Component Of Potassium Current Research Articles

High fat diet induces change in mitochondria respiration, activation of K-ATP channel and action potential shortening in mice

Obesity is associated with an increased risk of atrial fibrillation (AF). This epidemiologic observation may not be only due to shared co-morbidities but also from direct impacts of metabolic disorders on myocardium. To examine the impact of high fat diet (HFD) induced obesity on electrical properties in mice atria. Eight weeks old C57Bl/6 J mice were subjected to 4 months of HFD (60% fat) or normal diet (ND, 4% fat). Rapid burst atrial pacing were delivered using a trans-esophageal probe to induced AF in anesthetized animals. Action potential (AP) were recorded in left atrium using glass microelectrode technique. Potassium currents were recorded in isolated atrial myocytes using whole cell or perforated patch-clamp configurations. Mitochondrial respiration was studied in saponin-permeabilized atrial muscle fibers using a Clarke electrode. HFD mice showed higher atrial vulnerability to AF as indicated by longer AF episodes. APs were shorter in HFD versus ND mice (AP duration measured at 90% of repolarization: 49.3 ± 1.6 msec in HFD vs. 56.7 ± 2.3 msec in ND, P < 0.01). Glibenclamide, a KATP channel blocker, reversed AP shortening whereas cromakalim, a KATP channel activator, had no effect. Only in perforated patch-clamp configuration, without intracellular dialysis, KATP component of potassium current was enhanced in HFD mice (at +70 mV: 0.41 ± 0.12 pA/pF in HFD vs. 0.13 ± 0.08 pA/pF in ND, P < 0.05). There was no change in mitochondria respiration capacity between ND and HFD, however the ability of palmitoyl-CoA to support mitochondrial respiration was higher in HFD (25.99 ± 4.11% vs. 46.9 ± 3.96% in ND, P = 0.001). In addition, HADHA activity was enhanced in HFD mice. Obesity in mice is associated with atrial AP shortening which appears to result from increased activity of KATP-regulated currents and be associated with a metabolic shift. Shortening of AP duration could contribute to higher vulnerability to AF during obesity.

Pharmacological evaluation of R(+)-pulegone on cardiac excitability: Role of potassium current blockage and control of action potential waveform

IntroductionR(+)-pulegone is a ketone monoterpene and it is the main constituent of essential oils in several plants. Previous studies provided some evidence that R(+)-pulegone may act on isolated cardiac myocytes. In this study, we evaluated in extended detail, the pharmacological effects of R(+)-pulegone on cardiac tissue. MethodsUsing in vivo measurements of rat cardiac electrocardiogram (ECG) and patch-clamp technique in isolated myocytes we determinate the influence of R(+)-pulegone on cardiac excitability. ResultsR(+)-pulegone delayed action potential repolarization (APR) in a concentration-dependent manner (EC50=775.7±1.48, 325.0±1.30, 469.3±1.91μM at 10, 50 and 90% of APR respectively). In line with prolongation of APR R(+)-pulegone, in a concentration-dependent manner, blocked distinct potassium current components (transient outward potassium current (Ito), rapid delayed rectifier potassium current (IKr), inactivating steady state potassium current (Iss) and inward rectifier potassium current (IK1)) (EC50=1441±1.04; 605.0±1.22, 818.7±1.22; 1753±1.09μM for Ito, IKr, Iss and IK1, respectively). The inhibition occurred in a fast and reversible way, without changing the steady-state activation curve, but instead shifting to the left the steady-state inactivation curve (V1/2 from −56.92±0.35 to −67.52±0.19mV). In vivo infusion of 100mg/kg R(+)-pulegone prolonged the QTc (∼40%) and PR (∼62%) interval along with reducing the heart rate by ∼26%. ConclusionTaken together, R(+)-pulegone prolongs the APR by inhibiting several cardiomyocyte K+ current components in a concentration-dependent manner. This occurs through a direct block by R(+)-pulegone of the channel pore, followed by a left shift on the steady state inactivation curve. Finally, R(+)-pulegone induced changes in some aspects of the ECG profile, which are in agreement with its effects on potassium channels of isolated cardiomyocytes.

