Science.gov

Sample records for potassium katp channel

  1. Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse.

    PubMed

    Hong, Seung Hwa; Kyeong, Kyu-Sang; Kim, Chan Hyung; Kim, Young Chul; Choi, Woong; Yoo, Ra Young; Kim, Hun Sik; Park, Yeon Jin; Ji, Il Woon; Jeong, Eun-Hwan; Kim, Hak Soon; Xu, Wen-Xie; Lee, Sang Jin

    2016-08-01

    ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2.

  2. Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse

    PubMed Central

    HONG, Seung Hwa; KYEONG, Kyu-Sang; KIM, Chan Hyung; KIM, Young Chul; CHOI, Woong; YOO, Ra Young; KIM, Hun Sik; PARK, Yeon Jin; JI, Il Woon; JEONG, Eun-Hwan; KIM, Hak Soon; XU, Wen-Xie; LEE, Sang Jin

    2016-01-01

    ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2. PMID:27086859

  3. Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse.

    PubMed

    Hong, Seung Hwa; Kyeong, Kyu-Sang; Kim, Chan Hyung; Kim, Young Chul; Choi, Woong; Yoo, Ra Young; Kim, Hun Sik; Park, Yeon Jin; Ji, Il Woon; Jeong, Eun-Hwan; Kim, Hak Soon; Xu, Wen-Xie; Lee, Sang Jin

    2016-08-01

    ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2. PMID:27086859

  4. Heterogeneity and Function of KATP Channels in Canine Hearts

    PubMed Central

    Zhang, Hai Xia; Silva, Jonathan R.; Lin, Yu-Wen; Verbsky, John W.; Lee, Urvi S.; Kanter, Evelyn M.; Yamada, Kathryn A.; Schuessler, Richard B.; Nichols, Colin G.

    2013-01-01

    Background The concept that pore-forming Kir6.2 and regulatory SUR2A subunits form cardiac ATP-sensitive potassium (KATP) channels is challenged by recent reports that SUR1 is predominant in mouse atrial KATP channels. Objective To assess SUR subunit composition of KATP channels and consequence of KATP activation for action potential duration (APD) in dog heart. Methods Patch-clamp techniques were used on isolated dog cardiomyocytes to investigate KATP channel properties. Dynamic current-clamp, by injection of a linear K+ conductance to simulate activation of the native current, was employed to study consequences of KATP activation on APD. Results Metabolic inhibitor (MI)-activated current was not significantly different from pinacidil (SUR2A-specific)-activated current, and both currents were larger than diazoxide (SUR1- specific)-activated current, in both atrium and ventricle. Mean KATP conductance (activated by MI) did not differ significantly between chambers although, within the ventricle, both MI-induced and pinacidil-induced currents tended to decrease from epicardium to endocardium. Dynamic current-clamp results indicate that myocytes with longer baseline APDs are more susceptible to injected “KATP” current, a result reproduced in silico using a canine AP model to simulate Epi and Endo (HRd). Conclusions Even a small fraction of KATP activation significantly shortens APD in a manner that depends on existing heterogeneity in KATP current and APD. PMID:23871704

  5. KATP Channels in the Cardiovascular System.

    PubMed

    Foster, Monique N; Coetzee, William A

    2016-01-01

    KATP channels are integral to the functions of many cells and tissues. The use of electrophysiological methods has allowed for a detailed characterization of KATP channels in terms of their biophysical properties, nucleotide sensitivities, and modification by pharmacological compounds. However, even though they were first described almost 25 years ago (Noma 1983, Trube and Hescheler 1984), the physiological and pathophysiological roles of these channels, and their regulation by complex biological systems, are only now emerging for many tissues. Even in tissues where their roles have been best defined, there are still many unanswered questions. This review aims to summarize the properties, molecular composition, and pharmacology of KATP channels in various cardiovascular components (atria, specialized conduction system, ventricles, smooth muscle, endothelium, and mitochondria). We will summarize the lessons learned from available genetic mouse models and address the known roles of KATP channels in cardiovascular pathologies and how genetic variation in KATP channel genes contribute to human disease.

  6. Sarcolemmal KATP channel modulators and cardiac arrhythmias.

    PubMed

    Baczkó, I; Husti, Z; Lang, V; Leprán, I; Light, P E

    2011-01-01

    Cardiac atrial and ventricular arrhythmias are major causes of mortality and morbidity. Ischemic heart disease is the most common cause underlying 1) the development of ventricular fibrillation that results in sudden cardiac death and 2) atrial fibrillation that can lead to heart failure and stroke. Current pharmacological agents for the treatment of ventricular and atrial arrhythmias exhibit limited effectiveness and many of these agents can cause serious adverse effects - including the provocation of lethal ventricular arrhythmias. Sarcolemmal ATP-sensitive potassium channels (sarcK(ATP)) couple cellular metabolism to membrane excitability in a wide range of tissues. In the heart, sarcK(ATP) are activated during metabolic stress including myocardial ischemia, and both the opening of sarcK(ATP) and mitochondrial K(ATP) channels protect the ischemic myocardium via distinct mechanisms. Myocardial ischemia leads to a series of events that promote the generation of arrhythmia substrate eventually resulting in the development of life-threatening arrhythmias. In this review, the possible mechanisms of the anti- and proarrhythmic effects of sarcK(ATP) modulation as well as the influence of pharmacological K(ATP) modulators are discussed. It is concluded that in spite of the significant advances made in this field, the possible cardiovascular therapeutic utility of current sarcK(ATP) channel modulators is still hampered by the lack of chamber-specific selectivity. However, recent insights into the chamber-specific differences in the molecular composition of sarcKATP in addition to already existing cardioselective sarcK(ATP) channel modulators with sarcK(ATP) isoform selectivity holds the promise for the future development of pharmacological strategies specific for a variety of atrial and ventricular arrhythmias.

  7. Cardiovascular KATP channels and advanced aging

    PubMed Central

    Yang, Hua-Qian; Subbotina, Ekaterina; Ramasamy, Ravichandran; Coetzee, William A.

    2016-01-01

    With advanced aging, there is a decline in innate cardiovascular function. This decline is not general in nature. Instead, specific changes occur that impact the basic cardiovascular function, which include alterations in biochemical pathways and ion channel function. This review focuses on a particular ion channel that couple the latter two processes, namely the KATP channel, which opening is promoted by alterations in intracellular energy metabolism. We show that the intrinsic properties of the KATP channel changes with advanced aging and argue that the channel can be further modulated by biochemical changes. The importance is widespread, given the ubiquitous nature of the KATP channel in the cardiovascular system where it can regulate processes as diverse as cardiac function, blood flow and protection mechanisms against superimposed stress, such as cardiac ischemia. We highlight questions that remain to be answered before the KATP channel can be considered as a viable target for therapeutic intervention. PMID:27733235

  8. Subsecond regulation of striatal dopamine release by presynaptic KATP channels

    PubMed Central

    Patel, Jyoti C.; Witkovsky, Paul; Coetzee, William A.; Rice, Margaret E.

    2011-01-01

    ATP-sensitive K+ (KATP) channels are composed of pore-forming subunits, typically Kir6.2 in neurons, and regulatory sulfonylurea receptor subunits. In dorsal striatum, activity-dependent H2O2 produced from glutamatergic AMPA-receptor activation inhibits dopamine release via KATP channels. Sources of modulatory H2O2 include medium spiny neurons, but not dopaminergic axons. Using fast-scan cyclic voltammetry in guinea-pig striatal slices and immunohistochemistry, we determined the time window for H2O2/KATP-channel-mediated inhibition and assessed whether modulatory KATP channels are on dopaminergic axons. Comparison of paired-pulse suppression of dopamine release in the absence and presence of glibenclamide, a KATP-channel blocker, or mercaptosuccinate, a glutathione peroxidase inhibitor that enhances endogenous H2O2 levels, revealed a time window for inhibition of 500 to 1000 ms after stimulation. Immunohistochemistry demonstrated localization of Kir6.2 KATP-channel subunits on dopaminergic axons. Consistent with the presence of functional KATP channels on dopaminergic axons, KATP-channel openers, diazoxide and cromakalim, suppressed single-pulse evoked dopamine release. Although cholinergic interneurons that tonically regulate dopamine release also express KATP channels, diazoxide did not induce the enhanced frequency responsiveness of dopamine release seen with nicotinic-receptor blockade. Together, these studies reveal subsecond regulation of striatal dopamine release by endogenous H2O2 acting at KATP channels on dopaminergic axons, including a role in paired-pulse suppression. PMID:21689107

  9. Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density*

    PubMed Central

    Manna, Paul T.; Smith, Andrew J.; Taneja, Tarvinder K.; Howell, Gareth J.; Lippiat, Jonathan D.; Sivaprasadarao, Asipu

    2010-01-01

    Pancreatic ATP-sensitive potassium (KATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed KATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that KATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases KATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native KATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated KATP channel trafficking may play a role in the regulation of insulin secretion. PMID:20026601

  10. Muscle dysfunction caused by a KATP channel mutation in neonatal diabetes is neuronal in origin.

    PubMed

    Clark, Rebecca H; McTaggart, James S; Webster, Richard; Mannikko, Roope; Iberl, Michaela; Sim, Xiu Li; Rorsman, Patrik; Glitsch, Maike; Beeson, David; Ashcroft, Frances M

    2010-07-23

    Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel, cause neonatal diabetes. Many patients also suffer from hypotonia (weak and flaccid muscles) and balance problems. The diabetes arises from suppressed insulin secretion by overactive KATP channels in pancreatic beta-cells, but the source of the motor phenotype is unknown. By using mice carrying a human Kir6.2 mutation (Val59-->Met59) targeted to either muscle or nerve, we show that analogous motor impairments originate in the central nervous system rather than in muscle or peripheral nerves. We also identify locomotor hyperactivity as a feature of KATP channel overactivity. These findings suggest that drugs targeted against neuronal, rather than muscle, KATP channels are needed to treat the motor deficits and that such drugs require high blood-brain barrier permeability. PMID:20595581

  11. Altered KATP Channel Subunits Expression and Vascular Reactivity in Spontaneously Hypertensive Rats With Age

    PubMed Central

    Liu, Xiaojing; Duan, Peng; Hu, Xingxing; Li, Ruisheng

    2016-01-01

    Abstract: ATP-sensitive potassium (KATP) channels link membrane excitability to metabolic state to regulate a series of biological activities including the vascular tone. However, their ability to influence hypertension is controversial. Here we aim to investigate possible alteration of KATP channel in vascular smooth muscles (VSMs) during hypertension development process. In this study, we used 16-week-old spontaneously hypertensive rats (SHRs), 49-week-old SHRs, and their age-matched Wistar-Kyoto rats to study the expression of VSM KATP subunits at the mRNA and protein level and the function of VSM KATP by observing the relaxation reactivity of isolated aorta rings to KATP modulators. We found that the expression of VSM KATP subunits Kir6.1 and sulfonylurea receptor (SUR2B) decreased during hypertension. Moreover, the expression of SUR2B and Kir6.1 in 49-week-old SHRs decreased much more than that in 16-week-old SHRs. Furthermore, the aorta rings of 49-week-old SHRs showed lower reactivity to diazoxide than 16-week-old SHRs. This study suggests that KATP channels in VSM subunits Kir6.1 and SUR2B contribute to modify the functionality of this channel in hypertension with age. PMID:27035370

  12. Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic β-cells.

    PubMed

    Park, Sun-Hyun; Ryu, Shin-Young; Yu, Weon-Jin; Han, Young Eun; Ji, Young-Sun; Oh, Keunhee; Sohn, Jong-Woo; Lim, Ajin; Jeon, Jae-Pyo; Lee, Hyunsu; Lee, Kyu-Hee; Lee, Suk-Ho; Berggren, Per-Olof; Jeon, Ju-Hong; Ho, Won-Kyung

    2013-07-30

    Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic β-cells. In this study, we provide evidence that leptin modulates pancreatic β-cell functions by promoting K(ATP) channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K(ATP) channels were localized mostly to intracellular compartments of pancreatic β-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase β. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K(ATP) channel trafficking and hyperpolarization of pancreatic β-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and β-cell membrane potentials, suggesting that AMPK-dependent K(ATP) channel trafficking is a key mechanism for regulating β-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating β-cell excitability.

  13. Acute exposure of methylglyoxal leads to activation of KATP channels expressed in HEK293 cells

    PubMed Central

    Yang, Yang; Konduru, Anuhya S; Cui, Ningren; Yu, Lei; Trower, Timothy C; Shi, Weiwei; Shi, Yun; Jiang, Chun

    2014-01-01

    Aim: Highly reactive carbonyl methylglyoxal (MGO) is one of the metabolites excessively produced in diabetes. We have showed that prolonged exposure of vascular smooth muscle cells to MGO leads to instability of the mRNA encoding ATP-sensitive potassium (KATP) channel. In the present study we investigated the effects of MGO on the activity of KATP channels. Methods: Kir6.1/ SUR2B, Kir6.2/SUR2B or Kir6.2Δ36 (a truncated Kir6.2 isoform) alone was expressed in HEK293 cells. Whole-cell currents were recorded in the cells with an Axopatch 200B amplifier. Macroscopic currents and single-channel currents were recorded in giant inside-out patches and normal inside-out patches, respectively. Data were analyzed using Clampfit 9 software. Results: The basal activity of Kir6.1/SUR2B channels was low. The specific KATP channel opener pinacidil (10 μmol/L) could fully activate Kir6.1/SUR2B channels, which was inhibited by the specific KATP channel blocker glibenclamide (10 μmol/L). MGO (0.1-10 mmol/L) dose-dependently activated Kir6.1/SUR2B channels with an EC50 of 1.7 mmol/L. The activation of Kir6.1/SUR2B channels by MGO was reversible upon washout, and could be inhibited completely by glibenclamide. Kir6.2Δ36 channels expressed in HEK293 cells could open automatically, and the channel activity was enhanced in the presence of MGO (3 mmol/L). Single channel recordings showed that MGO (3 mmol/L) markedly increased the open probability of Kir6.1/SUR2B channels, leaving the channel conductance unaltered. Conclusion: Acute application of MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6 subunits. PMID:24122011

  14. Opening/blocking actions of pyruvate kinase antibodies on neuronal and muscular KATP channels.

    PubMed

    Mele, Antonietta; Buttiglione, Maura; Cannone, Gianluigi; Vitiello, Francesco; Camerino, Diana Conte; Tricarico, Domenico

    2012-11-01

    ATP-sensitive-K(+) (KATP) channels couple metabolism to the electrical activity of the cells. This channel is associated with glycolytic enzymes to form complexes regulating the channel activity in various tissues. The pyruvate-kinase (PK) enzyme is an antigen in the Paediatric Autoimmune Neuropsychiatric Disorders Associated Streptococcal infection known as PANDAS which is characterized by an abnormal production of auto-antibodies against PK. Here, the effects of the anti-pyruvate kinase antibody (anti-PK-ab) on the muscle and neuronal KATP channels were investigated in native rat skeletal muscle fibres and human neuroblastoma cell-line (SH-SY5Y), respectively. Furthermore, the interaction of PK with the inwardly rectifier potassium channel (Kir6.1/Kir6.2) subunits of the KATP channels was investigated by co-immunoprecipitation experiments in mouse brain using the anti-PK-ab. Patch-clamp experiments showed that the short-term incubation (1h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 enhanced the KATP current of 19.6% and 33.5%, respectively. As opposite, the long-term incubation (24h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 reduced the KATP current of 16% and 24%, respectively, reducing the diameter with atrophy. The direct application of the anti-PK-ab to the excised patches in the absence of intracellular ATP caused channel block, while in the presence of nucleotide channel opened. In neuronal cell line, in the short-term the anti-PK-ab potentiated KATP currents without affecting survival, while in the long-term the anti-PK-ab reduced KATP currents inducing neuronal death. Opening/blocking actions of the anti-PK antibodies on the KATP channels were observed, the blocking action causes fibre atrophy and neuronal death. We demonstrated that PK and Kir subunits are physically/functionally coupled in neurons. The KATP/PK complex can be proposed a novel target in the autoimmune diseases associated with anti

  15. Direct Activation of β-Cell KATP Channels with a Novel Xanthine Derivative

    PubMed Central

    Raphemot, Rene; Swale, Daniel R.; Dadi, Prasanna K.; Jacobson, David A.; Cooper, Paige; Wojtovich, Andrew P.; Banerjee, Sreedatta; Nichols, Colin G.

    2014-01-01

    ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry. PMID:24646456

  16. Cardiac K(ATP) channel alterations associated with acclimation to hypoxia in goldfish (Carassius auratus L.).

    PubMed

    Cameron, John S; DeWitt, Judy Park; Ngo, Thanh Thu; Yajnik, Tanya; Chan, Stefanie; Chung, Emma; Kang, Esther

    2013-04-01

    Goldfish (Carassius auratus L.) are highly tolerant of environmental hypoxia, and with appropriate acclimation may survive and remain active for several days in the complete absence of oxygen. Previous work suggests that the hypoxia-induced activation of cardiac ATP-sensitive potassium (KATP) channels serves to increase tolerance of low oxygen in many species. For goldfish, we have previously characterized a nitric oxide (NO)- and cGMP-dependent pathway by which this channel activation occurs in acute hypoxia. The purpose of the present study was to resolve alterations in KATP channel activity and relevant gene expression in response to acclimation under moderately hypoxic conditions (2.6mg O2/L for seven days at 22°C). Intracellular action potential duration in excised ventricles from hypoxia-acclimated animals was significantly (p<0.05) reduced at both 50% and 90% of full repolarization relative to those from normoxia-acclimated fish. In cell-attached ventricular membrane patches from hypoxia-acclimated goldfish, sarcolemmal KATP channel open probability (NPo) was significantly enhanced vs. control. Of the two genes coding for the pore-forming subunits of cardiac KATP channels (Kir6.1 and Kir6.2), mRNA transcription of kcnj8 (revealed by quantitative real-time PCR) was unchanged while kcnj11 was downregulated in response to chronic low oxygen. The mRNA levels for hif1a (hypoxia inducible factor 1α) in the hearts of hypoxia-acclimated fish were significantly enhanced, as was nitric oxide synthase (nos2) and the sulfonylurea receptor regulatory subunit (sur2, abcc9). These data suggest that prior whole-animal acclimation to chronic hypoxia enhances cardioprotective sarcolemmal KATP currents by altering transcription of regulatory proteins.

  17. Phosphatidic acid stimulates cardiac KATP channels like phosphatidylinositols, but with novel gating kinetics.

    PubMed

    Fan, Zheng; Gao, Lizhi; Wang, Wenxia

    2003-01-01

    Membrane-bound anionic phospholipids such as phosphatidylinositols have the capacity to modulate ATP-sensitive potassium (K(ATP)) channels through a mechanism involving long-range electrostatic interaction between the lipid headgroup and channel. However, it has not yet been determined whether the multiple effects of phosphatidylinositols reported in the literature all result from this general electrostatic interaction or require a specific headgroup structure. The present study investigated whether phosphatidic acid (PA), an anionic phospholipid substantially different in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native K(ATP) channels of rat heart and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2-1 mg/ml applied to the cytoplasmic side of the membrane) exhibited higher activity, lower sensitivity to ATP inhibition, less Mg(2+)-dependent nucleotide stimulation, and poor sulfonylurea inhibition. These effects match the spectrum of phosphatidylinositols' effects, but, in addition, PA also induced a novel pattern in gating kinetics, represented by a decreased mean open time (from 12.2 +/- 2.0 to 3.3 +/- 0.7 ms). This impact on gating kinetics clearly distinguishes PA's effects from those of phosphatidylinositols. Results indicate that multiple effects of anionic phospholipids on K(ATP) channels are related phenomena and can likely be attributed to a common mechanism, but additional specific effects due to other mechanisms may also coincide.

  18. Disruption of KATP Channel Expression in Skeletal Muscle by Targeted Oligonucleotide Delivery Promotes Activity-linked Thermogenesis

    PubMed Central

    Koganti, Siva Rama Krishna; Zhu, Zhiyong; Subbotina, Ekaterina; Gao, Zhan; Sierra, Ana; Proenza, Manuel; Yang, Liping; Alekseev, Alexey; Hodgson-Zingman, Denice; Zingman, Leonid

    2015-01-01

    Despite the medical, social, and economic impact of obesity, only a few therapeutic options, focused largely on reducing caloric intake, are currently available and these have limited success rates. A major impediment is that any challenge by caloric restriction is counterbalanced by activation of systems that conserve energy to prevent body weight loss. Therefore, targeting energy-conserving mechanisms to promote energy expenditure is an attractive strategy for obesity treatment. Here, in order to suppress muscle energy efficiency, we target sarcolemmal ATP-sensitive potassium (KATP) channels which have previously been shown to be important in maintaining muscle energy economy. Specifically, we employ intramuscular injections of cell-penetrating vivo-morpholinos to prevent translation of the channel pore-forming subunit. This intervention results in significant reduction of KATP channel expression and function in treated areas, without affecting the channel expression in nontargeted tissues. Furthermore, suppression of KATP channel function in a group of hind limb muscles causes a substantial increase in activity-related energy consumption, with little effect on exercise tolerance. These findings establish a proof-of-principle that selective skeletal muscle targeting of sarcolemmal KATP channel function is possible and that this intervention can alter overall bodily energetics without a disabling impact on muscle mechanical function. PMID:25648265

  19. Polyphenol (-)-Epigallocatechin Gallate during Ischemia Limits Infarct Size Via Mitochondrial KATP Channel Activation in Isolated Rat Hearts

    PubMed Central

    Song, Dae-Kyu; Kim, June Hong; Chun, Kook-Jin; Lee, Deokhee; Xu, Zhelong

    2010-01-01

    Polyphenol (-)-epigallocatechin gallate (EGCG), the most abundant catechin of green tea, appears to attenuate myocardial ischemia/reperfusion injury. We investigated the involvement of ATP-sensitive potassium (KATP) channels in EGCG-induced cardioprotection. Isolated rat hearts were subjected to 30 min of regional ischemia and 2 hr of reperfusion. EGCG was perfused for 40 min, from 10 min before to the end of index ischemia. A nonselective KATP channel blocker glibenclamide (GLI) and a selective mitochondrial KATP (mKATP) channel blocker 5-hydroxydecanoate (HD) were perfused in EGCG-treated hearts. There were no differences in coronary flow and cardiodynamics including heart rate, left ventricular developed pressure, rate-pressure product, +dP/dtmax, and -dP/dtmin throughout the experiments among groups. EGCG-treatment significantly reduced myocardial infarction (14.5±2.5% in EGCG 1 µM and 4.0±1.7% in EGCG 10 µM, P<0.001 vs. control 27.2±1.4%). This anti-infarct effect was totally abrogated by 10 µM GLI (24.6±1.5%, P<0.001 vs. EGCG). Similarly, 100 µM HD also aborted the anti-infarct effect of EGCG (24.1±1.2%, P<0.001 vs. EGCG ). These data support a role for the KATP channels in EGCG-induced cardioprotection. The mKATP channels play a crucial role in the cardioprotection by EGCG. PMID:20191036

  20. K(ATP) channels process nucleotide signals in muscle thermogenic response.

    PubMed

    Reyes, Santiago; Park, Sungjo; Terzic, Andre; Alekseev, Alexey E

    2010-12-01

    Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K(+) (K(ATP)) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, K(ATP) channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how K(ATP) channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the K(ATP) channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with K(ATP) channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal K(ATP) channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of K(ATP) channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of K(ATP) channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders.

  1. Metabolism Regulates the Spontaneous Firing of Substantia Nigra Pars Reticulata Neurons via KATP and Nonselective Cation Channels

    PubMed Central

    Lutas, Andrew; Birnbaumer, Lutz

    2014-01-01

    Neurons use glucose to fuel glycolysis and provide substrates for mitochondrial respiration, but neurons can also use alternative fuels that bypass glycolysis and feed directly into mitochondria. To determine whether neuronal pacemaking depends on active glucose metabolism, we switched the metabolic fuel from glucose to alternative fuels, lactate or β-hydroxybutyrate, while monitoring the spontaneous firing of GABAergic neurons in mouse substantia nigra pars reticulata (SNr) brain slices. We found that alternative fuels, in the absence of glucose, sustained SNr spontaneous firing at basal rates, but glycolysis may still be supported by glycogen in the absence of glucose. To prevent any glycogen-fueled glycolysis, we directly inhibited glycolysis using either 2-deoxyglucose or iodoacetic acid. Inhibiting glycolysis in the presence of alternative fuels lowered SNr firing to a slower sustained firing rate. Surprisingly, we found that the decrease in SNr firing was not mediated by ATP-sensitive potassium (KATP) channel activity, but if we lowered the perfusion flow rate or omitted the alternative fuel, KATP channels were activated and could silence SNr firing. The KATP-independent slowing of SNr firing that occurred with glycolytic inhibition in the presence of alternative fuels was consistent with a decrease in a nonselective cationic conductance. Although mitochondrial metabolism alone can prevent severe energy deprivation and KATP channel activation in SNr neurons, active glucose metabolism appears important for keeping open a class of ion channels that is crucial for the high spontaneous firing rate of SNr neurons. PMID:25471572

  2. Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells.

    PubMed

    Wu, Yi; Shyng, Show-Ling; Chen, Pei-Chun

    2015-12-11

    In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (KATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface KATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of KATP and Kv2.1 channels to coordinately inhibit insulin secretion.

  3. Mechanotransduction Drives Post Ischemic Revascularization Through KATP Channel Closure and Production of Reactive Oxygen Species

    PubMed Central

    Browning, Elizabeth; Wang, Hui; Hong, Nankang; Yu, Kevin; Buerk, Donald G.; DeBolt, Kristine; Gonder, Daniel; Sorokina, Elena M.; Patel, Puja; De Leon, Diva D.; Feinstein, Sheldon I.; Fisher, Aron B.

    2014-01-01

    Abstract Aims: We reported earlier that ischemia results in the generation of reactive oxygen species (ROS) via the closure of a KATP channel which causes membrane depolarization and NADPH oxidase 2 (NOX2) activation. This study was undertaken to understand the role of ischemia-mediated ROS in signaling. Results: Angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) was studied in vitro and in the hind limb in vivo. Flow adapted PMVEC injected into a Matrigel matrix showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of KATP channels or the NOX2. Blocking of hypoxia inducible factor-1 alpha (HIF-1α) accumulation completely abrogated the tube formation in wild-type (WT) PMVEC. With ischemia in vivo (femoral artery ligation), revascularization was high in WT mice and was significantly decreased in mice with knockout of KATP channel and in mice orally fed with a KATP channel agonist. In transgenic mice with endothelial-specific NOX2 expression, the revascularization observed was intermediate between that of WT and knockout of KATP channel or NOX2. Increased HIF-1α activation and vascular endothelial growth factor (VEGF) expression was observed in ischemic tissue of WT mice but not in KATP channel and NOX2 null mice. Revascularization could be partially rescued in KATP channel null mice by delivering VEGF into the hind limb. Innovation: This is the first report of a mechanosensitive ion channel (KATP channel) initiating endothelial signaling that drives revascularization. Conclusion: The KATP channel responds to the stop of flow and activates signals for revascularization to restore the impeded blood flow. Antioxid. Redox Signal. 20, 872–886. PMID:23758611

  4. Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice.

    PubMed

    Seino, S; Iwanaga, T; Nagashima, K; Miki, T

    2000-03-01

    The regulation of insulin secretion from pancreatic beta-cells depends critically on the activities of their plasma membrane ion channels. ATP-sensitive K+ channels (K(ATP) channels) are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. The activity of the K(ATP) channels in pancreatic beta-cells is regulated by changes in the ATP and ADP concentrations (ATP/ADP ratio) caused by glucose metabolism. Thus, the K(ATP) channels are the ATP and ADP sensors in the regulation of glucose-induced insulin secretion. K(ATP) channels are also the target of sulfonylureas, which are widely used in the treatment of type 2 diabetes. Molecular cloning of the two subunits of the pancreatic beta-cell K(ATP) channel, Kir6.2 (an inward rectifier K+ channel member) and SUR1 (a receptor for sulfonylureas), has provided great insight into its structure and function. Kir6.2 subunits form the K+ ion-permeable pore and primarily confer inhibition of the channels by ATP, while SUR1 subunits confer activation of the channels by MgADP and K+ channel openers, such as diazoxide, as well as inhibition by sulfonylureas. The SUR1 subunits also enhance the sensitivity of the channels to ATP. To determine the physiological roles of K(ATP) channels directly, we have generated two kinds of genetically engineered mice: mice expressing a dominant-negative form of Kir6.2 specifically in the pancreatic beta-cells (Kir6.2G132S Tg mice) and mice lacking Kir6.2 (Kir6.2 knockout mice). Studies of these mice elucidated various roles of the K(ATP) channels in endocrine pancreatic function: 1) the K(ATP) channels are the major determinant of the resting membrane potential of pancreatic beta-cells, 2) both glucose- and sulfonylurea-induced membrane depolarization of beta-cells require closure of the K(ATP) channels, 3) both glucose- and sulfonylurea-induced rises in intracellular calcium concentration in beta-cells require closure of the K(ATP

  5. Verrucotoxin inhibits KATP channels in cardiac myocytes through a muscarinic M3 receptor-PKC pathway.

    PubMed

    Wang, Jian-Wu; Yazawa, Kazuto; Hao, Li-Ying; Onoue, Yoshio; Kameyama, Masaki

    2007-06-01

    Verrucotoxin is the major component of venom from the stonefish (Synanceia verrucosa). Stings from the dorsal spines of the stonefish produce intensive pain, convulsions, hypotension, paralysis, respiratory weakness and collapse of the cardiovascular system, occasionally leading to death. It has been reported that verrucotoxin might modulate ATP-sensitive K+ (KATP) current in frog atrial fibers. However, the mechanism by which verrucotoxin acts on KATP current remains unclear. In this study, we examined whether verrucotoxin inhibited KATP current in guinea pig ventricular myocytes, using the patch clamp method. Verrucotoxin suppressed KATP current induced by pinacidil (KATP channel opener) in a concentration-dependent manner, with a half maximum concentration of 16.3 microg/ml. The effect of verrucotoxin on KATP current was suppressed by atropine (1 microM), a muscarinic receptor antagonist, or by 4-diphenylacetoxy-N-methylpiperidine (100 nM), a muscarinic M3 receptor antagonist. Furthermore, the effect of verrucotoxin on KATP current was attenuated by the protein kinase C (PKC) inhibitor chelerythrine (10 microM) and calphostin C (10 microM), yet not by the cAMP-dependent protein kinase (PKA) inhibitor H-89 (0.5 microM). These results suggest that verrucotoxin inhibits KATP current through the muscarinic M3 receptor-PKC pathway. These findings enhance our understanding of the toxic effects of verrucotoxin from the stonefish. PMID:17362922

  6. GLUCOSE-INDUCED INTESTINAL VASODILATION VIA ADENOSINE A1 RECEPTORS REQUIRES NITRIC OXIDE BUT NOT K+ATP CHANNELS

    PubMed Central

    Matheson, Paul J.; Li, Na; Harris, Patrick D.; Zakaria, El Rasheid; Garrison, R. Neal

    2010-01-01

    INTRODUCTION Both nitric oxide (NO) and adenosine A1 receptor activation mediate microvascular vasodilation during intestinal glucose absorption. Our overall hypothesis is that ATP utilization during glucose absorption would increase adenosine metabolite release, which acts on adenosine A1 receptors to alter endothelial production of NO and/or activate ATP-dependent potassium channels (K+ATP) to dilate intestinal microvessels. METHODS Intravital videomicroscopy of the rat jejunum was used to record the vascular responses of inflow (termed 1A) arterioles and proximal (p3A) and distal (d3A) premucosal arterioles during exposure to isotonic glucose or mannitol solutions alone or in the presence of the selective nitric oxide synthase (NOS) inhibitor (L-NMMA), an adenosine A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine (DPCPX)), or a K+ATP channel inhibitor (glibenclamide). RESULTS As expected, glucose exposure caused rapid dilation of both p3A and d3A arterioles, while mannitol exposure had no effect on microvascular diameters. Adenosine A1 receptor blockade completely prevented glucose-induced dilation of the premucosal arterioles. NOS inhibition significantly blunted the glucose-induced vasodilation of the premucosal arterioles, but had little effect in the mannitol group. Simultaneous application of both the NOS inhibitor and the adenosine A1 receptor antagonist gave the same reduction in glucose-induced dilation of the premucosal arterioles as the adenosine A1 receptor antagonist alone. Blockade of K+ATP channels with glibenclamide did not attenuate glucose-induced vasodilation of the premucosal arterioles. CONCLUSIONS These data suggest that glucose-induced vasodilation of premucosal jejunal arterioles is mediated through adenosine A1 receptors, and NO at least partially mediates the adenosine A1 receptor-induced vasodilation. In addition, K+ATP channels are not involved in premucosal arteriolar vasodilation during intestinal glucose exposure. PMID

  7. Leptin-stimulated KATP channel trafficking: a new paradigm for β-cell stimulus-secretion coupling?

    PubMed

    Holz, George G; Chepurny, Oleg G; Leech, Colin A

    2013-01-01

    Insulin secretion from pancreatic β-cells is initiated by the closure of ATP-sensitive K+ channels (KATP) in response to high concentrations of glucose, and this action of glucose is counteracted by the hormone leptin, an adipokine that signals through the Ob-Rb receptor to increase KATP channel activity. Despite intensive investigations, the molecular basis for KATP channel regulation remains uncertain, particularly from the standpoint of whether fluctuations in plasma membrane KATP channel content underlie alterations of KATP channel activity in response to glucose or leptin. Surprisingly, newly published findings reveal that leptin stimulates AMP-activated protein kinase (AMPK) in order to promote trafficking of KATP channels from cytosolic vesicles to the plasma membrane of β-cells. This action of leptin is mimicked by low concentrations of glucose that also activate AMPK and that inhibit insulin secretion. Thus, a new paradigm for β-cell stimulus-secretion coupling is suggested in which leptin exerts a tonic inhibitory effect on β-cell excitability by virtue of its ability to increase plasma membrane KATP channel density and whole-cell KATP channel current. One important issue that remains unresolved is whether high concentrations of glucose suppress AMPK activity in order to shift the balance of membrane cycling so that KATP channel endocytosis predominates over vesicular KATP channel insertion into the plasma membrane. If so, high concentrations of glucose might transiently reduce KATP channel density/current, thereby favoring β-cell depolarization and insulin secretion. Such an AMPK-dependent action of glucose would complement its established ability to generate an increase of ATP/ADP concentration ratio that directly closes KATP channels in the plasma membrane.

