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Aminophylline, a bronchodilator mainly used to treat severe asthma attacks, may induce arrhythmias. Unfortunately, the underlying mechanism is not well understood. We have recently described a significant, on average inhibitory effect of aminophylline on inward rectifier potassium current I K1 , known to substantially contribute to arrhythmogenesis, in rat ventricular myocytes at room temperature. This study was aimed to examine whether a similar effect may be observed under clinically relevant conditions. Experiments were performed using the whole cell patch clamp technique at 37°C on enzymatically isolated healthy porcine and failing human ventricular myocytes. The effect of clinically relevant concentrations of aminophylline (10–100 µM) on I K1 did not significantly differ in healthy porcine and failing human ventricular myocytes. I K1 was reversibly inhibited by ∼20 and 30 % in the presence of 30 and 100 µM aminophylline, respectively, at −110 mV; an analogical effect was observed at −50 mV. To separate the impact of I K1 changes on AP configuration, potentially interfering ionic currents were blocked (L-type calcium and delayed rectifier potassium currents). A significant prolongation of AP duration was observed in the presence of 100 µM aminophylline in porcine cardiomyocytes which well agreed with the effect of a specific I K1 inhibitor Ba2+ (10 µM) and with the result of simulations using a porcine ventricular cell model. We conclude that the observed effect of aminophylline on healthy porcine and failing human I K1 might be involved in its proarrhythmic action. To fully understand the underlying mechanism, potential aminophylline impact on other ionic currents should be explored. [Display omitted] • The bronchodilator aminophylline may induce arrhythmias. • The mechanism of the proarrhythmic action of aminophylline is not well understood. • Aminophylline interacts with I K1 channels causing their inhibition on average. • The effect was similar in healthy porcine and failing human cardiomyocytes. • The observed effect of aminophylline might be involved in its proarrhythmic action. [ABSTRACT FROM AUTHOR]

The distribution of data presented in many electrophysiological studies is presumed to be normal without any convincing evidence. To test this presumption, the cell membrane capacitance and magnitude of inward rectifier potassium currents were recorded by the whole-cell patch clamp technique in rat atrial myocytes. Statistical analysis of the data showed that these variables were not distributed normally. Instead, a positively skewed distribution appeared to be a better approximation of the real data distribution. Consequently, the arithmetic mean, used inappropriately in such data, may substantially overestimate the true mean value characterizing the central tendency of the data. Moreover, a large standard deviation describing the variance of positively skewed data allowed 95% confidence interval to include unrealistic negative values. We therefore conclude that the normality of the electrophysiological data should be tested in every experiment and, if rejected, the positively skewed data should be more accurately characterized by the median and interpercentile range or, if justified (namely in the case of log-normal and gamma data distribution), by the geometric mean and the geometric standard deviation. [ABSTRACT FROM AUTHOR]

Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to the Keppen–Lubinsky syndrome but without lipodystrophy. Whole-exome sequencing of the patient’s DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K + selectivity and gained Ca 2+ permeability. Notably, the inward current was inhibited by the Na + channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen–Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies. [ABSTRACT FROM AUTHOR]

Background: The failing heart exhibits an increased arrhythmia susceptibility that is often attributed to action potential (AP) prolongation due to significant ion channel remodeling. The inwardly rectifying K+ current (IK1) has been reported to be reduced, but its contribution to shaping the AP waveform and cell excitability in the failing heart remains unclear.Objective: The purpose of this study was to define the effect of IK1 suppression on the cardiac AP and excitability in the normal and failing hearts.Methods: We used electrophysiological and pharmacological approaches to investigate IK1 function in a swine tachy-pacing model of heart failure (HF).Results: Terminal repolarization of the AP (TRAP; the time constant of the exponential fit to terminal repolarization) was markedly prolonged in both myocytes and arterially perfused wedges from animals with HF. TRAP was increased by 54.1% in HF myocytes (P < .001) and 26.2% in HF wedges (P = .014). The increase in TRAP was recapitulated by the potent and specific IK1 inhibitor, PA-6 (pentamidine analog 6), indicating that IK1 is the primary determinant of the final phase of repolarization. Moreover, we find that IK1 suppression reduced the ratio of effective refractory period to AP duration at 90% of repolarization, permitting re-excitation before full repolarization, reduction of AP upstroke velocity, and likely promotion of slow conduction.Conclusion: Using an objective measure of terminal repolarization, we conclude that IK1 is the major determinant of the terminal repolarization time course. Moreover, suppression of IK1 prolongs repolarization and reduces postrepolarization refractoriness without marked effects on the overall AP duration. Collectively, these findings demonstrate how IK1 suppression may contribute to arrhythmogenesis in the failing heart. [ABSTRACT FROM AUTHOR]

