The inwardly-rectifying K+ current (IK1) is important for heart cell function because it sets the resting potential, influences cell excitability, and promotes repolarization of the action potential. My objective was to investigate the modulation of IK1 by extracellular K+ (K+o) and intracellular K+ (K+i). IK1 was recorded from whole-cell-configured guinea-pig ventricular myocytes that were dialyzed with Na+-free EGTA-buffered pipette-filling solution and bathed with Na+ or NMDG+ solution that contained agents that inhibit non-IK1 currents. Lowering K+o from standard 5.4 to 2 and 1 mM shifted the reversal potential (Erev) of IK1 in accord with calculated K+ equilibrium potential (EK), and altered IK1 amplitude in accord with conductance (GK1)? ?K+o. Surprisingly, myocytes bathed with 0-mM K+ solution had a small outward IK1 at holding potential (Vhold) ?85 mV. This IK1 was attributed to channel-activation by T-tubular K+ (K+T) whose concentration is sensitive to the flow of T-tubular IK1. K+T in myocytes bathed with 0-mM K+ solution was ? 3.2 mM at Vhold ?85 mV, but ? 0.3 mM following large K+T-depleting flows of inward IK1 at ?160 mV. Results consistent with interplay of IK1 and K+T were also obtained in experiments on myocytes bathed with 2-, 5.4-, and 15-mM K+ solution. Myocytes were dialyzed with pipette solutions that contained 0-140 mM K+ to investigate modulation by K+i. When IK1 at Vhold was kept small, Erev varied with pipette K+ in a near-Nernstian manner (i.e., Erev ? EK); however, when IK1 (Vhold) was large and inward, Erev was markedly negative to nominal EK. Findings in experiments that involved shifting Vhold, changing K+o, and application of Ba2+ and Cs+ suggest that the magnitude/direction of IK1 strongly affects the concentration of K+ in submembrane cytoplasm. Classical GK1-voltage parameters GK1max and V0.5 (but not slope factor) were affected by decreases in K+i: GK1max declined by ? 25% per decade decrease in K+i, and V0.5 shifted approximately as 0.5 ? EK-shift. The latter findings are discussed and compared with those of earlier studies on the dependence of inwardly-rectifying K+ conductance on K+i.

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Sepsis has deleterious effects on cardiac function including reduced contractility. We have shown previously that lipopolysaccharides (LPS) directly affect HL-1 cardiac myocytes by inhibiting Ca2+ regulation and by impairing pacemaker "funny" current, If. We now explore further cellular mechanisms whereby LPS inhibits excitability in HL-1 cells. LPS (1 jxg/ml) derived from Salmonella enteritidis decreased rate of firing of spontaneous action potentials in HL-1 cells, and it increased their pacemaker potential durations and decreased their rates of depolarization, all measured by whole cell current clamp. LPS also increased action potential durations and decreased their amplitude in cells paced at 1 Hz with 0.1 nA, and 20 min were necessary for maximal effect. LPS decreased the amplitude of a rapidly inactivating inward current attributed to Na+ and of an outward current attributed to K+; both were measured by whole cell voltage clamp. The K+ currents displayed a resurgent outward tail current, which is characteristic of the rapid delayed-rectifier K+ current, Ikr. LPS accordingly reduced outward currents measured with pipette Cs+ substituted for K+ to isolate Ikr. E-4031 (1 (xM) markedly inhibited Ikr in HL-1 cells and also increased action potential duration; however, the direct effects of E-4031 occurred minutes faster than the slow effects of LPS. We conclude that LPS increases action potential duration in HL-1 mouse cardiomyocytes by inhibition of Ikr and decreases their rate of firing by inhibition of Ina. This protracted time course points toward an intermediary metabolic event, which either decreases available mouse ether-a-go-go (mERG) and Na+ channels or potentiates their inactivation.

No / Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage-activated K+ (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin-1 (ET-1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose-free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC-IP), and Kv currents were further reduced in 10 mm glucose. In current-clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mmd-glucose, 10 nm ET-1 decreased peak Kv current amplitude at +60 mV from 23.5 ± 3.3 to 12.1 ± 3.1 pA pF−1 (n = 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET-1 was prevented by PKC-IP. When d-glucose was increased to 10 mm, ET-1 no longer inhibited Kv current (n = 6). Glucose metabolism was required for prevention of ET-1 inhibition of Kv currents, since fructose mimicked the effects of d-glucose, while l-glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mmd-glucose, since pinacidil-activated ATP-dependent K+ (KATP) currents were reduced by 10 nm ET-1. This inhibition was nearly abolished by PKC-IP, indicating that endothelin receptors could still activate PKC in 10 mmd-glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.

