amplitude of co-expressed HERG channel. These results demonstrate that the Golgi-associated Golgin-160 recognizes the cytoplasmic C-terminus of ROMK, thereby facilitating the transport of ROMK to the cell surface. However, the stimulatory effect on the activity of more distantly-related potassium channels suggests a more general role of Golgin-160 in the trafficking of plasma membrane proteins. [ABSTRACT FROM AUTHOR]

1. Sipatrigine (BW 619C89), a blocker of neuronal Na+ and Ca2+ channels that is structurally related to lamotrigine, has been shown to be neuroprotective in models of cortical ischaemia. Although associated with cardiovascular effects in animal models in vivo, there is no published information concerning the effects of sipatrigine on cardiac ion currents and action potentials (AP). 2. The aim of the present study was to examine the effects of sipatrigine on the delayed rectifier currents (IKr and IKs), the inward rectifier current (IK1), the L-type Ca2+ current (ICa,L) and the fast Na+ current (INa), as well as on AP duration at 30% (APD30) and 90% (APD90) repolarization, in guinea-pig isolated ventricular myocytes. 3. Each of the currents was inhibited by sipatrigine, demonstrating the drug to be a relatively broad-spectrum blocker of cation channels in the heart. However, sipatrigine was a comparatively more potent inhibitor of IKr (IC50 = 0.85 µmol/L) and IKs (IC50 = 0.92 µmol/L) than of IK1 (IC50 = 5.3 µmol/L), ICa,L (IC50 = 6.0 µmol/L) and INa (IC50 = 25.5 µmol/L). 4. Consistent with block of IKr, IKs and IK1, sipatrigine (1–30 µmol/L) produced a concentration-dependent prolongation of APD90. Although lower concentrations of sipatrigine (≤ 3 µmol/L) caused APD30 prolongation, higher concentrations (≥ 10 µmol/L) shortened APD30, consistent with an involvement of ICa,L blockade. The contrasting effects of sipatrigine on APD30 and APD90 at higher concentrations resulted in a marked concentration-dependent triangulation of the AP. 5. The results of the present study demonstrate that sipatrigine, at concentrations previously shown to be neuroprotective in vitro, modulates cardiac K+, Ca2+ and Na+ currents and repolarization of the cardiac ventricular action potential. [ABSTRACT FROM AUTHOR]

Dendritically placed, voltage-sensitive ion channels are key regulators of neuronal synaptic integration. In several cell types, hyperpolarization/cyclic nucleotide gated (HCN) cation channels figure prominently in dendritic mechanisms controlling the temporal summation of excitatory synaptic events. In prefrontal cortex, the sustained activity of pyramidal neurons in working memory tasks is thought to depend on the temporal summation of dendritic excitatory inputs. Yet we know little about how this is accomplished in these neurons and whether HCN channels play a role. To gain a better understanding of this process, layer V-VI pyramidal neurons in slices of mouse prelimbic and infralimbic cortex were studied. Somatic voltage-clamp experiments revealed the presence of rapidly activating and deactivating cationic currents attributable to HCN1/HCN2 channels. These channels were open at the resting membrane potential and had an apparent half-activation voltage near -90 mV. In the same voltage range, K+ currents attributable to Kir2.2/2.3 and K+-selective leak (Kleak) channels were prominent. Computer simulations grounded in the biophysical measurements suggested a dynamic interaction among Kir2, Kleak, and HCN channel currents in shaping membrane potential and the temporal integration of synaptic potentials. This inference was corroborated by experiment. Blockade of Kir2/Kleak channels caused neurons to depolarize, leading to the deactivation of HCN channels, the initiation of regular spiking (4-5 Hz), and enhanced temporal summation of EPSPs. These studies show that HCN channels are key regulators of synaptic integration in prefrontal pyramidal neurons but that their functional contribution is dependent on a partnership with Kir2 and Kleak channels. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is characterized by marked prolongation of action potential duration and reduction in cellular repolarization reserve. These changes are caused in large part by HF-induced K+ current downregulation. Molecular mechanisms underlying these changes remain unclear. We determined whether downregulation of K+ currents in a canine model of tachycardia-induced HF is caused by altered expression of underlying K+ charmel α- and β-subunits encoding these currents. K+ channel subunit expression was quantified in normal and failing dogs at the mRNA and protein levels in epicardial (Epi), midmyocardial (Mid), and endocardial (Endo) layers of left ventricle. Analysis of mRNA and protein levels of candidate genes encoding the transient outward K+ current (1to) revealed marked reductions in canine cKv4.3 expression in HF in Epi (44% mRNA, 39% protein), Mid (52% mRNA, 34% protein), and Endo (49% mRNA, 73% protein) layers and a paradoxical enhancement (41% Epi, 97% Mid, 113% Endo) in cKv1.4 protein levels, without significant changes in Kv channel-interacting protein cKChIP2 expression. Expression of cKir2.1, the gene underlying inward rectifier K+ current (1K1), was unaffected by HF at mRNA and protein levels despite significant reduction in 1K1, whereas canine ether-à-go-go-related gene (cERG), which encodes the rapidly activating component of the delayed rectifier current (IK), exhibited increased protein expression. HF was not accompanied by significant changes in cKvLQT1 or cMinK mRNA and protein levels. These data indicate that 1) downregulation of 1to in HF is associated with decreased cKv4.3 and not cKv1.4 or cKChIP2, and 2) alterations in both the rapidly activating and slowly activating components of 1K as well as 1K1 in nonischemic dilated cardiomyopathy are not caused by changes in either transcript or immunoreactive protein levels of relevant channel subunits, which suggests posttranslational modification of these currents by HF. [ABSTRACT FROM AUTHOR]