Inward rectifier K + channels of the Kir2.x subfamily play important roles in controlling the neuronal excitability. Although their cellular localization in the brain has been extensively studied, only a few studies have examined their expression in the spinal cord and peripheral nervous system. In this study, immunohistochemical analyses of Kir2.1, Kir2.2, and Kir2.3 expression were performed in rat dorsal root ganglion (DRG) and spinal cord using bright-field and confocal microscopy. In DRG, most ganglionic neurons expressed Kir2.1, Kir2.2 and Kir2.3, whereas satellite glial cells chiefly expressed Kir2.3. In the spinal cord, Kir2.1, Kir2.2 and Kir2.3 were all expressed highly in the gray matter of dorsal and ventral horns and moderately in the white matter also. Within the gray matter, the expression was especially high in the substantia gelatinosa (lamina II). Confocal images obtained using markers for neuronal cells, NeuN, and astrocytes, Sox9, showed expression of all three Kir2 subunits in both neuronal somata and astrocytes in lamina I–III of the dorsal horn and the lateral spinal nucleus of the dorsolateral funiculus. Immunoreactive signals other than those in neuronal and glial somata were abundant in lamina I and II, which probably located mainly in nerve fibers or nerve terminals. Colocalization of Kir2.1 and 2.3 and that of Kir2.2 and 2.3 were present in neuronal and glial somata. In the ventral horn, motor neurons and interneurons were also immunoreactive with the three Kir2 subunits. Our study suggests that Kir2 channels composed of Kir2.1–2.3 subunits are expressed in neuronal and glial cells in the DRG and spinal cord, contributing to sensory transduction and motor control. [ABSTRACT FROM AUTHOR]

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K+ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (IK1) and the carbacholine-induced inward rectifier (IKACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal IK1 of ventricular myocytes was blocked with apparent IC50-values of 4.6×10−6 M and 3.5×10−6 M for rat and fish, respectively. IKACh was almost an order of magnitude more sensitive to KB-R7943 than IK1 with IC50-values of 6.2×10−7 M for rat and 2.5×10−7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3×10−6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order IK1 ~INCX

Abstract: The shape of a neuron’s dendritic arbor is critical for its function as it determines the number of inputs the neuron can receive and how those inputs are processed. During development, a neuron initiates primary dendrites that branch to form a simple arbor. Subsequently, growth occurs by a process that combines the extension and retraction of existing dendrites, and the addition of new branches. The loss and addition of the fine terminal branches of retinal ganglion cells (RGCs) is dependent on afferent inputs from its synaptic partners, the amacrine and bipolar cells. It is unknown, however, whether neural activity regulates the initiation of primary dendrites and their initial branching. To investigate this, Xenopus laevis RGCs developing in vivo were made to express either a delayed rectifier type voltage-gated potassium (KV) channel, Xenopus Kv1.1, or a human inward rectifying channel, Kir2.1, shown previously to modulate the electrical activity of Xenopus spinal cord neurons. Misexpression of either potassium channel increased the number of branch points and the total length of all the branches. As a result, the total dendritic arbor was bigger than for control green fluorescent protein-expressing RGCs and those ectopically expressing a highly related mutant non-functional Kv1.1 channel. Our data indicate that membrane excitability regulates the earliest differentiation of RGC dendritic arbors. [Copyright &y& Elsevier]

Abstract: This work demonstrates that extracellular Na+ modulates the cloned inwardly rectifying K+ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na+. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na+]o and to investigate the regulatory mechanism of external cations further. Our results showed that Na+ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na+-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na+. The Na+ activation was voltage-independent, whereas the Na+-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na+on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms. [Copyright &y& Elsevier]

Abstract: Atrial fibrillation (AF) has been linked to increased inward rectifier potassium current, I K1, either due to AF-induced electrical remodelling, or from functional changes due to the Kir2.1 V93I mutation. The aim of this simulation study was to identify at cell and tissue levels'' mechanisms by which increased I K1 facilitates and perpetuates AF. The Courtemanche et al. human atrial cell action potential (AP) model was modified to incorporate reported changes in I K1 induced by the Kir2.1 V93I mutation in both heterozygous (Het) and homozygous (Hom) mutant forms. The modified models for wild type (WT), Het and Hom conditions were incorporated into homogeneous 1D, 2D and 3D tissue models. Restitution curves of AP duration (APD), effective refractory period (ERP) and conduction velocity (CV) were computed and both the temporal and the spatial vulnerability of atrial tissue to re-entry were measured. The lifespan and tip meandering pattern of re-entry were also characterised. For comparison, parallel simulations were performed by incorporating into the Courtmanche et al. model a linear increase in maximal I K1 conductance. It was found that the gain-in-function of V93I ‘mutant’I K1 led to abbreviated atrial APs and flattened APD, ERP and CV restitution curves. It also hyperpolarised atrial resting membrane potential and slowed down intra-atrial conduction. V93I ‘mutant’I K1 reduced the tissue''s temporal vulnerability but increased spatial vulnerability to initiate and sustain re-entry, resulting in an increased overall susceptibility of atrial tissue to arrhythmogenesis. In the 2D model, spiral waves self-terminated for WT (lifespan < 3.3 s) tissue, but persisted in Het and Hom tissues for the whole simulation period (lifespan > 10 s). The tip of the spiral wave meandered more in WT tissue than in Het and Hom tissues. Increased I K1 due to augmented maximal conductance produced similar results to those of Het and Hom Kir2.1 V93I mutant conditions. In the 3D model the dynamic behaviour of scroll waves was stabilized by increased I K1. In conclusion, increased I K1 current, either by the Kir2.1 V93I mutation or by augmented maximal conductance, increases atrial susceptibility to arrhythmia by increasing the lifespan of re-entrant spiral waves and the stability of scroll waves in 3D tissue, thereby facilitating initiation and maintenance of re-entrant circuits. [Copyright &y& Elsevier]

