External Potassium Concentration Research Articles

Calcium-activated chloride channels. Role of potassium ions

Using the patch-clamp method in the whole-cell configuration, it was shownthat external potassium ions play an important role in the regulation of calcium-activated chloride currents. A clear dependence of the amplitude of chloride currents on changes in the concentration of external potassium is shown. Changes in concentration of sodium, magnesium and calcium ions from membrane outside have no so significant effect, like outside potassium ions. The effect of potassium on the amplitudes of chloride currents is significantly greater than the effect it has on other cell ionic currents — sodium, potassium, cation. There is reason to believe that a change in the amplitudes of chloride currents contributes to the pathophysiological processes characteristic of hypokalemia and hyperkalemia.

Inhibition of HCN1 currents by norquetiapine, an active metabolite of the atypical anti-psychotic drug quetiapine.

Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate Ih, may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and its active metabolites 7-hydroxyquetiapine and norquetiapine on currents from HCN1 channels expressed in Xenopus laevis oocytes. Norquetiapine, but not quetiapine nor 7-hydroxyquetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC50 of 13.9 ± 0.8μM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine also inhibited HCN4 channels with reduced efficacy than HCN1 and had no effect on HCN2 channels. Therefore, HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors.

Tartrolon sensing and detoxification by the Listeria monocytogenes timABR resistance operon.

Listeria monocytogenes is a foodborne bacterium that naturally occurs in the soil. Originating from there, it contaminates crops and infects farm animals and their consumption by humans may lead to listeriosis, a systemic life-threatening infectious disease. The adaptation of L. monocytogenes to such contrastive habitats is reflected by the presence of virulence genes for host infection and other genes for survival under environmental conditions. Among the latter are ABC transporters for excretion of antibiotics produced by environmental competitors; however, most of these transporters have not been characterized. Here, we generated a collection of promoter-lacZ fusions for genes encoding ABC-type drug transporters of L. monocytogenes and screened this reporter strain collection for induction using a library of natural compounds produced by various environmental microorganisms. We found that the timABR locus (lmo1964-lmo1962) was induced by the macrodiolide antibiotic tartrolon B, which is synthesized by the soil myxobacterium Sorangium cellulosum. Tartrolon B resistance of L. monocytogenes was dependent on timAB, encoding the ATPase and the permease component of a novel ABC transporter. Moreover, transplantation of timAB was sufficient to confer tartrolon B resistance to Bacillus subtilis. Expression of the timABR locus was found to be auto-repressed by the TimR repressor, whose repressing activity was lost in the presence of tartrolon B. We also demonstrate that tartrolon sensitivity was suppressed by high external potassium concentrations, suggesting that tartrolon acts as potassium ionophore. Our results help to map the ecological interactions of an important human pathogen with its co-residing species within their joint natural reservoir.

Depolarization-induced bursts of miniature synaptic currents in individual synapses of developing cerebellum.

In central synapses, spontaneous transmitter release observed in the absence of action potential firing is often considered as a random process lacking time or space specificity. However, when studying miniature glutamatergic currents at cerebellar synapses between parallel fibers and molecular layer interneurons, we found that these currents were sometimes organized in bursts of events occurring at high frequency (about 30 Hz). Bursts displayed homogeneous quantal size amplitudes. Furthermore, in the presence of the desensitization inhibitor cyclothiazide, successive events within a burst displayed quantal amplitude occlusion. Based on these findings, we conclude that bursts originate in individual synapses. Bursts were enhanced by increasing either the external potassium concentration or the external calcium concentration, and they were strongly inhibited when blocking voltage-gated calcium channels by cadmium. Bursts were prevalent in elevated potassium concentration during the formation of the molecular layer but were infrequent later in development. Since postsynaptic AMPA receptors are largely calcium permeant in developing parallel fiber-interneuron synapses, we propose that bursts involve presynaptic calcium transients implicating presynaptic voltage-gated calcium channels, together with postsynaptic calcium transients implicating postsynaptic AMPA receptors. These simultaneous pre- and postsynaptic calcium transients may contribute to the formation and/or stabilization of synaptic connections.

Role of Potassium Ions in Regulation of Calcium-Activated Chloride Channels.

Using the patch-clamp method in the whole cell configuration, it was shown that external potassium ions play an important role in the regulation of calcium-activated chloride channels (CaCCs). A clear dependence of the conductivity of the СаССs on the external potassium concentration was shown. The effect of external potassium in the range 0-15 mM on the conductivity of chloride channels was significantly greater than the effect it had on other ionic currents (sodium or potassium). There is reason to believe that these changes in the conductivity of CaCCs may contribute to the development of pathophysiological processes such as hypokalemia or hyperkalemia.

Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate.

Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD.IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.

Low Ouabain Doses and AMP-Activated Protein Kinase as Factors Supporting Electrogenesis in Skeletal Muscle.

Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.

Adaptation of Bacillus subtilis to Life at Extreme Potassium Limitation.

ABSTRACTPotassium is the most abundant metal ion in every living cell. This ion is essential due to its requirement for the activity of the ribosome and many enzymes but also because of its role in buffering the negative charge of nucleic acids. As the external concentrations of potassium are usually low, efficient uptake and intracellular enrichment of the ion is necessary. The Gram-positive bacterium Bacillus subtilis possesses three transporters for potassium, KtrAB, KtrCD, and the recently discovered KimA. In the absence of the high-affinity transporters KtrAB and KimA, the bacteria were unable to grow at low potassium concentrations. However, we observed the appearance of suppressor mutants that were able to overcome the potassium limitation. All these suppressor mutations affected amino acid metabolism, particularly arginine biosynthesis. In the mutants, the intracellular levels of ornithine, citrulline, and arginine were strongly increased, suggesting that these amino acids can partially substitute for potassium. This was confirmed by the observation that the supplementation with positively charged amino acids allows growth of B. subtilis even at the extreme potassium limitation that the bacteria experience if no potassium is added to the medium. In addition, a second class of suppressor mutations allowed growth at extreme potassium limitation. These mutations result in increased expression of KtrAB, the potassium transporter with the highest affinity and therefore allow the acquisition and accumulation of the smallest amounts of potassium ions from the environment.

Reduced Efficacy of the KCC2 Cotransporter Promotes Epileptic Oscillations in a Subiculum Network Model.

Ion regulation in the brain is a major determinant of neural excitability. Intracellular chloride in neurons, a partial determinant of the resting potential and the inhibitory reversal potentials, is regulated together with extracellular potassium via kation chloride cotransporters. During temporal lobe epilepsy, the homeostatic regulation of intracellular chloride is impaired in pyramidal cells, yet how this dysregulation may lead to seizures has not been explored. Using a realistic neural network model describing ion mechanisms, we show that chloride homeostasis pathology provokes seizure activity analogous to recordings from epileptogenic brain tissue. We show that there is a critical percentage of pathological cells required for seizure initiation. Our model predicts that restoration of the chloride homeostasis in pyramidal cells could be a viable antiepileptic strategy.

Potassium and Sodium Transport in Yeast.

As the proper maintenance of intracellular potassium and sodium concentrations is vital for cell growth, all living organisms have developed a cohort of strategies to maintain proper monovalent cation homeostasis. In the model yeast Saccharomyces cerevisiae, potassium is accumulated to relatively high concentrations and is required for many aspects of cellular function, whereas high intracellular sodium/potassium ratios are detrimental to cell growth and survival. The fact that S. cerevisiae cells can grow in the presence of a broad range of concentrations of external potassium (10μM-2.5M) and sodium (up to 1.5M) indicates the existence of robust mechanisms that have evolved to maintain intracellular concentrations of these cations within appropriate limits. In this review, current knowledge regarding potassium and sodium transporters and their regulation will be summarized. The cellular responses to high sodium and potassium and potassium starvation will also be discussed, as well as applications of this knowledge to diverse fields, including antifungal treatments, bioethanol production and human disease.

Suppression of sensitivity to drugs and antibiotics by high external cation concentrations in fission yeast.

BackgroundPotassium ion homeostasis plays an important role in regulating membrane potential and therefore resistance to cations, antibiotics and chemotherapeutic agents in Schizosaccharomyces pombe and other yeasts. However, the precise relationship between drug resistance in S. pombe and external potassium concentrations (particularly in its natural habitats) remains unclear. S. pombe can tolerate a wide range of external potassium concentrations which in turn affect plasma membrane polarization. We thus hypothesized that high external potassium concentrations suppress the sensitivity of this yeast to various drugs.MethodsWe have investigated the effect of external KCl concentrations on the sensitivity of S. pombe cells to a wide range of antibiotics, antimicrobial agents and chemotherapeutic drugs. We employed survival assays, immunoblotting and microscopy for these studies.ResultsWe demonstrate that KCl, and to a lesser extent NaCl and RbCl can suppress the sensitivity of S. pombe to a wide range of antibiotics. Ammonium chloride and potassium hydrogen sulphate also suppressed drug sensitivity. This effect appears to depend in part on changes to membrane polarization and membrane transport proteins. Interestingly, we have found little relationship between the suppressive effect of KCl on sensitivity and the structure, polarity or solubility of the various compounds investigated.ConclusionsHigh concentrations of external potassium and other cations suppress sensitivity to a wide range of drugs in S. pombe. Potassium-rich environments may thus provide S. pombe a competitive advantage in nature. Modulating potassium ion homeostasis may sensitize pathogenic fungi to antifungal agents.