A diversity of ion channels contributes to the active properties of neuronal dendrites. From the apical dendrites of hippocampal CA1 pyramidal neurons, we recorded inwardly rectifying K+ channels with a single-channel conductance of 33 pS. The inwardly rectifying K+ channels were constitutively active at the resting membrane potential. The amount of constitutive channel activity was significantly larger in the apical dendrites than in the soma. Activities of these inwardly rectifying K+ channels were inhibited by Ba2+ (200 µM) and tertiapin (10 nM), both of which are believed to block G-protein-coupled inwardly rectifying K+ (GIRK) channels. Intracellularly applied GTPγS (20 µM) during dual dendritic recordings significantly increased constitutive channel activity. Baclofen (20 µM), an agonist for the G-protein-coupled GABAB receptor, also significantly increased the level of channel activity. Therefore, these channels are GIRK channels, which are constitutively active at rest in the apical dendrites of CA1 pyramidal neurons and can be further activated via G-protein-coupled neurotransmitter receptors. [ABSTRACT FROM AUTHOR]

TheArabidopsis thalianaKAT1, an inward-rectifying potassium channel, shares molecular features with theShakerfamily of outward rectifier K+ channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with theShakerK+ channel, we have inserted a flag epitope in the NH2 terminus or the S3-S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells,Xenopusoocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to theShakerK+ channel. [ABSTRACT FROM AUTHOR]

The Arabidopsis thaliana KATI, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K+ channels. The KATI amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KATI are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K+ channel, we have inserted a flag epitope in the NH, terminus or the S3-S4 loop. The KATI and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti- C terminal KATI determined the membrane localization of these epitopes and the orientation of the KATI channel in the membrane. Our data confirm that KATI in eukaryotic cells has an orientation similar to the Shaker K+ channel. [ABSTRACT FROM AUTHOR]

Normal rat kidney (NRK) fibroblasts change their excitability properties through the various stages of cell proliferation. The present mathematical model has been developed to explain excitability of quiescent (serum deprived) NRK cells. It includes as cell membrane components, on the basis of patch-clamp experiments, an inwardly rectifying potassium conductance (GKir), an L-type calcium conductance (GCaL), a leak conductance (Gleak), an intracellular calcium-activated chloride conductance [GCl(Ca)], and a gap junctional conductance (Ggj), coupling neighboring cells in a hexagonal pattern. This membrane model has been extended with simple intracellular calcium dynamics resulting from calcium entry via GCaL channels, intracellular buffering, and calcium extrusion. It reproduces excitability of single NRK cells and cell clusters and intercellular action potential (AP) propagation in NRK cell monolayers. Excitation can be evoked by electrical stimulation, external potassium-induced depolarization, or hormone-induced intracellular calcium release. Analysis shows the roles of the various ion channels in the ultralong (∼30 s) NRK cell AP and reveals the particular role of intracellular calcium dynamics in this AP. We support our earlier conclusion (De Roos A, Willems PH, van Zoelen EJ, and Theuvenet AP. Am J Physiol Cell Physiol 273: C1900-C1907, 1997) that AP generation and propagation may act as a rapid mechanism for the propagation of intracellular calcium waves, thus contributing to fast intercellular calcium signaling. The present model serves as a starting point to further analyze excitability changes during contact inhibition and cell transformation. [ABSTRACT FROM AUTHOR]

Activation of G protein-gated inwardly rectifying K+ (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of Gi/o-Coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several Gi/o G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A1, adrenergic α2A, dopamine D2S, M4 muscarinic, and GABAB1b/2 receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A1 receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPγS), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca2+ imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/ GTP exchange by using nonhydrolyzable guanosine 5'-O-(2-thiodiphosphate) (GDPβS) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific Ga subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle. [ABSTRACT FROM AUTHOR]