Yes / Introduction:The human ether-a-go-go related gene (hERG) encodes the pore forming protein which mediates the rapid delayed rectifier K+ current in the heart (IKr). Together with other ion channels hERG determines the cardiac action potential and regulates the heart beating. Dysfuction of the hERG ion channel will lead to acquired long QT syndrome (LQTS). Therefore, new drug candidates must pass the test for a potential inhibitory effect on the hERG current as a first step in a nonclinical testing strategy. Arrhythmias in patients with LQTS are typically triggered during physical or emotional stress, suggesting a link between sympathetic stimulation and arrhythmias. It is well known that potassium level can affect the QT interval through affecting IhERG both in vivo and in vitro.In this study, we try to find out whether the trigger effect still exist when K+ changes violently in a short time period. In other words, whether the risk of TdP aggravate when patients suffer from acute water electrolyte balance disorder, which is a common symptom in hot weather. Methods: HEK293 Cell line stably expressing hERG channel were cultured in DMEM supplemented with 10% of fetal bovine serum.Whole-cell patch-clamp method was applied for ionic current recordings. The compositions of pipette was (in mM) 125 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 5 Na-ATP adjusted to pH 7.2 with KOH. The bath solutions for recording the IhERG currents was 136 NaCl, 4 KCl, 1 MgCl2, 10 HEPES-Na, 1.8 CaCl2 and 10 glucose, pH 7.4 with NaOH. The low extracellular K+ solution was 115 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 10 Na-ATP adjusted to pH 7.2 with NaOH. Patch-clamp experiments were performed at room temperature (22 ± 1°C). The recording of low K+ current was carried out immediately after the original normal K+ solution has been totally replaced. Isoproterenol (ISO) 100nM was added into both kinds of K+ solution to apply the effect of β1-AR stimulation. Results: We found that low K+ solution increased IhERG from 907.39±18.68to 1620.08±249.44pA(n=30,P<0.05); Low K+also shifted the I-V curve to the left. IC50 in control is 10.31±5.52 mV, low K+ is -6.15±1.58 mV. When adding ISO 100nM to extracellular solution, same effects were shown for both groups.ISO decreased Imax for both group. In control group, Imax reduced from 907.39±18.68to493.16±54.41pA (n=30, P<0.01), while in low K+ group, I max decreased Imax from 1620.08±29.44to 488.48±81.87pA(n=30,P<0.05). At the same time, ISO shifts the I-V curve to the right for the control group and shift the curve to the left for low K+ group. IC50 in control when added ISO is 22.25±3.80 mV, while IC50 in low K+ group after adding 100nM ISO is -31.00±5.73 mV. Conclusion: The results from this study is contradict to those in our previous study where low K+ combined with ISO can lead to temporarily increase of QT interval in vivo.It is reported that an increase in net outward repolarizing current, due to a relatively large increase of IKs, is responsible for the changes of QT interval in response to beta-adrenergic stimulation in vivo(2). Therefore future studies need to co-transfect IKs channel to confirm this. References: 1. Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, et al. Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. The Journal of clinical investigation. 2009;119(9):2745- 57. 2. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. Journal of the American College of Cardiology. 2000;35(3):778-86.