Suppression of the hERG potassium channel response to premature stimulation by reduction in extracellular potassium concentration.

Potassium channels encoded by human ether‐à‐go‐go‐related gene (hERG) mediate the cardiac rapid delayed rectifier K+ current (IKr), which participates in ventricular repolarization and has a protective role against unwanted premature stimuli late in repolarization and early in diastole. Ionic current carried by hERG channels (IhERG) is known to exhibit a paradoxical dependence on external potassium concentration ([K+]e), but effects of acute [K+]e changes on the response of IhERG to premature stimulation have not been characterized. Whole‐cell patch‐clamp measurements of hERG current were made at 37°C from hERG channels expressed in HEK293 cells. Under conventional voltage‐clamp, both wild‐type (WT) and S624A pore‐mutant IhERG during depolarization to +20 mV and subsequent repolarization to −40 mV were decreased when superfusate [K+]e was decreased from 4 to 1 mmol/L. When [K+]e was increased from 4 to 10 mmol/L, pulse current was increased and tail IhERG was decreased. Increasing [K+]e produced a +10 mV shift in voltage‐dependent inactivation of WT IhERG and slowed inactivation time course, while lowering [K+]e from 4 to 1 mmol/L produced little change in inactivation voltage dependence, but accelerated inactivation time course. Under action potential (AP) voltage‐clamp, lowering [K+]e reduced the amplitude of IhERG during the AP and suppressed the maximal IhERG response to premature stimuli. Raising [K+]e increased IhERG early during the AP and augmented the IhERG response to premature stimuli. Our results are suggestive that during hypokalemia not only is the contribution of IKr to ventricular repolarization reduced but its ability to protect against unwanted premature stimuli also becomes impaired.

Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance

Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C 3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake ( trk1 trk2) or efflux ( nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells.

가족성 저칼륨성 주기성 마비에서 세포외 칼륨농도가 지연성 정류형 채널을 형성하는 KCNQ3와 KCNQ5 단백질에 미치는 효과

가족성 저칼륨성 주기성 마비란 상염색체 우성 유전 질환으로 저칼륨혈증을 동반한 간헐적인 가역적 이완성 근육 마비를 특징으로 한다. 세포내 저류된 칼륨으로 인해 저칼륨혈증이 지속되고 근세포 활성이상으로 인해 마비가 발생하는 것으로 알려져 있다. 이러한 증상발현의 분자생물학적 기전을 확인하기 위해 세포 내 칼륨이온을 세포 밖으로 이동시키는 지연성 정류형 채널 단백질의 일종인 KCNQ3와 KCNQ5를 대상으로, 정상인과 환자에서 채취한 골격근 세포를 생리적 세포외 정상 칼륨농도인 4 mM과 탈분극 유도를 위한 고칼륨농도인 50mM에 노출시켜 단백질의 양적 변화 유무를 확인하였다. 유전자 발현양상을 확인하기 위해 mRNA의 양적 변화를 확인한 결과 모든 조건에서 유의한 변화가 관찰되지 않아 정상 칼륨조건과 고칼륨조건이 두 유전자발현의 변화를 야기하지 않음을 확인하였다. 그러나 단백질 양을 관찰한 결과 환자의 골격근 세포가 50 mM의 칼륨농도에 노출되는 경우 KCNQ3 단백질은 세포질 내에서 증가하고 세포막 내에서 감소하였다. 이는 환자의 골격근 세포가 고농도의 세포외 칼륨에 의해 탈분극 되는 경우 재분극에 중요한 기능을 담당하는 KCNQ3 채널 단백질이 세포질 내로 이동하여 재분극 형성의 장애를 초래하고 이로 인해 근세포 활성이 일어나지 않게 되어 마비를 유발할 수 있음을 시사하는 결과로 본 질환의 새로운 발병 기전을 설명할 수 있는 근거로 생각된다. Familial hypokalemic periodic paralysis (HOKPP) is an autosomal dominant muscle disorder characterized by episodic attacks of muscle weakness with concomitant hypokalemia. Mutations in either a calcium channel gene (CACNA1S) or a sodium channel gene (SCN4A) have been shown to be responsible for this disease. The combination of sarcolemmal depolarization and hypokalemia has been attributed to abnormalities of the potassium conductance governing the resting membrane potential. To understand the pathophysiology of this disorder, we examined both mRNA and protein levels of delayed rectifier potassium channel genes, KCNQ3 and KCNQ5, in skeletal muscle fibers biopsied from patients with HOKOur results showed an increase in the cytoplasmic level of KCNQ3 protein in patients' cells exposed to 50 mM external concentration of potassium. However, mRNA levels of both channel genes did not show significant change in the same condition. Our results suggest that long term exposure of skeletal muscle cells in HOKPP patients to high extracellular potassium alters the KCNQ3 localization, which could possibly hinder the normal function of this channel protein. These findings may provide an important clue to understanding the molecular mechanism of familial hypokalemic periodic paralysis.

