Kinetics Of IK Research Articles

Nociceptin/orphanin FQ-induced inhibition of delayed rectifier potassium currents by calcium/calmodulin-dependent protein kinase type II.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been shown to inhibit delayed rectifier potassium current (IK) in acutely dissociated rat parietal cortical neurons. However, the detailed mechanism of N/OFQ-induced inhibition on IK is not clear. This study is the first to explore an involvement of calcium/calmodulin (CaM)-dependent protein kinase type II (CaMKII) in mediating N/OFQ-induced responses. Utilizing pharmacological inhibitors of CaM and CaMKII, we have investigated the contribution of CaMKII in N/OFQ-induced effects using the whole-cell patch-clamp technique. In whole-cell voltage clamp, W-7 (100 μM), an antagonist of CaM, as well as KN-62, an inhibitor of CaMKII activity, attenuated the inhibitory effects of N/OFQ on IK. Activation and inactivation analysis indicated that the kinetics of IK were altered by N/OFQ, with decreased activation and promoted inactivation of IK. W-7 and KN-62 (10 μM) partly abolished the activation and inactivation curves shift of IK induced by N/OFQ. These findings show that CaMKII plays a critical role in N/OFQ-induced inhibition of IK in acutely dissociated rat parietal cortical neurons.

Propofol inhibited the delayed rectifier potassium current (Ik) via activation of protein kinase C epsilon in rat parietal cortical neurons

Propofol has been shown to exert neuroprotective effects. Delayed rectifier potassium current (IK) was reported to be closely related to neuronal damage. This study was designed to test the effects of propofol on IK in rat parietal cortical neurons and the involvement of PKC in this activity. Whole-cell patch-clamp recordings were performed in rat parietal cortical neurons. The amplitudes of IK were recorded before and after the addition of different concentrations of propofol. Propofol concentration-dependently inhibited IK with an IC50 value of 36.3±2.7μM. Moreover, propofol caused a downward shift of the I–V curve of IK in a concentration dependent manner. The kinetics of IK was altered by propofol, with decreased activation and delayed recovery of IK. Pretreatment with calphostin-C (a non-selective inhibitor of PKC) or PKC epsilon translocation inhibitor peptide (PKC epsilon inhibitor) abrogated the inhibition of IK by propofol. In conclusion, propofol inhibited IK via the activation of PKC epsilon in rat cerebral parietal cortical neurons.

Effect of the Antifibrillatory Compound Tedisamil (KC-8857) on Transmembrane Currents in Mammalian Ventricular Myocytes

The cellular mechanism of action of tedisamil (KC-8857) (TED), a novel antiarrhythmic/antifibrillatory compound, was studied on transmembrane currents in guinea pig, rabbit and dog ventricular myocytes by applying the patch-clamp and the conventional microelectrode technique. In guinea pig myocytes the rapid component of the delayed rectifier potassium current (IKr) was largely diminished by 1 microM TED (from 0.88+/-0.17 to 0.23+/-0.07 pA/pF, n=5, p<0.05), while its slow component (IKs) was reduced only by 5 microM TED (from 8.1+/-0.3 to 4.23+/-0.07 pA/pF, n=5, p<0.05). TED did not significantly change the IKr and IKs kinetics. In rabbit myocytes 1 microM TED decreased the amplitude of the transient outward current (I(to)) from 20.3+/-4.9 to 13.9+/-2.8 pA/pF (n=5, p<0.05), accelerated its fast inactivation time constant from 8.3+/-0.6 to 3.5+/-0.5 ms (n=5, p<0.05) and reduced the ATP-activated potassium current (IKATP) from 38.2+/-11.8 to 18.4+/-4.7 pA/pF (activator: 50 microM cromakalim; n=5, p<0.05). In dog myocytes 2 microM TED blocked the fast sodium current (INa) with rapid onset and moderately slow offset kinetics, while the inward rectifier potassium (IK1), the inward calcium (ICa) and even the I(to) currents were not affected by TED in concentration as high as 10 microM. The differences in I(to) responsiveness between dog and rabbit are probably due to the different alpha-subunits of I(to) in these species. It is concluded that inhibition of several transmembrane currents, including IKr, IKs, I(to), IKATP and even INa, can contribute to the high antiarrhythmic/antifibrillatory potency of TED, underlying predominant Class III combined with I A/B type antiarrhythmic characteristics.

Temperature dependence of bistability in squid giant axons with alkaline intracellular pH.