Voltage-Gated Potassium Currents Are Targets of Diurnal Changes in Estradiol Feedback Regulation and Kisspeptin Action on Gonadotropin-Releasing Hormone Neurons in Mice1

Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, I(A), and a sustained current, I(K). Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either I(A) or I(K) in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, I(A) and I(K) were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased I(A) in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced I(A) regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized I(A) activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.

Role of a Tetraethylammonium-Sensitive Component of Potassium Currents in High-Frequency Tonic Impulsation Generated by Rat Retinal Ganglion Cells

Using the voltage/current clamp technique in the whole-cell configuration, we studied the role of the highly tetraethylammonium (TEA) -sensitive component of integral potassium current in the generation of high-frequency tonic impulsation by rat retinal ganglion cells (RGCs). Application of 0.5 mM TEA led to a decrease in the frequency of evoked tonic impulsation by RGCs by 63% (from 55 ± 10 sec–1 in the control to 26 ± 5 sec–1 in the presence of the blocker; n = 11). In this case, the duration of single action potentials at the level of 50% their amplitude increased by 64% (from 1.1 ± 0.1 to 1.8 ± 0.1 msec; n = 11), the rate of repolarization decreased by 54% (from −101 ± 9 to −46 ± 5 mV/msec; n = 11), and the amplitude of afterhyperpolarization dropped by 62% (from −16 ± 2 to −6 ± 2 mV; n = 11). Upon the action of 0.5 mM TEA, the amplitude of the integral potassium current in RGCs decreased; the current component sensitive to the above blocker was equal to 0.41 ± 0.05 nA (n = 6), while the respective value in the control was 1.62 ± 0.14 nA (n = 12). Thus, a moderate (on average, by 25%) decrease in the amplitude of the above potassium current significantly influenced the characteristics of impulse activity generated by RGCs. The TEA-sensitive component of the current was similar to the Kv3.1/Kv3.2 potassium current described earlier. The obtained data are indicative of the key role of the highly TEA-sensitive component of the potassium current (passed probably via Kv3.1/Kv3 channels) in high-frequency tonic activity generated by RGCs.

Probing glycine receptor stoichiometry in superficial dorsal horn neurones using thespasmodicmouse

Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of α and β subunits assembled in a 2:3 stoichiometry. The α1/βheteromer is considered the dominant GlyR isoform at 'native' adult synapses in the spinal cord and brainstem. However, the α3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I-II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the α1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH.We usedwhole cell recording (at 22-24◦C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg⁻¹, I.P.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic micewere reduced by 25% and 50%, respectively (42.0 ± 3.6 pA vs. 31.0 ± 1.8 pA, P <0.05 and 7.4 ± 0.5 ms vs. 5.0 ± 0.4 ms, P <0.05; means ± SEM, n =34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABAAergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50 =130 ± 20 μM vs. 64 ± 11 μM, respectively; n =8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of α1-containing GlyRs in SDHneurones.Glycine sensitivity, based on the response to 1-3 μM glycine, was reduced in>75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design therapies that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.

Isolation and possible role of fast and slow potassium current components in hair cells dissociated from frog crista ampullaris

Potassium-current inactivation and recovery kinetics are pivotal in sustaining dynamic processing of time-varying sensory signals in hair cells. We report a detailed analysis of K(+)-currents in isolated hair cells from the frog crista ampullaris. The single components were dissected using a novel procedure based on their differential kinetic properties: The fast IA component exhibited two processes of inactivation removal; the persistent I (KD) component (I (KV) + I (KCa)), unexpectedly displayed partial inactivation, removed by negative potentials with particularly slow, delayed kinetics. The physiological relevance of these observations was investigated by imposing sinusoidal membrane potential changes to mimic receptor response to hair bundle deflection. The excitatory phase elicited extra-currents (hysteresis) only if the off phase went sufficiently negative to remove IA inactivation. Native, resting hair cells are depolarised by receptor current; thus, voltage continuously modulates I(KD), whereas IA only transiently ensues when the receptor current vanishes (zero-current potential approximately -70 mV) and polarisation removes IA inactivation.