Dopamine (DA) modulation of excitability in medial prefrontal cortex (mPFC) pyramidal neurons has attracted considerable attention because of the involvement of mPFC DA in several neuronal disorders. Here, we focused on DA modulation of inwardly rectifying K+ current (IRKC) in pyramidal neurons acutely dissociated from rat mPFC. A Cs+-sensitive whole-cell IRKC was elicited by hyperpolarizing voltage steps from a holding potential of -50 mV. DA (20 µM) reduced IRKC amplitude, as did selective stimulation of DA D1 or D2 class receptors (D1Rs and D2Rs). D1Rs activate, whereas D2Rs inhibit, the adenylyl cyclase-cAMP-protein kinase A (PKA) signaling pathway. Suppression of IRKC by D2R stimulation was attributable to decreased PKA activity because similar inhibition was observed with PKA inhibitors, whereas enhancing PKA activity increased IRKC. This suggests that the DA D1R suppression of IRKC occurred through a PKA phosphorylation-independent process. Using outside-out patches of mPFC pyramidal neurons, which preclude involvement of cytosolic signaling molecules, we observed a Cs+-sensitive macroscopic IRKC that was suppressed by the membrane-permeable cyclic nucleotide Sp-cAMP but was unaffected by non-nucleotide modulators of PKA, suggesting direct interactions of the cyclic nucleotides with IRK channels. Our results indicate that DA suppresses IRKC through two mechanisms: D1R activation of cAMP and direct interactions of the nucleotide with IRK channels and D2R-mediated dephosphorylation of IRK channels. The DA modulation of IRKC indicates that ambient DA would tend to increase responsiveness to excitatory inputs when PFC neurons are near the resting membrane potential and may provide a mechanism by which DA impacts higher cognitive function. [ABSTRACT FROM AUTHOR]

Objective: The Andersen’s syndrome is a hereditary disease, which is characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Recently, mutations of the KCNJ2 gene, which encodes the inward rectifying potassium channel subunit Kir2.1, have been identified in affected individuals. However, the functional effects of these mutations have not yet been fully elucidated. Methods and Results: To clarify this situation we generated known Andersen disease mutants of KCNJ2 which did not yield any measurable K+ currents in CHO cells indicating that the Andersen mutants failed to form functional homomultimeric complexes. EGFP-tagged KCNJ2 wild-type and mutant channels distributed in a similar homogeneous pattern in the cell membrane suggesting that protein trafficking was not altered by the Andersen mutations but rather implicating that the mutations rendered the KCNJ2 channel non-functional. In heterologous coexpression experiments the Andersen mutants exerted a dominant-negative effect on wild-type KCNJ2. However, the extent of suppression varied between the different KCNJ2 mutants. Given our results in CHO cells, we expressed the disease mutant KCNJ2-S136F in neonate rat cardiomyocytes using adenoviral gene transfer to test the effect of Andersen mutants on native IK1. IK1 density was indeed significantly reduced in KCNJ2-S136F-infected cells (n=9) compared to control cells (n=9) over a voltage range from −70 to −150 mV (P<0.05). Conclusion: These results support that Kir2.x channels are a critical component of native IK1 in neonate rat cardiomyocytes and that a dominant-negative suppression of IK1 in native cells is the pathophysiological correlate of the Andersen’s syndrome. [Copyright &y& Elsevier]

To date, data regarding the cellular electrophysiology of the atrioventricular node (AVN) have derived from AVN cells isolated from the rabbit heart. The aim of this study was to characterise for the first time the electrophysiological properties of single cells isolated from the AVN of the guinea-pig heart. Cells were isolated from the AVN region by a combination of enzymatic and mechanical dispersion. Mean (±SEM) cell dimensions were 110.8±2.3 µm in length by 11.4±1.3 µm in width (n=76 cells). Electrophysiological recordings were made using the whole-cell patch-clamp technique at 37°C. Mean cell capacitance was 25.1±0.9 pF (n=43) and mean cell input resistance was 1,377±178 MΩ (n=21). Spontaneously active cells exhibited action potentials characteristic of cardiac pacemaker tissue. Under whole-cell voltage clamp, the mean 'zero current' potential was –39.7±4.1 mV (n=21). Voltage commands of 500 ms duration to a range of test potentials from a holding potential of –40 mV revealed a number of distinct current components. At potentials positive to –40 mV an early inward current was observed that exhibited a bell-shaped current voltage (I-V) relation, typical of L-type calcium current. A delayed outward current that increased progressively with test potential magnitude was also observed. Analysis of the outward current 'tails' on repolarisation to –40 mV showed this to be comprised of two components with half-maximal activation voltages of –17.2±1.8 mV, and +27.1±3.6 mV (n=13). 'Transient outward' current appeared absent from guinea-pig AVN cells. Hyperpolarising test pulses activated net inward current, for which three components could be observed: a barium-sensitive (100 µM Ba2+) inwardly rectifying current evident at the start of the voltage command and prominent at potentials negative to the diastolic potential range; a time-dependent, hyperpolarisation-activated current, and a residual background current. On repolarisation to –40 mV, a large inward current spike typical of cardiac Na current was observed in some cells. Notably, the following electrophysiological characteristics of guinea-pig AVN cells are distinct from those characteristics previously reported for the rabbit AVN: (1) an absence of transient outward current, (2) the presence of two components of delayed outward current, and (3) the presence of a Ba2+-sensitive inwardly rectifying current at negative voltages. The guinea-pig AVN isolated cell preparation may be valuable in providing additional insights into the cellular electrophysiology of this important region of the heart. [ABSTRACT FROM AUTHOR]