  8. ATP-sensitive potassium channel activation induces angiogenesis in vitro and in vivo.

    PubMed

    Umaru, Bukar; Pyriochou, Anastasia; Kotsikoris, Vasileios; Papapetropoulos, Andreas; Topouzis, Stavros

    2015-07-01

    Intense research is conducted to identify new molecular mechanisms of angiogenesis. Previous studies have shown that the angiogenic effects of hydrogen sulfide (H2S) depend on the activation of ATP-sensitive potassium channels (KATP) and that C-type natriuretic peptide (CNP), which can act through KATP, promotes endothelial cell growth. We therefore investigated whether direct KATP activation induces angiogenic responses and whether it is required for the endothelial responses to CNP or vascular endothelial growth factor (VEGF). Chick chorioallantoic membrane (CAM) angiogenesis was similarly enhanced by the direct KATP channel activator 2-nicotinamidoethyl acetate (SG-209) and by CNP or VEGF. The KATP inhibitors glibenclamide and 5-hydroxydecanoate (5-HD) reduced basal and abolished CNP-induced CAM angiogenesis. In vitro, the direct KATP openers nicorandil and SG-209 and the polypeptides VEGF and CNP increased proliferation and migration in bEnd.3 mouse endothelial cells. In addition, VEGF and CNP induced cord-like formation on Matrigel by human umbilical vein endothelial cells (HUVECs). All these in vitro endothelial responses were effectively abrogated by glibenclamide or 5-HD. In HUVECs, a small-interfering RNA-mediated decrease in the expression of the inwardly rectifying potassium channel (Kir) 6.1 subunit impaired cell migration and network morphogenesis in response to either SG-209 or CNP. We conclude that 1) direct pharmacologic activation of KATP induces angiogenic effects in vitro and in vivo, 2) angiogenic responses to CNP and VEGF depend on KATP activation and require the expression of the Kir6.1 KATP subunit, and 3) KATP activation may underpin angiogenesis to a variety of vasoactive stimuli, including H2S, VEGF, and CNP. PMID:25977483

  9. Cardioprotection afforded by chronic exercise is mediated by the sarcolemmal, and not the mitochondrial, isoform of the KATP channel in the rat

    PubMed Central

    Brown, David A; Chicco, Adam J; Jew, Korinne N; Johnson, Micah S; Lynch, Joshua M; Watson, Peter A; Moore, Russell L

    2005-01-01

    This study was conducted to examine the role of myocardial ATP-sensitive potassium (KATP) channels in exercise-induced protection from ischaemia–reperfusion (I–R) injury. Female rats were either sedentary (Sed) or exercised for 12 weeks (Tr). Hearts were excised and underwent a 1–2 h regional I–R protocol. Prior to ischaemia, hearts were subjected to pharmacological blockade of the sarcolemmal KATP channel with HMR 1098 (SedHMR and TrHMR), mitochondrial blockade with 5-hydroxydecanoic acid (5HD; Sed5HD and Tr5HD), or perfused with buffer containing no drug (Sed and Tr). Infarct size was significantly smaller in hearts from Tr animals (35.4 ± 2.3 versus 44.7 ± 3.0% of the zone at risk for Tr and Sed, respectively). Mitochondrial KATP blockade did not abolish the training-induced infarct size reduction (30.0 ± 3.4 versus 38.0 ± 2.6 in Tr5HD and Sed5HD, respectively); however, sarcolemmal KATP blockade completely eradicated the training-induced cardioprotection. Infarct size was 71.2 ± 3.3 and 64.0 ± 2.4% of the zone at risk for TrHMR and Sed HMR. The role of sarcolemmal KATP channels in Tr-induced protection was also supported by significant increases in both subunits of the sarcolemmal KATP channel following training. LV developed pressure was better preserved in hearts from Tr animals, and was not influenced by addition of HMR 1098. 5HD decreased pressure development regardless of training status, from 15 min of ischaemia through the duration of the protocol. This mechanical dysfunction was likely to be due to a 5HD-induced increase in myocardial Ca2+ content following I–R. The major findings of the present study are: (1) unlike all other known forms of delayed cardioprotection, infarct sparing following chronic exercise was not abolished by 5HD; (2) pharmacological blockade of the sarcolemmal KATP channel nullified the cardioprotective benefits of exercise training; and (3) increased expression of sarcolemmal KATP channels was observed following

  10. AMP kinase regulates ligand-gated K-ATP channels in substantia nigra dopamine neurons.

    PubMed

    Shen, Ke-Zhong; Wu, Yan-Na; Munhall, Adam C; Johnson, Steven W

    2016-08-25

    AMP-activated protein kinase (AMPK) is a master enzyme that regulates ATP-sensitive K(+) (K-ATP) channels in pancreatic beta-cells and cardiac myocytes. We used patch pipettes to record currents and potentials to investigate effects of AMPK on K-ATP currents in substantia nigra compacta (SNC) dopamine neurons in slices of rat midbrain. When slices were superfused repeatedly with the K-ATP channel opener diazoxide, we were surprised to find that diazoxide currents gradually increased in magnitude, reaching 300% of the control value 60min after starting whole-cell recording. However, diazoxide current increased significantly more, to 472% of control, when recorded in the presence of the AMPK activator A769662. Moreover, superfusing the slice with the AMPK blocking agent dorsomorphin significantly reduced diazoxide current to 38% of control. Control experiments showed that outward currents evoked by the K-ATP channel opener NN-414 also increased over time, but not currents evoked by the GABAB agonist baclofen. Delaying the application of diazoxide after starting whole-cell recording correlated with augmentation of current. Loose-patch recording showed that diazoxide produced a 34% slowing of spontaneous firing rate that did not intensify with repeated applications of diazoxide. However, superfusion with A769662 significantly augmented the inhibitory effect of diazoxide on firing rate. We conclude that K-ATP channel function is augmented by AMPK, which is activated during the process of making whole-cell recordings. Our results suggest that AMPK and K-ATP interactions may play an important role in regulating dopamine neuronal excitability.

  11. ATP-sensitive potassium channels: uncovering novel targets for treating depression.

    PubMed

    Fan, Yi; Kong, Hui; Ye, Xinhai; Ding, Jianhua; Hu, Gang

    2016-07-01

    ATP-sensitive potassium (K-ATP) channels have been shown to couple membrane electrical activity to energy metabolism in a variety of cells and are important in several physiological systems. In the brain, K-ATP channels are strongly expressed in the neuronal circuitry. The distributional profile and functional significance of K-ATP channels suggest that they may be involved in stress-induced depression. First, we showed that chronic mild stress (CMS) significantly increased the expression of hippocampal Kir6.2 and Kir6.1 subunits of K-ATP channels. Next, using Kir6.2 knockout (Kir6.2(-/-)) mice, we presented that Kir6.2 deficiency resulted in antidepressant-like behaviors under non-stress conditions, but aggravated depressive behaviors accompanied by the loss of CA3 neuron and the reduction of brain-derived neurotrophic factor in hippocampus under chronic stress. Finally, we demonstrated that the K-ATP channel opener iptakalim, as well as a classical antidepressant fluoxetine, can reverse CMS-induced depression-related behaviors and counteract the deleterious effects of stress on hippocampus in wild-type mice, but only partially alleviate these symptoms in Kir6.2(-/-) mice. Collectively, our findings demonstrate that K-ATP channels are involved in the pathogenesis of depression and may be a promising target for the therapy of depression.

  12. Differential mechanisms of Cantú syndrome-associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel.

    PubMed

    Cooper, Paige E; Sala-Rabanal, Monica; Lee, Sun Joo; Nichols, Colin G

    2015-12-01

    Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium ((86)Rb(+)) efflux assays, we show that K(ATP) channels formed with P429L, A475V, or C1039Y mutants enhance K(ATP) activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive K(ATP) channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation. PMID:26621776

  13. Differential mechanisms of Cantú syndrome–associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel

    PubMed Central

    Cooper, Paige E.; Sala-Rabanal, Monica; Lee, Sun Joo

    2015-01-01

    Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium (86Rb+) efflux assays, we show that KATP channels formed with P429L, A475V, or C1039Y mutants enhance KATP activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive KATP channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation. PMID:26621776

  14. KATP channel as well as SGLT1 participates in GIP secretion in the diabetic state.

    PubMed

    Ogata, Hidetada; Seino, Yusuke; Harada, Norio; Iida, Atsushi; Suzuki, Kazuyo; Izumoto, Takako; Ishikawa, Kota; Uenishi, Eita; Ozaki, Nobuaki; Hayashi, Yoshitaka; Miki, Takashi; Inagaki, Nobuya; Tsunekawa, Shin; Hamada, Yoji; Seino, Susumu; Oiso, Yutaka

    2014-08-01

    Glucose-dependent insulinotropic polypeptide (GIP), a gut hormone secreted from intestinal K-cells, potentiates insulin secretion. Both K-cells and pancreatic β-cells are glucose-responsive and equipped with a similar glucose-sensing apparatus that includes glucokinase and an ATP-sensitive K(+) (KATP) channel comprising KIR6.2 and sulfonylurea receptor 1. In absorptive epithelial cells and enteroendocrine cells, sodium glucose co-transporter 1 (SGLT1) is also known to play an important role in glucose absorption and glucose-induced incretin secretion. However, the glucose-sensing mechanism in K-cells is not fully understood. In this study, we examined the involvement of SGLT1 (SLC5A1) and the KATP channels in glucose sensing in GIP secretion in both normal and streptozotocin-induced diabetic mice. Glimepiride, a sulfonylurea, did not induce GIP secretion and pretreatment with diazoxide, a KATP channel activator, did not affect glucose-induced GIP secretion in the normal state. In mice lacking KATP channels (Kir6.2(-/-) mice), glucose-induced GIP secretion was enhanced compared with control (Kir6.2(+) (/) (+)) mice, but was completely blocked by the SGLT1 inhibitor phlorizin. In Kir6.2(-/-) mice, intestinal glucose absorption through SGLT1 was enhanced compared with that in Kir6.2(+) (/) (+) mice. On the other hand, glucose-induced GIP secretion was enhanced in the diabetic state in Kir6.2(+) (/) (+) mice. This GIP secretion was partially blocked by phlorizin, but was completely blocked by pretreatment with diazoxide in addition to phlorizin administration. These results demonstrate that glucose-induced GIP secretion depends primarily on SGLT1 in the normal state, whereas the KATP channel as well as SGLT1 is involved in GIP secretion in the diabetic state in vivo.

  15. Targeting potassium channels in cancer

    PubMed Central

    2014-01-01

    Potassium channels are pore-forming transmembrane proteins that regulate a multitude of biological processes by controlling potassium flow across cell membranes. Aberrant potassium channel functions contribute to diseases such as epilepsy, cardiac arrhythmia, and neuromuscular symptoms collectively known as channelopathies. Increasing evidence suggests that cancer constitutes another category of channelopathies associated with dysregulated channel expression. Indeed, potassium channel–modulating agents have demonstrated antitumor efficacy. Potassium channels regulate cancer cell behaviors such as proliferation and migration through both canonical ion permeation–dependent and noncanonical ion permeation–independent functions. Given their cell surface localization and well-known pharmacology, pharmacological strategies to target potassium channel could prove to be promising cancer therapeutics. PMID:25049269

  16. ATP-sensitive potassium channel modulation of the guinea pig ventricular action potential and contraction.

    PubMed

    Nichols, C G; Ripoll, C; Lederer, W J

    1991-01-01

    The role of ATP-sensitive potassium (KATP) channels in modulating the action potential and contraction of guinea pig ventricular myocytes was investigated. Under voltage clamp, the maximum whole-cell KATP channel conductance was estimated (195 +/- 10 nS, n = 6) by exposing the cells to complete metabolic blockade (2 mM cyanide in the presence of 10 mM 2-deoxy-glucose). In isolated inside-out membrane patches, the ATP dependence of KATP channel activity under relevant conditions was measured (half-maximal inhibition at 114 microM). Under current clamp (with intracellular ATP concentration = 5 mM), the effect of graded KATP channel activation on the action potential and the twitch was estimated by injection of a current (proportional to voltage) that simulated the KATP conductance. As this "conductance" was increased, the action potential was shortened, and contractile amplitude declined, as expected. From the results of these experiments, the quantitative dependence of the action potential duration on intracellular ATP concentration was estimated, without relying on a mathematical model of the cell membrane. The results imply that KATP-dependent action potential shortening is likely to occur if ATP concentration falls below normal levels (approximately 5 mM), as may happen regionally, or globally, during myocardial ischemia.

  17. KR-31762, a novel KATP channel opener, exerts cardioprotective effects by opening SarcKATP channels in rat models of ischemia/reperfusion-induced heart injury.

    PubMed

    Lee, Sung-Hun; Yang, Min-Kyu; Lim, Jong-Hyun; Seo, Ho-Won; Yi, Kyu-Yang; Yoo, Sung-Eun; Lee, Byung-Ho; Won, Hyung-Sik; Lee, Chang-Soo; Choi, Wahn-Soo; Shin, Hwa-Sup

    2008-04-01

    The cardioprotective effects of KR-31762, a newly synthesized K+(ATP) opener, were evaluated in rat models of ischemia/reperfusion (I/R) heart injury. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, KR-31762 (3 and 10 microM) significantly increased the left ventricular developed pressure (LVDP) and double product (heart rate x LVDP) after 30-min reperfusion in a concentration-dependent manner, while decreasing the left ventricular end-diastolic pressure (LVEDP). KR-31762 also significantly increased the time to contracture (TTC) during ischemic period (20.0, 22.4 and 26.4 min for control, 3 and 10 microM, respectively), while decreasing the release of lactate dehydrogenase (LDH) from the heart during 30 min reperfusion (30.4, 14.3 and 19.7 U/g heart weight, respectively). All these parameters except LDH release were reversed by glyburide (1 microM), a nonselective blocker of K+(ATP) channel, but not by 5-hydroxydecanoate, a selective blocker of mitoK+(ATP) channel. In anesthetized rats subjected to 45-min occlusion of left anterior descending coronary artery followed by 90-min reperfusion, KR-31762 significantly decreased the infarct size (60.8, 40.5 and 37.8% for control, 0.3 and 1.0 mg/kg, iv, respectively). KR-31762 slightly relaxed the isolated rat aorta precontracted with methoxamine (IC(50): 23.5 microM). These results suggest that KR-31762 exerts potent cardioprotective effects through the opening of sarcolemmal K(ATP) channel in rat hearts with the minimal vasorelaxant effects.

  18. Glibenclamide attenuates the antiarrhythmic effect of endotoxin with a mechanism not involving K(ATP) channels.

    PubMed

    Iskit, Alper B; Erkent, Ulkem; Ertunc, Mert; Guc, M Oguz; Ilhan, Mustafa; Onur, Rustu

    2007-02-01

    The role of K(ATP) channels in the antiarrhythmic effect of Escherichia coli endotoxin-induced nitric oxide synthase (iNOS) was examined in an anesthetised rat model of myocardial ischemia and reperfusion arrhythmia by using glibenclamide (1 mg kg(-1)), nateglinide (10 mg kg(-1)) and repaglinide (0.5 mg kg(-1)). Endotoxin (1 mg kg(-1)) was administered intraperitoneally 4 h before the occlusion of the left coronary artery and glibenclamide, nateglinide or repaglinide was administered 30 min before coronary artery occlusion. We also evaluated the effects of K(ATP) channel blockers and nonselective K(+) channel blocker tetraethylammonium (TEA) on cardiac action potential configuration in the atria obtained from endotoxemic rats. The mean arterial blood pressure of rats receiving endotoxin was lower during both the occlusion and reperfusion periods. Endotoxin significantly reduced the total number of ectopic beats and the duration of ventricular tachycardia. Glibenclamide, but not nateglinide and repaglinide, prevented the hypotension and antiarrhythmic effects of endotoxin. Atria obtained from endotoxin-treated rats had prolonged action potential duration. This effect was abolished with pretreatment of iNOS inhibitors, l-canavanine and dexamethasone and perfusion of glibenclamide, but not with TEA and non-sulfonylurea drug, nateglinide. We demonstrated that glibenclamide inhibits the antiarrhythmic effect of endotoxin and this effect does not appear to involve K(ATP) channels.

  19. Blockade of microglial KATP -channel abrogates suppression of inflammatory-mediated inhibition of neural precursor cells.

    PubMed

    Ortega, Francisco J; Vukovic, Jana; Rodríguez, Manuel J; Bartlett, Perry F

    2014-02-01

    Microglia positively affect neural progenitor cell physiology through the release of inflammatory mediators or trophic factors. We demonstrated previously that reactive microglia foster K(ATP) -channel expression and that blocking this channel using glibenclamide administration enhances striatal neurogenesis after stroke. In this study, we investigated whether the microglial K(ATP) -channel directly influences the activation of neural precursor cells (NPCs) from the subventricular zone using transgenic Csf1r-GFP mice. In vitro exposure of NPCs to lipopolysaccharide and interferon-gamma resulted in a significant decrease in precursor cell number. The complete removal of microglia from the culture or exposure to enriched microglia culture also decreased the precursor cell number. The addition of glibenclamide rescued the negative effects of enriched microglia on neurosphere formation and promoted a ∼20% improvement in precursor cell number. Similar results were found using microglial-conditioned media from isolated microglia. Using primary mixed glial and pure microglial cultures, glibenclamide specifically targeted reactive microglia to restore neurogenesis and increased the microglial production of the chemokine monocyte chemoattractant protein-1 (MCP-1). These findings provide the first direct evidence that the microglial K(ATP) -channel is a regulator of the proliferation of NPCs under inflammatory conditions.

  20. Evidence for glucagon-like peptide-1 receptor signaling to activate ATP-sensitive potassium channels in pancreatic beta cells.

    PubMed

    Kwon, Hye-Jung; Park, Hyun-Sun; Park, Sung-Hee; Park, Jae-Hyung; Shin, Su-Kyung; Song, Seung Eun; Hwang, Meeyul; Cho, Ho-Chan; Song, Dae-Kyu

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is a gut peptide that promotes insulin release from pancreatic beta cells. GLP-1 has been shown to confer glucose-insensitive beta cells with glucose sensitivity by modulation of the activity of the ATP-sensitive potassium (KATP) channel. The channel closing effect of GLP-1, interacting with corresponding G-protein-coupled receptors, has been well established; however, to our knowledge, no study has shown whether GLP-1 directly induces activation of beta-cell KATP channels. Here, we aimed to evaluate whether the activation of beta-cell KATP channels by GLP-1 exists and affects intracellular Ca(2+) levels ([Ca(2+)]i). KATP channel activity was measured in isolated rat pancreatic beta cells by whole-cell perforated patch-clamp recordings with a diazoxide-containing pipette solution. Changes in [Ca(2+)]i and the subcellular localization of KATP channels were observed using the calcium-sensitive dye fura-4/AM and anti-Kir6.2 antibodies in INS-1 beta cells, respectively. To eliminate the well-known inhibitory effects of GLP-1 on KATP channel activity, channels were fully inhibited by pretreatment with methyl pyruvate and epigallocatechin-3-gallate. In the pretreated beta cells, GLP-1 and exendin-4 promptly activated the channels, reducing [Ca(2+)]i. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 blocked the effects of GLP-1 on channel activity. Moreover, phosphatidylinositol-3,4,5-trisphosphate mimicked the effects of GLP-1. These results suggested that beta-cell GLP-1 receptor signaling involved activation of KATP channels via a PI3K-dependent pathway. This alternative mechanism of GLP-1 function may act as a negative feedback pathway, modulating the glucose-dependent GLP-1 inhibition on KATP channel activity. PMID:26655814

  1. Bisabolol-induced gastroprotection against acute gastric lesions: role of prostaglandins, nitric oxide, and KATP+ channels.

    PubMed

    Bezerra, S B; Leal, L K A M; Nogueira, N A P; Pinto, N A N; Campos, A R

    2009-12-01

    The effects of Matricaria recutita and alpha-bisabolol, a bioactive component from Chamomile species, were investigated against gastric damage induced by absolute ethanol (96%, 1 mL per animal) in rats. The effects of M. recutita extract and alpha-bisabolol on gastric mucosal damage were assessed by determination of changes in mean gastric lesion area. Mechanistic studies were carried out at with 100 mg=kg alpha-bisabolol. We further examined the possible participation of prostaglandins, nitric oxide, and KATP+ channels in its mechanism. M. recutita reduced gastric damage in all doses tested. Alpha-bisabolol at oral doses of 50 and 100 mg=kg markedly attenuated the gastric lesions induced by ethanol to the extent of 87% and 96%, respectively. Pretreatments with the nitric oxide antagonist N-nitro-l-arginine methyl ester (10 mg=kg, i.p.) or with indomethacin, an inhibitor of cyclooxygenase, failed to block effectively the gastroprotective effect of alpha-bisabolol. Furthermore, the alpha-bisabolol effect was significantly reduced in rats pretreated with glibenclamide, an inhibitor of KATP+ channel activation. Thus we provide evidence that alpha-bisabolol reduces the gastric damage induced by ethanol, at least in part, by the mechanism of activation of KATP+ channels. PMID:20041801

  2. Neuronal and Cardiovascular Potassium Channels as Therapeutic Drug Targets

    PubMed Central

    Humphries, Edward S. A.

    2015-01-01

    Potassium (K+) channels, with their diversity, often tissue-defined distribution, and critical role in controlling cellular excitability, have long held promise of being important drug targets for the treatment of dysrhythmias in the heart and abnormal neuronal activity within the brain. With the exception of drugs that target one particular class, ATP-sensitive K+ (KATP) channels, very few selective K+ channel activators or inhibitors are currently licensed for clinical use in cardiovascular and neurological disease. Here we review what a range of human genetic disorders have told us about the role of specific K+ channel subunits, explore the potential of activators and inhibitors of specific channel populations as a therapeutic strategy, and discuss possible reasons for the difficulty in designing clinically relevant K+ channel modulators. PMID:26303307

  3. KATP channels in the nodose ganglia mediate the orexigenic actions of ghrelin

    PubMed Central

    Grabauskas, Gintautas; Wu, Xiaoyin; Lu, Yuanxu; Heldsinger, Andrea; Song, Il; Zhou, Shi-Yi; Owyang, Chung

    2015-01-01

    Abstract Ghrelin is the only known hunger signal derived from the peripheral tissues. Ghrelin overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. The mechanisms by which ghrelin reduces the sensory signals evoked by anorexigenic hormones, which act via the vagus nerve to stimulate feeding, are unknown. Patch clamp recordings of isolated rat vagal neurons show that ghrelin hyperpolarizes neurons by activating K+ conductance. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition in vitro and in vivo. Patch clamp studies show that ghrelin inhibits currents evoked by leptin and CCK-8, which operate through independent ionic channels. The inhibitory actions of ghrelin were abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. In vivo gene silencing of PI3K and Erk1/2 in the nodose ganglia prevented ghrelin inhibition of leptin- or CCK-8-evoked vagal firing. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a–Gαi–PI3K–Erk1/2–KATP pathway. The resulting hyperpolarization renders the neurons less responsive to signals evoked by anorexigenic hormones. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways. Key points Ghrelin, a hunger signalling peptide derived from the peripheral tissues, overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. Using in vivo and in vitro electrophysiological

  4. Impairment of the Vascular KATP Channel Imposes Fatal Susceptibility to Experimental Diabetes Due to Multi-Organ Injuries.

    PubMed

    Li, Shan-Shan; Cui, Ningren; Yang, Yang; Trower, Timothy C; Wei, Yu-Min; Wu, Yang; Zhang, Shuang; Jin, Xin; Jiang, Chun

    2015-12-01

    The vascular isoform of ATP-sensitive K(+) (KATP ) channels regulates blood flow to all organs. The KATP channel is strongly inhibited by reactive oxygen and carbonyl species produced in diabetic tissue inflammation. To address how such channel inhibition impacts vascular regulation as well as tissue viability, we performed studies in experimental diabetic mice. Strikingly, we found that knockout of the Kcnj8 encoding Kir6.1 subunit (Kcnj8-KO) caused mice to be fatally susceptible to diabetes. Organ perfusion studies suggested that the lack of this vascular K(+) channel handicapped activity-dependent vasodilation, leading to hypoperfusion, tissue hypoxia, and multi-organ failure. Morphologically, Kcnj8-KO mice showed greater inflammatory cell infiltration, higher levels of expression of inflammation indicator proteins, more severe cell apoptosis, and worse tissue disruptions. These were observed in the kidney, liver, and heart under diabetic condition in parallel comparison to tissues from WT mice. Patch clamping and molecular studies showed that the KATP channel was S-glutathionylated in experimental diabetes contributing to the inhibition of channel activity as well as the reduced arterial responses to vasodilators. These results suggest that the vascular KATP channel is organ protective in diabetic condition, and since the channel is suppressed by diabetic oxidative stress, therapeutical interventions to the maintenance of functional KATP channels may help to lower or prevent diabetic organ dysfunction.

  5. Impairment of the Vascular KATP Channel Imposes Fatal Susceptibility to Experimental Diabetes Due to Multi-Organ Injuries.

    PubMed

    Li, Shan-Shan; Cui, Ningren; Yang, Yang; Trower, Timothy C; Wei, Yu-Min; Wu, Yang; Zhang, Shuang; Jin, Xin; Jiang, Chun

    2015-12-01

    The vascular isoform of ATP-sensitive K(+) (KATP ) channels regulates blood flow to all organs. The KATP channel is strongly inhibited by reactive oxygen and carbonyl species produced in diabetic tissue inflammation. To address how such channel inhibition impacts vascular regulation as well as tissue viability, we performed studies in experimental diabetic mice. Strikingly, we found that knockout of the Kcnj8 encoding Kir6.1 subunit (Kcnj8-KO) caused mice to be fatally susceptible to diabetes. Organ perfusion studies suggested that the lack of this vascular K(+) channel handicapped activity-dependent vasodilation, leading to hypoperfusion, tissue hypoxia, and multi-organ failure. Morphologically, Kcnj8-KO mice showed greater inflammatory cell infiltration, higher levels of expression of inflammation indicator proteins, more severe cell apoptosis, and worse tissue disruptions. These were observed in the kidney, liver, and heart under diabetic condition in parallel comparison to tissues from WT mice. Patch clamping and molecular studies showed that the KATP channel was S-glutathionylated in experimental diabetes contributing to the inhibition of channel activity as well as the reduced arterial responses to vasodilators. These results suggest that the vascular KATP channel is organ protective in diabetic condition, and since the channel is suppressed by diabetic oxidative stress, therapeutical interventions to the maintenance of functional KATP channels may help to lower or prevent diabetic organ dysfunction. PMID:25825210

  6. KATP-channels play a minor role in the protective hypoxic shut-down of cerebellar activity in eider ducks (Somateria mollissima).

    PubMed

    Geiseler, S J; Ludvigsen, S; Folkow, L P

    2015-01-22

    Eider duck (Somateria mollissima) cerebellar neurons are highly tolerant toward hypoxia in vitro, which in part is due to a hypoxia-induced depression of their spontaneous activity. We have studied whether this response involves ATP-sensitive potassium (KATP) channels, which are known to be involved in the hypoxic/ischemic defense of mammalian neural and muscular tissues, by causing hyperpolarization and reduced ATP demand. Extracellular recordings in the Purkinje layer of isolated normoxic eider duck cerebellar slices showed that their spontaneous neuronal activity decreased significantly compared to in control slices when the KATP channel opener diazoxide (600 μM) was added (F1,70=92.781, p<0.001). Adding the KATP channel blocker tolbutamide (400 μM) 5 min prior to diazoxide completely abolished its effect (F1,55=39.639, p<0.001), strongly suggesting that these drugs have a similar mode of action in this avian species as in mammals. The spontaneous activity of slices treated with tolbutamide in combined hypoxia/chemical anoxia (95% N2-5% CO2 and 2 mM NaCN) was not significantly different from that of control slices (F1,203=0.071, p=0.791). Recovery from hypoxia/anoxia was, however, slightly but significantly weaker in tolbutamide-treated slices than in control slices (F1,137=15.539, p<0.001). We conclude that KATP channels are present in eider duck cerebellar neurons and are activated in hypoxia/anoxia, but that they do not play a key role in the protective shut-down response to hypoxia/anoxia. PMID:25451290

  7. Alpha lipoic acid protects the heart against myocardial post ischemia-reperfusion arrhythmias via KATP channel activation in isolated rat hearts.

    PubMed

    Dudek, Magdalena; Knutelska, Joanna; Bednarski, Marek; Nowiński, Leszek; Zygmunt, Małgorzata; Bilska-Wilkosz, Anna; Iciek, Małgorzata; Otto, Monika; Żytka, Iwona; Sapa, Jacek; Włodek, Lidia; Filipek, Barbara

    2014-06-01

    The cardiovascular effects of alpha lipoic acid were evaluated in isolated rat hearts exposed to ischemia-reperfusion injury in vitro. Alpha-lipoic acid raised the level of sulfane sulfur playing an important role in the release of hydrogen sulfide. H2S was shown to prevent the post-reperfusion arrhythmias and to protect the cardiomyocytes from death caused by hypoxia. The activation of potassium ATP-sensitive channels (K(ATP) channels) is one of the most important mechanisms of action of hydrogen sulfide in the cardiovascular system. The aim of this study was to investigate whether alpha lipoic acid can prevent the occurrence of post-reperfusion arrhythmias in vitro using a Langendorff model of ischemia-reperfusion in rats affecting the K(ATP) channels. Alpha lipoic acid significantly improved post-reperfusion cardiac function (reducing incidence of arrhythmias), especially in a dose of 10(-7)M. These cardiovascular effects of this compound on the measured parameters were reversed by glibenclamide, a selective K(ATP) blocker. Alpha lipoic acid increased the level of sulfane sulfur in the hearts. This may suggest that the positive effects caused by alpha lipoic acid in the cardiovascular system are not only related to its strong antioxidant activity, and the influence on the activity of such enzymes as aldehyde dehydrogenase 2, as previously suggested, but this compound can affect K(ATP) channels. It is possible that this indirect effect of alpha lipoic acid is connected with changes in the release of sulfane sulfur and hydrogen sulfide.

  8. Influence of Thromboxane A2 on the Regulation of Adenosine Triphosphate-Sensitive Potassium Channels in Mouse Ventricular Myocytes

    PubMed Central

    Jeong, In Seok; Cho, Hwa Jin; Cho, Jeong Gwan; Kim, Sang Hyung; Na, Kook Joo

    2016-01-01

    Background and Objectives Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels play an important role in myocardial protection. We examined the effects of thromboxane A2 on the regulation of KATP channel activity in single ventricular myocytes. Subjects and Methods Single ventricular myocytes were isolated from the hearts of adult Institute of Cancer Research (ICR) mice by enzymatic digestion. Single channel activity was recorded by excised inside-out and cell-attached patch clamp configurations at −60 mV holding potential during the perfusion of an ATP-free K-5 solution. Results In the excised inside-out patches, the thromboxane A2 analog, U46619, decreased the KATP channel activity in a dose-dependent manner; however, the thromboxane A2 receptor antagonist, SQ29548, did not significantly attenuate the inhibitory effect of U46619. In the cell-attached patches, U46619 inhibited dinitrophenol (DNP)-induced KATP channel activity in a dose-dependent manner, and SQ29548 attenuated the inhibitory effects of U46619 on DNP-induced KATP channel activity. Conclusion Thromboxane A2 may inhibit KATP channel activity, and may have a harmful effect on ischemic myocardium. PMID:27482267

  9. K+ channel mechanisms underlying cholinergic cutaneous vasodilation and sweating in young humans: roles of KCa, KATP, and KV channels?

    PubMed

    Fujii, Naoto; Louie, Jeffrey C; McNeely, Brendan D; Zhang, Sarah Yan; Tran, My-An; Kenny, Glen P

    2016-09-01

    Acetylcholine released from cholinergic nerves is involved in heat loss responses of cutaneous vasodilation and sweating. K(+) channels are thought to play a role in regulating cholinergic cutaneous vasodilation and sweating, though which K(+) channels are involved in their regulation remains unclear. We evaluated the hypotheses that 1) Ca(2+)-activated K(+) (KCa), ATP-sensitive K(+) (KATP), and voltage-gated K(+) (KV) channels all contribute to cholinergic cutaneous vasodilation; and 2) KV channels, but not KCa and KATP channels, contribute to cholinergic sweating. In 13 young adults (24 ± 5 years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at intradermal microdialysis sites that were continuously perfused with: 1) lactated Ringer (Control), 2) 50 mM tetraethylammonium (KCa channel blocker), 3) 5 mM glybenclamide (KATP channel blocker), and 4) 10 mM 4-aminopyridine (KV channel blocker). At all sites, cholinergic cutaneous vasodilation and sweating were induced by coadministration of methacholine (0.0125, 0.25, 5, 100, and 2,000 mM, each for 25 min). The methacholine-induced increase in CVC was lower with the KCa channel blocker relative to Control at 0.0125 (1 ± 1 vs. 9 ± 6%max) and 5 (2 ± 5 vs. 17 ± 14%max) mM methacholine, whereas it was lower in the presence of KATP (69 ± 7%max) and KV (57 ± 14%max) channel blocker compared with Control (79 ± 6%max) at 100 mM methacholine. Furthermore, methacholine-induced sweating was lower at the KV channel blocker site (0.42 ± 0.17 mg·min(-1)·cm(-2)) compared with Control (0.58 ± 0.15 mg·min(-1)·cm(-2)) at 2,000 mM methacholine. In conclusion, we show that KCa, KATP, and KV channels play a role in cholinergic cutaneous vasodilation, whereas only KV channels contribute to cholinergic sweating in normothermic resting humans. PMID:27440718

  10. Activation of ATP-sensitive potassium channels in rat pancreatic beta-cells by linoleic acid through both intracellular metabolites and membrane receptor signalling pathway.

    PubMed

    Zhao, Yu-Feng; Pei, Jianming; Chen, Chen

    2008-09-01

    ATP-sensitive potassium channels (K(ATP) channels) determine the excitability of pancreatic beta-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to beta-cells, but the effects of exogenous FFAs on K(ATP) channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and K(ATP) channels were observed in primary cultured rat pancreatic beta-cells. LA (20 microM) induced hyperpolarization of MP and opening of K(ATP) channels, which was totally reversed and inhibited by tolbutamide, a K(ATP) channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 microM), partially inhibited LA-induced opening of K(ATP) channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 microM), induced an opening of K(ATP) channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of K(ATP) channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on K(ATP) channels. The present study demonstrates that LA induces hyperpolarization of MP by activating K(ATP) channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and beta-cell dysfunction induced by FFAs.

  11. cAMP-independent dilation of coronary arterioles to adenosine : role of nitric oxide, G proteins, and K(ATP) channels.

    PubMed

    Hein, T W; Kuo, L

    1999-10-01

    Adenosine is known to play an important role in the regulation of coronary blood flow during metabolic stress. However, there is sparse information on the mechanism of adenosine-induced dilation at the microcirculatory levels. In the present study, we examined the role of endothelial nitric oxide (NO), G proteins, cyclic nucleotides, and potassium channels in coronary arteriolar dilation to adenosine. Pig subepicardial coronary arterioles (50 to 100 microm in diameter) were isolated, cannulated, and pressurized to 60 cm H(2)O without flow for in vitro study. The arterioles developed basal tone and dilated dose dependently to adenosine. Disruption of endothelium, blocking of endothelial ATP-sensitive potassium (K(ATP)) channels by glibenclamide, and inhibition of NO synthase by N(G)-nitro-L-arginine methyl ester and of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Production of NO from coronary arterioles was significantly increased by adenosine. Pertussis toxin, but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Tetraethylammonium, glibenclamide, and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels by iberiotoxin had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. Furthermore, vasodilations to 8-Br-cAMP and sodium nitroprusside were not inhibited by glibenclamide, indicating that cAMP- and cGMP-induced dilations are not mediated by the activation of K(ATP) channels. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert

  12. Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction.

    PubMed

    Pfohl, Jeffrey L; Worley, Jennings F; Condreay, J Patrick; An, Gang; Apolito, Christopher J; Kost, Tom A; Truax, James F

    2002-01-01

    A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.