Abstract: Electrocytes are muscle-derived cells that generate the electric organ discharge (EOD) in most gymnotiform fish. We used an in vitro preparation to determine if the complex EOD of Gymnotus carapo was related to the membrane properties of electrocytes. We discovered that in addition to the three Na+-mediated conductances described in a recent paper [Sierra F, Comas V, Buño W, Macadar O (2005) Sodium-dependent plateau potentials in electrocytes of the electric fish Gymnotus carapo. J Comp Physiol A 191:1–11] there were four K+-dependent conductances. Membrane depolarization activated a delayed rectifier (IK) and an A-type (IA) current. IA displayed fast voltage-dependent activation-inactivation kinetics, was blocked by 4-aminopyridine (1 mM) and played a major role in action potential (AP) repolarization. Its voltage dependence and kinetics shape the brief AP that typifies Gymnotus electrocytes. The IK activated by depolarization contributed less to AP repolarization. Membrane hyperpolarization uncovered two inward rectifiers (IR1 and IR2) with voltage dependence and kinetics that correspond to the complex “hyperpolarizing responses” (HRs) described under current-clamp. IR1 shows “instantaneous” activation, is blocked by Ba2+ and Cs+ and displays a voltage and time dependent inactivation that matches the hyperpolarizing phase of the HR. The activation of IR2 is slower and at more negative potentials than IR1 and is resistant to Ba2+ and Cs+. This current fits the depolarizing phase of the HR. The EOD waveform of Gymnotus carapo is more complex than that of other gymnotiform fish species, the complexity originates in the voltage responses generated through the interactions of three Na+ and four K+ voltage- and time-dependent conductances although the innervation pattern also contributes [Trujillo-Cenóz O, Echagüe JA (1989) Waveform generation of the electric organ discharge in Gymnotus carapo. I. Morphology and innervation of the electric organ. J Comp Physiol A 165:343–351]. [Copyright &y& Elsevier]

Abstract: Inwardly rectifying K+ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2–Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2–Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E K were essentially time independent for Kir4.2 indicating rapid block by Mg2+. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity. [Copyright &y& Elsevier]

Background: Normal cardiac rhythm is critically dependent on the sinoatrial (SA) node, the natural biological pacemaker. Although recent studies have focused on the development of “artificial” biological pacemakers using gene transfer, less is known about the functional consequences of such interventions. Objective: The purpose of this study was to investigate the electrophysiological consequences of two approaches used to create a biological pacemaker: overexpression of the hyperpolarization-activated cyclic nucleotide gated channel (HCN “pacemaker” channels) and suppression of the inward-rectifier potassium current, IK1. Methods: We used a linear multicellular Luo-Rudy (LRd) AP model consisting of 130 ventricular cells connected by resistive gap junctions. To induce automaticity, IK1 current was reduced or If (HCN) current was introduced in endocardial and midmyocardial (M) cells. Results: Similar to the previously published results for a single LRd model, myocyte IK1 suppression induced automaticity in the fiber. While introduction of If also resulted in automaticity, the main differences between IK1 suppression and If expression were (1) a relatively more gradual phase 4 depolarization with HCN expression, (2) stabilization of cycle lengths during IK1 suppression, but not during HCN expression, and (3) responsiveness to β-adrenergic stimulation during HCN expression, but not during IK1 suppression. Upon further investigation, we found that cycle length instability during HCN expression was primarily due to a gradual reduction of intracellular potassium ([K+]i) from its baseline value of 142 mM to 120 mM in 600 beats and subsequent alteration of potassium-dependent ionic currents. A twofold increase in HCN expression also led to a similar behavior. We attribute this decrease in [K+]i to a large IK1 during phase 4 depolarization. When intracellular [K+]i loss was minimized, cycle lengths stabilized during HCN expression. Conclusions: Our results help to further understand the electrophysiologic consequences as well as some of the challenges associated with the creation of biological pacemakers using HCN and IK1 gene transfer strategies. [Copyright &y& Elsevier]