Biophysical Model of an Auditory Nerve Fiber With a Novel Adaptation Component

Recent data from feline auditory nerve fibers (ANFs) indicate that electrically stimulated fibers can undergo large degrees of rate adaptation to pulse-train stimuli using pulse rates within the range used by clinical auditory prostheses. However, the application of Hodgkin-Huxley-type models does not produce such adaptation, which occurs over time periods on the order of 100 ms. We describe our development of a computational ANF axon model that incorporates a time-changing external potassium concentration ( [K(+)](ext)) that depends on potassium currents produced by active nodal channel activity. This relatively simple and computationally tractable approach produces poststimulus time histograms that are similar to experimental (cat) data. Furthermore, this mechanism could be easily incorporated into other models to produce much more realistic estimates of the neural coding produced by repeated electric stimulation.

Ligand-Dependent Effects on the Conformational Equilibrium of the Na+,K+-ATPase As Monitored by Voltage Clamp Fluorometry

Voltage clamp fluorometry was used to monitor conformational changes associated with electrogenic partial reactions of the Na+,K+-ATPase after changes in the concentration of internal sodium (Na+i) or external potassium (K+o). To probe the effects of the Na+i concentration on the Na+ branch of the Na+,K+-ATPase, oocytes were depleted of Na+i and then loaded with external sodium (Na+o) using the amiloride-sensitive epithelial sodium channel. The K+ branch of the Na+,K+-ATPase was studied by exposing the oocytes to different K+o concentrations in the presence and absence of Na+o to obtain additional information on the apparent affinity for K+o. Our results demonstrate that lowering the concentration of Na+i or increasing the amount of K+o in the external solution shifts the equilibrium toward E1/E1P. Furthermore, the K+o-induced relocation toward E1 occurs at a much lower K+o concentration when Na+o is absent, indicating a higher apparent affinity. Finally, voltage-dependent steps associated with the K+ branch or the Na+ branch of the Na+,K+-ATPase are affected by the K+o concentration or the Na+i concentration, respectively.

A High-Throughput Assay for Evaluating State Dependence and Subtype Selectivity of Cav2 Calcium Channel Inhibitors

Cav2.2 channels play a critical role in pain signaling by controlling synaptic transmission between dorsal root ganglion neurons and dorsal horn neurons. The Cav2.2-selective peptide blocker ziconotide (Prialt, Elan Pharmaceuticals, Dublin, Ireland) has proven efficacious in pain relief, but has a poor therapeutic index and requires intrathecal administration. This has provided impetus for finding an orally active, state-dependent Cav2.2 inhibitor with an improved safety profile. Members of the Cav2 subfamily of calcium channels are the main contributors to central and peripheral synaptic transmission, but the pharmacological effects of blocking each subtype is not yet defined. Here we describe a high-throughput fluorescent assay using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA]) designed to quickly evaluate the state dependence and selectivity of inhibitors across the Cav2 subfamily. Stable cell lines expressing functional Cav2 channels (Ca(V)alpha, beta(3), and alpha(2)delta subunits) were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, could be controlled by external potassium concentration. Following cell incubation in drug with varying concentrations of potassium, a high potassium trigger was added to elicit calcium influx through available, unblocked channels. State-dependent inhibitors that preferentially bind to channels in the open or inactivated state can be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. Although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity.