  13. Potent Cardioprotective Effect of the 4-Anilinoquinazoline Derivative PD153035: Involvement of Mitochondrial KATP Channel Activation

    PubMed Central

    Rocco, Silvana A.; Cerqueira, Fernanda M.; Caldeira da Silva, Camille C.; Rittner, Roberto; Kowaltowski, Alicia J.; Vercesi, Anibal E.; Franchini, Kleber G.; Castilho, Roger F.

    2010-01-01

    Background The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function. Methodology/Principal Findings Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca2+ induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K+ transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K+ channels (mitoKATP). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoKATP activation. Conclusions/Significance We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoKATP activation. PMID:20498724

  14. Opening of ATP-sensitive K(+) (KATP) channels enhance hydroxyl radical generation induced by MPP(+) in rat striatum.

    PubMed

    Obata, Toshio; Nakashima, Michiko

    2016-07-15

    The present study examined whether opening of adenosine triphosphate (ATP) sensitive K(+) (KATP) channels can enhance 1-methyl-4-phenylpyridinium (MPP(+))-induced hydroxyl radical (OH) generation in rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5nmol/ml per min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2.3-dihydroxybenzoic acid (DHBA) in the striatum. MPP(+) (5mM) enhanced generation of OH with concomitant increased efflux of dopamine (DA). Cromakalim (100μM), a KATP channel opener, through the microdialysis probe significantly increased both DA efflux and OH formation induced by MPP(+). Another KATP channel opener, nicorandil (1mM), also increased the level DA or DHBA, but these changes were not significant. However, in the presence of glibenclamide (10μM), a KATP channel antagonist, and the increase of MPP(+)-induced DA or DHBA were not observed. Cromakalim (10, 50 and 100μM) increased MPP(+)-induced DHBA formation in a concentration-dependent manner. However, the effects of cromakalim in the presence of glibenclamide were abolished. These results suggest that opening of KATP channels may cause OH generation by MPP(+). PMID:27288802

  15. Vascular potassium channels in NVC.

    PubMed

    Yamada, K

    2016-01-01

    It has long been proposed that the external potassium ion ([K(+)]0) works as a potent vasodilator in the dynamic regulation of local cerebral blood flow. Astrocytes may play a central role for producing K(+) outflow possibly through calcium-activated potassium channels on the end feet, responding to a rise in the intracellular Ca(2+) concentration, which might well reflect local neuronal activity. A mild elevation of [K(+)]0 in the end feet/vascular smooth muscle space could activate Na(+)/K(+)-ATPase concomitant with inwardly rectifying potassium (Kir) channels in vascular smooth muscle cells, leading to a hyperpolarization of vascular smooth muscle and relaxation of smooth muscle actin-positive vessels. Also proposed notion is endothelial calcium-activated potassium channels and/or inwardly rectifying potassium channel-mediated hyperpolarization of vascular smooth muscle. A larger elevation of [K(+)]0, which may occur pathophysiologically in such as spreading depression or stroke, can trigger a depolarization of vascular smooth muscle cells and vasoconstriction instead. PMID:27130411

  16. Matrix volume measurements challenge the existence of diazoxide/glibencamide-sensitive KATP channels in rat mitochondria

    PubMed Central

    Das, Manika; Parker, Joanne E; Halestrap, Andrew P

    2003-01-01

    A mitochondrial sulphonylurea-sensitive, ATP-sensitive K+ channel (mitoKATP) that is selectively inhibited by 5-hydroxydecanoate (5-HD) and activated by diazoxide has been implicated in ischaemic preconditioning. Here we re-evaluate the evidence for the existence of this mitoKATP by measuring changes in light scattering (A520) in parallel with direct determination of mitochondrial matrix volumes using 3H2O and [14C]sucrose. Incubation of rat liver and heart mitochondria in KCl medium containing Mg2+ and inorganic phosphate caused a decrease in light scattering over 5 min, which was accompanied by a small (15–30 %) increase in matrix volume. The presence of ATP or ADP in the buffer from the start greatly inhibited the decline in A520, whilst addition after a period of incubation (1–5 min) induced a rapid increase in A520, especially in heart mitochondria. Neither response was accompanied by a change in matrix volume, as measured isotopically. However, the effects of ATP and ADP on A520 were abolished by carboxyatractyloside and bongkrekic acid, inhibitors of the adenine nucleotide translocase (ANT) that lock the transporter in two discrete conformations and cause distinct changes in A520 in their own right. These data suggest that rather than matrix volume changes, the effects of ATP and ADP on A520 reflect changes in mitochondrial shape induced by conformational changes in the ANT. Furthermore, we were unable to demonstrate either a decrease in A520 or increase in matrix volume with a range of ATP-sensitive K+ channel openers such as diazoxide. Nor did glibencamide or 5-HD cause any reduction of matrix volume, whereas the K+ ionophore valinomycin (0.2 nm), produced a 10–20 % increase in matrix volume that was readily detectable by both techniques. Our data argue against the existence of a sulphonylurea-inhibitable mitoKATP channel. PMID:12562892

  17. The Protective Effect of Remote Renal Preconditioning Against Hippocampal Ischemia Reperfusion Injury: Role of KATP Channels.

    PubMed

    Mehrjerdi, Fatemeh Zare; Aboutaleb, Nahid; Pazoki-Toroudi, Hamidreza; Soleimani, Mansoureh; Ajami, Marjan; Khaksari, Mehdi; Safari, Fatemeh; Habibey, Rouhollah

    2015-12-01

    Remote ischemic preconditioning (RIPC), which consists of several brief ischemia/reperfusion applied at the remote site of lethal ischemia reperfusion, can, through activating different mechanisms, increase the ability of the body's endogenous protection against prolonged ischemia/reperfusion. Recent studies have shown that RIPC has neuroprotective effects, but its mechanisms are not well elucidated. The present study aimed to determine whether activation of KATP channels in remote renal preconditioning decreases hippocampus damage induced by global cerebral ischemia. RIPC was induced by ischemia of the left renal artery (IPC); 24 h later, global cerebral ischemia reperfusion (IR) was induced by common carotid arteries occlusion. 5hydroxydecanoate (5HD) and glibenclamide (Gli) were injected before of IPC. The levels of malondialdehyde (MDA) and catalase (CAT) activity were assessed in hippocampus. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was assessed to detect apoptotic cells in hippocampus. RIPC inhibited apoptosis by decreasing positive TUNEL cells (P < 0.05). KATP channels blocking with 5HD and Gli markedly increased apoptosis in hippocampal cells in RIPC group (P < 0.001). RIPC decreased MDA level and increased CAT activity in ischemic hippocampus (P < 0.01). Also, 5HD and Gli inhibited the effect of RIPC on MDA level and CAT activity (P < 0.05). The present study shows that RIPC can effectively attenuate programmed cell death, increase activity of CAT, and reduce MDA levels. Blocking of KATP channels inhibited the protective effects of RIPC. PMID:26254913

  18. Bioenergetic and volume regulatory effects of mitoKATP channel modulators protect against hypoxia-reoxygenation-induced mitochondrial dysfunction.

    PubMed

    Onukwufor, John O; Stevens, Don; Kamunde, Collins

    2016-09-01

    The mitochondrial ATP-sensitive K(+) (mitoKATP) channel plays a significant role in mitochondrial physiology and protects against ischemic reperfusion injury in mammals. Although fish frequently face oxygen fluctuations in their environment, the role of the mitoKATP channel in regulating the responses to oxygen stress is rarely investigated in this class of animals. To elucidate whether and how the mitoKATP channel protects against hypoxia-reoxygenation (H-R)-induced mitochondrial dysfunction in fish, we first determined the mitochondrial bioenergetic effects of two key modulators of the channel, diazoxide and 5-hydroxydecanoate (5-HD), using a wide range of doses. Subsequently, the effects of low and high doses of the modulators on mitochondrial bioenergetics and volume under normoxia and after H-R using buffers with and without magnesium and ATP (Mg-ATP) were tested. In the absence of Mg-ATP (mitoKATP channel open), both low and high doses of diazoxide improved mitochondrial coupling, but only the high dose of 5-HD reversed the post-H-R coupling-enhancing effect of diazoxide. In the presence of Mg-ATP (mitoKATP channel closed), diazoxide at the low dose improved coupling post-H-R, and this effect was abolished by 5-HD at the low dose. Interestingly, both low and high doses of diazoxide reversed H-R-induced swelling under mitoKATP channel open conditions, but this effect was not sensitive to 5-HD. Under mitoKATP channel closed conditions, diazoxide at the low dose protected the mitochondria from H-R-induced swelling and 5-HD at the low dose reversed this effect. In contrast, diazoxide at the high dose failed to reduce the swelling caused by H-R, and the addition of the high dose of 5-HD enhanced mitochondrial swelling. Overall, our study showed that in the presence of Mg-ATP, both opening of mitoKATP channels and bioenergetic effects of diazoxide were protective against H-R in fish mitochondria, while in the absence of Mg-ATP only the bioenergetic effect of

  19. Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic β-cells

    PubMed Central

    Han, Young-Eun; Lim, Ajin; Park, Sun-Hyun; Chang, Sunghoe; Lee, Suk-Ho; Ho, Won-Kyung

    2015-01-01

    AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic β-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic β-cells. PMID:26471000

  20. Activation of Mitochondrial Uncoupling Protein 4 and ATP-Sensitive Potassium Channel Cumulatively Decreases Superoxide Production in Insect Mitochondria.

    PubMed

    Slocińska, Malgorzata; Rosinski, Grzegorz; Jarmuszkiewicz, Wieslawa

    2016-01-01

    It has been evidenced that mitochondrial uncoupling protein 4 (UCP4) and ATP-regulated potassium channel (mKATP channel) of insect Gromphadorhina coqereliana mitochondria decrease superoxide anion production. We elucidated whether the two energy-dissipating systems work together on a modulation of superoxide level in cockroach mitochondria. Our data show that the simultaneous activation of UCP4 by palmitic acid and mKATP channel by pinacidil revealed a cumulative effect on weakening mitochondrial superoxide formation. The inhibition of UCP4 by GTP (and/or ATP) and mKATP channel by ATP elevated superoxide production. These results suggest a functional cooperation of both energy-dissipating systems in protection against oxidative stress in insects.

  1. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system.

    PubMed

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system.

  2. ATP-sensitive potassium channel modulators and cardiac arrhythmias: an update.

    PubMed

    Muntean, Danina M; Kiss, Loránd; Jost, Norbert; Baczko, István

    2015-01-01

    Ischemia and heart failure-related cardiac arrhythmias, both atrial (e.g., atrial fibrillation) and ventricular (e.g., malignant tachyarrhythmias) represent a leading cause of morbidity and mortality worldwide. Despite the progress made in the last decade in understanding their pathophysiological mechanisms there is still an unmet need for safer and more efficacious pharmacological treatment, especially when considering the drawbacks and complications of implantable devices. Cardiac ATP-sensitive potassium channels located in the sarcolemmal membrane (sarcKATP) and the inner mitochondrial membrane (mitoKATP) have emerged as crucial controllers of several key cellular functions. In the past three decades a tremendous amount of research led to their structural and functional characterization unveiling both a protective role in cardiac adaptive responses to metabolic stress and a seemingly paradoxical role in promoting as well as protecting against atrial and ventricular arrhythmias. On the other hand, several KATP inhibitors have emerged as potential ischemia selective antiarrhythmic drugs. In this respect, cardioselective, chamber specific and combined sarcKATP and mitoKATP modulators currently represent a promising field for drug development.

  3. Isosteviol Sensitizes sarcKATP Channels towards Pinacidil and Potentiates Mitochondrial Uncoupling of Diazoxide in Guinea Pig Ventricular Myocytes

    PubMed Central

    Fan, Zhuo; Wen, Ting; Chen, Yaoxu; Huang, Lijie; Lin, Wei; Yin, Chunxia; Tan, Wen

    2016-01-01

    KATP channel is an important mediator or factor in physiological and pathological metabolic pathway. Activation of KATP channel has been identified to be a critical step in the cardioprotective mechanism against IR injury. On the other hand, desensitization of the channel to its opener or the metabolic ligand ATP in pathological conditions, like cardiac hypertrophy, would decrease the adaption of myocardium to metabolic stress and is a disadvantage for drug therapy. Isosteviol, obtained by acid hydrolysis of stevioside, has been demonstrated to play a cardioprotective role against diseases of cardiovascular system, like anti-IR injury, antihypertension, antihyperglycemia, and so forth. The present study investigated the effect of isosteviol (STV) on sarcKATP channel current induced by pinacidil and mitochondrial flavoprotein oxidation induced by diazoxide. Our results showed that preincubating cells with STV not only increased the current amplitude and activating rate of sarcKATP channels induced by pinacidil but also potentiated diazoxide-elicited oxidation of flavoprotein in mitochondria. PMID:26949448

  4. Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria-An Amazing Defense Tool Against Hyperosmotic Stress.

    PubMed

    Trono, Daniela; Laus, Maura N; Soccio, Mario; Alfarano, Michela; Pastore, Donato

    2015-01-01

    In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about 15 years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP). Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous) and tissues/organs (etiolated and green) have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel) are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs) and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane toward the matrix, so collapsing membrane potential (ΔΨ), the main component of the protonmotive force (Δp) in plant mitochondria; moreover, cooperation between PmitoKATP and the K(+)/H(+) antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS) production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate hyperosmotic stress

  5. Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria—An Amazing Defense Tool Against Hyperosmotic Stress

    PubMed Central

    Trono, Daniela; Laus, Maura N.; Soccio, Mario; Alfarano, Michela; Pastore, Donato

    2015-01-01

    In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about 15 years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP). Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous) and tissues/organs (etiolated and green) have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel) are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs) and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane toward the matrix, so collapsing membrane potential (ΔΨ), the main component of the protonmotive force (Δp) in plant mitochondria; moreover, cooperation between PmitoKATP and the K+/H+ antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS) production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate hyperosmotic stress

  6. Gene transfer of cystathionine β-synthase into RVLM increases hydrogen sulfide-mediated suppression of sympathetic outflow via KATP channel in normotensive rats.

    PubMed

    Duan, Xiao-cui; Guo, Rong; Liu, Shang-yu; Xiao, Lin; Xue, Hong-mei; Guo, Qi; Jin, Sheng; Wu, Yu-ming

    2015-03-15

    Hydrogen sulfide has been shown to have a sympathoinhibitory effect in the rostral ventrolateral medulla (RVLM). The present study examined the function of cystathionine β-synthase (CBS)/hydrogen sulfide system in the RVLM, which plays a crucial role in the control of blood pressure and sympathetic nerve activity. Adenovirus vectors encoding CBS (AdCBS) or enhanced green fluorescent protein (AdEGFP) were transfected into the RVLM in normotensive rats. Identical microinjection of AdCBS into the RVLM had no effect on systolic blood pressure and heart rate (HR) in conscious rats. Acute experiments were performed at day 7 after gene transfer in anesthetized rats. Microinjection of the CBS inhibitors hydroxylamine (HA) or amino-oxyacetate into the RVLM produced an increase in the renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and HR. There was a potentiation of the increases in RSNA, MAP, and HR because of the CBS inhibitors in AdCBS-injected rats compared with AdEGFP-injected rats. Pretreatment with pinacidil, a ATP-sensitive potassium (KATP) channel activator, abolished the effects of HA in two groups. Microinjection of glibenclamide, a KATP channel blocker, produced increases in RSNA, MAP, and HR in AdCBS-injected rats. No changes in behavior were observed in AdEGFP-injected rats. Furthermore, Western blot analysis indicated an increase in the expression of sulfonylurea receptor 2 and inward rectifier K(+) 6.1 in AdCBS-injected rats. These results suggest that the increase in KATP channels in the RVLM may be responsible for the greater sympathetic outflow and pressor effect of HA in AdCBS-injected rats compared with AdEGFP-injected rats.

  7. Random assembly of SUR subunits in K(ATP) channel complexes.

    PubMed

    Cheng, Wayland W L; Tong, Ailing; Flagg, Thomas P; Nichols, Colin G

    2008-01-01

    Sulfonylurea receptors (SURs) associate with Kir6.x subunits to form tetradimeric K(ATP) channel complexes. SUR1 and SUR2 confer differential channel sensitivities to nucleotides and pharmacological agents, and are expressed in specific, but overlapping, tissues. This raises the question of whether these different SUR subtypes can assemble in the same channel complex and generate channels with hybrid properties. To test this, we engineered dimeric constructs of wild type or N160D mutant Kir6.2 fused to SUR1 or SUR2A. Dimeric fusions formed functional, ATP-sensitive, channels. Coexpression of weakly rectifying SUR1-Kir6.2 (WTF-1) with strongly rectifying SUR1-Kir6.2[N160D] (NDF-1) in COSm6 cells results in mixed subunit complexes that exhibit unique rectification properties. Coexpression of NDF-1 and SUR2A-Kir6.2 (WTF-2) results in similar complex rectification, reflecting the presence of SUR1- and SUR2A-containing dimers in the same channel. The data demonstrate clearly that SUR1 and SUR2A subunits associate randomly, and suggest that heteromeric channels will occur in native tissues. PMID:18690055

  8. Exercise-induced expression of cardiac ATP-sensitive potassium channels promotes action potential shortening and energy conservation.

    PubMed

    Zingman, Leonid V; Zhu, Zhiyong; Sierra, Ana; Stepniak, Elizabeth; Burnett, Colin M-L; Maksymov, Gennadiy; Anderson, Mark E; Coetzee, William A; Hodgson-Zingman, Denice M

    2011-07-01

    Physical activity is one of the most important determinants of cardiac function. The ability of the heart to increase delivery of oxygen and metabolic fuels relies on an array of adaptive responses necessary to match bodily demand while avoiding exhaustion of cardiac resources. The ATP-sensitive potassium (K(ATP)) channel has the unique ability to adjust cardiac membrane excitability in accordance with ATP and ADP levels, and up-regulation of its expression that occurs in response to exercise could represent a critical element of this adaption. However, the mechanism by which K(ATP) channel expression changes result in a beneficial effect on cardiac excitability and function remains to be established. Here, we demonstrate that an exercise-induced rise in K(ATP) channel expression enhanced the rate and magnitude of action potential shortening in response to heart rate acceleration. This adaptation in membrane excitability promoted significant reduction in cardiac energy consumption under escalating workloads. Genetic disruption of normal K(ATP) channel pore function abolished the exercise-related changes in action potential duration adjustment and caused increased cardiac energy consumption. Thus, an expression-driven enhancement in the K(ATP) channel-dependent membrane response to alterations in cardiac workload represents a previously unrecognized mechanism for adaptation to physical activity and a potential target for cardioprotection.

  9. Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A

    PubMed Central

    Surah-Narwal, S; Xu, S Z; McHugh, D; McDonald, R L; Hough, E; Cheong, A; Partridge, C; Sivaprasadarao, A; Beech, D J

    1999-01-01

    A human aorta cDNA library was screened at low stringency with a rat pancreatic Kir6.1 cDNA probe and a homologue of Kir6.1 (hKir6.1) was isolated and sequenced.Metabolic poisoning of Xenopus laevis oocytes with sodium azide and application of the K+ channel opener drug diazoxide induced K+ channel currents in oocytes co-injected with cRNA for hKir6.1 and hamster sulphonylurea receptor (SUR1), but not in oocytes injected with water or cRNA for hKir6.1 or SUR1 alone.K+ channel currents due to hKir6.1+SUR1 or mouse Kir6.2+SUR1 were strongly inhibited by 1 μM glibenclamide. K+-current carried by hKir6.1+SUR1 was inhibited by the putative vascular-selective KATP channel inhibitor U37883A (IC50 32 μM) whereas current carried by Kir6.2+SUR1 or Shaker K+ channels was unaffected.The data support the hypothesis that hKir6.1 is a component of the vascular KATP channel, although the lower sensitivity of hKir6.1+SUR1 to U37883A compared with native vascular tissues suggests the need for another factor or subunit. Furthermore, the data suggest that pharmacology of KATP channels can be determined by the pore-forming subunit as well as the sulphonylurea receptor and point to a molecular basis for the pharmacological distinction between vascular and pancreatic/cardiac KATP channels. PMID:10516647

  10. Synergistic Potentials of Coffee on Injured Pancreatic Islets and Insulin Action via KATP Channel Blocking in Zebrafish.

    PubMed

    Nam, Youn Hee; Hong, Bin Na; Rodriguez, Isabel; Ji, Min Gun; Kim, Keonwoo; Kim, Ung-Jin; Kang, Tong Ho

    2015-06-17

    Pancreatic islets (PIs) are damaged under diabetic conditions, resulting in decreased PI size. This study examined the regenerative effects of coffee and its components (caffeine, CFI; trigonelline, TRG; chlorogenic acid, CGA) on zebrafish larval PIs and β-cells damaged by administration of alloxan (AX). In addition, the influence of coffee and its active components on KATP channels was investigated using diazoxide (DZ) as a KATP channel activator. PI size and fluorescence intensity were significantly increased in the coffee-treated group relative to the no-treatment group (P < 0.0001). In addition, coffee exerted significant regenerative effects on pancreatic β-cells (p = 0.006). Treatment with TRG and CGA rescued PI damage, and the combination of TRG/CGA had a synergistic effect. In conclusion, the results indicate that coffee has beneficial effects on AX-damaged PIs and may also be useful as a blocker of pancreatic β-cell K(+) channels.

  11. Non-cell autonomous cues for enhanced functionality of human embryonic stem cell-derived cardiomyocytes via maturation of sarcolemmal and mitochondrial KATP channels

    PubMed Central

    Keung, Wendy; Ren, Lihuan; Sen Li; Wong, Andy On-Tik; Chopra, Anant; Kong, Chi-Wing; Tomaselli, Gordon F.; Chen, Christopher S.; Li, Ronald A.

    2016-01-01

    Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs), but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs, sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study, we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK, ATP in single hESC-VCMs was dormant under baseline conditions, but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ~8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly, sarcIK, ATP displayed a ~3-fold increase after treatment with hypoxia (5% O2). MitoIK, ATP was absent in hESC-VCMs. However, the thyroid hormone T3 up-regulated mitoIK, ATP, conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system, T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK, ATP. PMID:27677332

  12. Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury.

    PubMed

    Gao, Zhan; Sierra, Ana; Zhu, Zhiyong; Koganti, Siva Rama Krishna; Subbotina, Ekaterina; Maheshwari, Ankit; Anderson, Mark E; Zingman, Leonid V; Hodgson-Zingman, Denice M

    2016-01-01

    The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (KATP) channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane KATP channels ensures a rapid energy-sparing reduction in action potential duration (APD) in response to metabolic challenges, while cellular signaling that reduces surface KATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kir6.2 KATP channel subunit initiating a cascade responsible for KATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB) model of heart failure is coupled with a 35-40% reduction in surface expression of KATP channels compared to hearts from sham-operated mice. Linkage between KATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of KATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial KATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by KATP channels. Recognition of Ca

  13. Prolonged exposure to methylglyoxal causes disruption of vascular KATP channel by mRNA instability

    PubMed Central

    Yang, Yang; Li, Shanshan; Konduru, Anuhya S.; Zhang, Shuang; Trower, Timothy C.; Shi, Weiwei; Cui, Ningren; Yu, Lei; Wang, Yali; Zhu, Daling

    2012-01-01

    Diabetes mellitus is characterized by hyperglycemia and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl species that can lead to cell injuries. Interacting with proteins, lipids, and DNA, excessive MGO can cause dysfunction of various tissues, especially the vascular walls where diabetic complications often take place. However, the potential vascular targets of excessive MGO remain to be fully understood. Here we show that the vascular Kir6.1/SUR2B isoform of ATP-sensitive K+ (KATP) channels is likely to be disrupted with an exposure to submillimolar MGO. Up to 90% of the Kir6.1/SUR2B currents were suppressed by 1 mM MGO with a time constant of ∼2 h. Consistently, MGO treatment caused a vast reduction of both Kir6.1 and SUR2B mRNAs endogenously expressed in the A10 vascular smooth muscle cells. In the presence of the transcriptional inhibitor actinomycin-D, MGO remained to lower the Kir6.1 and SUR2B mRNAs to the same degree as MGO alone, suggesting that the MGO effect is likely to compromise the mRNA stability. Luciferase reporter assays indicated that the 3′-untranslated regions (UTRs) of the Kir6.1 but not SUR2 mRNA were targeted by MGO. In contrast, the SUR2B mRNAs obtained with in vitro transcription were disrupted by MGO directly, while the Kir6.1 transcripts were unaffected. Consistent with these results, the constriction of mesenteric arterial rings was markedly augmented with an exposure to 1 mM MGO for 2 h, and such an MGO effect was totally eliminated in the presence of glibenclamide. These results therefore suggest that acting on the 3′-UTR of Kir6.1 and the coding region of SUR2B, MGO causes instability of Kir6.1 and SUR2B mRNAs, disruption of vascular KATP channels, and impairment of arterial function. PMID:22972803

  14. Single KATP channel opening in response to stimulation of AMPA/kainate receptors is mediated by Na+ accumulation and submembrane ATP and ADP changes

    PubMed Central

    Mollajew, R; Toloe, J; Mironov, S L

    2013-01-01

    Excessive stimulation of glutamatergic receptors (GluRs) can overexcite neurons. This can be dampened by KATP channels linking metabolic and neuronal activities, but the cross-talk has not yet been examined on the single channel level. In the brainstem and hippocampal neurons, GluR agonists augmented the open state probability (Popen) of KATP channels with relative efficacy: kainate ≍ AMPA > NMDA > t-ACPD. Inhibition of calcium influx and chelation of intracellular calcium did not modify the effects. Kainate did not augment production of reactive oxygen species measured with roGFP1. H2O2 slightly increased Popen, but GluR effects were not modified. GluR actions were abolished in Na+-free solutions and after blockade of Na+-K+-ATPase. KATP channels in open-cell patch-clamp measurements were inhibited by ATP, stimulated by ADP, and kainate was effective only in the presence of ATP. GluR stimulation enhanced ATP consumption that decreased submembrane ATP levels, whereas metabolic poisoning diminished bulk ATP. Modelling showed strong ATP depletion and ADP accumulation near the membrane, and both effects contributed to Popen increases after GluR stimulation. Kainate and hypoxia activated KATP channels in the functional brainstem slices. Inhibition of aerobic ATP production and GluR stimulation were about equally effective in KATP channel opening during hypoxia. Induction of seizure-like activity in hippocampal slices with Mg2+-free solutions was accompanied by ATP decrease and KATP channel opening. We propose that KATP channels and GluRs are functionally coupled that can regulate long-lasting changes of neuronal activity in the CNS neurons. PMID:23507878

  15. Involvement of ATP-sensitive potassium channels and the opioid system in the anticonvulsive effect of zolpidem in mice.

    PubMed

    Sheikhi, Mehdi; Shirzadian, Armin; Dehdashtian, Amir; Amiri, Shayan; Ostadhadi, Sattar; Ghasemi, Mehdi; Dehpour, Ahmad Reza

    2016-09-01

    Zolpidem is a hypnotic medication that mainly exerts its function through activating γ-aminobutyric acid (GABA)A receptors. There is some evidence that zolpidem may have anticonvulsive effects. However, the mechanisms underlying this effect have not been elucidated yet. In the present study, we used the pentylentetrazole (PTZ)-induced generalized seizure model in mice to investigate whether zolpidem can affect seizure threshold. We also further evaluated the roles of ATP-sensitive potassium (KATP) channels as well as μ-opioid receptors in the effects of zolpidem on seizure threshold. Our data showed that zolpidem in a dose-dependent manner increased the PTZ-induced seizure threshold. The noneffective (i.e., did not significantly alter the PTZ-induced seizure threshold by itself) doses of KATP channel blocker (glibenclamide) and nonselective opioid receptor antagonist (naloxone) were able to inhibit the anticonvulsive effect of zolpidem. Additionally, noneffective doses of either KATP channel opener (cromakalim) or nonselective μ-opioid receptor agonist (morphine) in combination with a noneffective dose of zolpidem exerted a significant anticonvulsive effect on PTZ-induced seizures in mice. A combination of noneffective doses of naloxone and glibenclamide, which separately did not affect zolpidem effect on seizure threshold, inhibited the anticonvulsive effects of zolpidem. These results suggest a role for KATP channels and the opioid system, alone or in combination, in the anticonvulsive effects of zolpidem. PMID:27521722

  16. Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit

    PubMed Central

    Antcliff, Jennifer F; Haider, Shozeb; Proks, Peter; Sansom, Mark S P; Ashcroft, Frances M

    2005-01-01

    ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the ‘slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state. PMID:15650751

  17. Decreasing Cx36 Gap Junction Coupling Compensates for Overactive KATP Channels to Restore Insulin Secretion and Prevent Hyperglycemia in a Mouse Model of Neonatal Diabetes

    PubMed Central

    Nguyen, Linda M.; Pozzoli, Marina; Hraha, Thomas H.; Benninger, Richard K.P.

    2014-01-01

    Mutations to the ATP-sensitive K+ channel (KATP channel) that reduce the sensitivity of ATP inhibition cause neonatal diabetes mellitus via suppression of β-cell glucose-stimulated free calcium activity ([Ca2+]i) and insulin secretion. Connexin-36 (Cx36) gap junctions also regulate islet electrical activity; upon knockout of Cx36, β-cells show [Ca2+]i elevations at basal glucose. We hypothesized that in the presence of overactive ATP-insensitive KATP channels, a reduction in Cx36 would allow elevations in glucose-stimulated [Ca2+]i and insulin secretion to improve glucose homeostasis. To test this, we introduced a genetic knockout of Cx36 into mice that express ATP-insensitive KATP channels and measured glucose homeostasis and islet metabolic, electrical, and insulin secretion responses. In the normal presence of Cx36, after expression of ATP-insensitive KATP channels, blood glucose levels rapidly rose to >500 mg/dL. Islets from these mice showed reduced glucose-stimulated [Ca2+]i and no insulin secretion. In mice lacking Cx36 after expression of ATP-insensitive KATP channels, normal glucose levels were maintained. Islets from these mice had near-normal glucose-stimulated [Ca2+]i and insulin secretion. We therefore demonstrate a novel mechanism by which islet function can be recovered in a monogenic model of diabetes. A reduction of gap junction coupling allows sufficient glucose-stimulated [Ca2+]i and insulin secretion to prevent the emergence of diabetes. PMID:24458355

  18. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro

    PubMed Central

    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-01-01

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson’s disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe2+-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment. PMID:27646472

  19. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro.

    PubMed

    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-09-20

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson's disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe(2+)-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment.

  20. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro.

    PubMed

    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-01-01

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson's disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe(2+)-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment. PMID:27646472

  1. The Vasorelaxant Effect of p-Cymene in Rat Aorta Involves Potassium Channels

    PubMed Central

    Silva, Martapolyana T. M.; Ribeiro, Fernanda P. R. A.; Medeiros, Maria Alice M. B.; Sampaio, Pedrita A.; Silva, Yonara M. S.; Silva, Morganna T. A.; Quintans, Jullyana S. S.; Quintans-Júnior, Lucindo J.; Ribeiro, Luciano A. A.

    2015-01-01

    The monoterpenes are the main constituents of most essential oils and p-cymene is a monoterpene commonly found in various species of aromatic herbs, which has been reported for anti-inflammatory, antinociceptive, and antimicrobial activities. However, there is no report concerning its pharmacological activity on the vascular smooth muscle. The aim of current work was to investigate the effects of p-cymene in isolated rat aorta and also study its mechanism of action. In this work, we show that p-cymene has a relaxant effect, in a dose-dependent way, on the vascular smooth muscle, regardless of the presence of the endothelium. Using a nonselective potassium channel blocker, the CsCl, the relaxant effect of p-cymene was attenuated. In the presence of more selective potassium channels blockers, such as TEA or 4-AP, no change in the relaxant effect of p-cymene was evidenced, indicating that BKCa and KV channels are not involved in that relaxant effect. However, in the presence of glibenclamide or BaCl2, KATP and Kir blockers, respectively, the relaxant effect of p-cymene was attenuated. The data presented indicate that p-cymene has a relaxing effect on rat aorta, regardless of the endothelium, but with the participation of the KATP and Kir channels. PMID:25667938

  2. KR-31761, a novel K+(ATP)-channel opener, exerts cardioprotective effects by opening both mitochondrial K+(ATP) and Sarcolemmal K+(ATP) channels in rat models of ischemia/reperfusion-induced heart injury.

    PubMed

    Yang, Min-Kyu; Lee, Sung-Hun; Seo, Ho-Won; Yi, Kyu-Yang; Yoo, Sung-Eun; Lee, Byung-Ho; Chung, Hun-Jong; Won, Hyung-Sik; Lee, Chang-Soo; Kwon, Suk-Hyung; Choi, Wahn-Soo; Shin, Hwa-Sup

    2009-02-01

    The cardioprotective effects of KR-31761, a newly synthesized K+(ATP) opener, were evaluated in rat models of ischemia/reperfusion (I/R) heart injury. In isolated rat hearts subjected to 30-min global ischemia/30-min reperfusion, KR-31761 perfused prior to ischemia significantly increased both the left ventricular developed pressure (% of predrug LVDP: 17.8, 45.1, 54.2, and 62.6 for the control, 1 microM, 3 microM, and 10 microM, respectively) and double product (DP: heart rate x LVDP; % of predrug DP: 17.5, 44.9, 56.2, and 64.5 for the control, 1 microM, 3 microM, and 10 microM, respectively) at 30-min reperfusion while decreasing the left ventricular end-diastolic pressure (LVEDP). KR-31761 (10 microM) significantly increased the time to contracture during the ischemic period, whereas it concentration-dependently decreased the lactate dehydrogenase release during reperfusion. All these parameters were significantly reversed by 5-hydroxydecanoate (5-HD, 100 microM) and glyburide (1 microM), selective and nonselective blockers of the mitochondrial K+(ATP) (mitoK+(ATP)) channel and K+(ATP) channel, respectively. In anesthetized rats subjected to 30-min occlusion of left anterior descending coronary artery/2.5-h reperfusion, KR-31761 administered 15 min before the onset of ischemia significantly decreased the infarct size (72.2%, 55.1%, and 47.1% for the control, 0.3 mg/kg, i.v., and 1.0 mg/kg, i.v., respectively); and these effects were completely and almost completely abolished by 5-HD (10 mg/kg, i.v.) and HMR-1098, a selective blocker of sarcolemmal K+(ATP) (sarcK+(ATP)) channel (6 mg/kg, i.v.) administered 5 min prior to KR-31761 (72.3% and 67.9%, respectively). KR-31761 only slightly relaxed methoxamine-precontracted rat aorta (IC50: > 30.0 microM). These results suggest that KR-31761 exerts potent cardioprotective effects through the opening of both mitoK+(ATP) and sarcK+(ATP) channels in rat hearts with a minimal vasorelaxant effect.

  3. Effects of ATP-sensitive potassium channel blockers on vascular hyporeactivity, mesenteric blood flow, and survival in lipopolysaccharide-induced septic shock model.