Abstract: In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP2. In cells loaded via patch pipettes with varying concentrations of Ca2+ buffered by 4mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca2+ (maximum at “100nM”, ∼10-fold inhibition at <10nM or ∼1μM). However, experiments where the total BAPTA concentration was varied whilst free [Ca2+] was maintained constant indicated that inhibition of PLC at higher (>100nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 ∼8mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 ∼1mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca2+ chelators, whilst non-physiologically low (<100nM) levels of Ca2+ suppress both basal and light-induced PLC activity. [Copyright &y& Elsevier]

The possible involvement of potassium channels in central chemosensitivity, with special reference to the Kir1.1 potassium channel, was investigated by studying the CO2 response of presympathetic neurons in the rostroventrolateral medulla (RVLM) in the absence or presence of various K+ channel inhibitors. Synaptic input to RVLM neurons was blocked by local injection of ω-agatoxin and ω-conotoxin. Activity of RVLM neurons was measured by recording the electrical activity in preganglionic (WR-T3) or postganglionic (renal) sympathetic nerves after perfusion of the lower brainstem via the left vertebral artery with CO2-enriched saline solution. Unspecific K+ channel blockade by BaCl2 reduced the excitatory response of sympathetic activity after CO2-perfusion to 56% of control. A quantitatively similar inhibition of the central CO2 response was obtained after administration of 9-fluorenylmethylchloroformate (FMOC-Cl) which eliminates pH sensitivity of Kir1 and Kir4.1. Furthermore, two structurally different Kir1 inhibiting toxins, tertiapin and Lq2, also reduced the central CO2 response to ∼50% of control. In contrast, charybdotoxin (CTX) had no effect on the CO2 response. Using RT-PCR the expression of mRNA homologous to rat Kir1 mRNA was identified in the cat medulla oblongata. These data suggest that a modulation of potassium channel activity possibly via Kir1 may contribute to central chemosensitivity. [Copyright &y& Elsevier]

Inhibition by GABAB receptors comprises activation of K+ conductance and inhibition of Ca2+ conductance, thereby reducing action potential dependent transmitter release and silencing neuronal activity. We compared epileptiform activity and its inhibition by the activation of GABAB receptors in homozygous weaver (wv/wv) and wild type (+/+) CA3 neurons disinhibited by GABAA receptor blockade. In wv/wv mice GABAB receptors have lost their ability to activate K+ conductance (J. Neurosci. 18 (1998) 4001). Spontaneous synchronous burst discharges in elevated [K+]o displayed only subtle differences in +/+ and wv/wv slices, except that the GABAB receptor agonist R-baclofen in low concentration (0.1 μM) strongly reduced the frequency of synchronous bursts in +/+ CA3 neurons, but not in wv/wv CA3 neurons. A high affinity GABAB antagonist, CGP55845A (0.5 μM) promoted the incidence of bursts in low [K+]o. Concentration dependence of the reduction of evoked EPSCs was identical in wv/wv and +/+ neurons (IC50=0.3 μM). Amplitudes of evoked IPSCs were reduced by 0.01 μM R-baclofen in +/+, but not in wv/wv CA3 neurons. The effect of the low concentration was abolished by Ba2+, which is known to block Kir conductance. The data suggest that activation of Kir conductance is important for the control of GABA release by GABAB autoreceptors in the CA3 network. We conclude that the loss of a contribution of Kir conductance to GABAB receptor-mediated autoinhibition reduces the inclination towards spontaneous bursts of wv/wv CA3 pyramidal neurons. [Copyright &y& Elsevier]

Potassium channel dysfunction has been indicated in Alzheimer''s disease. In the present study, spatial memory, the activity of cortical acetylcholinesterase (AChE) and the expressions of inward rectifier potassium channels (Kir) were measured after ibotenic acid lesions of nucleus basalis magnocellularis (nbm) in rats. Expressions of Kir (Kir2.1, Kir3.1, Kir6.1 and Kir6.2) at mRNA level were assessed using reverse transcription-polymerase chain reaction. At 28 days after ibotenic acid injection, the spatial memory of rats was significantly impaired accompanied by a 32% reduction of cortical AChE activity. Furthermore, the expression of Kir6.2 was increased by 79.3% in cortex, and that of Kir6.1 was increased by 172.1% in hippocampus, while no obvious changes in the mRNA expression of Kir2.1 and Kir3.1 were detected. This study indicated that the expression of adenosine triphosphate-sensitive potassium channel was area and channel subtype specifically increased following nbm lesions. [Copyright &y& Elsevier]