Abstract 1367: Altered Transmural Expression Of I Kr In Cardiac Memory Is Regulated By The Transcription Factor, Activator Protein-1

Introduction: We previously reported that the transient outward current (I to ) and mRNA and protein levels of its accessory subunit KChIP2 decrease in ventricular pacing-induced cardiac memory (CM). These changes appear dependent on a decrease of the cAMP response element binding protein (CREB). We have also reported that the transmural gradients of the delayed rectifier potassium current component, I kr and the mRNA and protein of its subunit ERG (all showing epicardium (EPI) &gt; endocardium (ENDO)), are diminished in CM. However, there is no CREB binding site in the ERG promoter region to explain the change in ERG. We now tested the hypothesis that 1) the normal transmural gradient of ERG expression in canine left ventricle (LV) is paralleled by a transmural gradient of the Activator protein-1 (AP-1) components c-Jun and c-Fos, and 2) altered transmural expression of c-Jun and c-Fos parallels the altered transmural ERG in CM. Methods: We screened 2000bp upstream from the first exon of ERG for transcription factor binding sites. Then 10 dogs were chronically instrumented with LV posterior wall and LA pacing electrodes. Five underwent 3w AV-sequential pacing (CM), and 5, 3w of atrial pacing (Sham). CM was quantified as change in T wave vector displacement (TVD). At 3w biopsies were taken 1–2 cm from the LV pacing site. Real time PCR was used to determine c-Jun and c-Fos. Results: Screening revealed an AP-1 response element upstream to the ERG promoter region, supporting the hypothesis that c-Jun and c-Fos might be reasonable candidate transcription factors here. TVD increased in CM compared to Sham. As previously, mRNA and protein transmural gradients of ERG decreased in CM. C-jun and c-Fos exhibited transmural gradients, EPI&gt;ENDO, in Sham (Table ). This gradient was lost in CM. Conclusion AP-1 is a transcription factor for ERG and the altered ERG transmural gradient in CM may be regulated by the altered c-Jun and c-Fos gradient. T vector displacement (TVD) and mRNA of c-Jun and c-Fos

Azimilide inhibits multiple cardiac potassium currents in human atrial myocytes.

Studies in animal cell preparations suggest that azimilide may produce a more desirable rate-dependent profile of class III action as a result of its effects on both the slowly (I(Ks)) and rapidly (I(Kr)) activating components of potassium current (I(K)). However, relatively little is known about the effects of azimilide on K(+) currents in human atrial cells. The present study investigated the effect of azimilide on the inward rectifier potassium current (I(K1)), delayed rectifier potassium current (I(K)), ultrarapid delayed rectifier current (I(Kur)), and transient outward potassium current (I(to)) in isolated single human atrial myocytes. The tight-seal, whole-cell voltage clamp technique was used to investigate the acute effects of azimilide on K(+) currents in single human atrial myocytes. The cells were isolated enzymatically from atrial tissues that were obtained from patients undergoing open-heart surgeries, with the approval of the local Institutional Review Board. The average cell capacitance of the human atrial myocytes was 77.5 +/- 2.8 pF (Mean +/- standard error of mean, total 28 cells from 17 patients). We found that 100 microM of azimilide in the extracellular solution significantly inhibited the inward rectifier potassium current (12.3 +/- 3.1 vs 6.7 +/- 2.0 pA/pF, n = 12, P < 0.05) at the testing potential of -100 mV. Superfusion with 100 microM of azimilide for 10 minutes inhibited I(K) by 51.7 +/- 5.1% (from 3.4 +/- 0.5 to 1.6 +/- 0.2 pA/pF, n = 9, P < 0.01) at the clamping membrane potential of +40 mV. Human atrial cell I(Kur) was inhibited with 100 microM of azimilide by 38.6 +/- 4.4% (from 3.9 +/- 0.5 to 2.3 +/- 0.2 pA/pF, n = 9, P < 0.01, test potential = 40 mV). We also found that the average peak current amplitude of I(to) in these cells was significantly inhibited with 100 microM of azimilide by 60.3 +/- 5.9% (from 10.3 +/- 1.5 to 3.6 +/- 0.3 pA/pF, n = 6, P < 0.01, test potential = 50 mV). The present study provides direct evidence that azimilide inhibits multiple cellular transmembrane K(+) currents in freshly isolated human atrial myocytes. Inhibition of these K(+) currents by azimilide, especially of I(Ks) and I(Kur) is likely to be the electrophysiologic basis for the prolongation of the action potential duration in the human atria which mediates its known antifibrillatory effects in atrial fibrillation and flutter.