    PubMed

    Boz, Mustafa; Atilla, Pergin; Iskit, Alper B; Ilhan, Mustafa

    2016-08-01

    In this study, the possible therapeutic effects of various ATP-sensitive potassium channel (KATP) blockers (glibenclamide, repaglinide, 5-HD, HMR-1098) have been tested in experimental septic shock model. Rats were given lipopolysaccharide (1 mg·kg(-1)) to create experimental shock model and 4 h later, under 400 mg·kg(-1) chloral hydrate anesthesia, parameters such as blood pressure, mesenteric blood flow, the response of mesenteric circulation to phenylephrine (vasoconstrictor stimulation), and organ and oxidative damage were analyzed. Also 75 mg·kg(-1) lethal dose of lipopolysaccharide was given to mice and effects of KATP blockers on survival have been tested. Non-selective blocker glibenclamide with sulphonylurea structure and sarcolemmal KATP channel blocker HMR-1098, which have the similar chemical structure, have improved the pathological parameters such as decrease in mesenteric blood flow, vascular hyporeactivity, but could not prevent the decrease in blood pressure, and oxidative and organ damage that were observed in the shock model. Also, both blockers have decreased the mortality rate from 80% to 40%-50%. Similar (preventive) therapeutic effects were not observed with non-selective blocker repaglinide and mitochondrial KATP channel blocker 5-HD, which were non-sulphonylurea structure. As a result, only KATP channel blockers that have sulphonylurea structure can be a new therapeutic approach in septic shock. PMID:27239899

  4. Permeation in potassium channels: implications for channel structure.

    PubMed

    Yellen, G

    1987-01-01

    The SR K+ channel is a single-ion channel with a tunnel that is not very selective, while the DR and CaK channels are both more selective, multi-ion channels. The permeation mechanisms of the three channels are probably most systematically distinguished by the length of their tunnels; the SR has the shortest and the DR the longest. Although different in their mechanisms of activation, the DR and CaK channels have very similar permeation characteristics, down to the details of selectivity and blockade. The longer tunnel and reduced conductance (perhaps a result of the extra tunnel length) of the DR K+ channel are the main differences. The selectivity of the rate-limiting barriers and the binding sites within the channels, however, are strikingly similar. A successful potassium channel must satisfy two criteria: It must let potassium ions through and not much else, and it must let many potassium ions through. To be selective the channel must have a narrow selectivity filter, so that an ion must shed some of its waters of hydration to pass through. Sodium ions are excluded because they are more reluctant to lose their water, and they are not adequately compensated for this loss by interaction with the selectivity filter. To carry a large current the narrow region must be short, with wide antechambers to reduce the diffusional access resistance (48). Energetically, the channel must strike a balance. There must be enough binding energy to compensate the ions for their lost hydration energy, so that the energy barrier to permeation is small. If the channel binds the ion too tightly, however, the ion will not be able to exit, and the current will be small. Some of the shared properties of different potassium channels are probably consequences of these requirements; others may be incidental to function, suggesting a common origin. Barium ions have almost exactly the same radius as potassium ions but twice the charge, so it is perhaps not surprising that barium can block

  5. The cardioprotective effect of naringenin against ischemia-reperfusion injury through activation of ATP-sensitive potassium channel in rat.

    PubMed

    Meng, Li-Min; Ma, Hui-Jie; Guo, Hui; Kong, Qian-Qian; Zhang, Yi

    2016-09-01

    Naringenin (Nari) has antioxidative and anti-atherosclerosis effects, and activation of ATP-sensitive potassium channel (KATP) can offer cardiac protection. We hypothesized that Nari protects the heart against ischemia-reperfusion (I-R) injury through activation of KATP. Isolated hearts from adult male Sprague-Dawley rats experienced a 30-min global ischemia followed by 60-min reperfusion (120 min for the infarct size determination). The hearts were treated with Nari (NARI); Nari plus glibenclamide (GLI), a non-specific ATP-sensitive potassium channel blocker (NARI+GLI); and Nari plus 5-hydroxy decanoic acid (5-HD), a mitochondrial membrane ATP-sensitive potassium channel blocker (NARI+5-HD). The left ventricular pressure, lactate dehydrogenates (LDH) in coronary effluent, superoxide dismutase (SOD) and malondialdehyde (MDA) in myocardium, and myocardial infarct area were measured. Nari above 2.5 μmol/L improved the recovery of left ventricular function, decreased LDH in coronary effluent, and reduced myocardial infarct area. The SOD activity was increased and MDA was decreased in Nari-treated myocardium. The cardioprotective effect of Nari was canceled by GLI and 5-HD. In conclusion, Nari has a cardioprotective effect against I-R injury, which may be carried out through activating ATP-sensitive potassium channels in both cell and mitochondrial membrane, and enhancing myocardial antioxidant capacity. PMID:27408985

  6. Hypotensive effect of S-adenosyl-L-methionine in hypertensive rats is reduced by autonomic ganglia and KATP channel blockers.

    PubMed

    Sikora, Mariusz; Pham, Kinga; Ufnal, Marcin

    2016-07-01

    S-adenosyl-L-methionine (SAM) is an amino acid involved in a number of physiological processes in the nervous system. Some evidence suggests a therapeutic potential of SAM in hypertension. In this study we investigated the effect of intracerebroventricular (ICV) infusions of SAM on arterial blood pressure in rats. Mean arterial blood pressure (MABP) and heart rate (HR) were measured at baseline and during ICV infusion of either SAM or vehicle (aCSF; controls) in conscious, male normotensive Wistar Kyoto rats (WKY) and Spontaneously Hypertensive Rats (SHR). MABP and HR were not affected by the vehicle. WKY rats infused with SAM (10 μM, 100 μM and 1 mM) showed a biphasic hemodynamic response i.e., mild hypotension and bradycardia followed by a significant increase in MABP and HR. On the contrary, SHR infused with SAM showed a dose-dependent hypotensive response. In separate series of experiments, pretreatment with hexamethonium, a ganglionic blocker as well as pretreatment with glibenclamide, a KATP channel blocker reduced the hemodynamic effects of SAM. SAM may affect the nervous control of arterial blood pressure via the autonomic nervous system and KATP channel-dependent mechanisms. PMID:27108137

  7. Researches toward potassium channels on tumor progressions.

    PubMed

    Shen, Zheng; Yang, Qian; You, Qidong

    2009-01-01

    As trans-membrane proteins located in cytoplasm and organelle membrane, potassium (K(+)) channels are generally divided into four super-families: voltage-gated K(+) channels (K(v)), Ca(2+)-activated K(+) channels (K(Ca)), inwardly rectifying K(+) channels (K(ir)) and two-pore domain K(+) channels (K(2P)). Since dysfunctions of K(+) channels would induce many diseases, various studies toward their functions in physiologic and pathologic process have been extensively launched. This review focuses on the recent advances of K(+) channels in tumor progression, including the brief introduction of K(+) channels, the role of K(+) channels in tumor cells, the possible mechanism of action at cellular level, and the possible application of K(+) channel modulators in cancer chemotherapy.

  8. Clofilium inhibits Slick and Slack potassium channels

    PubMed Central

    de los Angeles Tejada, Maria; Stolpe, Kathleen; Meinild, Anne-Kristine; Klaerke, Dan A

    2012-01-01

    Slick and Slack high-conductance potassium channels have been recently discovered, and are found in the central nervous system and in the heart. Both channels are activated by Na+ and Cl−, and Slick channels are also inhibited by adenosine triphospate (ATP). An important role of setting the resting membrane potential and controlling the basal excitability of neurons has been suggested for these channels. In addition, no specific blockers for these channels are known up to the present. With the purpose of studying the pharmacological characteristics of Slick and Slack channels, the effects of exposure to the antiarrhythmic compound clofilium were evaluated. Clofilium was able to modulate the activity of Slick and Slack channels effectively, with a stronger effect on Slack than Slick channels. In order to evaluate the pharmacological behavior of Slick and Slack channels further, 38 commonly used potassium channel blockers were tested. Screening of these compounds did not reveal any modulators of Slick and Slack channels, except for clofilium. The present study provides a first approach towards elucidating the pharmacological characteristics of Slick and Slack channels and could be the basis for future studies aimed at developing potent and specific blockers and activators for these channels. PMID:23271893

  9. Taurine supplementation increases K(ATP) channel protein content, improving Ca2+ handling and insulin secretion in islets from malnourished mice fed on a high-fat diet.

    PubMed

    Vettorazzi, Jean F; Ribeiro, Rosane A; Santos-Silva, Junia C; Borck, Patricia C; Batista, Thiago M; Nardelli, Tarlliza R; Boschero, Antonio C; Carneiro, Everardo M

    2014-09-01

    Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

  10. Potassium channels in pulmonary arterial hypertension.

    PubMed

    Boucherat, Olivier; Chabot, Sophie; Antigny, Fabrice; Perros, Frédéric; Provencher, Steeve; Bonnet, Sébastien

    2015-10-01

    Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder with various origins. All forms of PAH share a common pulmonary arteriopathy characterised by vasoconstriction, remodelling of the pre-capillary pulmonary vessel wall, and in situ thrombosis. Although the pathogenesis of PAH is recognised as a complex and multifactorial process, there is growing evidence that potassium channels dysfunction in pulmonary artery smooth muscle cells is a hallmark of PAH. Besides regulating many physiological functions, reduced potassium channels expression and/or activity have significant effects on PAH establishment and progression. This review describes the molecular mechanisms and physiological consequences of potassium channel modulation. Special emphasis is placed on KCNA5 (Kv1.5) and KCNK3 (TASK1), which are considered to play a central role in determining pulmonary vascular tone and may represent attractive therapeutic targets in the treatment of PAH. PMID:26341985

  11. Adenosine Triphosphate-Sensitive Potassium Channel Kir Subunits Implicated in Cardioprotection by Diazoxide

    PubMed Central

    Henn, Matthew C; Janjua, M Burhan; Kanter, Evelyn M; Makepeace, Carol M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

    2015-01-01

    Background ATP-sensitive potassium (KATP) channel openers provide cardioprotection in multiple models. Ion flux at an unidentified mitochondrial KATP channel has been proposed as the mechanism. The renal outer medullary kidney potassium channel subunit, potassium inward rectifying (Kir)1.1, has been implicated as a mitochondrial channel pore-forming subunit. We hypothesized that subunit Kir1.1 is involved in cardioprotection (maintenance of volume homeostasis and contractility) of the KATP channel opener diazoxide (DZX) during stress (exposure to hyperkalemic cardioplegia [CPG]) at the myocyte and mitochondrial levels. Methods and Results Kir subunit inhibitor Tertiapin Q (TPN-Q) was utilized to evaluate response to stress. Mouse ventricular mitochondrial volume was measured in the following groups: isolation buffer; 200 μmol/L of ATP; 100 μmol/L of DZX+200 μmol/L of ATP; or 100 μmol/L of DZX+200 μmol/L of ATP+TPN-Q (500 or 100 nmol/L). Myocytes were exposed to Tyrode’s solution (5 minutes), test solution (Tyrode’s, cardioplegia [CPG], CPG+DZX, CPG+DZX+TPN-Q, Tyrode’s+TPN-Q, or CPG+TPN-Q), N=12 for all (10 minutes); followed by Tyrode’s (5 minutes). Volumes were compared. TPN-Q, with or without DZX, did not alter mitochondrial or myocyte volume. Stress (CPG) resulted in myocyte swelling and reduced contractility that was prevented by DZX. TPN-Q prevented the cardioprotection afforded by DZX (volume homeostasis and maintenance of contractility). Conclusions TPN-Q inhibited myocyte cardioprotection provided by DZX during stress; however, it did not alter mitochondrial volume. Because TPN-Q inhibits Kir1.1, Kir3.1, and Kir3.4, these data support that any of these Kir subunits could be involved in the cardioprotection afforded by diazoxide. However, these data suggest that mitochondrial swelling by diazoxide does not involve Kir1.1, 3.1, or 3.4. PMID:26304939

  12. Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis.

    PubMed

    Zhang, Xiaohui; Zhang, Xiaohua; Xiong, Yiqun; Xu, Chaoying; Liu, Xinliang; Lin, Jian; Mu, Guiping; Xu, Shaogang; Liu, Wenhe

    2016-09-01

    The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction. PMID:27430376

  13. Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis

    PubMed Central

    Zhang, Xiaohui; Zhang, Xiaohua; Xiong, Yiqun; Xu, Chaoying; Liu, Xinliang; Lin, Jian; Mu, Guiping; Xu, Shaogang; Liu, Wenhe

    2016-01-01

    The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction. PMID:27430376

  14. Cardiac Strong Inward Rectifier Potassium Channels

    PubMed Central

    Anumonwo, Justus MB; Lopatin, Anatoli N

    2009-01-01

    Cardiac IK1 and IKACh are the major potassium currents displaying classical strong inward rectification, a unique property that is critical for their roles in cardiac excitability. In the last fifteen years, research on IK1 and IKACh has been propelled by the cloning of the underlying inwardly rectifying potassium (Kir) channels, the discovery of the molecular mechanism of strong rectification and the linking of a number of disorders of cardiac excitability to defects in genes encoding Kir channels. Disease-causing mutations in Kir genes have been shown experimentally to affect one or more of the following channel properties: structure, assembly, trafficking and regulation, with the ultimate effect of a gain-, or a loss-of-function of the channel. It is now established that IK1 and IKACh channels are heterotetramers of Kir2 and Kir3 subunits, respectively. Each homomeric Kir channel has distinct biophysical and regulatory properties, and individual Kir subunits often display different patterns of regional, cellular and membrane distribution. These differences are thought to underlie important variations in the physiological properties of IK1 and IKACh. It has become increasingly clear that the contribution of IK1 and IKACh channels to cardiac electrical activity goes beyond their long recognized role in the stabilization of resting membrane potential and shaping the late phase of action potential repolarization in individual myocytes, but extends to being critical elements determining the overall electrical stability of the heart. PMID:19703462

  15. Effects of sodium metabisulfite on the expression of BK(Ca), K(ATP), and L-Ca(2+) channels in rat aortas in vivo and in vitro.

    PubMed

    Zhang, Quanxi; Bai, Yunlong; Tian, Jingjing; Lei, Xiaodong; Li, Mei; Yang, Zhenhua; Meng, Ziqiang

    2015-03-01

    Sodium metabisulfite (SMB) is most commonly used as the preservative in many food preparations and drugs. So far, few studies about its negative effects were reported. The purpose of this study was to investigate the effect of SMB on the expression of big-conductance Ca(2+)-activated K(+) (BKCa), ATP-sensitive K(+) (KATP), and L-type calcium (L-Ca(2+)) channels in rat aorta in vivo and in vitro. The results showed that the mRNA and protein levels of the BKCa channel subunits α and β1 of aorta in rats were increased by SMB in vivo and in vitro. Similarly, the expression of the KATP channel subunits Kir6.1, Kir6.2, and SUR2B were increased by SMB. However, SMB at the highest concentration significantly decreased the expression of the L-Ca(2+) channel subunits Cav1.2 and Cav1.3. These results suggest that SMB can activate BKCa and KATP channels by increasing the expression of α, β1, and Kir6.1, Kir6.2, SUR2B respectively, while also inhibit L-Ca(2+) channels by decreasing the expression of Cav1.2 and Cav1.3 of aorta in rats. The molecular mechanism of SMB-induced vasorelaxant effect might be related to the expression changes of BKCa, KATP, and L-Ca(2+) channels subunits. Further work is needed to determine the relative contribution of each channel in SMB-mediated vasorelaxant effect.

  16. Effects of sodium metabisulfite on the expression of BK(Ca), K(ATP), and L-Ca(2+) channels in rat aortas in vivo and in vitro.

    PubMed

    Zhang, Quanxi; Bai, Yunlong; Tian, Jingjing; Lei, Xiaodong; Li, Mei; Yang, Zhenhua; Meng, Ziqiang

    2015-03-01

    Sodium metabisulfite (SMB) is most commonly used as the preservative in many food preparations and drugs. So far, few studies about its negative effects were reported. The purpose of this study was to investigate the effect of SMB on the expression of big-conductance Ca(2+)-activated K(+) (BKCa), ATP-sensitive K(+) (KATP), and L-type calcium (L-Ca(2+)) channels in rat aorta in vivo and in vitro. The results showed that the mRNA and protein levels of the BKCa channel subunits α and β1 of aorta in rats were increased by SMB in vivo and in vitro. Similarly, the expression of the KATP channel subunits Kir6.1, Kir6.2, and SUR2B were increased by SMB. However, SMB at the highest concentration significantly decreased the expression of the L-Ca(2+) channel subunits Cav1.2 and Cav1.3. These results suggest that SMB can activate BKCa and KATP channels by increasing the expression of α, β1, and Kir6.1, Kir6.2, SUR2B respectively, while also inhibit L-Ca(2+) channels by decreasing the expression of Cav1.2 and Cav1.3 of aorta in rats. The molecular mechanism of SMB-induced vasorelaxant effect might be related to the expression changes of BKCa, KATP, and L-Ca(2+) channels subunits. Further work is needed to determine the relative contribution of each channel in SMB-mediated vasorelaxant effect. PMID:25463229

  17. Mouth breathing increases the pentylenetetrazole-induced seizure threshold in mice: a role for ATP-sensitive potassium channels.

    PubMed

    Niaki, Seyed Esfandiar Akhavan; Shafaroodi, Hamed; Ghasemi, Mehdi; Shakiba, Bijan; Fakhimi, Ali; Dehpour, Ahamd Reza

    2008-08-01

    Nasal obstruction and consequent mouth breathing have been shown to change the acid-base balance, producing respiratory acidosis. Additionally, there exists a large body of evidence maintaining that acidosis affects the activity of ATP-sensitive potassium (K(ATP)) channels, which play a crucial role in the function of the central nervous system (CNS), for example, in modulating seizure threshold. Thus, in the study described here, we examined whether mouth breathing, induced by surgical ligation of nostrils, could affect the seizure threshold induced by pentylenetetrazole in male NMRI mice. Using the selective K(ATP) channel opener (diazoxide) and blocker (glibenclamide), we also evaluated the possible role of K(ATP) channels in this process. Our data revealed that seizure threshold was increased 6 to 72 hours after nasal obstruction, reaching a peak 48 hours afterward, compared with either control or sham-operated mice (P<0.01). There was a significant decrease in pH of arterial blood samples and increase in CO(2) partial pressure (PCO(2)) during this time. Systemic injection of glibenclamide (1 and 2mg/kg, ip, daily) significantly prevented the increase in seizure threshold in 48-hour bilaterally nasally obstructed mice, whereas it had no effect on seizure threshold in sham-operated mice. Systemic injection of diazoxide (25mg/kg, ip, daily) had no effect on seizure threshold in all groups, whereas higher doses (50 and 100mg/kg, ip, daily) significantly increased seizure threshold in both 48-hour-obstructed and sham-operated mice. The decrease in seizure threshold induced by glibenclamide (2mg/kg, ip, daily) was prevented by diazoxide (25mg/kg, ip, daily). These results demonstrate for the first time that mouth breathing, which could result in respiratory acidosis, increases seizure threshold in mice and K(ATP) channels may play a role in this effect.

  18. The insulin sensitizing effect of topiramate involves KATP channel activation in the central nervous system

    PubMed Central

    Coomans, C P; Geerling, J J; van den Berg, S A A; van Diepen, H C; Garcia-Tardón, N; Thomas, A; Schröder-van der Elst, J P; Ouwens, D M; Pijl, H; Rensen, P C N; Havekes, L M; Guigas, B; Romijn, J A

    2013-01-01

    BACKGROUND AND PURPOSE Topiramate improves insulin sensitivity, in addition to its antiepileptic action. However, the underlying mechanism is unknown. Therefore, the present study was aimed at investigating the mechanism of the insulin-sensitizing effect of topiramate both in vivo and in vitro. EXPERIMENTAL APPROACH Male C57Bl/6J mice were fed a run-in high-fat diet for 6 weeks, before receiving topiramate or vehicle mixed in high-fat diet for an additional 6 weeks. Insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamp. The extent to which the insulin sensitizing effects of topiramate were mediated through the CNS were determined by concomitant i.c.v. infusion of vehicle or tolbutamide, an inhibitor of ATP-sensitive potassium channels in neurons. The direct effects of topiramate on insulin signalling and glucose uptake were assessed in vivo and in cultured muscle cells. KEY RESULTS In hyperinsulinaemic-euglycaemic clamp conditions, therapeutic plasma concentrations of topiramate (∼4 μg·mL−1) improved insulin sensitivity (glucose infusion rate + 58%). Using 2-deoxy-D-[3H]glucose, we established that topiramate improved the insulin-mediated glucose uptake by heart (+92%), muscle (+116%) and adipose tissue (+586%). Upon i.c.v. tolbutamide, the insulin-sensitizing effect of topiramate was completely abrogated. Topiramate did not directly affect glucose uptake or insulin signalling neither in vivo nor in cultured muscle cells. CONCLUSION AND IMPLICATIONS In conclusion, topiramate stimulates insulin-mediated glucose uptake in vivo through the CNS. These observations illustrate the possibility of pharmacological modulation of peripheral insulin resistance through a target in the CNS. PMID:23957854

  19. The shifting landscape of KATP channelopathies and the need for 'sharper' therapeutics.

    PubMed

    Kharade, Sujay V; Nichols, Colin; Denton, Jerod S

    2016-05-01

    ATP-sensitive potassium (KATP) channels play fundamental roles in the regulation of endocrine, neural and cardiovascular function. Small-molecule inhibitors (e.g., sulfonylurea drugs) or activators (e.g., diazoxide) acting on SUR1 or SUR2 have been used clinically for decades to manage the inappropriate secretion of insulin in patients with Type 2 diabetes, hyperinsulinism and intractable hypertension. More recently, the discovery of rare disease-causing mutations in KATP channel-encoding genes has highlighted the need for new therapeutics for the treatment of certain forms of neonatal diabetes mellitus, congenital hyperinsulinism and Cantu syndrome. Here, we provide a high-level overview of the pathophysiology of these diseases and discuss the development of a flexible high-throughput screening platform to enable the development of new classes of KATP channel modulators. PMID:27161588

  20. The shifting landscape of KATP channelopathies and the need for 'sharper' therapeutics.

    PubMed

    Kharade, Sujay V; Nichols, Colin; Denton, Jerod S

    2016-05-01

    ATP-sensitive potassium (KATP) channels play fundamental roles in the regulation of endocrine, neural and cardiovascular function. Small-molecule inhibitors (e.g., sulfonylurea drugs) or activators (e.g., diazoxide) acting on SUR1 or SUR2 have been used clinically for decades to manage the inappropriate secretion of insulin in patients with Type 2 diabetes, hyperinsulinism and intractable hypertension. More recently, the discovery of rare disease-causing mutations in KATP channel-encoding genes has highlighted the need for new therapeutics for the treatment of certain forms of neonatal diabetes mellitus, congenital hyperinsulinism and Cantu syndrome. Here, we provide a high-level overview of the pathophysiology of these diseases and discuss the development of a flexible high-throughput screening platform to enable the development of new classes of KATP channel modulators.

  1. Fractal Behavior of the Pancreatic β-Cell Near the Percolation Threshold: Effect of the KATP Channel On the Electrical Response.

    PubMed

    Bahlouli, S; Mokaddem, A; Hamdache, F; Riane, H; Kameche, M

    2016-01-01

    The molecular system built with true chemical bonds or strong molecular interaction can be described using conceptual mathematical tools. Modeling of the natural generated ionic currents on the human pancreatic β-cell activity had been already studied using complicated analytical models. In our present contribution, we prove the same using our simple electrical model. The ionic currents are associated with different proteins membrane channels (K-Ca, K(v), K(ATP), Ca(v)-L) and Na/Ca Exchanger (NCX). The proteins are Ohmic conductors and are modeled by conductance randomly distributed. Switches are placed in series with conductances in order to highlight the channel activity. However, the KATP channel activity is stimulated by glucose, and the NCX's conductance change according to the intracellular calcium concentration. The percolation threshold of the system is calculated by the fractal nature of the infinite cluster using the Tarjan's depth-first-search algorithm. It is shown that the behavior of the internal concentration of Ca(2+) and the membrane potential are modulated by glucose. The results confirm that the inhibition of KATP channels depolarizes the membrane and increases the influx of [Ca(2+)]i through NCX and Ca(v)-L channel for high glucose concentrations. PMID:26886736

  2. Sea Anemone Toxins Affecting Potassium Channels

    NASA Astrophysics Data System (ADS)

    Diochot, Sylvie; Lazdunski, Michel

    The great diversity of K+ channels and their wide distribution in many tissues are associated with important functions in cardiac and neuronal excitability that are now better understood thanks to the discovery of animal toxins. During the past few decades, sea anemones have provided a variety of toxins acting on voltage-sensitive sodium and, more recently, potassium channels. Currently there are three major structural groups of sea anemone K+ channel (SAK) toxins that have been characterized. Radioligand binding and electrophysiological experiments revealed that each group contains peptides displaying selective activities for different subfamilies of K+ channels. Short (35-37 amino acids) peptides in the group I display pore blocking effects on Kv1 channels. Molecular interactions of SAK-I toxins, important for activity and binding on Kv1 channels, implicate a spot of three conserved amino acid residues (Ser, Lys, Tyr) surrounded by other less conserved residues. Long (58-59 amino acids) SAK-II peptides display both enzymatic and K+ channel inhibitory activities. Medium size (42-43 amino acid) SAK-III peptides are gating modifiers which interact either with cardiac HERG or Kv3 channels by altering their voltage-dependent properties. SAK-III toxins bind to the S3C region in the outer vestibule of Kv channels. Sea anemones have proven to be a rich source of pharmacological tools, and some of the SAK toxins are now useful drugs for the diagnosis and treatment of autoimmune diseases.

  3. The potassium channel: Structure, selectivity and diffusion

    NASA Astrophysics Data System (ADS)

    Allen, T. W.; Bliznyuk, A.; Rendell, A. P.; Kuyucak, S.; Chung, S.-H.

    2000-05-01

    We employ the entire experimentally determined protein structure for the KcsA potassium channel from Streptomyces lividans in molecular dynamics calculations to observe hydrated channel protein structure, ion solvation, selectivity, multiple ion configurations, and diffusion. Free energy perturbation calculations display a significant ion discrimination of ˜9 kT in favor of the larger K+ ion. The protein forming the channel is very flexible yet is unable to fully solvate the Na+ ion because of its smaller size and large solvation energy. There is evidence that acidic and basic sidechains may dissociate in the presence of multiple K+ ions to explain experimental ion density maps. K+ diffusion is found to vary from approximately 10%-90% of bulk, supporting the high channel currents observed experimentally.

  4. Modulation of Potassium Channels Inhibits Bunyavirus Infection*

    PubMed Central

    Hover, Samantha; King, Barnabas; Hall, Bradley; Loundras, Eleni-Anna; Taqi, Hussah; Daly, Janet; Dallas, Mark; Peers, Chris; Schnettler, Esther; McKimmie, Clive; Kohl, Alain; Barr, John N.; Mankouri, Jamel

    2016-01-01

    Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease. PMID:26677217

  5. Modulation of Potassium Channels Inhibits Bunyavirus Infection.

    PubMed

    Hover, Samantha; King, Barnabas; Hall, Bradley; Loundras, Eleni-Anna; Taqi, Hussah; Daly, Janet; Dallas, Mark; Peers, Chris; Schnettler, Esther; McKimmie, Clive; Kohl, Alain; Barr, John N; Mankouri, Jamel

    2016-02-12

    Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

  6. Regulation of Substantia Nigra Pars Reticulata GABAergic Neuron Activity by H2O2 via Flufenamic Acid-Sensitive Channels and KATP Channels

    PubMed Central

    Lee, Christian R.; Witkovsky, Paul; Rice, Margaret E.

    2011-01-01

    Substantia nigra pars reticulata (SNr) GABAergic neurons are key output neurons of the basal ganglia. Given the role of these neurons in motor control, it is important to understand factors that regulate their firing rate and pattern. One potential regulator is hydrogen peroxide (H2O2), a reactive oxygen species that is increasingly recognized as a neuromodulator. We used whole-cell current clamp recordings of SNr GABAergic neurons in guinea-pig midbrain slices to determine how H2O2 affects the activity of these neurons and to explore the classes of ion channels underlying those effects. Elevation of H2O2 levels caused an increase in the spontaneous firing rate of SNr GABAergic neurons, whether by application of exogenous H2O2 or amplification of endogenous H2O2 through inhibition of glutathione peroxidase with mercaptosuccinate. This effect was reversed by flufenamic acid (FFA), implicating transient receptor potential (TRP) channels. Conversely, depletion of endogenous H2O2 by catalase, a peroxidase enzyme, decreased spontaneous firing rate and firing precision of SNr neurons, demonstrating tonic control of firing rate by H2O2. Elevation of H2O2 in the presence of FFA revealed an inhibition of tonic firing that was prevented by blockade of ATP-sensitive K+ (KATP) channels with glibenclamide. In contrast to guinea-pig SNr neurons, the dominant effect of H2O2 elevation in mouse SNr GABAergic neurons was hyperpolarization, indicating a species difference in H2O2-dependent regulation. Thus, H2O2 is an endogenous modulator of SNr GABAergic neurons, acting primarily through presumed TRP channels in guinea-pig SNr, with additional modulation via KATP channels to regulate SNr output. PMID:21503158

  7. Remote ischemic preconditioning mitigates myocardial and neurological dysfunction via K(ATP) channel activation in a rat model of hemorrhagic shock.

    PubMed

    Hu, Xianwen; Yang, Zhengfei; Yang, Min; Qian, Jie; Cahoon, Jena; Xu, Jiefeng; Sun, Shijie; Tang, Wanchun

    2014-09-01

    Severe hemorrhagic shock and resuscitation is a state of global body ischemia and reperfusion that causes myocardial and cerebral dysfunction. We investigated whether remote ischemic preconditioning (RIPC) would reduce myocardial and cerebral ischemia and reperfusion injuries after hemorrhagic shock as the result of the K(ATP) channel activation. Twenty-one male rats were randomized into three groups: RIPC, RIPC with K(ATP) channel blocker, and control. Remote ischemic preconditioning was induced by four cycles of 5 min of limb ischemia followed by reperfusion for 5 min. Hemorrhagic shock was induced by removing 50% of the estimated total blood volume during an interval of 1 h. Thirty minutes after the completion of bleeding, the animals were reinfused with shed blood during the ensuing 30 min. The animals were monitored for 2 h and observed for an additional 72 h. Myocardial function was measured by echocardiography, and sublingual microcirculation was measured by a sidestream dark-field imaging device at baseline, 1 h after bleeding, 30 min after the completion of bleeding, 30 min after reinfusion, and hourly intervals thereafter. The survival and neurological function were evaluated at 12, 24, 48, and 72 h after reinfusion. At 2 h after reinfusion, ejection fraction and myocardial performance index were significantly better in the RIPC group than in the control group (P < 0.01). The sublingual microvascular flow index and perfused vessel density were significantly greater after reinfusion in the RIPC group than that in the control group (P < 0.01). The duration of survival was significantly longer, and neurological deficit score was significantly better in the RIPC group than the control animals (P < 0.01). Pretreatment with the K(ATP) channel blocker (glibenclamide) completely abolished the myocardial and cerebral protective effects of RIPC. We demonstrate, for the first time, that after severe hemorrhagic shock and resuscitation, RIPC mitigated myocardial and

  8. Remote ischemic preconditioning mitigates myocardial and neurological dysfunction via K(ATP) channel activation in a rat model of hemorrhagic shock.

    PubMed

    Hu, Xianwen; Yang, Zhengfei; Yang, Min; Qian, Jie; Cahoon, Jena; Xu, Jiefeng; Sun, Shijie; Tang, Wanchun

    2014-09-01

    Severe hemorrhagic shock and resuscitation is a state of global body ischemia and reperfusion that causes myocardial and cerebral dysfunction. We investigated whether remote ischemic preconditioning (RIPC) would reduce myocardial and cerebral ischemia and reperfusion injuries after hemorrhagic shock as the result of the K(ATP) channel activation. Twenty-one male rats were randomized into three groups: RIPC, RIPC with K(ATP) channel blocker, and control. Remote ischemic preconditioning was induced by four cycles of 5 min of limb ischemia followed by reperfusion for 5 min. Hemorrhagic shock was induced by removing 50% of the estimated total blood volume during an interval of 1 h. Thirty minutes after the completion of bleeding, the animals were reinfused with shed blood during the ensuing 30 min. The animals were monitored for 2 h and observed for an additional 72 h. Myocardial function was measured by echocardiography, and sublingual microcirculation was measured by a sidestream dark-field imaging device at baseline, 1 h after bleeding, 30 min after the completion of bleeding, 30 min after reinfusion, and hourly intervals thereafter. The survival and neurological function were evaluated at 12, 24, 48, and 72 h after reinfusion. At 2 h after reinfusion, ejection fraction and myocardial performance index were significantly better in the RIPC group than in the control group (P < 0.01). The sublingual microvascular flow index and perfused vessel density were significantly greater after reinfusion in the RIPC group than that in the control group (P < 0.01). The duration of survival was significantly longer, and neurological deficit score was significantly better in the RIPC group than the control animals (P < 0.01). Pretreatment with the K(ATP) channel blocker (glibenclamide) completely abolished the myocardial and cerebral protective effects of RIPC. We demonstrate, for the first time, that after severe hemorrhagic shock and resuscitation, RIPC mitigated myocardial and

  9. Inhibition of the ATP-sensitive potassium channel from mouse pancreatic β-cells by surfactants

    PubMed Central

    Smith, Paul A; Proks, Peter

    1998-01-01

    We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the KATP channel in the pancreatic β-cell from mouse.All three detergents blocked KATP channel activity with the following order of potency: Tween 80 (Ki<∼83 nM)>Triton X100 (Ki=350 nM)>Cremophor. In all cases the block was poorly reversible.Single-channel studies suggested that at low doses, the detergents act as slow blockers of the KATP channel.Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg2+-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel.The detergents could block the pore-forming subunit, Kir6.2ΔC26, which can be expressed independently of SUR1 (the regulatory subunit of the KATP channel). These data suggest that the detergents act on Kir6.2 and not SUR1.The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1).Voltage-dependent K-currents in the β-cell were reversibly blocked by the detergents with a far lower potency than that found for the KATP channel.Like other insulin secretagogues that act by blocking the KATP channel, Cremophor elevated intracellular Ca2+ in single β-cells to levels that would be expected to elicit insulin secretion.Given the role of the KATP channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the KATP channel PMID:9647478

  10. Role of the KATP channel in the protective effect of nicorandil on cyclophosphamide-induced lung and testicular toxicity in rats.

    PubMed

    Ahmed, Lamiaa A; El-Maraghy, Shohda A; Rizk, Sherine M

    2015-01-01

    This study is the first to investigate the role of the KATP channel in the possible protection mediated by nicorandil against cyclophosphamide-induced lung and testicular toxicity in rats. Animals received cyclophosphamide (150 mg/kg/day, i.p.) for 2 consecutive days and then were untreated for the following 5 days. Nicorandil (3 mg/kg/day, p.o.) was administered starting from the day of cyclophosphamide injection with or without glibenclamide (5 mg/kg/day, p.o.). Nicorandil administration significantly reduced the cyclophosphamide-induced deterioration of testicular function, as demonstrated by increases in the level of serum testosterone and the activities of the testicular 3β- hydroxysteroid, 17β-hydroxysteroid and sorbitol dehydrogenases. Furthermore, nicorandil significantly alleviated oxidative stress (as determined by lipid peroxides and reduced glutathione levels and total antioxidant capacity), as well as inflammatory markers (tumour necrosis factor-α and interleukin-1β), in bronchoalveolar lavage fluid and testicular tissue. Finally, the therapy decreased the levels of fibrogenic markers (transforming growth factor-β and hydroxyproline) and ameliorated the histological alterations (as assessed by lung fibrosis grading and testicular Johnsen scores). The co-administration of glibenclamide (a KATP channel blocker) blocked the protective effects of nicorandil. In conclusion, KATP channel activation plays an important role in the protective effect of nicorandil against cyclophosphamide-induced lung and testicular toxicity. PMID:26403947

  11. Role of the KATP channel in the protective effect of nicorandil on cyclophosphamide-induced lung and testicular toxicity in rats

    PubMed Central

    Ahmed, Lamiaa A.; EL-Maraghy, Shohda A.; Rizk, Sherine M.