Computational analysis of the effects of the hERG channel opener NS1643 in a human ventricular cell model

Dysfunction or pharmacologic inhibition of repolarizing cardiac ionic currents can lead to fatal arrhythmias. The hERG potassium channel underlies the repolarizing current I(Kr), and mutations therein can produce both long and short QT syndromes (LQT2 and SQT1). We previously reported on the diphenylurea compound NS1643, which acts on hERG channels in two distinct ways: by increasing overall conductance and by shifting the inactivation curve in the depolarized direction. The purpose of this study was to determine which of the two components contributes more to the antiarrhythmic effects of NS1643 under normokalemic and hypokalemic conditions. The study consisted of mathematical simulation of action potentials in a human ventricular ionic cell model in single cell and string of 100 cells. Regardless of external potassium concentration or diastolic interval used, NS1643 decreases action potential duration and triangulation. For single cells, NS1643 increases the postrepolarization refractory time but shortens the absolute refractory period. In one dimensional simulations, NS1643 increases the vulnerable window for unidirectional block but suppresses the emergence of premature action potentials and unidirectional blocks around APD(90). During normokalemia, shifting the inactivation curve has greater impact than increasing conductance, whereas the opposite occurs during hypokalemia. Increased hERG conductance and the depolarizing shift of the inactivation curve both contribute to the antiarrhythmic actions of NS1643, with relative effects dependent on external K(+) concentration.

Dopamine Induces a Slow Afterdepolarization in Lateral Amygdala Neurons

The amygdala and dopaminergic innervation thereunto are considered to cooperatively regulate emotional states and behaviors. The present experiments examined effects of dopamine on lateral amygdala (LA) neuron excitability by whole cell recordings. Bath application of dopamine induced slow afterdepolarization (sADP). This sADP lasted for >5 s, and its magnitude varied in a concentration-dependent manner. Co-application of the D1 receptor antagonist SKF83566 reduced its amplitude. The D1 receptor agonist SKF38393, applied alone, induced sADP of a smaller amplitude. Induction of the full sADP required 5-HT(2) and noradrenalin alpha(1) receptor activation as well. D2 receptor activation or blockade did not affect sADP induction. The calcium channel blocker cadmium or intracellular calcium chelator bis-(o-aminophenoxy)-N,N,N',N' tetraacetic acid (BAPTA) blocked induction of the sADP, which was suggested to be triggered by calcium influx. Under voltage clamp, membrane conductance decreased at the peak of sADP current (I(sADP)). I(sADP) was suppressed by cesium included in pipettes. The I-V curve of the net I(sADP) was shifted as the external concentration of potassium was raised, and the reversal potential was identical to that of potassium, suggesting that dopamine decreases potassium conductance to induce the sADP. The present sADP may serve as a positive-feedback regulator of excitability in LA neurons.

ALTERING EXTRACELLULAR POTASSIUM CONCENTRATION DOES NOT MODULATE DRUG BLOCK OF HUMAN ETHER‐A‐GO‐GO‐RELATED GENE (hERG) CHANNELS

1. Drug-induced block of the rapidly activating delayed rectifier K+ current (I(Kr)), encoded by human ether-a-go-go-related gene (hERG), has been linked to acquired long QT syndrome (aLQTS). Hypokalaemia is a recognized risk factor in aLQTS. To further understand why hypokalaemia is a risk factor in aLQTS, we examined the effect of [K+]o on drug block of the hERG potassium channel stably expressed in human embryonic kidney (HEK-293) cells using whole-cell voltage-clamp techniques. 2. The effects of selected [K+]o (1-20 mmol/L) on hERG block with four structurally diverse compounds (dofetilide, mesoridazine, quinidine and terfenadine) from different therapeutic classes were evaluated. Reducing [K+]o from 20 to 1 mmol/L had little effect on IC50 values for hERG current block for all four compounds. For example, evaluating quinidine in external potassium concentrations of 20, 10, 5 and 1 mmol/L resulted in IC50 values of 1.82 +/- 0.33, 2.04 +/- 0.28, 1.57 +/- 0.52 and 1.14 +/- 0.21 mmol/L, respectively. No statistically significant difference (P > 0.35, anova) was observed between drug block of hERG in different external potassium concentrations. These data are in contrast with previously reported results examining hERG channel modulation expressed in AT-1 cells under similar experimental conditions. 3. These results demonstrate that [K+]o does not directly modulate drug block of hERG channels expressed in an HEK-293 cell line. The enhanced risk of Torsades de Pointes associated with hypokalaemia in aLQTS may be due to reduction of other (non-hERG) potassium currents, further reducing the repolarization reserve, and not due to direct modulation of hERG block by [K+]o.