Potassium Currents during the Action Potential of Hippocampal CA3 Neurons

Central neurons have multiple types of voltage-dependent potassium channels, whose activation during action potentials shapes spike width and whose activation and inactivation at subthreshold voltages modulate firing frequency. We characterized the voltage-dependent potassium currents flowing during the action potentials of hippocampal CA3 pyramidal neurons and examined the susceptibility of the underlying channel types to inactivation at subthreshold voltages. Using acutely dissociated neurons that permitted rapid voltage clamp, action potentials recorded previously were used as the command voltage waveform, and individual components of potassium current were identified by pharmacological sensitivity. The overall voltage-dependent potassium current in the neurons could be split into three major components based on pharmacology and kinetics during step voltage pulses: I(D) (fast activating, slowly inactivating, and sensitive to 4-aminopyridine at 30 microm), I(A) (fast activating, fast inactivating, and sensitive to 4-aminopyridine at 3 mm), and I(K) (slowly activating, noninactivating, and sensitive to external TEA at 3-25 mm). The potassium current during the action potential was composed of approximately equal contributions of I(D) and I(A), with a negligible contribution of I(K). I(D) and I(A) had nearly identical trajectories of activation and deactivation during the action potential. Both I(A) and I(D) showed steady-state inactivation at subthreshold voltages, but maximal inactivation at such voltages was incomplete for both currents. Because of the major contribution of both I(D) and I(A) to spike repolarization, it is likely that modulation or partial inactivation at subthreshold voltages of either current can influence spike timing with minimal effect on spike width.

Effects of imipramine on ion channels and proliferation of IGR1 melanoma cells.

Human IGR1 cells are a model for malignant melanoma. Since progression through the cell cycle is accompanied by transient cell hyperpolarization, we studied the properties of potassium and chloride ion channels and their impact on cell growth. The major potassium current components were mediated by outward rectifying ether à go-go (hEAG) channels and Ca2+-activated channels (KCa) of the IK/SK type. The major chloride channel component was activated by osmotic cell swelling (Clvol). To infer about the contribution of these channels to proliferation, specific inhibitors are required. Since there is no specific blocker for hEAG available, we used the tricyclic antidepressant imipramine, which blocked all channels mentioned, in combination with blockers for KCa (charybdotoxin) and Clvol (DIDS and pamoic acid). Incubation of IGR1 cells for 48 hr in 10-15 mM imipramine reduced DNA synthesis and metabolism without significant effects on apoptosis. hEAG channels were most sensitive to imipramine (IC50: 3.4 microM at +50 mV), followed by KCa (13.8 microM at +50 mV) and Clvol (12 microM at -100 mV), indicating that hEAG expression may be of importance for proliferation of melanoma cells. The contribution of KCa channels could be excluded, as 500 nM charybdotoxin, which completely blocked KCa, had no effect on proliferation. The impact of Clvol also seems to be minor, because 500 microM pamoic acid, which completely blocked Clvol, did not affect proliferation either.

GH3 Cells, Ionic Currents and Cell Killing: Photomodification Sensitized by Rose Bengal

Photosensitization using Rose Bengal (RB) modifies membrane ionic currents and kills cultured mouse pituitary, GH3, cells. Here we investigate the dose-response relationship for ionic current modification and for cell killing to assess a possible causal link. When exposed to 0.5 microM RB and 6.5 mW/cm2 of visible light, calcium current was blocked in 1.9 +/- 0.2 min (mean +/- SEM; 0.74 +/- 0.08 J/cm2; n = 18), a transient component of potassium current, tentatively identified as a delayed-rectifier potassium current, disappeared in 52 +/- 8 s (0.34 +/- 0.05 J/cm2; n = 10) and a steady-state component of potassium current, largely a calcium-activated potassium current, disappeared in 3.5 +/- 0.4 min (1.37 +/- 0.16 J/cm2; n = 11). Conversely, the background leak current increased in magnitude. At 5 min of illumination, the longest time studied here, it continued to increase nearly linearly, making it the only current component studied that is still changing after 5 min of light. Under the conditions used, cell killing increased to 100% in the exposure range of 4-10 min of illumination (1.6 J/cm2 to 3.9 J/cm2) when assessed using fluorescent markers, ethidium homodimer and calcein and required slightly longer exposure times when assessed using trypan blue. Thus, it is difficult to ascribe a causal role in cell killing by photosensitization to alterations of standard ion channels and known ionic currents. However, the increase in leak current has the correct dose-response characteristics to be involved.

Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia.

The effects of brief (2-4 min) hypoxia on presumed dopaminergic "principal" neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode (Vh -60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 +/- 15.2 (SE) pA (n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 +/- 10.6 pA (n = 18). Although the hypoOUT reversed at -83.7 +/- 3.8 mV (n = 18), the posthypoOUT did not reverse. The K+ATP-blocking sulphonylureas tolbutamide (100 microM) and glibenclamide (30 microM), significantly reduced the peak of the hypoOUT by 47.6 +/- 7.7% (n = 16) and 54.18 +/- 7.5% (n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 microM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10-50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings (Vh -60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 +/- 14.5 pA that reversed polarity at -87.9 +/- 5.1 mV (n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 microM), glibenclamide (10-30 microM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 microM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.

Elimination of potassium channel expression by antisense oligonucleotides in a pituitary cell line.

The clonal rat pituitary cell line GH4C1 expresses the genes for several voltage-dependent potassium channels including Kv1.5 and Kv1.4. Dexamethasone, a glucocorticoid agonist, induces a slowly inactivating potassium current in these cells but does not alter the amplitude of a rapidly inactivating component of potassium current. We have found that the induction of the slowly inactivating current can be blocked by an antisense phosphorothioate deoxyoligonucleotide to the Kv1.5 mRNA sequence. In contrast, antisense deoxyoligonucleotides against Kv1.4 mRNA specifically decrease the expression of the dexamethasone-insensitive rapidly inactivating current. These results demonstrate the usefulness of antisense oligonucleotides in correlating potassium currents with specific potassium channel proteins in the cell types in which they are naturally expressed.

Potassium currents in isolated CA1 neurons of the rat after kindling epileptogenesis

Daily tetanic stimulation of the Schaffer collaterals generates an epileptogenic focus in area CA1 of the rat hippocampus, ultimately leading to generalized tonic-clonic convulsions (kindling). Potassium currents were measured under voltage-clamp conditions in pyramidal neurons, acutely dissociated from the focus of fully kindled rats, one day and six weeks after the last generalized seizure. Their amplitude, kinetics, voltage dependence and calcium dependence were compared with controls. With Ca2+ influx blocked by 0.5 mM Ni2+, the sustained current (delayed rectifier) and the transient current (A-current) were not different after kindling. Calcium influx evoked an additional fast transient current component. This transient calcium-dependent current component was increased by 154%, but only immediately after the seizure. A second, slow calcium-dependent potassium current component was dependent on the intracellular calcium level, set by the pipette as well as on calcium influx. The peak amplitude of this slow calcium-dependent current was under optimal calcium conditions not different after kindling, but we found indications that either calcium homeostasis or the calcium sensitivity of the potassium channels was affected by the kindling process. In contrast to the previously described enhancement of calcium current, kindling epileptogenesis did not change the total potassium current amplitude. The minor changes that were observed can be related either to changes in calcium current or to changes in intracellular calcium homeostasis.

Enhancement of a slow potassium current component by uranyl nitrate and its relation to the antagonism on β-bungarotoxin in the mouse motor nerve terminal