    2015-01-01

    This study is the first to investigate the role of the KATP channel in the possible protection mediated by nicorandil against cyclophosphamide-induced lung and testicular toxicity in rats. Animals received cyclophosphamide (150 mg/kg/day, i.p.) for 2 consecutive days and then were untreated for the following 5 days. Nicorandil (3 mg/kg/day, p.o.) was administered starting from the day of cyclophosphamide injection with or without glibenclamide (5 mg/kg/day, p.o.). Nicorandil administration significantly reduced the cyclophosphamide-induced deterioration of testicular function, as demonstrated by increases in the level of serum testosterone and the activities of the testicular 3β- hydroxysteroid, 17β-hydroxysteroid and sorbitol dehydrogenases. Furthermore, nicorandil significantly alleviated oxidative stress (as determined by lipid peroxides and reduced glutathione levels and total antioxidant capacity), as well as inflammatory markers (tumour necrosis factor-α and interleukin-1β), in bronchoalveolar lavage fluid and testicular tissue. Finally, the therapy decreased the levels of fibrogenic markers (transforming growth factor-β and hydroxyproline) and ameliorated the histological alterations (as assessed by lung fibrosis grading and testicular Johnsen scores). The co-administration of glibenclamide (a KATP channel blocker) blocked the protective effects of nicorandil. In conclusion, KATP channel activation plays an important role in the protective effect of nicorandil against cyclophosphamide-induced lung and testicular toxicity. PMID:26403947

  12. What do we not know about mitochondrial potassium channels?

    PubMed

    Laskowski, Michał; Augustynek, Bartłomiej; Kulawiak, Bogusz; Koprowski, Piotr; Bednarczyk, Piotr; Jarmuszkiewicz, Wieslawa; Szewczyk, Adam

    2016-08-01

    In this review, we summarize our knowledge about mitochondrial potassium channels, with a special focus on unanswered questions in this field. The following potassium channels have been well described in the inner mitochondrial membrane: ATP-regulated potassium channel, Ca(2+)-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the two-pore domain TASK-3 potassium channel. The primary functional roles of these channels include regulation of mitochondrial respiration and the alteration of membrane potential. Additionally, they modulate the mitochondrial matrix volume and the synthesis of reactive oxygen species by mitochondria. Mitochondrial potassium channels are believed to contribute to cytoprotection and cell death. In this paper, we discuss fundamental issues concerning mitochondrial potassium channels: their molecular identity, channel pharmacology and functional properties. Attention will be given to the current problems present in our understanding of the nature of mitochondrial potassium channels. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

  13. Extracellular potassium inhibits Kv7.1 potassium channels by stabilizing an inactivated state.

    PubMed

    Larsen, Anders Peter; Steffensen, Annette Buur; Grunnet, Morten; Olesen, Søren-Peter

    2011-08-17

    Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1-50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC(50) of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2-5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels. PMID:21843472

  14. Closure of mitochondrial potassium channels favors opening of the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey M; Brailovskaya, Irina V; Shumakov, Anton R; Emelyanova, Larisa V

    2015-06-01

    It is known that a closure of ATP sensitive (mitoKATP) or BK-type Ca(2+) activated (mitoKCa) potassium channels triggers opening of the mitochondrial permeability transition pore (MPTP) in cells and isolated mitochondria. We found earlier that the Tl(+)-induced MPTP opening in Ca(2+)-loaded rat liver mitochondria was accompanied by a decrease of 2,4-dinitrophenol-uncoupled respiration and increase of mitochondrial swelling and ΔΨmito dissipation in the medium containing TlNO3 and KNO3. On the other hand, our study showed that the mitoKATP inhibitor, 5-hydroxydecanoate favored the Tl(+)-induced MPTP opening in the inner membrane of Ca(2+)-loaded rat heart mitochondria (Korotkov et al. 2013). Here we showed that 5-hydroxydecanoate increased the Tl(+)-induced MPTP opening in the membrane of rat liver mitochondria regardless of the presence of mitoKATP modulators (diazoxide and pinacidil). This manifested in more pronounced decrease in the uncoupled respiration and acceleration of both the swelling and the ΔΨmito dissipation in isolated rat liver mitochondria, incubated in the medium containing TlNO3, KNO3, and Ca(2+). A slight delay in Ca(2+)-induced swelling of the mitochondria exposed to diazoxide could be result of an inhibition of succinate oxidation by the mitoKATP modulator. Mitochondrial calcium retention capacity (CRC) was markedly decreased in the presence of the mitoKATP inhibitor (5-hydroxydecanoate) or the mitoKCa inhibitor (paxilline). We suggest that the closure of mitoKATP or mitoKCa in calcium loaded mitochondria favors opening of the Tl(+)-induced MPTP in the inner mitochondrial membrane.

  15. Nicorandil improves ischemic changes in epicardial ECG during short-term coronary occlusion by opening ATP-sensitive potassium channels in pigs.

    PubMed

    Takaoka, A; Nakae, I; Liu, Q; Yamamoto, K; Ito, M; Kinoshita, M

    1997-12-01

    The aims of this study were to investigate whether nicorandil (NIC), an ATP-sensitive potassium channel (KATP) opener and nitrate, has antiischemic effects during transient ischemia in pigs, and to investigate whether its effects are due to its KATP-opening action or nitrate action. Myocardial ischemia was induced by ligating the proximal portion of the left anterior descending coronary artery for 1 minute in anesthetized open-chest pigs, and was measured as the magnitude of ST-segment elevation on epicardial electrocardiogram (ECG). Epicardial ST-segment elevation during coronary occlusion was significantly reduced by pretreatment with NIC (3 mg, intracoronary [i.c.]), but not by pretreatment with nitroglycerin (NTG, 0.2 mg, i.c.). Pretreatment with glibenclamide (GLB, a KATP blocker, 6 mg, i.c.) significantly augmented the ST-segment elevation during coronary occlusion. The augmentation of ST-segment elevation by GLB was significantly reduced by subsequent administration of NIC, but not by that of NTG (0.2 mg, i.c.). There were no significant differences between hemodynamic variables immediately before coronary occlusion with and without pretreatment. The intracoronary administration of NIC (3 mg) significantly shortened the endocardial monophasic action potential durations at 50% (MAPD50) and 90% repolarization (MAPD90) by 28.3 +/- 6.9% and 17.0 +/- 4.7%, respectively. These results suggest that the intracoronary administration of NIC has antiischemic effects during transient ischemia via KATP activation in myocardium.

  16. A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels*

    PubMed Central

    Zhang, Roger S.; Wright, Jordan D.; Pless, Stephan A.; Nunez, John-Jose; Kim, Robin Y.; Li, Jenny B. W.; Yang, Runying; Ahern, Christopher A.; Kurata, Harley T.

    2015-01-01

    ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels. PMID:25934393

  17. A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

    PubMed

    Zhang, Roger S; Wright, Jordan D; Pless, Stephan A; Nunez, John-Jose; Kim, Robin Y; Li, Jenny B W; Yang, Runying; Ahern, Christopher A; Kurata, Harley T

    2015-06-19

    ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

  18. Bioinspired Artificial Sodium and Potassium Ion Channels.

    PubMed

    Rodríguez-Vázquez, Nuria; Fuertes, Alberto; Amorín, Manuel; Granja, Juan R

    2016-01-01

    In Nature, all biological systems present a high level of compartmentalization in order to carry out a wide variety of functions in a very specific way. Hence, they need ways to be connected with the environment for communication, homeostasis equilibrium, nutrition, waste elimination, etc. The biological membranes carry out these functions; they consist of physical insulating barriers constituted mainly by phospholipids. These amphipathic molecules spontaneously aggregate in water to form bilayers in which the polar groups are exposed to the aqueous media while the non-polar chains self-organize by aggregating to each other to stay away from the aqueous media. The insulating properties of membranes are due to the formation of a hydrophobic bilayer covered at both sides by the hydrophilic phosphate groups. Thus, lipophilic molecules can permeate the membrane freely, while the small charged or very hydrophilic molecules require the assistance of other membrane components in order to overcome the energetic cost implied in crossing the non-polar region of the bilayer. Most of the large polar species (such as oligosaccharides, polypeptides or nucleic acids) cross into and out of the cell via endocytosis and exocytosis, respectively. Nature has created a series of systems (carriers and pores) in order to control the balance of small hydrophilic molecules and ions. The most important structures to achieve these goals are the ionophoric proteins that include the channel proteins, such as the sodium and potassium channels, and ionic transporters, including the sodium/potassium pumps or calcium/sodium exchangers among others. Inspired by these, scientists have created non-natural synthetic transporting structures to mimic the natural systems. The progress in the last years has been remarkable regarding the efficient transport of Na(+) and K(+) ions, despite the fact that the selectivity and the ON/OFF state of the non-natural systems remain a present and future challenge

  19. Bioinspired Artificial Sodium and Potassium Ion Channels.

    PubMed

    Rodríguez-Vázquez, Nuria; Fuertes, Alberto; Amorín, Manuel; Granja, Juan R

    2016-01-01

    In Nature, all biological systems present a high level of compartmentalization in order to carry out a wide variety of functions in a very specific way. Hence, they need ways to be connected with the environment for communication, homeostasis equilibrium, nutrition, waste elimination, etc. The biological membranes carry out these functions; they consist of physical insulating barriers constituted mainly by phospholipids. These amphipathic molecules spontaneously aggregate in water to form bilayers in which the polar groups are exposed to the aqueous media while the non-polar chains self-organize by aggregating to each other to stay away from the aqueous media. The insulating properties of membranes are due to the formation of a hydrophobic bilayer covered at both sides by the hydrophilic phosphate groups. Thus, lipophilic molecules can permeate the membrane freely, while the small charged or very hydrophilic molecules require the assistance of other membrane components in order to overcome the energetic cost implied in crossing the non-polar region of the bilayer. Most of the large polar species (such as oligosaccharides, polypeptides or nucleic acids) cross into and out of the cell via endocytosis and exocytosis, respectively. Nature has created a series of systems (carriers and pores) in order to control the balance of small hydrophilic molecules and ions. The most important structures to achieve these goals are the ionophoric proteins that include the channel proteins, such as the sodium and potassium channels, and ionic transporters, including the sodium/potassium pumps or calcium/sodium exchangers among others. Inspired by these, scientists have created non-natural synthetic transporting structures to mimic the natural systems. The progress in the last years has been remarkable regarding the efficient transport of Na(+) and K(+) ions, despite the fact that the selectivity and the ON/OFF state of the non-natural systems remain a present and future challenge.

  20. KATP channel blocker does not abolish the protective effect of NHE1 inhibition against ischaemia/reperfusion in aged myocardium

    PubMed Central

    Liu, Hong; Moore, Peter G

    2010-01-01

    Background and objective Aging is associated with an increase in myocardial susceptibility to ischaemia/reperfusion (I/R) injury. Na+/H+ exchange (NHE) inhibition and anaesthetic preconditioning (APC) are shown to protect myocardium from I/R injury. We set out to investigate if NHE inhibition can induce protection against I/R injury and whether KATP channel inhibition can enhance this effect in aged rat myocardium. Methods Hearts from 24-month old rats were assigned to 4 groups: (1) Control group; (2) APC group perfused with 2.5% sevoflurane before ischemia; (3) HOE group perfused with (3-methylsulfonyl-4-piperidinobenzoyl) guanidine methanesulfonate (HOE-694) prior to ischaemia; (4) HOE+5HD group perfused with both HOE and 5-hydroxydecanoic acid before ischaemia. We measured intracellular Na+ and Ca++ to quantitate the severity of myocardial injury. Results Both intracellular Na+ and Ca++ were significantly increased at the end of ischaemia and both were attenuated by NHE inhibition. Intracellular Na+ was 134±12 mEq/kg/dry weight in control group and 55±7 in HOE group (p<0.05). Intracellular Ca++ was 1764±142 nM in control group and 694±213 in HOE group (p<0.05). Infarct size was measured at 28±4% in control group vs. 17±2% in HOE group (p<0.05). High-energy phosphates and myocardial function were better preserved in HOE group compared to control (p<0.05). The beneficial effects of HOE on myocardial preservation was not blocked by 5HD nor were there any differences between APC and control groups. Conclusions NHE inhibition was effective in protecting myocardium from I/R injury in aged rats whereas APC was not. 5HD failed to block the protective effect of NHE inhibition. PMID:20216068

  1. Intractable hyperkalemia due to nicorandil induced potassium channel syndrome.

    PubMed

    Chowdhry, Vivek; Mohanty, B B

    2015-01-01

    Nicorandil is a commonly used antianginal agent, which has both nitrate-like and ATP-sensitive potassium (K ATP ) channel activator properties. Activation of potassium channels by nicorandil causes expulsion of potassium ions into the extracellular space leading to membrane hyperpolarization, closure of voltage-gated calcium channels and finally vasodilatation. However, on the other hand, being an activator of K ATP channel, it can expel K + ions out of the cells and can cause hyperkalemia. Here, we report a case of nicorandil induced hyperkalemia unresponsive to medical treatment in a patient with diabetic nephropathy.

  2. Gain-of-Function Mutation, S422L, in the KCNJ8-Encoded Cardiac KATP Channel Kir6.1 as a Pathogenic Substrate for J Wave Syndromes

    PubMed Central

    Medeiros-Domingo, Argelia; Tan, Bi-Hua; Crotti, Lia; Tester, David J.; Eckhardt, Lee; Cuoretti, Alessandra; Kroboth, Stacie L.; Song, Chunhua; Zhou, Qing; Kopp, Doug; Schwartz, Peter J.; Makielski, Jonathan C.; Ackerman, Michael J.

    2011-01-01

    Background J Wave Syndromes have emerged conceptually to encompass the pleiotropic expression of J point abnormalities including Brugada syndrome (BrS) and early repolarization syndrome (ERS). Recently, KCNJ8, which encodes the cardiac KATP Kir6.1 channel, has been implicated in ERS following the identification of a functionally uncharacterized missense mutation, S422L. Here, we sought to further explore KCNJ8 as a novel susceptibility gene for J wave syndromes. Methods Using PCR, DHPLC, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of KCNJ8 was performed in 101 unrelated patients with J wave syndromes including 87 with BrS and 14 with ERS. 600 healthy individuals were examined to assess allelic frequency for all variants detected. KCNJ8 mutation(s) were engineered by site directed mutagenesis and co-expressed heterologously with SUR2A in COS-1 cells. Ion currents were recorded using whole cell configuration of the patch-clamp technique. Results One BrS case and one ERS case hosted the identical missense mutation, S422L that was reported previously. KCNJ8-S422L involves a highly conserved residue and was absent in 1200 reference alleles. Both cases were negative for mutations in all known BrS- and ERS-susceptibility genes. The KATP current of Kir6.1-S422L mutation was increased significantly over the voltage range of 0 mV to 40 mV compared to Kir6.1-WT channels (p < 0.05, n=16-21). Conclusions These findings further implicate KCNJ8 as a novel J wave syndrome-susceptibility gene and a marked gain-of-function in the cardiac KATP Kir6.1 channel secondary to KCNJ8-S422L as a novel pathogenic mechanism for the phenotypic expression of both BrS and ERS. PMID:20558321

  3. Involvements of calcium channel and potassium channel in Danshen and Gegen decoction induced vasodilation in porcine coronary LAD artery.

    PubMed

    Hu, Fan; Koon, Chi Man; Chan, Judy Yuet Wa; Lau, Kit Man; Kwan, Y W; Fung, Kwok Pui

    2012-09-15

    Danshen (Salviae Miltiorrhizae Radix) and Gegen (Puerariae Lobatae Radix) have been widely used in treating cardiovascular diseases for thousands of years in China. The present study was carried out to evaluate the effects of a Danshen and Gegen decoction (DG) on the vascular reactivity of a porcine isolated coronary artery and the underlying mechanisms involved. Porcine coronary rings were precontracted with 15 nM U46619. The involvement of endothelium-dependent mechanisms was explored by removing the endothelium; the involvement of potassium channels was investigated by the pretreatment of the artery rings with various blockers, and the involvement of the calcium channels was investigated by incubating the artery rings with Ca²⁺-free buffer and priming them with high [K⁺] prior to adding CaCl₂ to elicit contraction. The involvement of Ca²⁺ sensitization was explored by evaluating the Rho-activity expression. The results revealed that DG elicited a concentration-dependent relaxation on a U46619-precontracted coronary artery ring. These relaxation responses were not altered by the pretreatment of inhibitors of endothelium-related dilator synthases, cGMP and cAMP pathway inhibitors, potassium channel (BK(Ca), SK(Ca), K(V) and K(ATP)) blockers and endothelium removal. The K(IR) channel blocker BaCl₂ only slightly attenuated the DG-induced relaxation. However, the Ca²⁺-induced artery contraction was inhibited by DG. Additionally, the expression of the phosphorylated myosin light chain was inhibited by DG whereas the activity of RhoA was not affected. Therefore, DG could be a useful cardioprotective agent for vasodilation in patients who have hypertension.

  4. Potassium channel conductance as a control mechanism in hair follicles.

    PubMed

    Buhl, A E; Conrad, S J; Waldon, D J; Brunden, M N

    1993-07-01

    The opening of intracellular potassium channels is a common mechanism of action for a set of anti-hypertensive drugs that includes the hair-growth-inducing agent minoxidil. Recent work suggests potassium channel openers (PCOs) also influence hair growth. Correlative studies demonstrate that a series of PCOs including minoxidil, pinacidil, P-1075, an active pinacidil analog, RP-49,356, cromakalim, and nicorandil maintain hair growth in cultured vibrissa follicles. Studies using balding stumptail macaques verify that minoxidil, P-1075, and cromakalim but not RP-49,356 stimulate hair growth. The definition of potassium channels and documentation of drug effects on these channels is classically done using electrophysiologic techniques. Such studies require the identification and isolation of target cells. Both these are among the unsolved problems in the area of hair biology. Estimating K+ flux using 86Rb+ as a K+ tracer is an accepted method of assessing potassium channel conductance in other organ systems. Both pinacidil and RP-49,356 induce measurable Rb+ flux in isolated vibrissa follicles and a hair epithelial cell line whereas neither minoxidil nor minoxidil sulfate had measurable effects. Potassium channels have been studied successfully in other organ systems using specific pharmacologic blockers for the various channel subtypes. Blockers including glyburide, tetraethylammonium, and procaine failed to inhibit minoxidil stimulation of cultured follicles. The current explosion of knowledge on potassium channel biology, cloning of channels, and continued progress in hair biology promise to clarify the role of K+ ions in the control of hair follicles.

  5. Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

    PubMed Central

    Proks, Peter; Capener, Charlotte E.; Jones, Phillippa; Ashcroft, Frances M.

    2001-01-01

    The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings. PMID:11585848

  6. Cumulative Activation of Voltage-Dependent KVS-1 Potassium Channels

    PubMed Central

    Rojas, Patricio; Garst-Orozco, Jonathan; Baban, Beravan; de Santiago-Castillo, Jose Antonio; Covarrubias, Manuel; Salkoff, Lawrence

    2008-01-01

    In this study, we reveal the existence of a novel use-dependent phenomenon in potassium channels, which we refer to as cumulative activation (CA). CA consists of an increase in current amplitude in response to repetitive depolarizing step pulses to the same potential. CA persists for up to 20 s and is similar to a phenomenon called “voltage-dependent facilitation” observed in some calcium channels. The KVS-1 K+ channel, which exhibits CA, is a rapidly activating and inactivating voltage-dependent potassium channel expressed in chemosensory and other neurons of Caenorhabditis elegans. It is unusual in being most closely related to the Shab (Kv2) family of potassium channels, which typically behave like delayed rectifier K+ channels in other species. The magnitude of CA depends on the frequency, voltage, and duration of the depolarizing step pulse. CA also radically changes the activation and inactivation kinetics of the channel, suggesting that the channel may undergo a physical modification in a use-dependent manner; thus, a model that closely simulates the behavior of the channel postulates the existence of two populations of channels, unmodified and modified. Use-dependent changes in the behavior of potassium channels, such as CA observed in KVS-1, could be involved in functional mechanisms of cellular plasticity such as synaptic depression that represent the cellular basis of learning and memory. PMID:18199775

  7. A novel potassium channel in skeletal muscle mitochondria.

    PubMed

    Skalska, Jolanta; Piwońska, Marta; Wyroba, Elzbieta; Surmacz, Liliana; Wieczorek, Rafal; Koszela-Piotrowska, Izabela; Zielińska, Joanna; Bednarczyk, Piotr; Dołowy, Krzysztof; Wilczynski, Grzegorz M; Szewczyk, Adam; Kunz, Wolfram S

    2008-01-01

    In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

  8. Characterization of the potassium channels involved in EDHF-mediated relaxation in cerebral arteries

    PubMed Central

    Petersson, Jesper; Zygmunt, Peter M; Högestätt, Edward D

    1997-01-01

    In the presence of NG-nitro-L-arginine (L-NOARG, 0.3 mM) and indomethacin (10 μM), the relaxations induced by acetylcholine and the calcium (Ca) ionophore A23187 are considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF) in the guinea-pig basilar artery.Inhibitors of adenosine 5′-triphosphate (ATP)-sensitive potassium (K)-channels (KATP; glibenclamide, 10 μM), voltage-sensitive K-channels (KV; dendrotoxin-I, 0.1 μM or 4-aminopyridine, 1 mM), small (SKCa; apamin, 0.1 μM) and large (BKCa; iberiotoxin, 0.1 μM) conductance Ca-sensitive K-channels did not affect the L-NOARG/indomethacin-resistant relaxation induced by acetylcholine.Synthetic charybdotoxin (0.1 μM), an inhibitor of BKCa and KV, caused a rightward shift of the concentration-response curve for acetylcholine and reduced the maximal relaxation in the presence of L-NOARG and indomethacin, whereas the relaxation induced by A23187 was not significantly inhibited.A combination of charybdotoxin (0.1 μM) and apamin (0.1 μM) abolished the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and A23187. However, the acetylcholine-induced relaxation was not affected by a combination of iberiotoxin (0.1 μM) and apamin (0.1 μM).Ciclazindol (10 μM), an inhibitor of KV in rat portal vein smooth muscle, inhibited the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and A23187, and the relaxations were abolished when ciclazindol (10 μM) was combined with apamin (0.1 μM).Human pial arteries from two out of four patients displayed an L-NOARG/indomethacin-resistant relaxation in response to substance P. This relaxation was abolished in both cases by pretreatment with the combination of charybdotoxin (0.1 μM) and apamin (0.1 μM), whereas each toxin had little effect alone.The results suggest that KV, but not KATP and BKCa, is involved in the EDHF-mediated relaxation in the guinea-pig basilar artery. The synergistic

  9. Photochromic potassium channel blockers: design and electrophysiological characterization.

    PubMed

    Mourot, Alexandre; Fehrentz, Timm; Kramer, Richard H

    2013-01-01

    Voltage-gated potassium (K v) channels are membrane proteins that open a selective pore upon membrane depolarization, allowing K(+) ions to flow down their electrochemical gradient. In neurons, K v channels play a key role in repolarizing the membrane potential during the falling phase of the action potential, often resulting in an after hyperpolarization. Opening of K v channels results in a decrease of cellular excitability, whereas closing (or pharmacological block) has the opposite effect, increased excitability. We have developed a series of photosensitive blockers for K v channels that enable reversible, optical regulation of potassium ion flow. Such molecules can be used for remote control of neuronal excitability using light as an on/off switch. Here we describe the design and electrophysiological characterization of photochromic blockers of ion channels. Our focus is on K v channels but in principle, the techniques described here can be applied to other ion channels and signaling proteins.

  10. Photochromic Potassium Channel Blockers: Design and Electrophysiological Characterization

    PubMed Central

    Mourot, Alexandre; Fehrentz, Timm; Kramer, Richard H.

    2016-01-01

    Voltage-gated potassium (Kv) channels are membrane proteins that open a selective pore upon membrane depolarization, allowing K+ ions to flow down their electrochemical gradient. In neurons, Kv channels play a key role in repolarizing the membrane potential during the falling phase of the action potential, often resulting in an after hyperpolarization. Opening of Kv channels results in a decrease of cellular excitability, whereas closing (or pharmacological block) has the opposite effect, increased excitability. We have developed a series of photosensitive blockers for Kv channels that enable reversible, optical regulation of potassium ion flow. Such molecules can be used for remote control of neuronal excitability using light as an on/off switch. Here we describe the design and electrophysiological characterization of photochromic blockers of ion channels. Our focus is on Kv channels but in principle, the techniques described here can be applied to other ion channels and signaling proteins. PMID:23494374

  11. Cardiac Delayed Rectifier Potassium Channels in Health and Disease.

    PubMed

    Chen, Lei; Sampson, Kevin J; Kass, Robert S

    2016-06-01

    Cardiac delayed rectifier potassium channels conduct outward potassium currents during the plateau phase of action potentials and play pivotal roles in cardiac repolarization. These include IKs, IKr and the atrial specific IKur channels. In this article, we will review their molecular identities and biophysical properties. Mutations in the genes encoding delayed rectifiers lead to loss- or gain-of-function phenotypes, disrupt normal cardiac repolarization and result in various cardiac rhythm disorders, including congenital Long QT Syndrome, Short QT Syndrome and familial atrial fibrillation. We will also discuss the prospect of using delayed rectifier channels as therapeutic targets to manage cardiac arrhythmia. PMID:27261823

  12. Potassium channels of pig articular chondrocytes are blocked by propofol.

    PubMed

    Mozrzymas, J W; Visintin, M; Vittur, F; Ruzzier, F

    1994-07-15

    The effect of propofol on the voltage-activated potassium channels in pig articular chondrocytes was investigated. Propofol was found to reversibly block the potassium channels in a dose-dependent manner. The blocking effect was voltage-independent and the Hill coefficient was 1.85 +/- 0.18. No changes either in the slope conductance or in the single channel kinetics were observed. The half-blocking concentration (Ec50) was 6.0 +/- 0.49 microM which is much lower than the concentrations used to observe the scavenging effect of the drug in an artificial synovial fluid. Interestingly, Ec50 found in our experiments is also smaller than the blood concentration of propofol used in anaesthesia. These results show that propofol may strongly affect the potassium channels in some non-excitable cells.

  13. Myorelaxant action of fluorine-containing pinacidil analog, flocalin, in bladder smooth muscle is mediated by inhibition of L-type calcium channels rather than activation of KATP channels.

    PubMed

    Philyppov, Igor B; Golub, Andriy А; Boldyriev, Oleksiy I; Shtefan, Natalia L; Totska, Khrystyna; Voitychuk, Oleg I; Shuba, Yaroslav M

    2016-06-01

    Flocalin (FLO) is a new ATP-sensitive K(+) (KATP) channel opener (KCO) derived from pinacidil (PIN) by adding fluorine group to the drug's structure. FLO acts as a potent cardioprotector against ischemia-reperfusion damage in isolated heart and whole animal models primarily via activating cardiac-specific Kir6.2/SUR2A KATP channels. Given that FLO also confers relaxation on several types of smooth muscles and can partially inhibit L-type Ca(2+) channels, in this study, we asked what is the mechanism of FLO action in bladder detrusor smooth muscle (DSM). The actions of FLO and PIN on contractility of rat and guinea pig DSM strips and membrane currents of isolated DSM cells were compared by tensiometry and patch clamp. Kir6 and SUR subunit expression in rat DSM was assayed by reverse transcription PCR (RT-PCR). In contrast to PIN (10 μM), FLO (10 μM) did not produce glibenclamide-sensitive DSM strips' relaxation and inhibition of spontaneous and electrically evoked contractions. However, FLO, but not PIN, inhibited contractions evoked by high K(+) depolarization. FLO (40 μM) did not change the level of isolated DSM cell's background K(+) current, but suppressed by 20 % L-type Ca(2+) current. Determining various Kir6 and SUR messenger RNA (mRNA) expressions in rat DSM by RT-PCR indicated that dominant KATP channel in rat DSM is of vascular type involving association of Kir6.1 and SUR2B subunits. Myorelaxant effects of FLO in bladder DSM are explained by partial blockade of L-type Ca(2+) channel-mediated Ca(2+) influx rather than by hyperpolarization associated with increased K(+) permeability. Thus, insertion of fluorine group in PIN's structure made the drug more discriminative between Kir6.2/SUR2A cardiac- and Kir6.1/SUR2B vascular-type KATP channels and rendered it partial L-type Ca(2+) channel-blocking potency.

  14. Effects of cytokines on potassium channels in renal tubular epithelia.

    PubMed

    Nakamura, Kazuyoshi; Komagiri, You; Kubokawa, Manabu

    2012-02-01

    Renal tubular potassium (K(+)) channels play important roles in the formation of cell-negative potential, K(+) recycling, K(+) secretion, and cell volume regulation. In addition to these physiological roles, it was reported that changes in the activity of renal tubular K(+) channels were involved in exacerbation of renal cell injury during ischemia and endotoxemia. Because ischemia and endotoxemia stimulate production of cytokines in immune cells and renal tubular cells, it is possible that cytokines would affect K(+) channel activity. Although the regulatory mechanisms of renal tubular K(+) channels have extensively been studied, little information is available about the effects of cytokines on these K(+) channels. The first report was that tumor necrosis factor acutely stimulated the single channel activity of the 70 pS K(+) channel in the rat thick ascending limb through activation of tyrosine phosphatase. Recently, it was also reported that interferon-γ (IFN-γ) and interleukin-1β (IL-1β) modulated the activity of the 40 pS K(+) channel in cultured human proximal tubule cells. IFN-γ exhibited a delayed suppression and an acute stimulation of K(+) channel activity, whereas IL-1β acutely suppressed the channel activity. Furthermore, these cytokines suppressed gene expression of the renal outer medullary potassium channel. The renal tubular K(+) channels are functionally coupled to the coexisting transporters. Therefore, the effects of cytokines on renal tubular transporter activity should also be taken into account, when interpreting their effects on K(+) channel activity. PMID:22042037

  15. Circulating insulin stimulates fatty acid retention in white adipose tissue via KATP channel activation in the central nervous system only in insulin-sensitive mice[S

    PubMed Central

    Coomans, Claudia P.; Geerling, Janine J.; Guigas, Bruno; van den Hoek, Anita M.; Parlevliet, Edwin T.; Ouwens, D. Margriet; Pijl, Hanno; Voshol, Peter J.; Rensen, Patrick C. N.; Havekes, Louis M.; Romijn, Johannes A.

    2011-01-01

    Insulin signaling in the central nervous system (CNS) is required for the inhibitory effect of insulin on glucose production. Our aim was to determine whether the CNS is also involved in the stimulatory effect of circulating insulin on the tissue-specific retention of fatty acid (FA) from plasma. In wild-type mice, hyperinsulinemic-euglycemic clamp conditions stimulated the retention of both plasma triglyceride-derived FA and plasma albumin-bound FA in the various white adipose tissues (WAT) but not in other tissues, including brown adipose tissue (BAT). Intracerebroventricular (ICV) administration of insulin induced a similar pattern of tissue-specific FA partitioning. This effect of ICV insulin administration was not associated with activation of the insulin signaling pathway in adipose tissue. ICV administration of tolbutamide, a KATP channel blocker, considerably reduced (during hyperinsulinemic-euglycemic clamp conditions) and even completely blocked (during ICV administration of insulin) WAT-specific retention of FA from plasma. This central effect of insulin was absent in CD36-deficient mice, indicating that CD36 is the predominant FA transporter in insulin-stimulated FA retention by WAT. In diet-induced insulin-resistant mice, these stimulating effects of insulin (circulating or ICV administered) on FA retention in WAT were lost. In conclusion, in insulin-sensitive mice, circulating insulin stimulates tissue-specific partitioning of plasma-derived FA in WAT in part through activation of KATP channels in the CNS. Apparently, circulating insulin stimulates fatty acid uptake in WAT but not in BAT, directly and indirectly through the CNS. PMID:21700834

  16. Dopamine midbrain neurons in health and Parkinson's disease: emerging roles of voltage-gated calcium channels and ATP-sensitive potassium channels.

    PubMed

    Dragicevic, E; Schiemann, J; Liss, B

    2015-01-22

    Dopamine (DA) releasing midbrain neurons are essential for multiple brain functions, such as voluntary movement, working memory, emotion and cognition. DA midbrain neurons within the substantia nigra (SN) and the ventral tegmental area (VTA) exhibit a variety of distinct axonal projections and cellular properties, and are differentially affected in diseases like schizophrenia, attention deficit hyperactivity disorder, and Parkinson's disease (PD). Apart from having diverse functions in health and disease states, DA midbrain neurons display distinct electrical activity patterns, crucial for DA release. These activity patterns are generated and modulated by specific sets of ion channels. Recently, two ion channels have been identified, not only contributing to these activity patterns and to functional properties of DA midbrain neurons, but also seem to render SN DA neurons particularly vulnerable to degeneration in PD and its animal models: L-type calcium channels (LTCCs) and ATP-sensitive potassium channels (K-ATPs). In this review, we focus on the emerging physiological and pathophysiological roles of these two ion channels (and their complex interplay with other ion channels), particularly in highly vulnerable SN DA neurons, as selective degeneration of these neurons causes the major motor symptoms of PD. PMID:25450964

  17. Dopamine midbrain neurons in health and Parkinson's disease: emerging roles of voltage-gated calcium channels and ATP-sensitive potassium channels.

    PubMed

    Dragicevic, E; Schiemann, J; Liss, B

    2015-01-22

    Dopamine (DA) releasing midbrain neurons are essential for multiple brain functions, such as voluntary movement, working memory, emotion and cognition. DA midbrain neurons within the substantia nigra (SN) and the ventral tegmental area (VTA) exhibit a variety of distinct axonal projections and cellular properties, and are differentially affected in diseases like schizophrenia, attention deficit hyperactivity disorder, and Parkinson's disease (PD). Apart from having diverse functions in health and disease states, DA midbrain neurons display distinct electrical activity patterns, crucial for DA release. These activity patterns are generated and modulated by specific sets of ion channels. Recently, two ion channels have been identified, not only contributing to these activity patterns and to functional properties of DA midbrain neurons, but also seem to render SN DA neurons particularly vulnerable to degeneration in PD and its animal models: L-type calcium channels (LTCCs) and ATP-sensitive potassium channels (K-ATPs). In this review, we focus on the emerging physiological and pathophysiological roles of these two ion channels (and their complex interplay with other ion channels), particularly in highly vulnerable SN DA neurons, as selective degeneration of these neurons causes the major motor symptoms of PD.