Der neuronale Zelluntergang bei einer Vielzahl von Krankheiten des ZNS, wie z.B. Morbus Alzheimer (AD) und Temporallappenepilepsie (TLE), wird mit oxidativem Stress sowie Fehlfunktionen von Kaliumkanälen in Verbindung gebracht. In dieser Studie soll die selektive neuronale Sensitivität auf oxidativen Stress durch die Messung der oxidativen Modulation von Kaliumströmen untersucht werden. Dabei werden sternförmige Neuronen der zweiten Schicht des entorhinalen Kortex (EC) (bei AD bereits früh geschädigt) mit pyramidalen Neuronen der dritten Schicht des EC (früh geschädigt bei TLE) sowie hippocampalen pyramidalen Neuronen der CA1 Region (bei AD und TLE erst spät geschädigt) miteinander verglichen. Mittels patch-clamp Ganzzellmessung zeigt diese Studie die differentielle Hemmung spannungsabhängiger transienter (IA) und „delayed-rectifier“ K+-Ströme (IK(V)) durch Arachidonsäure (AA) und Wasserstoffperoxid (H2O2). Die intrazelluläre Applikation von AA (1 pM) reduzierte IA in Neuronen des entorhinalen Kortex signifikant stärker verglichen mit Neuronen des CA1. ETYA imitiert diesen Effekt, dies schliesst die Metabolite der AA als Mediatoren des Effekts auf Kaliumkanäle aus. Weder AA noch ETYA reduzierten IK(V). Im Gegensatz dazu reduzierte H2O2 IA in Neuronen des CA1 effektiver als in Neuronen der Schichten II und III des entorhinalen Kortex. Die Reduktion des IA, vermittelt durch AA, wurde durch Radikalfänger (Glutathion, Ascorbinsäure, Vitamin E Analogon Trolox) blockiert. Dabei verstärkten manche dieser Antioxidantien den Effekt der AA, dies legt eine komplexere Modulation dieser Ströme in Schnitten verglichen mit Kulturen nahe. Dies sollte bei der Entwicklung antioxidativer Therapien von AD und TLE berücksichtigt werden. Bei der heterologer Expression von Kv1.4 und Kv4.2 in HEK-293 Zellen wurden funktionelle Kanäle gebildet und A-Typ Ströme ausgelöst. Diese Ströme wurden nach der Applikation von 1 pM AA stark reduziert. ROS scheinen neben ihrer zellschädigenden Wirkung physiologische Prozesse zu regulieren, indem sie eine Reihe von Signalwegen beeinflussen. Da spannungsabhängige Kaliumkanäle vielen wichtigen zellulären Funktionen zugrundeliegen, könnte die Modulation dieser Kanäle durch ROS einen Mechanismus für die Feinabstimmung zellulärer Prozesse darstellen. / Oxidative stress and dysfunction of potassium channels are believed to play a role in neuronal death in a number of CNS diseases (e.g. Alzheimer’s disease, epilepsy). The present study addresses selective neuronal vulnerability to oxidative stress by studying oxidative modulation of potassium channels in entorhinal cortex (EC) layer II stellate neurons (cell loss early in AD) and layer III pyramidal neurons (early damage in TLE), in comparison to hippocampal CA1 pyramidal neurons (late damage in TLE and AD). Using whole-cell patch-clamp, differential inhibition of transient IA and delayed rectifier K+-currents IK(V) by arachidonic acid (AA) and H2O2 was demonstrated. Intracellular AA (1 pM) reduced IA in EC neurons significantly stronger than in CA1 neurons. AA affected the voltage dependence of steady-state inactivation as well. ETYA mimicked the effect of AA, excluding its metabolites as mediators of IA modulation. Neither AA nor ETYA reduced IK(V). In contrast, a non-lipid oxidizing agent, H2O2 reduced IA more effectively and robustly attenuated IK(V) in CA1, compared to EC neurons. AA-mediated reduction of IA was blocked by free radical scavengers (glutathione, ascorbic acid, Trolox). Antioxidants did not simply inhibit AA and H2O2 effects. In particular, they even enhanced AA effects, suggesting more complex modulation of these currents in slices, compared to culture. Moreover, intracellular antioxidants, themselves, influenced maximal conductance and voltage-conductance characteristics of IA and IK(V). This should be considered in design of anti-oxidative therapies in AD and TLE. Heterologous expression of Kv1.4 and of Kv4.2 cDNA in HEK-293 cells formed functional channels and elicited A-type currents, which shared similar biophysical characteristics with native IA from the hippocampus. These currents were strongly decreased upon administration of 1pM AA, demonstrating that at least one of multiple sites for AA action is situated on the pore-forming alfa-subunit of the A-channel. In conclusion, beside contribution to cell damage, ROS may regulate physiological processes by acting on different signalling pathways. Since voltage-gated K+-channels underlie many important cellular functions modulation of these channels by ROS would represent a mechanism for fine tuning of cellular processes.