Uranyl nitrate (UO 2(NO 3) 2) has been shown to be capable of increasing transmitter release from the motor nerve accompanied by the potentiation of nerve evoked muscle contraction. In this paper, we have demonstrated that UO 2 2+ induced an initial twitch depression followed by a later twitch potentiation in low (0.35 mM) Ca 2+ medium. Although UO 2 2+ has been identified as a K +-channel blocker, we have found it only partially blocked the fast K +-current (IK (f)) as recorded in the perineurial sheath of the mouse triangularis sterni preparation. Increasing the concentration of UO 2 2+ to a high concentration of 0.4 mM did not further inhibit IK (f) but markedly prolonged the duration of the outward current of the nerve terminals. From the time course of its appearance together with the specific inhibition by 4-aminopyridine, dendrotoxin and β-bungarotoxin, which has been shown to be capable of blocking the K +-current of the motor nerve terminal, it was proposed that UO 2 2+ prolonged the duration of the nerve terminal spikes by an enhancement of an IK (s)-like current, which was further characterized by its susceptibility to be enhanced by low K +, low Ca 2+ and Cd 2+ but attenuated by high K + and high Ca 2+. These cation effects not only supported UO 2 2+-induced IK (s) current but also excluded the possibility of an enhancement of Ca 2+-activated K +-current induced by UO 2 2+ plus TEA. The significance of this enhancement of IK (s) induced by UO 2 2+ has been elucidated by the finding that dendrotoxin inhibited but tetraethylammonium potentiated not only UC 2 2+-induced IK (s) but also UO 2 2+-induced twitch depression. Therefore, it is concluded that in addition to the blockade of IK (f), UO 2 2+ can also enhance IK (s)-like current which accounts for presynaptic inhibition of the twitch contractions. Moreover, UO 2(NO 3) 2 has been found to be a selective potent antagonist of β-bungarotoxin (β-BuTX) in blocking the neuromuscular transmission of mouse phrenic nerve diaphragm. In accordance with their effects on UO 2 2+-induced IK (s), TEA markedly potentiated UO 2 2+ in antagonizing the neuromuscular blocking action of β-BuTX, while 4-AP, DTX, high K + and Ca 2+ all reversed the antagonistic action of UO 2 2+ plus TEA against β-BuTX. These findings provide more evidence that IK (s) plays a role in the regulation of transmitter release and UO 2 2+ antagonizes the neuromuscular blocking action of β-BuTX through their interaction at least in part on the slow K +-channels of the motor nerve terminals.

Regulation of voltage-gated ion channels by NGF and ciliary neurotrophic factor in SK-N-SH neuroblastoma cells

Neurotrophic factors have powerful effects on neuronal differentiation and the maintenance of neuronal phenotype, but understanding of their regulation of one important aspect of neuronal function, excitability, remains limited. We have examined the regulation of voltage-gated ion channels by two unrelated neurotrophic factors, NGF and ciliary neurotrophic factor (CNTF), in the SK-N-SH neuroblastoma cell line that is responsive to both factors. NGF and CNTF have strikingly different neuronal specificities and distributions in the nervous system, and might be expected to have significantly different effects on neuronal function. Using whole-cell, perforated-patch, and single-channel recording, we found that treatment with NGF increased levels of voltage-gated sodium, calcium, and potassium currents. In contrast, CNTF treatment increased levels of potassium currents only. NGF and CNTF appeared to regulate the same delayed-rectifier potassium current; in addition, NGF treatment resulted in increased levels of a second potassium current component. Such differential effects of neurotrophic factors on the expression of voltage-gated ion channels would have profound effects on the excitability of target neurons in vivo.

Two components of potassium current activated by depolarization of single smooth muscle cells from the rabbit portal vein.