  18. The involvement of ATP-sensitive potassium channels in β2-adrenoceptor agonist-induced vasodilatation on rat diaphragmatic microcirculation

    PubMed Central

    Chang, Han-Yu

    1997-01-01

    The effects of glibenclamide (GLB), a blocker of ATP-sensitive potassium (KATP) channels, on diaphragmatic microcirculation in male Sprague-Dawley rats were assessed under basal conditions and after β2-adrenoceptor-agonist stimulation. In addition, forskolin was used to bypass β-adrenoceptors and GTP-binding proteins (G-protein) to explore the possible mechanism of GLB effects. For comparison, the relationships between KATP channel activity and cyclic GMP-mediated vasodilator responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were also assessed. Male Sprague-Dawley rats were anaesthetized with urethane and mechanically ventilated. The left hemi-diaphragm of each rat was prepared and microvascular blood flow (QLDF) was recorded with laser-Doppler flowmetry during continuous superfusion with bicarbonate-buffered, prewarmed Ringer solution. The drugs were topically applied to the surface of the hemi-diaphragm. Salbutamol (0.32–32 μM), terbutaline (0.32 μM–0.32 μM) and forskolin (0.32–10 μM) each elicited a concentration-dependent increase in QLDF. Baseline microvascular blood flow was unaffected by a 30 min suffusion of 1 μM GLB (295±51 mV vs 325±62 mV, P=0.738). The vasodilator response elicited by salbutamol (0.32 μM, 1 μM and 3.2 μM), was significantly attenuated by a 30 min superfusion with 1 μM GLB; this salbutamol-induced vasodilatation was mediated via an interaction with β-adrenoceptor receptors, as in other experiments it was greatly inhibited by 30-min superfusion with propranolol (10 μM). Similarly, following 30-min superfusion with GLB (1 μM), the terbutaline (1 μM, 3.2 μM and 10 μM)-induced vasodilator response was almost abolished and the vasodilator responses induced by incremental concentrations of forskolin (0.32 μM, 1 μM and 3.2 μM) were also significantly attenuated. Cromakalim (1.5 μM, 3 μM and 3.2 μM) produced an increase of QLDF in a dose-dependent manner

  19. A family of putative potassium channel genes in Drosophila.

    PubMed

    Butler, A; Wei, A G; Baker, K; Salkoff, L

    1989-02-17

    Mutant flies in which the gene coding for the Shaker potassium channel is deleted still have potassium currents similar to those coded by the Shaker gene. This suggests the presence of a family of Shaker-like genes in Drosophila. By using a Shaker complementary DNA probe and low-stringency hybridization, three additional family members have now been isolated, Shab, Shaw, and Shal. The Shaker family genes are not clustered in the genome. The deduced proteins of Shab, Shaw, and Shal have high homology to the Shaker protein; the sequence identity of the integral membrane portions is greater than 50 percent. These genes are organized similarly to Shaker in that only a single homology domain containing six presumed membrane-spanning segments common to all voltage-gated ion channels is coded by each messenger RNA. Thus, potassium channel diversity could result from an extended gene family, as well as from alternate splicing of the Shaker primary transcript.

  20. Pore size matters for potassium channel conductance.

    PubMed

    Naranjo, David; Moldenhauer, Hans; Pincuntureo, Matías; Díaz-Franulic, Ignacio

    2016-10-01

    Ion channels are membrane proteins that mediate efficient ion transport across the hydrophobic core of cell membranes, an unlikely process in their absence. K(+) channels discriminate K(+) over cations with similar radii with extraordinary selectivity and display a wide diversity of ion transport rates, covering differences of two orders of magnitude in unitary conductance. The pore domains of large- and small-conductance K(+) channels share a general architectural design comprising a conserved narrow selectivity filter, which forms intimate interactions with permeant ions, flanked by two wider vestibules toward the internal and external openings. In large-conductance K(+) channels, the inner vestibule is wide, whereas in small-conductance channels it is narrow. Here we raise the idea that the physical dimensions of the hydrophobic internal vestibule limit ion transport in K(+) channels, accounting for their diversity in unitary conductance.

  1. A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.

    PubMed

    Allebrandt, K V; Amin, N; Müller-Myhsok, B; Esko, T; Teder-Laving, M; Azevedo, R V D M; Hayward, C; van Mill, J; Vogelzangs, N; Green, E W; Melville, S A; Lichtner, P; Wichmann, H-E; Oostra, B A; Janssens, A C J W; Campbell, H; Wilson, J F; Hicks, A A; Pramstaller, P P; Dogas, Z; Rudan, I; Merrow, M; Penninx, B; Kyriacou, C P; Metspalu, A; van Duijn, C M; Meitinger, T; Roenneberg, T

    2013-01-01

    Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism. PMID:22105623

  2. A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.

    PubMed

    Allebrandt, K V; Amin, N; Müller-Myhsok, B; Esko, T; Teder-Laving, M; Azevedo, R V D M; Hayward, C; van Mill, J; Vogelzangs, N; Green, E W; Melville, S A; Lichtner, P; Wichmann, H-E; Oostra, B A; Janssens, A C J W; Campbell, H; Wilson, J F; Hicks, A A; Pramstaller, P P; Dogas, Z; Rudan, I; Merrow, M; Penninx, B; Kyriacou, C P; Metspalu, A; van Duijn, C M; Meitinger, T; Roenneberg, T

    2013-01-01

    Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism.

  3. Slack, Slick, and Sodium-Activated Potassium Channels

    PubMed Central

    Kaczmarek, Leonard K.

    2013-01-01

    The Slack and Slick genes encode potassium channels that are very widely expressed in the central nervous system. These channels are activated by elevations in intracellular sodium, such as those that occur during trains of one or more action potentials, or following activation of nonselective cationic neurotransmitter receptors such as AMPA receptors. This review covers the cellular and molecular properties of Slack and Slick channels and compares them with findings on the properties of sodium-activated potassium currents (termed KNa currents) in native neurons. Human mutations in Slack channels produce extremely severe defects in learning and development, suggesting that KNa channels play a central role in neuronal plasticity and intellectual function. PMID:24319675

  4. State-dependent inactivation of the Kv3 potassium channel.

    PubMed Central

    Marom, S; Levitan, I B

    1994-01-01

    Inactivation of Kv3 (Kv1.3) delayed rectifier potassium channels was studied in the Xenopus oocyte expression system. These channels inactivate slowly during a long depolarizing pulse. In addition, inactivation accumulates in response to a series of short depolarizing pulses (cumulative inactivation), although no significant inactivation occurs within each short pulse. The extent of cumulative inactivation does not depend on the voltage during the depolarizing pulse, but it does vary in a biphasic manner as a function of the interpulse duration. Furthermore, the rate of cumulative inactivation is influenced by changing the rate of deactivation. These data are consistent with a model in which Kv3 channel inactivation is a state-dependent and voltage-independent process. Macroscopic and single channel experiments indicate that inactivation can occur from a closed (silent) state before channel opening. That is, channels need not open to inactivate. The transition that leads to the inactivated state from the silent state is, in fact, severalfold faster then the observed inactivation of current during long depolarizing pulses. Long pulse-induced inactivation appears to be slow, because its rate is limited by the probability that channels are in the open state, rather than in the silent state from which they can inactivate. External potassium and external calcium ions alter the rates of cumulative and long pulse-induced inactivation, suggesting that antagonistic potassium and calcium binding steps are involved in the normal gating of the channel. PMID:7948675

  5. Genome-Wide Expression Profiling of Anoxia/Reoxygenation in Rat Cardiomyocytes Uncovers the Role of MitoKATP in Energy Homeostasis

    PubMed Central

    Cao, Song; Liu, Yun; Sun, Wenting; Zhao, Li; Zhang, Lin; Liu, Xinkui; Yu, Tian

    2015-01-01

    Mitochondrial ATP-sensitive potassium channel (mitoKATP) is a common end effector of many protective stimuli in myocardial ischemia-reperfusion injury (MIRI). However, the specific molecular mechanism underlying its myocardial protective effect is not well elucidated. We characterized an anoxia/reoxygenation (A/R) model using freshly isolated adult rat cardiomyocytes. MitoKATP status was interfered with its specific opener diazoxide (DZ) or blocker 5-hydroxydecanote (5-HD). Digital gene expression (DGE) and bioinformatic analysis were deployed. Three energy metabolism related genes (MT-ND6, Idh2, and Acadl) were upregulated when mitoKATP opened. In addition, as many as 20 differentially expressed genes (DEGs) were significantly enriched in five energy homeostasis correlated pathways (PPAR, TCA cycle, fatty acid metabolism, and peroxisome). These findings indicated that mitoKATP opening in MIRI resulted in energy mobilization, which was confirmed by measuring ATP content in cardiomyocytes. These causal outcomes could be a molecular mechanism of myocardial protection of mitoKATP and suggested that the mitoKATP opening plays a physiologic role in triggering cardiomyocytes' energy homeostasis during MIRI. Strategies of modulating energy expenditure during myocardial ischemia-reperfusion may be promising approaches to reduce MIRI. PMID:26171116

  6. The analgesic effect of dipyrone in peripheral tissue involves two different mechanisms: neuronal K(ATP) channel opening and CB(1) receptor activation.

    PubMed

    dos Santos, Gilson Gonçalves; Dias, Elayne Vieira; Teixeira, Juliana Maia; Athie, Maria Carolina Pedro; Bonet, Ivan José Magayewski; Tambeli, Cláudia Herrera; Parada, Carlos Amilcar

    2014-10-15

    Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

  7. Effects of fractal gating of potassium channels on neuronal behaviours

    NASA Astrophysics Data System (ADS)

    Zhao, De-Jiang; Zeng, Shang-You; Zhang, Zheng-Zhen

    2010-10-01

    The classical model of voltage-gated ion channels assumes that according to a Markov process ion channels switch among a small number of states without memory, but a bunch of experimental papers show that some ion channels exhibit significant memory effects, and this memory effects can take the form of kinetic rate constant that is fractal. Obviously the gating character of ion channels will affect generation and propagation of action potentials, furthermore, affect generation, coding and propagation of neural information. However, there is little previous research on this series of interesting issues. This paper investigates effects of fractal gating of potassium channel subunits switching from closed state to open state on neuronal behaviours. The obtained results show that fractal gating of potassium channel subunits switching from closed state to open state has important effects on neuronal behaviours, increases excitability, rest potential and spiking frequency of the neuronal membrane, and decreases threshold voltage and threshold injected current of the neuronal membrane. So fractal gating of potassium channel subunits switching from closed state to open state can improve the sensitivity of the neuronal membrane, and enlarge the encoded strength of neural information.

  8. Ion permeation mechanism of the potassium channel

    NASA Astrophysics Data System (ADS)

    Åqvist, Johan; Luzhkov, Victor

    2000-04-01

    Ion-selective channels enable the specific permeation of ions through cell membranes and provide the basis of several important biological functions; for example, electric signalling in the nervous system. Although a large amount of electrophysiological data is available, the molecular mechanisms by which these channels can mediate ion transport remain a significant unsolved problem. With the recently determined crystal structure of the representative K+ channel (KcsA) from Streptomyces lividans, it becomes possible to examine ion conduction pathways on a microscopic level. K+ channels utilize multi-ion conduction mechanisms, and the three-dimensional structure also shows several ions present in the channel. Here we report results from molecular dynamics free energy perturbation calculations that both establish the nature of the multiple ion conduction mechanism and yield the correct ion selectivity of the channel. By evaluating the energetics of all relevant occupancy states of the selectivity filter, we find that the favoured conduction pathway involves transitions only between two main states with a free difference of about 5 kcal mol-1. Other putative permeation pathways can be excluded because they would involve states that are too high in energy.

  9. Magnetic and electric fields across sodium and potassium channels

    NASA Astrophysics Data System (ADS)

    Soares, Marília A. G.; Cruz, Frederico A. O.; Silva, Dilson

    2015-12-01

    We determined the magnetic field around sodium and potassium ionic channels based on a physico-mathematical model that took into account charges in the surface bilayer. For the numerical simulation, we applied the finite element method. Results show that each channel produces its specific and individual response to the ion transport, according to its individual intrinsic properties. The existence of a number of active Na+-channels in a given membrane region seems not to interfere directly in the functioning of K+-channel located among them, and vice-versa.

  10. Potassium channels in cell cycle and cell proliferation

    PubMed Central

    Urrego, Diana; Tomczak, Adam P.; Zahed, Farrah; Stühmer, Walter; Pardo, Luis A.

    2014-01-01

    Normal cell-cycle progression is a crucial task for every multicellular organism, as it determines body size and shape, tissue renewal and senescence, and is also crucial for reproduction. On the other hand, dysregulation of the cell-cycle progression leading to uncontrolled cell proliferation is the hallmark of cancer. Therefore, it is not surprising that it is a tightly regulated process, with multifaceted and very complex control mechanisms. It is now well established that one of those mechanisms relies on ion channels, and in many cases specifically on potassium channels. Here, we summarize the possible mechanisms underlying the importance of potassium channels in cell-cycle control and briefly review some of the identified channels that illustrate the multiple ways in which this group of proteins can influence cell proliferation and modulate cell-cycle progression. PMID:24493742

  11. Potassium Channels and Human Epileptic Phenotypes: An Updated Overview

    PubMed Central

    Villa, Chiara; Combi, Romina

    2016-01-01

    Potassium (K+) channels are expressed in almost every cells and are ubiquitous in neuronal and glial cell membranes. These channels have been implicated in different disorders, in particular in epilepsy. K+ channel diversity depends on the presence in the human genome of a large number of genes either encoding pore-forming or accessory subunits. More than 80 genes encoding the K+ channels were cloned and they represent the largest group of ion channels regulating the electrical activity of cells in different tissues, including the brain. It is therefore not surprising that mutations in these genes lead to K+ channels dysfunctions linked to inherited epilepsy in humans and non-human model animals. This article reviews genetic and molecular progresses in exploring the pathogenesis of different human epilepsies, with special emphasis on the role of K+ channels in monogenic forms. PMID:27064559

  12. SUMOylation and Potassium Channels: Links to Epilepsy and Sudden Death.

    PubMed

    Wu, Hongmei; Chen, Xu; Cheng, Jinke; Qi, Yitao

    2016-01-01

    Neuronal potassium ion channels play an essential role in the generation of the action potential and excitability of neurons. The dysfunction of ion channel subunits can cause channelopathies, which are associated in some cases with sudden unexplained death in epilepsy SUDEP. The physiological roles of neuronal ion channels have been largely determined, but little is known about the molecular mechanisms underlying neurological channelopathies, especially the determinants of the channels' regulation. SUMO (small ubiquitin-like modifier) proteins covalently conjugate lysine residues in a large number of target proteins and modify their functions. SUMO modification (SUMOylation) has emerged as an important regulatory mechanism for protein stability, function, subcellular localization, and protein-protein interactions. Since SUMO was discovered almost 20 years ago, the biological contribution of SUMOylation has not fully understood. It is until recently that the physiological impacts of SUMOylation on the regulation of neuronal potassium ion channels have been investigated. It is well established that SUMOylation controls many aspects of nuclear function, but it is now clear that it is also a key determinant in the function of potassium channels, and SUMOylation has also been implicated in a wide range of channelopathies, including epilepsy and sudden death. PMID:26920693

  13. TRESK potassium channel in human T lymphoblasts

    SciTech Connect

    Sánchez-Miguel, Dénison Selene; García-Dolores, Fernando; Rosa Flores-Márquez, María; Delgado-Enciso, Iván; Pottosin, Igor; Dobrovinskaya, Oxana

    2013-05-03

    Highlights: • TRESK (KCNK18) mRNA is present in different T lymphoblastic cell lines. • KCNK18 mRNA was not found in resting peripheral blood lymphocytes. • Clinical samples of T lymphoblastic leukemias and lymphomas were positive for TRESK. • TRESK in T lymphoblasts has dual localization, in plasma membrane and intracellular. -- Abstract: TRESK (TWIK-related spinal cord K{sup +}) channel, encoded by KCNK18 gene, belongs to the double-pore domain K{sup +} channel family and in normal conditions is expressed predominantly in the central nervous system. In our previous patch-clamp study on Jurkat T lymphoblasts we have characterized highly selective K{sup +} channel with pharmacological profile identical to TRESK. In the present work, the presence of KCNK18 mRNA was confirmed in T lymphoblastic cell lines (Jurkat, JCaM, H9) but not in resting peripheral blood lymphocytes of healthy donors. Positive immunostaining for TRESK was demonstrated in lymphoblastic cell lines, in germinal centers of non-tumoral lymph nodes, and in clinical samples of T acute lymphoblastic leukemias/lymphomas. Besides detection in the plasma membrane, intracellular TRESK localization was also revealed. Possible involvement of TRESK channel in lymphocyte proliferation and tumorigenesis is discussed.

  14. Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

    PubMed Central

    Zhou, Qing; Garin, Intza; Castaño, Luis; Argente, Jesús; Muñoz-Calvo, Ma. Teresa; Perez de Nanclares, Guiomar; Shyng, Show-Ling

    2010-01-01

    Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating. PMID:20810569

  15. Potassium channels as multi-ion single-file pores

    PubMed Central

    1978-01-01

    A literature review reveals many lines of evidence that both delayed rectifier and inward rectifier potassium channels are multi-ion pores. These include unidirectional flux ratios given by the 2--2.5 power of the electrochemical activity ratio, very steeply voltage-dependent block with monovalent blocking ions, relief of block by permeant ions added to the side opposite from the blocking ion, rectification depending on E--EK, and a minimum in the reversal potential or conductance as external K+ ions are replaced by an equivalent concentration of T1+ ions. We consider a channel with a linear sequence of energy barriers and binding sites. The channel can be occupied by more than one ion at a time, and ions hop in single file into vacant sites with rate constants that depend on barrier heights, membrane potential, and interionic repulsion. Such multi-ion models reproduce qualitatively the special flux properties of potassium channels when the barriers for hopping out of the pore are larger than for hopping between sites within the pore and when there is repulsion between ions. These conditions also produce multiple maxima in the conductance-ion activity relationship. In agreement with Armstrong's hypothesis (1969. J. Gen. Physiol. 54:553--575), inward rectification may be understood in terms of block by an internal blocking cation. Potassium channels must have at least three sites and often contain at least two ions at a time. PMID:722275

  16. Mechanisms of Activation of Voltage-Gated Potassium Channels

    PubMed Central

    Grizel, A. V.; Glukhov, G. S.; Sokolova, O. S.

    2014-01-01

    Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date. PMID:25558391

  17. Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels

    PubMed Central

    Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

    2015-01-01

    All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

  18. Loss of endothelial KATP channel-dependent, NO-mediated dilation of endocardial resistance coronary arteries in pigs with left ventricular hypertrophy.

    PubMed

    Gendron, Marie-Eve; Thorin, Eric; Perrault, Louis P

    2004-09-01

    The influence of left ventricular hypertrophy (LVH) on the endothelial function of resistance endocardial arteries is not well established. The aim of this study was to characterise the mechanisms responsible for UK-14,304 (alpha(2)-adrenoreceptor agonist)-induced endothelium-dependent dilation in pig endocardial arteries isolated from hearts with or without LVH. LVH was induced by aortic banding 2 months before determining endothelial function. Following euthanasia, hearts were harvested and endocardial resistance arteries were isolated and pressurised to 100 mmHg in no-flow conditions. Vessels were preconstricted with acetylcholine (ACh) or high external K(+) (40 mmol l(-1) KCl). Results are expressed as mean+/-s.e.m. UK-14,304 induced a maximal dilation representing 79+/-6% (n=8) of the maximal diameter. NO synthase (l-NNA, 10 micromol l(-1), n=7) or guanylate cyclase (ODQ, 10 micromol l(-1), n=4) inhibition reduced (P<0.05) UK-14,304-dependent dilation to 35+/-6 and 18+/-7%, respectively. Apamin and charybdotoxin reduced (P<0.05) to 39+/-8% (n=4) the dilation induced by UK-14,304. In depolarised conditions, however, this dilation was prevented (P<0.05). UK-14,304-induced dilation was reduced (P<0.05) by glibenclamide (Glib, 1 micromol l(-1)), a K(ATP) channel blocker, either alone (35+/-10%, n=5) or in combination with l-NNA (34+/-9%, n=4). In LVH, UK-14,304-induced maximal dilation was markedly reduced (25+/-4%, P<0.05) compared to control; it was insensitive to l-NNA (21+/-5%) but prevented either by the combination of l-NNA, apamin and charybdotoxin, or by 40 mmol l(-1) KCl. Activation of endothelial alpha(2)-adrenoreceptor induces an endothelium-dependent dilation of pig endocardial resistance arteries. This dilation is in part dependent on NO, the release of which appears to be dependent on the activation of endothelial K(ATP) channels. This mechanism is blunted in LVH, leading to a profound reduction in UK-14,304-dependent dilation. PMID:15326036

  19. Hydrogen Sulfide as Endothelial Derived Hyperpolarizing Factor Sulfhydrates Potassium Channels

    PubMed Central

    Mustafa, Asif K.; Sikka, Gautam; Gazi, Sadia K.; Steppan, Jochen; Jung, Sung M.; Bhunia, Anil K.; Barodka, Viachaslau M.; Gazi, Farah K.; Barrow, Roxanne K.; Wang, Rui; Amzel, L. Mario; Berkowitz, Dan E.; Snyder, Solomon H.

    2011-01-01

    Rationale Nitric oxide, the classic endothelial derived relaxing factor (EDRF), acts via cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelial derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H2S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine γ-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine. Objective The purpose of this study is to determine if H2S is a major physiologic EDHF. Methods and Results We now show that H2S is a major EDHF, as in blood vessels of CSE deleted mice hyperpolarization is virtually abolished. H2S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H2S-elicited hyperpolarization. The endothelial intermediate conductance (IKCa) and small conductance (SKCa) potassium channels mediate in part the effects of H2S, as selective IKCa and SKCa channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide insensitive H2S induced vasorelaxation. Conclusions H2S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. As EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H2S biosynthesis offer therapeutic potential. PMID:21980127

  20. Targeting a mitochondrial potassium channel to fight cancer.

    PubMed

    Leanza, Luigi; Venturini, Elisa; Kadow, Stephanie; Carpinteiro, Alexander; Gulbins, Erich; Becker, Katrin Anne

    2015-07-01

    Although chemotherapy is able to cure many patients with malignancies, it still also often fails. Therefore, novel approaches and targets for chemotherapeutic treatment of malignancies are urgently required. Recent studies demonstrated the expression of several potassium channels in the inner mitochondrial membrane. Among them the voltage gated potassium channel Kv1.3 and the big-potassium (BK) channel were shown to directly function in cell death by serving as target for pro-apoptotic Bax and Bak proteins. Here, we discuss the role of mitochondrial potassium channel Kv1.3 (mitoKv1.3) in cell death and its potential function in treatment of solid tumors, leukemia and lymphoma. Bax and Bak inhibit mitoKv1.3 by directly binding into the pore of the channel, by a toxin-like mechanism. Inhibition of mitoKv1.3 results in an initial hyperpolarization of the inner mitochondrial membrane that triggers the production of reactive oxygen species (ROS). ROS in turn induce a release of cytochrome c from the cristae of the inner mitochondrial membrane and an activation of the permeability transition pore, resulting in opening of the intrinsic apoptotic cell death. Since mitoKv1.3 functions downstream of pro-apoptotic Bax and Bak, compounds that directly inhibit mitoKv1.3 may serve as a new class of drugs for treatment of tumors, even with an altered expression of either pro- or anti-apoptotic Bcl-2 protein family members. This was successfully proven by the in vivo treatment of mouse melanoma and ex vivo human chronic leukemia B cells with inhibitors of mitoKv1.3.

  1. Optogenetics. Engineering of a light-gated potassium channel.

    PubMed

    Cosentino, Cristian; Alberio, Laura; Gazzarrini, Sabrina; Aquila, Marco; Romano, Edoardo; Cermenati, Solei; Zuccolini, Paolo; Petersen, Jan; Beltrame, Monica; Van Etten, James L; Christie, John M; Thiel, Gerhard; Moroni, Anna

    2015-05-01

    The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K(+)) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.

  2. Plasmodium falciparum: growth response to potassium channel blocking compounds.

    PubMed

    Waller, Karena L; Kim, Kami; McDonald, Thomas V

    2008-11-01

    Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K(+) channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca(2+)-activated K(+) channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K(+) channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca(2+)-activated K(+) channel blocking compounds. PMID:18703053

  3. Low glucose-induced ghrelin secretion is mediated by an ATP-sensitive potassium channel.

    PubMed

    Oya, Manami; Kitaguchi, Tetsuya; Harada, Kazuki; Numano, Rika; Sato, Takahiro; Kojima, Masayasu; Tsuboi, Takashi

    2015-07-01

    Ghrelin is synthesized in X/A-like cells of the gastric mucosa, which plays an important role in the regulation of energy homeostasis. Although ghrelin secretion is known to be induced by neurotransmitters or hormones or by nutrient sensing in the ghrelin-secreting cells themselves, the mechanism of ghrelin secretion is not clearly understood. In the present study, we found that changing the extracellular glucose concentration from elevated (25  mM) to optimal (10 mM) caused an increase in the intracellular Ca2+ concentration ([Ca2+]i) in ghrelin-secreting mouse ghrelinoma 3-1 (MGN3-1) cells (n=32, P<0.01), whereas changing the glucose concentration from elevated to lowered (5 or 1 mM) had little effect on [Ca2+]i increase. Overexpression of a closed form of an ATP-sensitive K+ (KATP) channel mutant suppressed the 10  mM glucose-induced [Ca2+]i increase (n=8, P<0.01) and exocytotic events (n=6, P<0.01). We also found that a low concentration of a KATP channel opener, diazoxide, with 25  mM glucose induced [Ca2+]i increase (n=23, P<0.01) and ghrelin secretion (n≥3, P<0.05). In contrast, the application of a low concentration of a KATP channel blocker, tolbutamide, significantly induced [Ca2+]i increase (n=15, P<0.01) and ghrelin secretion (n≥3, P<0.05) under 5 mM glucose. Furthermore, the application of voltage-dependent Ca2+ channel inhibitors suppressed the 10 mM glucose-induced [Ca2+]i increase (n≥26, P<0.01) and ghrelin secretion (n≥5, P<0.05). These findings suggest that KATP and voltage-dependent Ca2+ channels are involved in glucose-dependent ghrelin secretion in MGN3-1 cells.

  4. Diazoxide Attenuates Postresuscitation Brain Injury in a Rat Model of Asphyxial Cardiac Arrest by Opening Mitochondrial ATP-Sensitive Potassium Channels

    PubMed Central

    Wang, Peng; Li, Yi; Wu, Manhui; Lin, Jiali

    2016-01-01

    Objective. We investigated whether and how diazoxide can attenuate brain injury after cardiopulmonary resuscitation (CPR) by selective opening of mitochondrial ATP-sensitive potassium (mitoKATP) channels. Methods. Adult male Sprague-Dawley rats with induced cerebral ischemia (n = 10 per group) received an intraperitoneal injection of 0.1% dimethyl sulfoxide (1 mL; vehicle group), diazoxide (10 mg/kg; DZ group), or diazoxide (10 mg/kg) plus 5-hydroxydecanoate (5 mg/kg; DZ + 5-HD group) 30 min after CPR. The control group (sham group, n = 5) underwent sham operation, without cardiac arrest. Mitochondrial respiratory control rate (RCR) was determined. Brain cell apoptosis was assessed using TUNEL staining. Expression of Bcl-2, Bax, and protein kinase C epsilon (PKCε) in the cerebral cortex was determined by Western blotting and immunohistochemistry. Results. The neurological deficit scores (NDS) in the vehicle group decreased significantly at 24 h and 48 h after CPR. Diazoxide significantly improved NDS and mitochondrial RCR after CPR at both time points; 5-HD cotreatment abolished these effects. Diazoxide decreased TUNEL-positive cells following CPR, upregulated Bcl-2 and PKCε, downregulated Bax, and increased the Bcl-2/Bax ratio; 5-HD cotreatment reversed these effects. Conclusions. Diazoxide attenuates postresuscitation brain injury, protects mitochondrial function, inhibits brain cell apoptosis, and activates the PKC pathway by opening mitoKATP channels.

  5. Diazoxide Attenuates Postresuscitation Brain Injury in a Rat Model of Asphyxial Cardiac Arrest by Opening Mitochondrial ATP-Sensitive Potassium Channels.

    PubMed

    Wu, Haidong; Wang, Peng; Li, Yi; Wu, Manhui; Lin, Jiali; Huang, Zitong

    2016-01-01

    Objective. We investigated whether and how diazoxide can attenuate brain injury after cardiopulmonary resuscitation (CPR) by selective opening of mitochondrial ATP-sensitive potassium (mitoKATP) channels. Methods. Adult male Sprague-Dawley rats with induced cerebral ischemia (n = 10 per group) received an intraperitoneal injection of 0.1% dimethyl sulfoxide (1 mL; vehicle group), diazoxide (10 mg/kg; DZ group), or diazoxide (10 mg/kg) plus 5-hydroxydecanoate (5 mg/kg; DZ + 5-HD group) 30 min after CPR. The control group (sham group, n = 5) underwent sham operation, without cardiac arrest. Mitochondrial respiratory control rate (RCR) was determined. Brain cell apoptosis was assessed using TUNEL staining. Expression of Bcl-2, Bax, and protein kinase C epsilon (PKCε) in the cerebral cortex was determined by Western blotting and immunohistochemistry. Results. The neurological deficit scores (NDS) in the vehicle group decreased significantly at 24 h and 48 h after CPR. Diazoxide significantly improved NDS and mitochondrial RCR after CPR at both time points; 5-HD cotreatment abolished these effects. Diazoxide decreased TUNEL-positive cells following CPR, upregulated Bcl-2 and PKCε, downregulated Bax, and increased the Bcl-2/Bax ratio; 5-HD cotreatment reversed these effects. Conclusions. Diazoxide attenuates postresuscitation brain injury, protects mitochondrial function, inhibits brain cell apoptosis, and activates the PKC pathway by opening mitoKATP channels. PMID:27648441

  6. Diazoxide Attenuates Postresuscitation Brain Injury in a Rat Model of Asphyxial Cardiac Arrest by Opening Mitochondrial ATP-Sensitive Potassium Channels

    PubMed Central

    Wang, Peng; Li, Yi; Wu, Manhui; Lin, Jiali

    2016-01-01

    Objective. We investigated whether and how diazoxide can attenuate brain injury after cardiopulmonary resuscitation (CPR) by selective opening of mitochondrial ATP-sensitive potassium (mitoKATP) channels. Methods. Adult male Sprague-Dawley rats with induced cerebral ischemia (n = 10 per group) received an intraperitoneal injection of 0.1% dimethyl sulfoxide (1 mL; vehicle group), diazoxide (10 mg/kg; DZ group), or diazoxide (10 mg/kg) plus 5-hydroxydecanoate (5 mg/kg; DZ + 5-HD group) 30 min after CPR. The control group (sham group, n = 5) underwent sham operation, without cardiac arrest. Mitochondrial respiratory control rate (RCR) was determined. Brain cell apoptosis was assessed using TUNEL staining. Expression of Bcl-2, Bax, and protein kinase C epsilon (PKCε) in the cerebral cortex was determined by Western blotting and immunohistochemistry. Results. The neurological deficit scores (NDS) in the vehicle group decreased significantly at 24 h and 48 h after CPR. Diazoxide significantly improved NDS and mitochondrial RCR after CPR at both time points; 5-HD cotreatment abolished these effects. Diazoxide decreased TUNEL-positive cells following CPR, upregulated Bcl-2 and PKCε, downregulated Bax, and increased the Bcl-2/Bax ratio; 5-HD cotreatment reversed these effects. Conclusions. Diazoxide attenuates postresuscitation brain injury, protects mitochondrial function, inhibits brain cell apoptosis, and activates the PKC pathway by opening mitoKATP channels. PMID:27648441

  7. Pharmacodynamics of potassium channel openers in cultured neuronal networks.

    PubMed

    Wu, Calvin; V Gopal, Kamakshi; Lukas, Thomas J; Gross, Guenter W; Moore, Ernest J

    2014-06-01

    A novel class of drugs - potassium (K(+)) channel openers or activators - has recently been shown to cause anticonvulsive and neuroprotective effects by activating hyperpolarizing K(+) currents, and therefore, may show efficacy for treating tinnitus. This study presents measurements of the modulatory effects of four K(+) channel openers on the spontaneous activity and action potential waveforms of neuronal networks. The networks were derived from mouse embryonic auditory cortices and grown on microelectrode arrays. Pentylenetetrazol was used to create hyperactivity states in the neuronal networks as a first approximation for mimicking tinnitus or tinnitus-like activity. We then compared the pharmacodynamics of the four channel activators, retigabine and flupirtine (voltage-gated K(+) channel KV7 activators), NS1619 and isopimaric acid ("big potassium" BK channel activators). The EC50 of retigabine, flupirtine, NS1619, and isopimaric acid were 8.0, 4.0, 5.8, and 7.8µM, respectively. The reduction of hyperactivity compared to the reference activity was significant. The present results highlight the notion of re-purposing the K(+) channel activators for reducing hyperactivity of spontaneously active auditory networks, serving as a platform for these drugs to show efficacy toward target identification, prevention, as well as treatment of tinnitus.

  8. Two-pore Domain Potassium Channels in Astrocytes

    PubMed Central

    Ryoo, Kanghyun

    2016-01-01

    Two-pore domain potassium (K2P) channels have a distinct structure and channel properties, and are involved in a background K+ current. The 15 members of the K2P channels are identified and classified into six subfamilies on the basis of their sequence similarities. The activity of the channels is dynamically regulated by various physical, chemical, and biological effectors. The channels are expressed in a wide variety of tissues in mammals in an isoform specific manner, and play various roles in many physiological and pathophysiological conditions. To function as channels, the K2P channels form dimers, and some isoforms form heterodimers that provide diversity in channel properties. In the brain, TWIK1, TREK1, TREK2, TRAAK, TASK1, and TASK3 are predominantly expressed in various regions, including the cerebral cortex, dentate gyrus, CA1-CA3, and granular layer of the cerebellum. TWIK1, TREK1, and TASK1 are highly expressed in astrocytes, where they play specific cellular roles. Astrocytes keep leak K+ conductance, called the passive conductance, which mainly involves TWIK1-TREK1 heterodimeric channel. TWIK1 and TREK1 also mediate glutamate release from astrocytes in an exocytosis-independent manner. The expression of TREK1 and TREK2 in astrocytes increases under ischemic conditions, that enhance neuroprotection from ischemia. Accumulated evidence has indicated that astrocytes, together with neurons, are involved in brain function, with the K2P channels playing critical role in these astrocytes. PMID:27790056

  9. Proinflammatory Cytokines and Potassium Channels in the Kidney

    PubMed Central

    Nakamura, Kazuyoshi; Hayashi, Hikaru; Kubokawa, Manabu

    2015-01-01

    Proinflammatory cytokines affect several cell functions via receptor-mediated processes. In the kidney, functions of transporters and ion channels along the nephron are also affected by some cytokines. Among these, alteration of activity of potassium ion (K+) channels induces changes in transepithelial transport of solutes and water in the kidney, since K+ channels in tubule cells are indispensable for formation of membrane potential which serves as a driving force for the transepithelial transport. Altered K+ channel activity may be involved in renal cell dysfunction during inflammation. Although little information was available regarding the effects of proinflammatory cytokines on renal K+ channels, reports have emerged during the last decade. In human proximal tubule cells, interferon-γ showed a time-dependent biphasic effect on a 40 pS K+ channel, that is, delayed suppression and acute stimulation, and interleukin-1β acutely suppressed the channel activity. Transforming growth factor-β1 activated KCa3.1 K+ channel in immortalized human proximal tubule cells, which would be involved in the pathogenesis of renal fibrosis. This review discusses the effects of proinflammatory cytokines on renal K+ channels and the causal relationship between the cytokine-induced changes in K+ channel activity and renal dysfunction. PMID:26508816

  10. Gliadin fragments and a specific gliadin 33-mer peptide close KATP channels and induce insulin secretion in INS-1E cells and rat islets of langerhans.