Raising the intracellular pH (pHi) above 7.7 in intracellularly perfused squid giant axons causes spontaneous firing of action potentials. The firing frequency ranged from 20 Hz at 0 degrees C to 200 Hz at 23 degrees C. Above 23 degrees C, the axons were quiescent. They were bistable for 13 <T <23 degrees C. That is, they were either quiescent or spontaneously firing. Below 13 degrees C, spontaneous firing was the only stable element. The primary effects of changes in temperature on the underlying ionic currents were on gating of the delayed rectifier potassium channel IK, and the sodium ion channel INa. The kinetics of IK had a Q10 of 3.63. The effect of T on INa was more complicated in that the peak INa amplitude increased with T, as demonstrated in earlier reports. This effect, as well as the changes in INa kinetics produced by changes in T, were mimicked in the context of a model of INa gating in which activation and inactivations are coupled. Electrical activity was simulated in a model of the action potential with appropriate temperature-dependent modifications for INa and IK. The model predicts a change from monostability (spontaneous firing) at relatively low temperatures to bistability (quiescence and spontaneous firing) as the temperature is raised, followed by change back to monostability (quiescence) as the temperature is further increased, which is consistent with the experimental results.

Overexpressed A 1 Adenosine Receptors Reduce Activation of Acetylcholine-Sensitive K + Current by Native Muscarinic M 2 Receptors in Rat Atrial Myocytes

In adult rat atrial myocytes, muscarinic acetylcholine (ACh)-sensitive K(+) current activated by a saturating concentration of adenosine (I(K(ACh),(Ado))) via A(1) receptors (A(1)Rs) amounts to only 30% of the current activated by a saturating concentration of ACh (I(K(ACh),(ACh))) via muscarinic M(2) receptors. The half-time of activation of I(K(ACh),(Ado)) on a rapid exposure to agonist was approximately 4-fold longer than that of I(K(ACh),(ACh)). Furthermore, I(K(ACh),(Ado)) never showed fast desensitization. To study the importance of receptor density for A(1)R-I(K(ACh),(Ado)) signaling, adult atrial myocytes in vitro were transfected with cDNA encoding for rat brain A(1)R and enhanced green fluorescent protein (EGFP) as a reporter. Whole-cell current was measured on days 3 and 4 after transfection. Time-matched cells transfected with only the EGFP vector served as controls. In approximately 30% of EGFP-positive cells (group I), the density of I(K(ACh),(Ado)) was increased by 72%, and its half-time of activation was reduced. Density and kinetic properties of I(K(ACh),(ACh)) were not affected in this fraction. In approximately 70% of transfection-positive myocytes (group II), the density of I(K(ACh),(ACh)) was significantly reduced, its activation was slowed, and the fast desensitizing component was lost. Adenosine-induced currents were larger in group II than in group I, their activation rate was further increased, and a fast desensitizing component developed. These data indicate that in native myocytes the amplitude and activation kinetics of I(K(ACh),(Ado)) are limited by the expression of A(1)R. Overexpression of A(1)R negatively interferes with signal transduction via the muscarinic M(2) receptor-linked pathway, which might reflect a competition of receptors with a common pool of G proteins. Negative interference of an overexpressed receptor with physiological regulation of a target protein by a different receptor should be considered in attempts to use receptor overexpression for gene therapy.

K(+)-sensitive gating of the K+ outward rectifier in Vicia guard cells.

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1-30 mM K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of -200 mV, clamp steps to voltages between -150 and +50 mV in 0.1 mM K+ activated current through outward-rectifying K+ channels (IK,out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]o shifted the voltage-sensitivity of IK,out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]o was evident in the kinetics of IK,out activation and deactivation, as well as the steady-state conductance-(g kappa-) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near -130 mV in 0.1 mM K+, -80 mV in 1.0 mM K+, and -20 mV in 10 mM K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+ > or = Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mM was without effect on the voltage-dependence of g kappa or on IK,out activation kinetics, although 10 mM [Ca2+]o accelerated current deactivation at voltages negative of -75 mV. At any one voltage, increasing [K+]o suppressed g kappa completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mM with clamp voltages near -100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of IK,out gating; the results implicate K(+)-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K+ ions outside affects the distribution between closed states of the channel.

Fosinoprilate prolongs the action potential: reduction of iK and enhancement of the L-type calcium current in guinea pig ventricular myocytes.