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells. [Copyright &y& Elsevier]

ATP-sensitive K+ channels are an octameric assembly of two proteins, a sulfonylurea receptor (SUR1) and an ion conducting subunit (Kir 6.0). We have examined the role of the C-terminus of SUR1 by expressing a series of truncation mutants together with Kir6.2 stably in HEK293 cells. Biochemical analyses using coimmunoprecipitation indicate that SUR1 deletion mutants and Kir6.2 assemble and that a SUR1 deletion mutant binds glibenclamide with high affinity. Electrophysiological recordings indicate that ATP sensitivity is normal but the response of the mutant channel complexes to tolbutamide, MgADP and diazoxide is disturbed. Quantitative immunofluorescence and cell surface biotinylation supports the idea that there is little disturbance in the efficiency of trafficking. Our data show that deletions of the C-terminal most cytoplasmic domain of SUR1, can result in functional channels at the plasma membrane in mammalian cells that have an abnormal response to physiological and pharmacological agents. [ABSTRACT FROM AUTHOR]

To obtain a clearer understanding of the mechanisms by which somatostatin modulates stimulus-secretion coupling in neuroendocrine cells, we investigated the pharmacology of the somatostatin-activated inward rectifier in mouse pituitary tumour cells (AtT-20 corticotrophs). Individual AtT-20 cells displayed spontaneous, long-lasting action potentials that caused transient spikes in cytosolic [Ca2+] ([Ca]i). Application of 1–10 nM somatostatin led to membrane hyperpolarization and loss of [Ca]i spiking activity. Voltage-clamp recordings revealed that the somatostatin-induced hyperpolarization was due to an inwardly rectifying K+ current. Tetrabutylammonium (TBA+) inhibited both outward and inward currents through the inward rectifier, whereas Cs+ blocked only inward current and tetraethylammonium (TEA+) was completely ineffective in blocking somatostatin-activated currents. However TEA+, but neither TBA+ nor Cs+, blocked voltage-gated outward currents. Correspondingly, TBA+ abolished the hyperpolarizing effects of somatostatin and, of the three K+ channel blockers, only TBA+ prevented the somatostatin-induced inhibition of [Ca]i spiking. TBA+ may thus prove a useful tool in elucidating the underlying mechanisms by which somatostatin affects the secretory activity of neuroendocrine cells. [ABSTRACT FROM AUTHOR]

RP58866 is a potent antiarrhythmic drug that maintains its antiarrhythmic properties during ischemia. Since interstitial concentrations of adenosine increase during ischemia, we examined the properties of the drug with respect to the muscarinic K⁺ current (IK(ACh)), with a main emphasis on the adenosine (Ado) -induced current (IK(Ado))- Using different Gi-coupled receptors (M₂, A₁, sphingolipid), we studied the effect of RP58866 in isolated guinea-pig atrial myocytes by the whole-cell voltage clamp technique. Application of 50 μM RP58866 resulted in complete inhibition of the muscarinic K⁺ current. Inhibition was observed during activation of IK(Ach) by each of the three receptors. IC₅₀ was ∼2.0 μM. GTP-γ-S induced IK(ACh) was reduced by RP58866. The drug was active from the outside only, and its intracellular application via the patch pipet had no inhibitory effect. Despite the structural homologies between inward rectifying K⁺ channels, the adenosine triphosphate-sensitive K⁺ current (IK(ATP)) was not inhibited by the compound. It is concluded that muscarinic K⁺ current is inhibited by RP58866, an inhibition not limited to IK₁, Ito, and IKr. High interstitial adenosine concentrations during ischemia are expected to increase the participation of IK(Ado) on repolarization. RP58866-induced inhibition of IK(Ado) would, therefore, be of particular relevance during ischemia. The high sensitivity of IK(Ado) to RP58866 may partially explain the unique properties of the drug toward arrhythmias developing in the ischemic myocardium. [ABSTRACT FROM AUTHOR]