    PubMed

    Dall, Morten; Calloe, Kirstine; Haupt-Jorgensen, Martin; Larsen, Jesper; Schmitt, Nicole; Josefsen, Knud; Buschard, Karsten

    2013-01-01

    In non-obese diabetic (NOD) mice, diabetes incidence is reduced by a gluten-free diet. Gluten peptides, such as the compound gliadin, can cross the intestinal barrier and may directly affect pancreatic beta cells. We investigated the effects of enzymatically-digested gliadin in NOD mice, INS-1E cells and rat islets. Six injections of gliadin digest in 6-week-old NOD mice did not affect diabetes development, but increased weight gain (20% increase by day 100). In INS-1E cells, incubation with gliadin digest induced a dose-dependent increase in insulin secretion, up to 2.5-fold after 24 hours. A similar effect was observed in isolated rat islets (1.6-fold increase). In INS-1E cells, diazoxide reduced the stimulatory effect of gliadin digest. Additionally, gliadin digest was shown to decrease current through KATP-channels. A specific gliadin 33-mer had a similar effect, both on current and insulin secretion. Finally, INS-1E incubation with gliadin digest potentiated palmitate-induced insulin secretion by 13% compared to controls. Our data suggest that gliadin fragments may contribute to the beta-cell hyperactivity observed prior to the development of type 1 diabetes. PMID:23785500

  11. Voltage-gated Potassium Channels as Therapeutic Drug Targets

    PubMed Central

    Wulff, Heike; Castle, Neil A.; Pardo, Luis A.

    2009-01-01

    The human genome contains 40 voltage-gated potassium channels (KV) which are involved in diverse physiological processes ranging from repolarization of neuronal or cardiac action potentials, over regulating calcium signaling and cell volume, to driving cellular proliferation and migration. KV channels offer tremendous opportunities for the development of new drugs for cancer, autoimmune diseases and metabolic, neurological and cardiovascular disorders. This review first discusses pharmacological strategies for targeting KV channels with venom peptides, antibodies and small molecules and then highlights recent progress in the preclinical and clinical development of drugs targeting KV1.x, KV7.x (KCNQ), KV10.1 (EAG1) and KV11.1 (hERG) channels. PMID:19949402

  12. Potassium Versus Sodium Selectivity in Monovalent Ion Channel Selectivity Filters.

    PubMed

    Lim, Carmay; Dudev, Todor

    2016-01-01

    Transport of Na(+) and K(+) ions across the cell membrane is carried out by specialized pore-forming ion channel proteins, which exert tight control on electrical signals in cells by regulating the inward/outward flow of the respective cation. As Na(+) and K(+) ions are both present in the body fluids, their respective ion channels should discriminate with high fidelity between the two competing metal ions, conducting the native cation while rejecting its monovalent contender (and other ions present in the cellular/extracellular milieu). Indeed, monovalent ion channels are characterized by remarkable metal selectivity. This striking ion selectivity of monovalent ion channels is astonishing in view of the close similarity between Na(+) and K(+): both are spherical alkali cations with the same charge, analogous chemical and physical properties, and similar ionic radii. The monovalent ion channel selectivity filters (SFs), which dictate the selectivity of the channel, differ in oligomericity, composition, overall charge, pore size, and solvent accessibility. This diversity of SFs raises the following intriguing questions: (1) What factors govern the metal competition in these SFs? (2) Which of these factors are exploited in achieving K(+) or Na(+) selectivity in the different types of monovalent channel SFs? These questions are addressed herein by summarizing results from recent studies. The results show that over billions of years of evolution, the SFs of potassium and sodium ion channels have adapted to the specific physicochemical properties of the cognate ion, using various strategies to enable them to efficiently select the native ion among its contenders.

  13. Chloride and potassium channels in cystic fibrosis airway epithelia

    NASA Astrophysics Data System (ADS)

    Welsh, Michael J.; Liedtke, Carole M.

    1986-07-01

    Cystic fibrosis, the most common lethal genetic disease in Caucasians, is characterized by a decreased permeability in sweat gland duct and airway epithelia. In sweat duct epithelium, a decreased Cl- permeability accounts for the abnormally increased salt content of sweat1. In airway epithelia a decreased Cl- permeability, and possibly increased sodium absorption, may account for the abnormal respiratory tract fluid2,3. The Cl- impermeability has been localized to the apical membrane of cystic fibrosis airway epithelial cells4. The finding that hormonally regulated Cl- channels make the apical membrane Cl- permeable in normal airway epithelial cells5 suggested abnormal Cl- channel function in cystic fibrosis. Here we report that excised, cell-free patches of membrane from cystic fibrosis epithelial cells contain Cl- channels that have the same conductive properties as Cl- channels from normal cells. However, Cl- channels from cystic fibrosis cells did not open when they were attached to the cell. These findings suggest defective regulation of Cl- channels in cystic fibrosis epithelia; to begin to address this issue, we performed two studies. First, we found that isoprenaline, which stimulates Cl- secretion, increases cellular levels of cyclic AMP in a similar manner in cystic fibrosis and non-cystic fibrosis epithelial cells. Second, we show that adrenergic agonists open calcium-activated potassium channels, indirectly suggesting that calcium-dependent stimulus-response coupling is intact in cystic fibrosis. These data suggest defective regulation of Cl- channels at a site distal to cAMP accumulation.

  14. Mechanism of Electromechanical Coupling in Voltage-Gated Potassium Channels

    PubMed Central

    Blunck, Rikard; Batulan, Zarah

    2012-01-01

    Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion – sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3–4e+ each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4–S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during

  15. Chaotic dynamics in cardiac aggregates induced by potassium channel block

    NASA Astrophysics Data System (ADS)

    Quail, Thomas; McVicar, Nevin; Aguilar, Martin; Kim, Min-Young; Hodge, Alex; Glass, Leon; Shrier, Alvin

    2012-09-01

    Chaotic rhythms in deterministic models can arise as a consequence of changes in model parameters. We carried out experimental studies in which we induced a variety of complex rhythms in aggregates of embryonic chick cardiac cells using E-4031 (1.0-2.5 μM), a drug that blocks the hERG potassium channel. Following the addition of the drug, the regular rhythm evolved to display a spectrum of complex dynamics: irregular rhythms, bursting oscillations, doublets, and accelerated rhythms. The interbeat intervals of the irregular rhythms can be described by one-dimensional return maps consistent with chaotic dynamics. A Hodgkin-Huxley-style cardiac ionic model captured the different types of complex dynamics following blockage of the hERG mediated potassium current.

  16. α-Synuclein forms non-selective cation channels and stimulates ATP-sensitive potassium channels in hippocampal neurons

    PubMed Central

    Mironov, Sergej L

    2015-01-01

    In Parkinson's disease and several other neurodegenerative diseases, the protein α-synuclein (αS) is produced within neurons and accumulates in the extracellular fluid. Several mechanisms of αS action are proposed, one of which is the formation of cation-permeable pores that may mediate toxicity. αS induces non-selective cation channels in lipid bilayers, but whether this occurs in living neurons and which properties the channels possess have not yet been examined. In this study the properties of αS channels in dissociated hippocampal neurons are documented. In cell-attached recordings the incorporation of αS into membranes was driven by applied negative potentials. These channels exhibited multiple levels of conductance (30, 70 and 120 pS at −100 mV) and inward rectification. The persistent activity of αS channels induced local changes in intracellular Na+ and Ca2+, depolarized neurons and augmented bursting activity. αS channels formed by adding αS to the intracellular membrane in inside-out patches exhibited outward rectification. αS channels were equally permeable to Na+, K+ and Ca2+. These channels were also observed in neurons transfected with wild-type or mutant A53T αS, and after extracellular application of wild-type or mutant A53T αS proteins. Opening of αS channels stimulated opening of ATP-sensitive K+ (KATP ) channels and did not interfere with the activity of delayed rectifier K+ channels. The properties of αS channels in neuronal membranes suggest stronger toxicity of extracellularly applied αS than intracellular αS. Enhancement of neuronal excitability and distortions in ion homeostasis may underlie the toxic effects of αS that can be dampened by KATP channels. PMID:25556793

  17. Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry.

    PubMed Central

    Krezel, A. M.; Kasibhatla, C.; Hidalgo, P.; MacKinnon, R.; Wagner, G.

    1995-01-01

    The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel beta-sheet and a single helix covering one face of the beta-sheet. The cysteine side chains connecting the beta-sheet and the helix form the core of the molecule. One edge of the beta-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, Arg24, Lys27, and Arg31 of the beta-sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions. PMID:8520473

  18. A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

    PubMed

    Kajma, Anna; Szewczyk, Adam

    2012-10-01

    Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

  19. Voltage-Dependent Gating of hERG Potassium Channels

    PubMed Central

    Cheng, Yen May; Claydon, Tom W.

    2012-01-01

    The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv) channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4–S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-à-go-go related gene, hERG), which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure–function relationships underlying activation and deactivation gating in Shaker and hERG channels, with a focus on the roles of the voltage-sensing domain and the S4–S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter-charge interactions. More recent data suggest that key amino acid differences in the hERG voltage-sensing unit and S4–S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor. PMID:22586397

  20. The Walter B. Cannon Physiology in Perspective Lecture, 2007. ATP-sensitive K+ channels and disease: from molecule to malady.

    PubMed

    Ashcroft, Frances M

    2007-10-01

    This essay is based on a lecture given to the American Physiological Society in honor of Walter B. Cannon, an advocate of homeostasis. It focuses on the role of the ATP-sensitive potassium K(+) (K(ATP)) channel in glucose homeostasis and, in particular, on its role in insulin secretion from pancreatic beta-cells. The beta-cell K(ATP) channel comprises pore-forming Kir6.2 and regulatory SUR1 subunits, and mutations in either type of subunit can result in too little or too much insulin release. Here, I review the latest information on the relationship between K(ATP) channel structure and function, and consider how mutations in the K(ATP) channel genes lead to neonatal diabetes or congenital hyperinsulinism.

  1. The Walter B. Cannon Physiology in Perspective Lecture, 2007. ATP-sensitive K+ channels and disease: from molecule to malady.

    PubMed

    Ashcroft, Frances M

    2007-10-01

    This essay is based on a lecture given to the American Physiological Society in honor of Walter B. Cannon, an advocate of homeostasis. It focuses on the role of the ATP-sensitive potassium K(+) (K(ATP)) channel in glucose homeostasis and, in particular, on its role in insulin secretion from pancreatic beta-cells. The beta-cell K(ATP) channel comprises pore-forming Kir6.2 and regulatory SUR1 subunits, and mutations in either type of subunit can result in too little or too much insulin release. Here, I review the latest information on the relationship between K(ATP) channel structure and function, and consider how mutations in the K(ATP) channel genes lead to neonatal diabetes or congenital hyperinsulinism. PMID:17652156

  2. Potassium channels mediate killing by human natural killer cells

    SciTech Connect

    Schlichter, L.; Sidell N.; Hagiwara, S.

    1986-01-01

    Human natural killer (NK) cells in peripheral blood spontaneously recognize and kill a wide variety of target cells. It has been suggested that ion channels are involved in the killing process because there is a Ca-dependent stage and because killing by presensitized cytotoxic T lymphocytes, which in many respects resembles NK killing, is associated with changes in K and Na transport in the target cell. Using the whole-cell variation of the patch-clamp technique, the authors found a voltage-dependent potassium (K/sup +/) current in NK cells. The K/sup +/ current was reduced in a dose-dependent manner by the K-channel blockers 4-aminopyridine and quinidine and by the traditional Ca-channel blockers verapamil and Cd/sup 2 +/. They tested the effects of ion-channel blockers on killing of two commonly used target cell lines: K562, which is derived from a human myeloid leukemia, and U937, which is derived from a human histiocytic leukemia. Killing of K562 target cells, determined in a standard /sup 51/Cr-release assay, was inhibited in a dose-dependent manner by verapamil, quinidine, Cd/sup 2 +/, and 4-aminopyridine at concentrations comparable to those that blocked the K/sup +/ current in NK cells. In K562 target cells only a voltage-dependent Na= current was found and it was blocked by concentrations of tetrodotoxin that had no effect on killing. Killing of U937 target cells was also inhibited by the two ion-channel blockers tested, quinidine and verapamil. In this cell line only a small K/sup +/ current was found that was similar to the one in NK cells. The findings show that there are K channels in NK cells and that these channels play a necessary role in the killing process.

  3. Open-state models of a potassium channel.

    PubMed Central

    Biggin, Philip C; Sansom, Mark S P

    2002-01-01

    The structure of the bacterial potassium channel, KcsA, corresponds to the channel in a closed state. Two lines of evidence suggest that the channel must widen its intracellular mouth when in an open state: 1) internal block by a series of tetraalkylammonium ions and 2) spin labeling experiments. Thus it is known that the protein moves in this region, but it is unclear by how much and the mechanisms that are involved. To address this issue we have applied a novel approach to generate plausible open-state models of KcsA. The approach can be thought of as placing a balloon inside the channel and gradually inflating it. Only the protein sees the balloon, and so water is free to move in and out of the channel. The balloon is a van der Waals sphere whose parameters change by a small amount at each time step, an approach similar to methods used in free energy perturbation calculations. We show that positioning of this balloon at various positions along the pore axis generates similar open-state models, thus indicating that there may be a preferred pathway to an open state. We also show that the resulting structures from this process are conformationally unstable and need to undergo a relaxation process for up to 4 ns. We show that the channel can relax into a new state that has a larger pore radius at the region of the intracellular mouth. The resulting models may be useful in exploring models of the channel in the context of ion permeation and blocking agents. PMID:12324408

  4. Potential use of potassium efflux-deficient yeast for studying trafficking signals and potassium channel functions.

    PubMed

    Bernstein, Joshua D; Okamoto, Yukari; Kim, Minjee; Shikano, Sojin

    2013-01-01

    The activity of potassium (K(+)) channels critically depends on their density on the cell surface membrane, which is regulated by dynamic protein-protein interactions that often involve distinct trafficking signals on the cargo proteins. In this paper we explored the possibility of utilizing the Saccharomyces cerevisiae strain B31 for identification of the signal motifs that regulate surface expression of membrane proteins and for studying structure-function relationships of K(+) channels. B31 cells lack the K(+) efflux system and were reported to show overloaded K(+)-mediated growth inhibition in high K(+) media upon heterologous expression of a mammalian inwardly rectifying K(+) channel (Kir2.1). We show that while the expression of wild-type Kir2.1 channel inhibits the growth of B31 cells in high K(+) media, the human disease-causing mutations of Kir2.1 that abolish K(+) conduction (V302M) or surface trafficking (Δ314/315) fully restores the growth. The expression of two-pore-domain K(+) channel KCNK3 or KCNK9 also inhibited the growth of B31 in high K(+) media while C-terminal mutations that reduce their 14-3-3 protein-dependent cell surface trafficking restored the growth of B31. Finally, the expression of Kir2.1 channels that were C-terminally fused with known sequence motifs including ER retention/retrieval signals and an endocytosis signal allowed the growth of B31 in high K(+) media. These results demonstrate the potential of B31 yeast strain as a unique biological tool to screen the random peptide libraries for novel sequence signals that down-regulate surface expression of membrane proteins, as well as to systematically identify the structural determinants for cell surface trafficking and/or ion conductance of K(+) channels.

  5. Trypsin-Sensitive, Rapid Inactivation of a Calcium-Activated Potassium Channel

    NASA Astrophysics Data System (ADS)

    Solaro, Christopher R.; Lingle, Christopher J.

    1992-09-01

    Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

  6. A heme-binding domain controls regulation of ATP-dependent potassium channels.

    PubMed

    Burton, Mark J; Kapetanaki, Sofia M; Chernova, Tatyana; Jamieson, Andrew G; Dorlet, Pierre; Santolini, Jérôme; Moody, Peter C E; Mitcheson, John S; Davies, Noel W; Schmid, Ralf; Raven, Emma L; Storey, Nina M

    2016-04-01

    Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

  7. Potassium Channels Mediate Killing by Human Natural Killer Cells

    NASA Astrophysics Data System (ADS)

    Schlichter, Lyanne; Sidell, Neil; Hagiwara, Susumu

    1986-01-01

    Human natural killer (NK) cells in peripheral blood spontaneously recognize and kill a wide variety of target cells. It has been suggested that ion channels are involved in the killing process because there is a Ca-dependent stage and because killing by presensitized cytotoxic T lymphocytes, which in many respects resembles NK killing, is associated with changes in K and Na transport in the target cell. However, no direct evidence exists for ion channels in NK cells or in their target cells. Using the whole-cell variation of the patch-clamp technique, we found a voltage-dependent potassium (K+) current in NK cells. The K+ current was reduced in a dose-dependent manner by the K-channel blockers 4-aminopyridine and quinidine and by the traditional Ca-channel blockers verapamil and Cd2+. We tested the effects of ion-channel blockers on killing of two commonly used target cell lines: K562, which is derived from a human myeloid leukemia, and U937, which is derived from a human histiocytic leukemia. Killing of K562 target cells, determined in a standard 51Cr-release assay, was inhibited in a dose-dependent manner by verapamil, quinidine, Cd2+, and 4-aminopyridine at concentrations comparable to those that blocked the K+ current in NK cells. In K562 target cells only a voltage-dependent Na+ current was found and it was blocked by concentrations of tetrodotoxin that had no effect on killing. Killing of U937 target cells was also inhibited by the two ion-channel blockers tested, quinidine and verapamil. In this cell line only a small K+ current was found that was similar to the one in NK cells. We could not find any evidence of a Ca2+ current in target cells or in NK cells; therefore, our results cannot explain the Ca dependence of killing. Our findings show that there are K channels in NK cells and that these channels play a necessary role in the killing process. In contrast, the endogenous channel type in the target cell is probably not a factor in determining target cell

  8. Slo1 is the principal potassium channel of human spermatozoa

    PubMed Central

    Mannowetz, Nadja; Naidoo, Natasha M; Choo, Seung-A Sara; Smith, James F; Lishko, Polina V

    2013-01-01

    Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K+ efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca2+] to produce hyperactivation and allowing sperm to fertilize an oocyte. DOI: http://dx.doi.org/10.7554/eLife.01009.001 PMID:24137539

  9. Sleep disturbances in voltage-gated potassium channel antibody syndrome.

    PubMed

    Barone, Daniel A; Krieger, Ana C

    2016-05-01

    Voltage-gated potassium channels (VGKCs) are a family of membrane proteins responsible for controlling cell membrane potential. The presence of antibodies (Ab) against neuronal VGKC complexes aids in the diagnosis of idiopathic and paraneoplastic autoimmune neurologic disorders. The diagnosis of VGKC Ab-associated encephalopathy (VCKC Ab syndrome) should be suspected in patients with subacute onset of disorientation, confusion, and memory loss in the presence of seizures or a movement disorder. VGKC Ab syndrome may present with sleep-related symptoms, and the purpose of this communication is to alert sleep and neurology clinicians of this still-under-recognized condition. In this case, we are presenting the VGKC Ab syndrome which improved after treatment with solumedrol. The prompt recognition and treatment of this condition may prevent the morbidity associated with cerebral atrophy and the mortality associated with intractable seizures and electrolyte disturbances.

  10. Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells.

    PubMed

    Pappone, P A; Lucero, M T

    1988-06-01

    We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.

  11. Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

    PubMed

    Dal-Secco, Daniela; Cunha, Thiago M; Freitas, Andressa; Alves-Filho, José Carlos; Souto, Fabrício O; Fukada, Sandra Y; Grespan, Renata; Alencar, Nylane M N; Neto, Alberto F; Rossi, Marcos A; Ferreira, Sérgio H; Hothersall, John S; Cunha, Fernando Q

    2008-09-15

    In this study, we have addressed the role of H(2)S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H(2)S synthesis inhibitors, dl-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H(2)S donors, NaHS or Lawesson's reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB(4). Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K(ATP)(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K(ATP)(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H(2)S augments neutrophil adhesion and locomotion, by a mechanism dependent on K(ATP)(+) channels. PMID:18768887

  12. Free RCK arrangement in Kch, a putative escherichia coli potassium channel, as suggested by electron crystallography.

    PubMed

    Kuang, Qie; Purhonen, Pasi; Jegerschöld, Caroline; Koeck, Philip J B; Hebert, Hans

    2015-01-01

    The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.

  13. Molecular Basis of Cardiac Delayed Rectifier Potassium Channel Function and Pharmacology.

    PubMed

    Wu, Wei; Sanguinetti, Michael C

    2016-06-01

    Human cardiomyocytes express 3 distinct types of delayed rectifier potassium channels. Human ether-a-go-go-related gene (hERG) channels conduct the rapidly activating current IKr; KCNQ1/KCNE1 channels conduct the slowly activating current IKs; and Kv1.5 channels conduct an ultrarapid activating current IKur. Here the authors provide a general overview of the mechanistic and structural basis of ion selectivity, gating, and pharmacology of the 3 types of cardiac delayed rectifier potassium ion channels. Most blockers bind to S6 residues that line the central cavity of the channel, whereas activators interact with the channel at 4 symmetric binding sites outside the cavity. PMID:27261821

  14. Hydrogen sulfide prevents ethanol-induced gastric damage in mice: role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons.

    PubMed

    Medeiros, Jand Venes R; Bezerra, Víctor H; Gomes, Antoniella S; Barbosa, André Luiz R; Lima-Júnior, Roberto César P; Soares, Pedro Marcos G; Brito, Gerly Anne C; Ribeiro, Ronaldo A; Cunha, Fernando Q; Souza, Marcellus H L P

    2009-09-01

    The aim of this study was to evaluate the protective effect of hydrogen sulfide (H(2)S) on ethanol-induced gastric lesions in mice and the influence of ATP-sensitive potassium (K(ATP)) channels, capsaicin-sensitive sensory afferent neurons, and transient receptor potential vanilloid (TRPV) 1 receptors on such an effect. Saline and L-cysteine alone or with propargylglycine, sodium hydrogen sulfide (NaHS), or Lawesson's reagent were administrated for testing purposes. For other experiments, mice were pretreated with glibenclamide, neurotoxic doses of capsaicin, or capsazepine. Afterward, mice received L-cysteine, NaHS, or Lawesson's reagent. After 30 min, 50% ethanol was administrated by gavage. After 1 h, mice were sacrificed, and gastric damage was evaluated by macroscopic and microscopic analyses. L-cysteine, NaHS, and Lawesson's reagent treatment prevented ethanol-induced macroscopic and microscopic gastric damage in a dose-dependent manner. Administration of propargylglycine, an inhibitor of endogenous H(2)S synthesis, reversed gastric protection induced by L-cysteine. Glibenclamide reversed L-cysteine, NaHS, or Lawesson's reagent gastroprotective effects against ethanol-induced macroscopic damage in a dose-dependent manner. Chemical ablation of sensory afferent neurons by capsaicin reversed gastroprotective effects of L-cysteine or H(2)S donors (NaHS or Lawesson's reagent) in ethanol-induced macroscopic gastric damage. Likewise, in the presence of the TRPV1 antagonist capsazepine, the gastroprotective effects of L-cysteine, NaHS, or Lawesson's reagent were also abolished. Our results suggest that H(2)S prevents ethanol-induced gastric damage. Although there are many mechanisms through which this effect can occur, our data support the hypothesis that the activation of K(ATP) channels and afferent neurons/TRPV1 receptors is of primary importance. PMID:19491326

  15. Cardioprotective Benefits of Adenosine Triphosphate: Sensitive Potassium Channel Opener Diazoxide Are Lost with Administration after the Onset of Stress in Mouse and Human Myocytes

    PubMed Central

    Janjua, M Burhan; Makepeace, Carol M; Anastacio, Melissa M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

    2014-01-01

    Background Adenosine triphosphate - sensitive (KATP) potassium channel opener diazoxide (DZX) maintains myocyte volume and contractility during stress via an unknown mechanism when administered at the onset of stress. This study was performed to investigate the cardioprotective potential of DZX when added after the onset of the stresses of hyperkalemic cardioplegia, metabolic inhibition, and hypo osmotic stress. Study Design Isolated mouse ventricular and human atrial myocytes were exposed to control Tyrode’s solution (TYR) for 10–20 min, test solution for 30 min (hypothermic hyperkalemic cardioplegia (CPG), CPG + 100uM diazoxide (CPG+DZX), metabolic inhibition (MI), MI+DZX, mild hypo osmotic stress (0.9T), or 0.9T + DZX) with DZX added after 10 or 20 min stress, followed by 20 min re-exposure to TYR (+/− DZX). Myocyte volume (human + mouse) and contractility (mouse) were compared. Results Mouse and human myocytes demonstrated significant swelling during exposure to CPG, MI, and hypo osmotic stress that was not prevented by DZX when administered either at 10 or 20 min after the onset of stress. Contractility following the stress of CPG in mouse myocytes significantly declined when DZX was administered 20 min after the onset of stress (p<0.05 vs. TYR). Contractility following hypo osmotic stress in mouse myocytes was not altered by the addition of DZX. Conclusions To maintain myocyte volume homeostasis and contractility during stress (hyperkalemic cardioplegia, metabolic inhibition, and hypo osmotic stress), KATP channel opener diazoxide requires administration at the onset of stress in this isolated myocyte model. These data have potential implications for any future clinical application of diazoxide. PMID:25158912

  16. Effect of mitochondrial potassium channel on the renal protection mediated by sodium thiosulfate against ethylene glycol induced nephrolithiasis in rat model

    PubMed Central

    Baldev, N.; Sriram, R.; Prabu, P.C.; Gino, A. Kurian

    2015-01-01

    ABSTRACT Purpose: Sodium thiosulfate (STS) is clinically reported to be a promising drug in preventing nephrolithiasis. However, its mechanism of action remains unclear. In the present study, we investigated the role of mitochondrial KATP channel in the renal protection mediated by STS. Materials and Methods: Nephrolithiasis was induced in Wistar rats by administrating 0.4% ethylene glycol (EG) along with 1% ammonium chloride for one week in drinking water followed by only 0.75% EG for two weeks. Treatment groups received STS, mitochondrial KATP channel opener and closer exclusively or in combination with STS for two weeks. Results: Animals treated with STS showed normal renal tissue architecture, supported by near normal serum creatinine, urea and ALP activity. Diazoxide (mitochondria KATP channel opening) treatment to the animal also showed normal renal tissue histology and improved serum chemistry. However, an opposite result was shown by glibenclamide (mitochondria KATP channel closer) treated rats. STS administered along with diazoxide negated the renal protection rendered by diazoxide alone, while it imparted protection to the glibenclamide treated rats, formulating a mitochondria modulated STS action. Conclusion: The present study confirmed that STS render renal protection not only through chelation and antioxidant effect but also by modulating the mitochondrial KATP channel for preventing urolithiasis. PMID:26742969

  17. Role of potassium ion channels in detrusor smooth muscle function and dysfunction

    PubMed Central

    Petkov, Georgi V.

    2013-01-01

    Contraction and relaxation of the detrusor smooth muscle (DSM), which makes up the wall of the urinary bladder, facilitates the storage and voiding of urine. Several families of K+ channels, including voltage-gated K+ (KV) channels, Ca2+-activated K+ (KCa) channels, inward-rectifying ATP-sensitive K+ (Kir, KATP) channels, and two-pore-domain K+ (K2P) channels, are expressed and functional in DSM. They control DSM excitability and contractility by maintaining the resting membrane potential and shaping the action potentials that determine the phasic nature of contractility in this tissue. Defects in DSM K+ channel proteins or in the molecules involved in their regulatory pathways may underlie certain forms of bladder dysfunction, such as overactive bladder. K+ channels represent an opportunity for novel pharmacological manipulation and therapeutic intervention in human DSM. Modulation of DSM K+ channels directly or indirectly by targeting their regulatory mechanisms has the potential to control urinary bladder function. This Review summarizes our current state of knowledge of the functional role of K+ channels in DSM in health and disease, with special emphasis on current advancements in the field. PMID:22158596

  18. Potassium Channels and Uterine Vascular Adaptation to Pregnancy and Chronic Hypoxia

    PubMed Central

    Zhu, Ronghui; Xiao, DaLiao; Zhang, Lubo

    2014-01-01

    During a normal course of pregnancy, uterine vascular tone is significantly decreased resulting in a striking increase in uterine blood flow, which is essential for fetal development and fetal growth. Chronic hypoxia during gestation may adversely affect the normal adaptation of uterine vascular tone and increase the risk of preeclampsia and fetal intrauterine growth restriction. In this review, we present evidence that the regulation of K+ channels is an important mechanism in the adaptation of uterine vascular tone to pregnancy and hypoxia. There are four types of K+ channels identified in arterial smooth muscle cells: 1) voltage-dependent K+ (Kv) channels, 2) Ca2+-activated K+ (KCa) channels, 3) inward rectifier K+ (KIR) channels, and 4) ATP-sensitive K+ (KATP) channels. Pregnancy differentially augments the expression and activity of K+ channels via downregulation of protein kinase C signaling in uterine and other vascular beds, leading to decreased uterine vascular tone and increased uterine blood flow. Sex steroid hormones play an important role in the pregnancy-mediated alteration of K+ channels in the uterine vasculature. In addition, chronic hypoxia alters uterine vascular K+ channels expression and activities via modulation of steroid hormones/receptors-mediated signaling, resulting in increased uterine vascular tone during pregnancy. PMID:24063385

  19. Phenylephrine preconditioning in embryonic heart H9c2 cells is mediated by up-regulation of SUR2B/Kir6.2: A first evidence for functional role of SUR2B in sarcolemmal KATP channels and cardioprotection.

    PubMed

    Jovanović, Sofija; Ballantyne, Thomas; Du, Qingyou; Blagojević, Miloš; Jovanović, Aleksandar

    2016-01-01

    ATP-sensitive K(+) (KATP) channels were originally described in cardiomyocytes, where physiological levels of intracellular ATP keep them in a closed state. Structurally, these channels are composed of pore-forming inward rectifier, Kir6.1 or Kir6.2, and a regulatory, ATP-binding subunit, SUR1, SUR2A or SUR2B. SUR1 and Kir6.2 form pancreatic type of KATP channels, SUR2A and Kir6.2 form cardiac type of KATP channels, SUR2B and Kir6.1 form vascular smooth muscle type of KATP channels. The presence of SUR2B has been described in cardiomyocytes, but its functional significance and role has remained unknown. Pretreatment with phenylephrine (100nM) for 24h increased mRNA levels of SUR2B and Kir6.2, without affecting those levels of SUR1, SUR2A and Kir6.1 in embryonic heart H9c2 cells. Such increase was associated with increased K(+) current through KATP channels and Kir6.2/SUR2B protein complexes as revealed by whole cell patch clamp electrophysiology and immunoprecipitation/Western blotting respectively. Pretreatment with phenylephrine (100nM) generated a cellular phenotype that acquired resistance to chemical hypoxia induced by 2,4-dinitrophenol (DNP; 10mM), which was accompanied by increased in K(+) current in response to DNP (10mM). Cytoprotection afforded by phenylephrine (100nM) was abolished by infection of H9c2 cells with adenovirus containing Kir6.2AFA, a mutant form of Kir6.2 with largely reduced K(+) conductance. Taking all together, the present findings demonstrate that the activation of α1-adrenoceptors up-regulates SUR2B/Kir6.2 to confer cardioprotection. This is the first account of possible physiological role of SUR2B in cardiomyocytes.

  20. Kalium: a database of potassium channel toxins from scorpion venom.

    PubMed

    Kuzmenkov, Alexey I; Krylov, Nikolay A; Chugunov, Anton O; Grishin, Eugene V; Vassilevski, Alexander A

    2016-01-01

    Kalium (http://kaliumdb.org/) is a manually curated database that accumulates data on potassium channel toxins purified from scorpion venom (KTx). This database is an open-access resource, and provides easy access to pages of other databases of interest, such as UniProt, PDB, NCBI Taxonomy Browser, and PubMed. General achievements of Kalium are a strict and easy regulation of KTx classification based on the unified nomenclature supported by researchers in the field, removal of peptides with partial sequence and entries supported by transcriptomic information only, classification of β-family toxins, and addition of a novel λ-family. Molecules presented in the database can be processed by the Clustal Omega server using a one-click option. Molecular masses of mature peptides are calculated and available activity data are compiled for all KTx. We believe that Kalium is not only of high interest to professional toxinologists, but also of general utility to the scientific community.Database URL:http://kaliumdb.org/. PMID:27087309

  1. Kalium: a database of potassium channel toxins from scorpion venom

    PubMed Central

    Kuzmenkov, Alexey I.; Krylov, Nikolay A.; Chugunov, Anton O.; Grishin, Eugene V.; Vassilevski, Alexander A.

    2016-01-01

    Kalium (http://kaliumdb.org/) is a manually curated database that accumulates data on potassium channel toxins purified from scorpion venom (KTx). This database is an open-access resource, and provides easy access to pages of other databases of interest, such as UniProt, PDB, NCBI Taxonomy Browser, and PubMed. General achievements of Kalium are a strict and easy regulation of KTx classification based on the unified nomenclature supported by researchers in the field, removal of peptides with partial sequence and entries supported by transcriptomic information only, classification of β-family toxins, and addition of a novel λ-family. Molecules presented in the database can be processed by the Clustal Omega server using a one-click option. Molecular masses of mature peptides are calculated and available activity data are compiled for all KTx. We believe that Kalium is not only of high interest to professional toxinologists, but also of general utility to the scientific community. Database URL: http://kaliumdb.org/ PMID:27087309

  2. Overexpression of the rice AKT1 potassium channel affects potassium nutrition and rice drought tolerance

    PubMed Central

    Ahmad, Izhar; Mian, Afaq; Maathuis, Frans J. M.

    2016-01-01

    Potassium (K+) is the most important cationic nutrient for all living organisms and has roles in most aspects of plant physiology. To assess the impact of one of the main K+ uptake components, the K+ inward rectifying channel AKT1, we characterized both loss of function and overexpression of OsAKT1 in rice. In many conditions, AKT1 expression correlated with K+ uptake and tissue K+ levels. No salinity-related growth phenotype was observed for either loss or gain of function mutants. However, a correlation between AKT1 expression and root Na+ when the external Na/K ratio was high suggests that there may be a role for AKT1 in Na+ uptake in such conditions. In contrast to findings with Arabidopsis thaliana, we did not detect any change in growth of AKT1 loss of function mutants in the presence of NH4 +. Nevertheless, NH4 +-dependent inhibition was detected during K+ uptake assays in loss of function and wild type plants, depending on pre-growth conditions. The most prominent result of OsAKT1 overexpression was a reduction in sensitivity to osmotic/drought stress in transgenic plants: the data suggest that AKT1 overexpression improved rice osmotic and drought stress tolerance by increasing tissue levels of K+, especially in the root. PMID:26969743

  3. G-protein-coupled inward rectifier potassium channels involved in corticostriatal presynaptic modulation.

    PubMed

    Meneses, David; Mateos, Verónica; Islas, Gustavo; Barral, Jaime

    2015-09-01

    Presynaptic modulation has been associated mainly with calcium channels but recent data suggests that inward rectifier potassium channels (K(IR)) also play a role. In this work we set to characterize the role of presynaptic K(IR) channels in corticostriatal synaptic transmission. We elicited synaptic potentials in striatum by stimulating cortical areas and then determined the synaptic responses of corticostriatal synapsis by using paired pulse ratio (PPR) in the presence and absence of several potassium channel blockers. Unspecific potassium channels blockers Ba(2+) and Cs(+) reduced the PPR, suggesting that these channels are presynaptically located. Further pharmacological characterization showed that application of tertiapin-Q, a specific K(IR)3 channel family blocker, also induced a reduction of PPR, suggesting that K(IR)3 channels are present at corticostriatal terminals. In contrast, exposure to Lq2, a specific K(IR)1.1 inward rectifier potassium channel, did not induce any change in PPR suggesting the absence of these channels in the presynaptic corticostriatal terminals. Our results indicate that K(IR)3 channels are functionally expressed at the corticostriatal synapses, since blockage of these channels result in PPR decrease. Our results also help to explain how synaptic activity may become sensitive to extracellular signals mediated by G-protein coupled receptors. A vast repertoire of receptors may influence neurotransmitter release in an indirect manner through regulation of K(IR)3 channels.