The aim was to study the effect of fosinoprilate, a new ACE inhibitor, on the action potential and plateau currents of cardiac muscle. Whole cell patch technique was used to record action potentials (n = 6), the L-type iCa (iCaL; n = 5), in some cases (n = 4) also using Cs+ loaded pipettes; with 5 mM Co2+, the time dependent K+ current (IK) underlying delayed rectification was analysed in guinea pig ventricular myocytes (n = 3). Fosinoprilate prolonged the 50% repolarisation (APD50) from 440(SEM 50) ms to 485(48) ms (0.1 microM), to 525(46) ms (0.3 microM), to 632(58) ms (1 microM), and to 702(69) ms (3.0 microM). The APD90 was delayed from 510(63) ms to 540(45) ms (0.1 microM), to 583(42) ms (0.3 microM), to 702(62) ms (1.0 microM), and to 765(72) ms (3.0 microM). Higher concentrations (10-100 microM) caused early afterdepolarisations, very long action potentials, and irregular oscillations. ICaL was enhanced by up to 183%, showing a Kd of 0.2 microM; in contrast to the steady state activation (d infinity), the inactivation curve f infinity was shifted in the depolarising direction, considerably enlarging the Ca2+ window. Slow inactivation time course was unchanged, whereas the fast time constant (tau f) was accelerated. Fosinoprilate reduced the outward current during depolarising clamps from 1.7(0.2) nA to 1.41(0.11) nA with a 0.1 microM dose, and to 0.54(0.14) nA with a 1.0 microM dose; the tails were decreased from 0.39(0.03) nA to 0.27(0.03) nA with 0.1 microM and to 0.13(0.02) nA with 1.0 microM. Kinetics of IK were unaltered. Computer simulations based on these data using the OXSOFT-HEART program mimicked the results rather closely. The results suggest that fosinoprilate prolongs the plateau due to a partial block of iK and an extension of the Ca2+ window by 10 mV, causing a class III antiarrhythmic effect. High concentrations further open the Ca2+ window resulting in early afterdepolarisations and plateau oscillations and may cause an inward transport of Ca2+ ions by the Na-Ca exchange.

Insensitivity of guinea pig ventricular delayed rectifier IK to intracellular trypsin: implications for channel structure and function.

Intracellular application of proteolytic agents modifies the function of many voltage gated ion channels. The presence of a trypsin sensitive inhibitory domain in a channel protein may be important for G protein dependent activation. Guinea pig ventricular IK is modulated by a direct G protein pathway. The aim was to determine if guinea pig ventricular IK is also modified by intracellularly applied trypsin. Whole cell and excised inside out configurations of patch clamp were used to record IK from guinea pig ventricular myocytes before and after cytosolic application of trypsin (1 mg.ml-1). We used previously reported effects of trypsin on the L type calcium current (ICa) to monitor dialysis time and enzyme activity in whole cell experiments where IK and ICa were measured concomitantly. Addition of trypsin to the solution bathing the cytosolic face of excised membrane patches had no effect on the amplitude or kinetics of IK. When added to the pipette solution and introduced by cell dialysis, trypsin had no effect on whole cell IK, even when significant effects on the amplitude and kinetics of ICa were evident. Guinea pig ventricular IK is not enhanced or otherwise altered by intracellularly applied trypsin. Therefore direct phosphorylation independent enhancement of IK by guanine nucleotides cannot depend on interactions between G protein subunits and trypsin sensitive inhibitory channel domains. The lack of trypsin modification of cardiac ventricular IK suggests that the structure of the endogenous delayed rectifier K+ channel may be different than that of other voltage gated channels.

Quinidine‐induced inhibition of the fast transient outward K+ current in rat melanotrophs

1. The effect of quinidine on the fast-activating, fast-inactivating potassium current (IK(f] in acutely dissociated melanotrophs of the adult rat pituitary was examined. Macroscopic currents were measured by use of the whole-cell configuration of the patch clamp technique. 2. Bath application of quinidine caused a dose-dependent reduction of the peak amplitude of IK(f). The Kd for blockade of IK(f) at 0 mV was estimated to be 41 +/- 5.6 microM. 3. Quinidine elicited a dose-dependent increase of the rate of the decay of IK(f) and this effect was enhanced by membrane depolarization. The possibility that this phenomenon reflects an open channel blocking reaction is discussed. 4. Quinidine also caused a 5 mV hyperpolarizing shift of the steady-state inactivation curve and increased the half-time for recovery from inactivation. Quinidine did not affect the onset of inactivation measured at -30 mV. 5. Internal quinidine did not appear substantially to affect either the peak amplitude or kinetics of IK(f). 6. A study of some structural analogues showed that hydroquinidine and quinacrine had effects similar to those of quinidine. The effect of quinacrine on the amplitude and kinetics of IK(f) was also pH-dependent. Cinchonine, which bears a close structural resemblance to quinidine, was much less effective as a blocker of IK(f).

Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents.

beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase.

Inhibitory action of 711389-S on the electrical activity of rabbit sinoatrial node.

The effects of 711389-S (0.1-10 mumol.litre-1) on membrane potentials and currents of rabbit sinoatrial node preparations were studied using a conventional double microelectrode voltage-clamp method. The agent 711389-S decreased the heart rate, the maximum rate of depolarisation, and the amplitude of the action potential and increased the action potential duration in a dose dependent manner. The slope of the diastolic depolarisation was also reduced. Of the current systems of sinoatrial node, 711389-S depressed the slow inward current (Isi), the potassium outward current (IK), and the hyperpolarisation activated current (Ih) dose dependently. The kinetics of IK were not altered significantly by the drug. It is concluded that 711389-S does not have an effect on a single current system but that the drug exerts a depressant effect on the electrical activity of the sinoatrial node.

Differentiation of voltage‐gated potassium current and modulation of excitability in cultured amphibian spinal neurones.

Gigaohm-seal whole-cell voltage-clamp techniques were used to study the development of ionic currents in the membrane of embryonic amphibian (Ambystoma) spinal neurones during in vitro differentiation. Dissociated neural plate cells, some of which are neuronal precursor cells, were placed into culture. Cells became excitable at the time of neurite outgrowth, 2-3 days later, and over the next 2-10 days the duration of the action potential shortened from about 100 ms to about 1 ms. Voltage-clamp recordings demonstrated that at the time of appearance of neurites, activatable Na, Ca and voltage-gated K channels were present in the membrane (Ca-dependent K channels were not studied). Over succeeding days in culture, records of total membrane current indicated that the amplitudes of peak inward and steady-state outward currents both increased. As a result of these increases, the pattern of total membrane current came to be increasingly dominated by outward currents. With inward Na and Ca currents blocked, a voltage-gated K current (IK(V] could be studied in isolation. The reversal potential of this current varied in good agreement with the equilibrium potential for K ions predicted by the Nernst relation. The wave form of IK(V) activation was sigmoidal. Activation was more rapid at more positive voltages (relative to the usual holding potential of -70 mV), and deactivation was more rapid at more negative voltages. The amplitude of IK(V) increased during neural development, while cell size remained approximately constant. Increases in rates of activation and deactivation were observed in parallel with the increase in current density. When measured at 0 mV, cells studied on day 4 of culture or earlier showed steady-state chord conductances (gK(V] of less than 20 nS, and one-half activation times (t1/2) of 2 X 5-10 ms. Older cells showed gK(V)s of 10-80 nS, and t1/2s of 0 X 8-2 X 5 ms. As Na, and to a lesser extent Ca, current amplitudes were also increasing during differentiation, these observations concerning IK(V) suggested that its amplitude and kinetic changes might in part be responsible for the observed decrease in action potential duration during development. This hypothesis was tested by modelling Na, Ca and voltage-gated K currents and testing the effects of changes in amplitude and kinetics of IK(V) on the duration and ionic dependence of reconstructed action potentials. The results obtained using this model suggested that the increase in IK(V) amplitude and activation rate was sufficient to change action potential duration and apparent ionic sensitivity.

Studies on the bradycardia induced by aprindine in rabbit sinoatrial node cells.

The effects of aprindine (1 X 10(-7) to 4 X 10(-6) M) were examined on membrane potential and current of rabbit sinoatrial node by means of conventional microelectrode and double microelectrode voltage clamp methods. Aprindine decreased, in a dose-dependent manner, the spontaneously firing frequency, the maximum rate of depolarization and the action potential amplitude, and prolonged the action potential duration at half-amplitude. The slope of the diastolic depolarization was also reduced by the drug. In the voltage clamp experiment, aprindine reduced the slow inward current (Isi), the time-dependent potassium current (Ik) and the hyperpolarization activated current (Ih). The recovery time constant of Isi was prolonged by aprindine, while the kinetics of Ik was not altered. It is indicated that aprindine does not have an effect on a specific conductance or a single current system, but that the drug exerts an inhibitory effect on the electrical activity of sinoatrial node.