1. Using the patch-clamp technique at 20-23 degrees C membrane currents were recorded from single smooth muscle cells enzymatically isolated from the rabbit portal vein. Single-channel currents were observed in outside-out patches excised from these. 2. Outward current elicited upon depolarization from -70 mV was not activated as a result of Ca2+ influx. It could be divided into two components: an inactivating, 4-aminopyridine- and phencyclidine-sensitive low-noise current (IdK), and a non-inactivating, tetraethylammonium (TEA)- and charybdotoxin-sensitive high-noise current (IcK). 3. IdK activated with a threshold around -40 mV and was carried by K+. It was substantially inhibited by 4-aminopyridine (5 mM) or phencyclidine (0.1 mM) but was insensitive to TEA+ (4 mM), charybdotoxin (0.1 microM) or apamin (0.1 microM). Upon stepping to 0 mV it reached a maximum within about 0.2 s. The time course of its activation could be described by a fourth-order single exponential; the time constants of these exponentials changed e-fold every 56 mV. It inactivated in a time- and voltage-dependent manner with a fast and slow component, and was about 50% available at -30 mV. From single-channel recordings in isolated patches single channels underlying this current have a small unitary conductance (around 5 pS). 4. IcK did not inactivate significantly over 6 s. It activated with a less negative threshold than IdK, usually near 0 mV when the pipette solution contained 0.8 mM-EGTA with no added calcium. It was blocked by TEA (4 mM) or charybdotoxin (0.1 microM), but not by 4-aminopyridine (5 mM), phencyclidine (0.1 mM) or apamin (0.1 microM). Estimates of the single-channel conductance from the noise variance of the whole-cell current IcK indicated a value at +80 mV of 115 pS, very similar to that of the large-conductance Ca2(+)-activated K+ channels studied in these cells using single-channel recording. 5. The results suggest that outward current evoked by depolarization from the resting potential can be carried by 100 pS Ca2(+)-activated K+ channels and by small-conductance delayed-rectifier K+ channels. It is likely that opening of both types of channel contributes to the repolarization phase of the action potential in this smooth muscle.

Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons

Whole-cell voltage-clamp techniques were used to study the effects of the protein kinase C (PKC) activators phorbol esters and OAG on Ca and K currents in differentiated neurons acutely dissociated from adult hippocampus and in tissue-cultured neurons from fetal hippocampus. PKC activators had selective depressant effects on K currents, with persistent currents (IK and IK-Ca) being reduced and transient current (IA) being unaffected. In both cell types we recorded both high-voltage-activated, noninactivating (L-type) and high-voltage-activated, rapidly inactivating (N-type) Ca current. A low-voltage-activated, rapidly inactivating (T-type) Ca current was also recorded in tissue-cultured neurons but not in acutely dissociated neurons. PKC activators markedly reduced N-type current with less effect on L-type and no effect on T-type Ca current. Effects of PKC activators could be reversed with washing or with application of PKC inhibitors H-7 or polymyxin-B, an effect that could not be attributed to inhibition of cAMP-dependent protein kinase. The Ca/calmodulin inhibitor calmidazolium was ineffective in reversing the actions of PKC activators. Using whole-cell voltage-clamp techniques, we have demonstrated that hippocampal neurons possess 3 distinguishable components of calcium current. Distinct K currents were also observed. Our data strongly support the hypothesis that both Ca and K currents are selectively regulated by PKC and that these effects occur directly on the postsynaptic neuron.

Blocking action of calmidazolium and chlorpromazine on outward potassium current dependent on extra-cellular calcium ions

The effects of the calmodulin antagonists, calmidazolium (R 24571) and chlorpromazine on delayed outward potassium current at the somatic membrane were investigated in non-identified intracellularly perfused neurons isolated fromHelix pomatia. Voltage was clamped at the membrane. Extracellular application of these substances produced effective depression of the outward current. This effect even occurred at test substance concentrations of 10−9–10−8 M. Block-ade of delayed outward current was produced mainly as a result of suppressing the potassium current component dependent on intracellular potassium ions (Ik(Ca/in)). The possibility that the receptor for intracellular calcium responsible for modulating this current may be of a calmodulin-like nature is discussed.

Both barium and calcium activate neuronal potassium currents.

Amphibian spinal neurons in culture possess both rapidly inactivating and sustained calcium-dependent potassium current components, similar to those described for other cells. Divalent cation-dependent whole-cell outward currents were isolated by subtracting the voltage-dependent potassium currents recorded from Xenopus laevis neurons in the presence of impermeant cadmium (100-500 microM) from the currents produced without cadmium but in the presence of permeant divalent cations (50-100 microM). These concentrations of permeant ions were low enough to avoid contamination by macroscopic inward currents through calcium channels. Calcium-dependent potassium currents were reduced by 1 microM tetraethylammonium. These currents can also be activated by barium or strontium. Barium as well as calcium activated outward currents in young neurons (6-8 hr) and in relatively mature neurons (19-26 hr in vitro). However, barium influx appeared to suppress the sustained voltage-dependent potassium current in most cells. Barium also activated at least one class of potassium channels observed in excised membrane patches, while blocking others. The blocking action may have masked and hindered detection of the stimulatory action of barium in other systems.