  4. EAG2 potassium channel with evolutionarily conserved function as a brain tumor target

    PubMed Central

    Huang, Xi; He, Ye; Dubuc, Adrian M.; Hashizume, Rintaro; Zhang, Wei; Reimand, Jüri; Yang, Huanghe; Wang, Tongfei A.; Stehbens, Samantha J.; Younger, Susan; Barshow, Suzanne; Zhu, Sijun; Cooper, Michael K.; Peacock, John; Ramaswamy, Vijay; Garzia, Livia; Wu, Xiaochong; Remke, Marc; Forester, Craig M.; Kim, Charles C.; Weiss, William A.; James, C. David; Shuman, Marc A.; Bader, Gary D.; Mueller, Sabine; Taylor, Michael D.; Jan, Yuh Nung; Jan, Lily Yeh

    2015-01-01

    Over 20% of the drugs for treating human diseases target ion channels, however, no cancer drug approved by the U.S. Food and Drug Administration (FDA) is intended to target an ion channel. Here, we demonstrate the evolutionarily conserved function of EAG2 potassium channel in promoting brain tumor growth and metastasis, delineate downstream pathways and uncover a mechanism for different potassium channels to functionally corporate and regulate mitotic cell volume and tumor progression. We show that EAG2 potassium channel is enriched at the trailing edge of migrating MB cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identify the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings thus illustrate the potential of targeting ion channels in cancer treatment. PMID:26258683

  5. Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.

    PubMed

    Wang, Shizhen; Makhina, Elena N; Masia, Ricard; Hyrc, Krzysztof L; Formanack, Mary Lynn; Nichols, Colin G

    2013-02-01

    K(ATP) channels link cell metabolism to excitability in many cells. They are formed as tetramers of Kir6.2 subunits, each associated with a SUR1 subunit. We used mutant GFP-based FRET to assess domain organization in channel complexes. Full-length Kir6.2 subunits were linked to YFP or cyan fluorescent protein (CFP) at N or C termini, and all such constructs, including double-tagged YFP-Kir6.2-CFP (Y6.2C), formed functional K(ATP) channels. In intact COSm6 cells, background emission of YFP excited by 430-nm light was ∼6%, but the Y6.2C construct expressed alone exhibited an apparent FRET efficiency of ∼25%, confirmed by trypsin digestion, with or without SUR1 co-expression. Similar FRET efficiency was detected in mixtures of CFP- and YFP-tagged full-length Kir6.2 subunits and transmembrane domain only constructs, when tagged at the C termini but not at the N termini. The FRET-reported Kir6.2 tetramer domain organization was qualitatively consistent with Kir channel crystal structures: C termini and M2 domains are centrally located relative to N termini and M1 domains, respectively. Additional FRET analyses were performed on cells in which tagged full-length Kir6.2 and tagged SUR1 constructs were co-expressed. These analyses further revealed that 1) NBD1 of SUR1 is closer to the C terminus of Kir6.2 than to the N terminus; 2) the Kir6.2 cytoplasmic domain is not essential for complexation with SUR1; and 3) the N-terminal half of SUR1 can complex with itself in the absence of either the C-terminal half or Kir6.2.

  6. Synthesis and structure-activity relationships of a novel series of 2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide K(ATP) channel openers: discovery of (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9- hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a potent K(ATP) opener that selectively inhibits spontaneous bladder contractions.

    PubMed

    Carroll, William A; Altenbach, Robert J; Bai, Hao; Brioni, Jorge D; Brune, Michael E; Buckner, Steven A; Cassidy, Christopher; Chen, Yiyuan; Coghlan, Michael J; Daza, Anthony V; Drizin, Irene; Fey, Thomas A; Fitzgerald, Michael; Gopalakrishnan, Murali; Gregg, Robert J; Henry, Rodger F; Holladay, Mark W; King, Linda L; Kort, Michael E; Kym, Philip R; Milicic, Ivan; Tang, Rui; Turner, Sean C; Whiteaker, Kristi L; Yi, Lin; Zhang, Henry; Sullivan, James P

    2004-06-01

    Structure-activity relationships were investigated on a novel series of sulfonyldihydropyridine-containing K(ATP) openers. Ring sizes, absolute stereochemistry, and aromatic substitution were evaluated for K(ATP) activity in guinea pig bladder cells using a fluorescence-based membrane potential assay and in a pig bladder strip assay. The inhibition of spontaneous bladder contractions in vitro was also examined for a select group of compounds. All compounds studied showed greater potency to inhibit spontaneous bladder contractions relative to their potencies to inhibit contractions elicited by electrical stimulation. In an anesthetized pig model of myogenic bladder overactivity, compound 14 and (-)-cromakalim 1 were found to inhibit spontaneous bladder contractions in vivo at plasma concentrations lower than those that affected hemodynamic parameters. Compound 14 showed approximately 5-fold greater selectivity than 1 in vivo and supports the concept that bladder-selective K(ATP) channel openers may have utility in the treatment of overactive bladder.

  7. The Sodium-Activated Potassium Channel Slack Is Required for Optimal Cognitive Flexibility in Mice

    ERIC Educational Resources Information Center

    Bausch, Anne E.; Dieter, Rebekka; Nann, Yvette; Hausmann, Mario; Meyerdierks, Nora; Kaczmarek, Leonard K.; Ruth, Peter; Lukowski, Robert

    2015-01-01

    "Kcnt1" encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual…

  8. Cardioprotective effects of sarcolemmal and mitochondrial K-ATP channel openers in an experimental model of autoimmune myocarditis. Role of the reduction in calcium overload during acute heart failure.

    PubMed

    Niwano, Shinichi; Hirasawa, Shoji; Niwano, Hiroe; Sasaki, Sae; Masuda, Ray; Sato, Kiyotaka; Masuda, Takashi; Izumi, Tohru

    2012-01-01

    It has been reported that K-ATP channel openers have a cardioprotective effect in acute ischemia as a pharmacological preconditioning effect. In the present study, the chronic effects of clinical K-ATP channel openers, ie, nicorandil (Nic) and mexiletine (Mex), on cardiac function were evaluated in a rat model of experimental autoimmune myocarditis (EAM). Nicorandil (3 or 10 mg/kg/day) or Mex (10 or 25 mg/kg/day) was administered to the EAM rats, and the effects were compared with those in untreated EAM rats (control EAM) and sham rats without EAM on day 21 (acute phase) or day 60 (chronic phase). In the acute phase, the control EAM rats exhibited a reduced left ventricular ejection fraction (LVEF) and prolonged monophasic action potential duration (MAPD). Neither drug had an affect on the LVEF or degree of myocarditis, but Mex 25 mg suppressed the MAPD prolongation. In the chronic phase, EAM+Nic and EAM+Mex 25 mg exhibited a higher LVEF than the control EAM. Although the control EAM exhibited sustained MAPD prolongation, the other groups showed recovery of the MAPD in the chronic phase. The mitochondorial redox state was lower in the control EAM than in the sham, and EAM+Nic exhibited a similar level of the redox state as the sham in the chronic phase. Nicorandil exhibited a cardioprotective effect through the protection of mitochondrial function. Mexiletine exhibited a cardioprotective effect possibly through a reduction in the calcium overload by shortening the MAPD in the acute phase. PMID:22688320

  9. Interactions of phospholipids with the potassium channel KcsA.

    PubMed

    Williamson, Ian M; Alvis, Simon J; East, J Malcolm; Lee, Anthony G

    2002-10-01

    The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer. PMID:12324421

  10. Differential blockage of two types of potassium channels in the crab giant axon.

    PubMed

    Soria, B; Arispe, N; Quinta-Ferreira, M E; Rojas, E

    1985-01-01

    Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crabs Carcinus maenas and Cancer pagirus. The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate types of potassium channels exist in these axons (M.E. Quinta-Ferreira, E. Rojas and N. Arispe, J. Membrane Biol. 66:171-181, 1982). It is shown here that, with small concentrations of conventional K+-channel blockers, it is possible to differentially inhibit these channels. The potassium channels with activation and fast inactivation gating (m3h, Hodgkin-Huxley kinetics) were blocked by external application of 4 amino-pyridine (4-AP). The potassium channels with standard gating (n4, Hodgkin-Huxley kinetics) were preferentially inhibited by externally applied tetraethylammonium (TEA). The differential blockage of the two types of potassium conductance changes suggests that they represent two different populations of potassium channels. It is further shown here that blocking the early transient conductance increase leads to the inhibition of the repetitive electrical activity induced by constant depolarizing current injection in fibers from Cardisoma guanhumi.

  11. Influence of the alkylsulfonylamino substituent located at the 6-position of 2,2-dimethylchromans structurally related to cromakalim: from potassium channel openers to calcium entry blockers?

    PubMed

    Florence, Xavier; Desvaux, Vincent; Goffin, Eric; de Tullio, Pascal; Pirotte, Bernard; Lebrun, Philippe

    2014-06-10

    The present study described the synthesis of original R/S-6-alkylsulfonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans bearing a 3- or 4-substituted phenylthiourea or phenylurea moiety at the 4-position. Their biological effects were evaluated both on insulin-secreting and smooth muscle cells and were compared to those of reference KATP channel activators such as (±)-cromakalim, diazoxide and previously synthesized cromakalim analogues. The study aimed at exploring the influence of the introduction of an alkylsulfonylamino substituent at the 6-position of 2,2-dimethylchromans in order to improve biological activity, tissue selectivity but also hydrophilicity of dihydrobenzopyran derivatives. Several compounds were found to be equipotent or even more potent than (±)-cromakalim and diazoxide at inhibiting the insulin releasing process. Most of the newly synthesized and more hydrophilic dihydrobenzopyrans also exhibited a marked vasorelaxant activity although they were less potent than (±)-cromakalim. Additional pharmacological and radioisotopic investigations suggested that R/S-N-3-chlorophenyl-N'-(3,4-dihydro-6-methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (21) did not act as a potassium channel opener but rather as a Ca(2+) entry blocker.

  12. Potassium

    MedlinePlus

    Potassium is essential for the proper functioning of the heart, kidneys, muscles, nerves, and digestive system. Usually the food you eat supplies all of the potassium you need. However, certain diseases (e.g., kidney ...

  13. Comparison of Single Channel Potassium Current in Biological and Synthetic Systems --- Dependence on Voltage

    NASA Astrophysics Data System (ADS)

    Siwy, Zuzanna; Mercik, Szymon; Weron, Karina; Spohr, Reimar; Wolf, Alexander; Grzywna, Zbigniew

    2000-05-01

    The influence of an external field on an ion current pattern in biological and synthetic systems was investigated. The patch clamp recordings of potassium current through a big conductance locust potassium channel (BK-channel) and a track-etched polyethylene terephthalate membrane were examined by the power spectrum, fractal analysis and relative dispersion analysis. A similar dependence of potassium current behaviour on the external voltage in both systems was found. The generalized dimension formalism is redefined to make it applicable to the analysis of time series.

  14. Substrate Regulation of Single Potassium and Chloride Ion Channels in Arabidopsis Plasma Membrane

    PubMed Central

    Lew, Roger R.

    1991-01-01

    Patch clamp measurements of excised inside-out patches of Arabidopsis thaliana plasma membrane reveal at least two ion channels which conduct either potassium or chloride. The conductance of the potassium channel ranged from 5 to 70 picosiemens depending on KCl concentration. The conductance increased linearly with increasing cytoplasmic-side [KCl]; the extent of this dependence declined as extracytoplasmic-side [KCl] was increased. This indicates that substrate regulation of the potassium channel is a consequence of the molecular architecture of the channel: in particular, multi-ion binding sites within the channel pore. The chloride channel conductance (ranging from 5-40 picosiemens) was independent of cytoplasmic-side [KCl] until a threshold concentration of about 300 millimolar was reached. Such behavior is expected only if the channel is allosterically regulated by cytoplasmic-side K+ and/or Cl−. The median open times of either channel (about 200 milliseconds for the potassium channel and 20 milliseconds for the chloride channel) were unaffected by substrate concentrations. PMID:16668031

  15. Stimulation of Oxytocin Receptor during Early Reperfusion Period Protects the Heart against Ischemia/Reperfusion Injury: the Role of Mitochondrial ATP-Sensitive Potassium Channel, Nitric Oxide, and Prostaglandins.

    PubMed

    Imani, Alireza; Khansari, Maryam; Azizi, Yaser; Rakhshan, Kamran; Faghihi, Mahdieh

    2015-08-01

    Postconditioning is a simple and safe strategy for cardioprotection and infarct size limitation. Our previous study showed that oxytocin (OT) exerts postconditioning effect on ischemic/reperfused isolated rat heart. The aim of this study was to investigate the involvement of OT receptor, mitochondrial ATP-sensitive potassium channel (mKATP), nitric oxide (NO) and cyclooxygenase (COX) pathways in OT postconditioning. Isolated rat hearts were divided into10 groups and underwent 30 min of regional ischemia followed by 120 min of reperfusion (n =6). In I/R (ischemia/reperfusion) group, ischemia and reperfusion were induced without any treatment. In OT group, oxytocin was perfused 5 min prior to beginning of reperfusion for 25 min. In groups 3-6, atosiban (oxytocin receptor blocker), L-NAME (N-Nitro-L-Arginine Methyl Ester, non-specific nitric oxide synthase inhibitor), 5-HD (5-hydroxydecanoate, mKATP inhibitor) and indomethacin (cyclooxygenase inhibitor) were infused prior to oxytocin administration. In others, the mentioned inhibitors were perfused prior to ischemia without oxytocin infusion. Infarct size, ventricular hemodynamic, coronary effluent, malondialdehyde (MDA) and lactate dehydrogenase (LDH) were measured at the end of reperfusion. OT perfusion significantly reduced infarct size, MDA and LDH in comparison with IR group. Atosiban, 5HD, L-NAME and indomethacin abolished the postconditioning effect of OT. Perfusion of the inhibitors alone prior to ischemia had no effect on infarct size, hemodynamic parameters, coronary effluent and biochemical markers as compared with I/R group. In conclusion, this study indicates that postconditioning effects of OT are mediated by activation of mKATP and production of NO and Prostaglandins (PGs).

  16. Unique properties of the ATP-sensitive K⁺ channel in the mouse ventricular cardiac conduction system.

    PubMed

    Bao, Li; Kefaloyianni, Eirini; Lader, Joshua; Hong, Miyoun; Morley, Gregory; Fishman, Glenn I; Sobie, Eric A; Coetzee, William A

    2011-12-01

    Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia.

  17. Modulation of hERG potassium channel gating normalizes action potential duration prolonged by dysfunctional KCNQ1 potassium channel

    PubMed Central

    Zhang, Hongkang; Zou, Beiyan; Yu, Haibo; Moretti, Alessandra; Wang, Xiaoying; Yan, Wei; Babcock, Joseph J.; Bellin, Milena; McManus, Owen B.; Tomaselli, Gordon; Nan, Fajun; Laugwitz, Karl-Ludwig; Li, Min

    2012-01-01

    Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, ∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as IKs and IKr. The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of IKr by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if IKs is reduced. Using a unique specific chemical activator for IKr that has a primary effect of causing a right shift of V1/2 for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an IKr chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent. PMID:22745159

  18. Modeling of the Binding of Peptide Blockers to Voltage-Gated Potassium Channels: Approaches and Evidence

    PubMed Central

    Novoseletsky, V. N.; Volyntseva, A. D.; Shaitan, K. V.; Kirpichnikov, M. P.; Feofanov, A. V.

    2016-01-01

    Modeling of the structure of voltage-gated potassium (KV) channels bound to peptide blockers aims to identify the key amino acid residues dictating affinity and provide insights into the toxin-channel interface. Computational approaches open up possibilities for in silico rational design of selective blockers, new molecular tools to study the cellular distribution and functional roles of potassium channels. It is anticipated that optimized blockers will advance the development of drugs that reduce over activation of potassium channels and attenuate the associated malfunction. Starting with an overview of the recent advances in computational simulation strategies to predict the bound state orientations of peptide pore blockers relative to KV-channels, we go on to review algorithms for the analysis of intermolecular interactions, and then take a look at the results of their application. PMID:27437138

  19. Regulation of Arterial Tone by Activation of Calcium-Dependent Potassium Channels

    NASA Astrophysics Data System (ADS)

    Brayden, Joseph E.; Nelson, Mark T.

    1992-04-01

    Blood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) vascular tone. However, many of the molecular determinants of this response are unknown. Evidence is now presented that the degree of myogenic tone is regulated in part by the activation of large-conductance calcium-activated potassium channels in arterial smooth muscle. Tetraethylammonium ion (TEA^+) and charybdotoxin (CTX), at concentrations that block calcium-activated potassium channels in smooth muscle cells isolated from cerebral arteries, depolarized and constricted pressurized cerebral arteries with myogenic tone. Both TEA^+ and CTX had little effect on arteries when intracellular calcium was reduced by lowering intravascular pressure or by blocking calcium channels. Elevation of intravascular pressure through membrane depolarization and an increase in intracellular calcium may activate calcium-activated potassium channels. Thus, these channels may serve as a negative feedback pathway to control the degree of membrane depolarization and vasoconstriction.

  20. Allosteric coupling of the inner activation gate to the outer pore of a potassium channel.

    PubMed

    Peters, Christian J; Fedida, David; Accili, Eric A

    2013-10-23

    In potassium channels, functional coupling of the inner and outer pore gates may result from energetic interactions between residues and conformational rearrangements that occur along a structural path between them. Here, we show that conservative mutations of a residue near the inner activation gate of the Shaker potassium channel (I470) modify the rate of C-type inactivation at the outer pore, pointing to this residue as part of a pathway that couples inner gate opening to changes in outer pore structure and reduction of ion flow. Because they remain equally sensitive to rises in extracellular potassium, altered inactivation rates of the mutant channels are not secondary to modified binding of potassium to the outer pore. Conservative mutations of I470 also influence the interaction of the Shaker N-terminus with the inner gate, which separately affects the outer pore.

  1. Calcium channels responsible for potassium-induced transmitter release at rat cerebellar synapses.

    PubMed

    Momiyama, A; Takahashi, T

    1994-04-15

    The effects of calcium channel blockers on potassium-induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole-cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma-aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high-potassium solution. In the presence of the L-type Ca2+ channel blocker nicardipine (10 microM), the potassium-induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTX, 3 microM) had no effect on the frequency of potassium-induced mIPSCs. The P-type Ca2+ channel blocker omega-Aga-IVA (200 nM) suppressed the potassium-induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium-induced transmitter release may be different from those mediating fast synaptic transmission.

  2. A heme-binding domain controls regulation of ATP-dependent potassium channels

    PubMed Central

    Burton, Mark J.; Kapetanaki, Sofia M.; Chernova, Tatyana; Jamieson, Andrew G.; Dorlet, Pierre; Santolini, Jérôme; Mitcheson, John S.; Davies, Noel W.; Schmid, Ralf; Raven, Emma L.; Storey, Nina M.

    2016-01-01

    Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels. PMID:27006498

  3. EXPRESSION AND DISTRIBUTION OF Kv4 POTASSIUM CHANNEL SUBUNITS AND POTASSIUM CHANNEL INTERACTING PROTEINS IN SUBPOPULATIONS OF INTERNEURONS IN THE BASOLATERAL AMYGDALA

    PubMed Central

    DABROWSKA, J.; RAINNIE, D. G.

    2010-01-01

    Kv4 potassium channel α subunits, Kv4.1, Kv4.2, and Kv4.3, determine some of the fundamental physiological properties of neurons in the CNS. Kv4 subunits are associated with auxiliary β-subunits, such as the potassium channel interacting proteins (KChIP1 – 4), which are thought to regulate the trafficking and gating of native Kv4 potassium channels. Intriguingly, KChIP1 is thought to show cell type-selective expression in GABA-ergic inhibitory interneurons, while other β-subunits (KChIP2–4) are associated with principal glutamatergic neurons. However, nothing is known about the expression of Kv4 family α- and β-subunits in specific interneurons populations in the BLA. Here, we have used immunofluorescence, co-immunoprecipitation, and Western Blotting to determine the relative expression of KChIP1 in the different interneuron subtypes within the BLA, and its co-localization with one or more of the Kv4 α subunits. We show that all three α-subunits of Kv4 potassium channel are found in rat BLA neurons, and that the immuno-reactivity of KChIP1 closely resembles that of Kv4.3. Indeed, Kv4.3 showed almost complete co-localization with KChIP1 in the soma and dendrites of a distinct subpopulation of BLA neurons. Dual-immunofluorescence studies revealed this to be in BLA interneurons immunoreactive for parvalbumin, cholecystokin-8, and somatostatin. Finally, co-immunoprecipitation studies showed that KChIP1 was associated with all three Kv4 α subunits. Together our results suggest that KChIP1 is selectively expressed in BLA interneurons where it may function to regulate the activity of A-type potassium channels. Hence, KChIP1 might be considered as a cell type-specific regulator of GABAergic inhibitory circuits in the BLA. PMID:20849929

  4. Genomic organization and chromosomal localization of the murine 2 P domain potassium channel gene Kcnk8: conservation of gene structure in 2 P domain potassium channels.

    PubMed

    Bockenhauer, D; Nimmakayalu, M A; Ward, D C; Goldstein, S A; Gallagher, P G

    2000-12-31

    A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.

  5. Calcium-Activated Potassium Channels: Potential Target for Cardiovascular Diseases.

    PubMed

    Dong, De-Li; Bai, Yun-Long; Cai, Ben-Zhi

    2016-01-01

    Ca(2+)-activated K(+) channels (KCa) are classified into three subtypes: big conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) KCa channels. The three types of KCa channels have distinct physiological or pathological functions in cardiovascular system. BKCa channels are mainly expressed in vascular smooth muscle cells (VSMCs) and inner mitochondrial membrane of cardiomyocytes, activation of BKCa channels in these locations results in vasodilation and cardioprotection against cardiac ischemia. IKCa channels are expressed in VSMCs, endothelial cells, and cardiac fibroblasts and involved in vascular smooth muscle proliferation, migration, vessel dilation, and cardiac fibrosis. SKCa channels are widely expressed in nervous and cardiovascular system, and activation of SKCa channels mainly contributes membrane hyperpolarization. In this chapter, we summarize the physiological and pathological roles of the three types of KCa channels in cardiovascular system and put forward the possibility of KCa channels as potential target for cardiovascular diseases.

  6. Involvement of potassium channels in the progression of cancer to a more malignant phenotype.

    PubMed

    Comes, Nuria; Serrano-Albarrás, Antonio; Capera, Jesusa; Serrano-Novillo, Clara; Condom, Enric; Ramón Y Cajal, Santiago; Ferreres, Joan Carles; Felipe, Antonio

    2015-10-01

    Potassium channels are a diverse group of pore-forming transmembrane proteins that selectively facilitate potassium flow through an electrochemical gradient. They participate in the control of the membrane potential and cell excitability in addition to different cell functions such as cell volume regulation, proliferation, cell migration, angiogenesis as well as apoptosis. Because these physiological processes are essential for the correct cell function, K+ channels have been associated with a growing number of diseases including cancer. In fact, different K+ channel families such as the voltage-gated K+ channels, the ether à-go-go K+ channels, the two pore domain K+ channels and the Ca2+-activated K+ channels have been associated to tumor biology. Potassium channels have a role in neoplastic cell-cycle progression and their expression has been found abnormal in many types of tumors and cancer cells. In addition, the expression and activity of specific K+ channels have shown a significant correlation with the tumor malignancy grade. The aim of this overview is to summarize published data on K+ channels that exhibit oncogenic properties and have been linked to a more malignant cancer phenotype. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

  7. Large-Conductance Calcium-Activated Potassium Channels in Glomerulus: From Cell Signal Integration to Disease

    PubMed Central

    Tao, Jie; Lan, Zhen; Wang, Yunman; Hei, Hongya; Tian, Lulu; Pan, Wanma; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca2+, possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus. PMID:27445840

  8. Large-Conductance Calcium-Activated Potassium Channels in Glomerulus: From Cell Signal Integration to Disease.

    PubMed

    Tao, Jie; Lan, Zhen; Wang, Yunman; Hei, Hongya; Tian, Lulu; Pan, Wanma; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca(2+), possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus. PMID:27445840

  9. Energetics of Multi-Ion Conduction Pathways in Potassium Ion Channels

    PubMed Central

    2013-01-01

    Potassium ion channels form pores in cell membranes, allowing potassium ions through while preventing the passage of sodium ions. Despite numerous high-resolution structures, it is not yet possible to relate their structure to their single molecule function other than at a qualitative level. Over the past decade, there has been a concerted effort using molecular dynamics to capture the thermodynamics and kinetics of conduction by calculating potentials of mean force (PMF). These can be used, in conjunction with the electro-diffusion theory, to predict the conductance of a specific ion channel. Here, we calculate seven independent PMFs, thereby studying the differences between two potassium ion channels, the effect of the CHARMM CMAP forcefield correction, and the sensitivity and reproducibility of the method. Thermodynamically stable ion–water configurations of the selectivity filter can be identified from all the free energy landscapes, but the heights of the kinetic barriers for potassium ions to move through the selectivity filter are, in nearly all cases, too high to predict conductances in line with experiment. This implies it is not currently feasible to predict the conductance of potassium ion channels, but other simpler channels may be more tractable. PMID:24353479

  10. Voltage-gated and ATP-sensitive K+ channels are associated with cell proliferation and tumorigenesis of human glioma.

    PubMed

    Ru, Qin; Tian, Xiang; Wu, Yu-Xiang; Wu, Ri-Hui; Pi, Ming-Shan; Li, Chao-Ying

    2014-02-01

    Increasing evidence indicates that potassium (K+) channels play important roles in the growth and development of human cancer. In the present study, we investigated the contribution of and the mechanism by which K+ channels control the proliferation and tumor development of U87-MG human glioma cells. A variety of K+ channel blockers and openers were used to differentiate the critical subtype of K+ channels involved. The in vitro data demonstrated that selective blockers of voltage-gated K+ (K(V)) channels or ATP-sensitive K+ (K(ATP)) channels significantly inhibited the proliferation of U87-MG cells, blocked the cell cycle at the G0/G1 phase and induced apoptosis. In the U87-MG xenograft model in nude mice, K(V) or K(ATP) channel blockers markedly suppressed tumor growth in vivo. Furthermore, electrophysiological results showed that KV or KATP channel blockers inhibited K(V)/K(ATP) channel currents as well as cell proliferation and tumor growth over the same concentration range. In contrast, iberiotoxin, a selective blocker of calcium-activated K+ channels, had no apparent effect on the cell proliferation, cell cycle or apoptosis of U87-MG cells. In addition, the results of fluorescence assays indicated that blockers of K(V) or K(ATP) channels attenuated intracellular Ca2+ signaling by blocking Ca2+ influx in U87-MG cells. Taken together, these data suggest that K(V) and K(ATP) channels play important roles in the proliferation of U87-MG cells and that the influence of K(V) and K(ATP) channels may be mediated by a Ca2+-dependent mechanism. PMID:24284968

  11. Mechanisms contributing to myocardial potassium channel diversity, regulation and remodeling.

    PubMed

    Yang, Kai-Chien; Nerbonne, Jeanne M

    2016-04-01

    In the mammalian heart, multiple types of K(+) channels contribute to the control of cardiac electrical and mechanical functioning through the regulation of resting membrane potentials, action potential waveforms and refractoriness. There are similarly vast arrays of K(+) channel pore-forming and accessory subunits that contribute to the generation of functional myocardial K(+) channel diversity. Maladaptive remodeling of K(+) channels associated with cardiac and systemic diseases results in impaired repolarization and increased propensity for arrhythmias. Here, we review the diverse transcriptional, post-transcriptional, post-translational, and epigenetic mechanisms contributing to regulating the expression, distribution, and remodeling of cardiac K(+) channels under physiological and pathological conditions. PMID:26391345

  12. The antifungal plant defensin AtPDF2.3 from Arabidopsis thaliana blocks potassium channels.

    PubMed

    Vriens, Kim; Peigneur, Steve; De Coninck, Barbara; Tytgat, Jan; Cammue, Bruno P A; Thevissen, Karin

    2016-01-01

    Scorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαβ-motif. These toxins contain a toxin signature (K-C4-X-N) in their amino acid sequence, and based on in silico analysis of 18 plant defensin sequences, we noted the presence of a toxin signature (K-C5-R-G) in the amino acid sequence of the Arabidopsis thaliana defensin AtPDF2.3. We found that recombinant (r)AtPDF2.3 blocks Kv1.2 and Kv1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C5-R-N), is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and Kv channel inhibitory function are not linked. PMID:27573545

  13. The antifungal plant defensin AtPDF2.3 from Arabidopsis thaliana blocks potassium channels

    PubMed Central

    Vriens, Kim; Peigneur, Steve; De Coninck, Barbara; Tytgat, Jan; Cammue, Bruno P. A.; Thevissen, Karin

    2016-01-01

    Scorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαβ-motif. These toxins contain a toxin signature (K-C4-X-N) in their amino acid sequence, and based on in silico analysis of 18 plant defensin sequences, we noted the presence of a toxin signature (K-C5-R-G) in the amino acid sequence of the Arabidopsis thaliana defensin AtPDF2.3. We found that recombinant (r)AtPDF2.3 blocks Kv1.2 and Kv1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C5-R-N), is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and Kv channel inhibitory function are not linked. PMID:27573545

  14. Molecular Diversity and Regulation of Renal Potassium Channels

    PubMed Central

    HEBERT, STEVEN C.; DESIR, GARY; GIEBISCH, GERHARD; WANG, WENHUI

    2010-01-01

    K+ channels are widely distributed in both plant and animal cells where they serve many distinct functions. K+ channels set the membrane potential, generate electrical signals in excitable cells, and regulate cell volume and cell movement. In renal tubule epithelial cells, K+ channels are not only involved in basic functions such as the generation of the cell-negative potential and the control of cell volume, but also play a uniquely important role in K+ secretion. Moreover, K+ channels participate in the regulation of vascular tone in the glomerular circulation, and they are involved in the mechanisms mediating tubuloglomerular feedback. Significant progress has been made in defining the properties of renal K+ channels, including their location within tubule cells, their biophysical properties, regulation, and molecular structure. Such progress has been made possible by the application of single-channel analysis and the successful cloning of K+ channels of renal origin. PMID:15618483

  15. Potassium

    MedlinePlus

    ... blackberries Root vegetables, such as carrots and potatoes Citrus fruits, such as oranges and grapefruit Your kidneys help to keep the right amount of potassium in your body. If you have chronic kidney disease, your kidneys may not remove extra potassium from ...

  16. Scorpion Potassium Channel-blocking Defensin Highlights a Functional Link with Neurotoxin.

    PubMed

    Meng, Lanxia; Xie, Zili; Zhang, Qian; Li, Yang; Yang, Fan; Chen, Zongyun; Li, Wenxin; Cao, Zhijian; Wu, Yingliang

    2016-03-25

    The structural similarity between defensins and scorpion neurotoxins suggests that they might have evolved from a common ancestor. However, there is no direct experimental evidence demonstrating a functional link between scorpion neurotoxins and defensins. The scorpion defensin BmKDfsin4 from Mesobuthus martensiiKarsch contains 37 amino acid residues and a conserved cystine-stabilized α/β structural fold. The recombinant BmKDfsin4, a classical defensin, has been found to have inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteusas well as methicillin-resistant Staphylococcus aureus Interestingly, electrophysiological experiments showed that BmKDfsin4,like scorpion potassium channel neurotoxins, could effectively inhibit Kv1.1, Kv1.2, and Kv1.3 channel currents, and its IC50value for the Kv1.3 channel was 510.2 nm Similar to the structure-function relationships of classical scorpion potassium channel-blocking toxins, basic residues (Lys-13 and Arg-19) of BmKDfsin4 play critical roles in peptide-Kv1.3 channel interactions. Furthermore, mutagenesis and electrophysiological experiments demonstrated that the channel extracellular pore region is the binding site of BmKDfsin4, indicating that BmKDfsin4 adopts the same mechanism for blocking potassium channel currents as classical scorpion toxins. Taken together, our work identifies scorpion BmKDfsin4 as the first invertebrate defensin to block potassium channels. These findings not only demonstrate that defensins from invertebrate animals are a novel type of potassium channel blockers but also provide evidence of a functional link between defensins and neurotoxins.

  17. Novel treatment strategies for smooth muscle disorders: Targeting Kv7 potassium channels.

    PubMed

    Haick, Jennifer M; Byron, Kenneth L

    2016-09-01

    Smooth muscle cells provide crucial contractile functions in visceral, vascular, and lung tissues. The contractile state of smooth muscle is largely determined by their electrical excitability, which is in turn influenced by the activity of potassium channels. The activity of potassium channels sustains smooth muscle cell membrane hyperpolarization, reducing cellular excitability and thereby promoting smooth muscle relaxation. Research over the past decade has indicated an important role for Kv7 (KCNQ) voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells. Expression of multiple Kv7 channel subtypes has been demonstrated in smooth muscle cells from viscera (gastrointestinal, bladder, myometrial), from the systemic and pulmonary vasculature, and from the airways of the lung, from multiple species, including humans. A number of clinically used drugs, some of which were developed to target Kv7 channels in other tissues, have been found to exert robust effects on smooth muscle Kv7 channels. Functional studies have indicated that Kv7 channel activators and inhibitors have the ability to relax and contact smooth muscle preparations, respectively, suggesting a wide range of novel applications for the pharmacological tool set. This review summarizes recent findings regarding the physiological functions of Kv7 channels in smooth muscle, and highlights potential therapeutic applications based on pharmacological targeting of smooth muscle Kv7 channels throughout the body.

  18. Parametrisation of the free energy of ATP binding to wild-type and mutant Kir6.2 potassium channels.

    PubMed

    Moran, Oscar; Grottesi, Alessandro; Chadburn, Andrew J; Tammaro, Paolo

    2013-01-01

    ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.x and regulatory SURx subunits, play important roles in many cellular functions; because of their sensitivity to inhibition by intracellular ATP, K(ATP) channels provide a link between cell metabolism and membrane electrical activity. We constructed structural homology models of Kir6.2 and a series of Kir6.2 channels carrying mutations within the putative ATP-binding site. Computational docking was carried out to determine the conformation of ATP in its binding site. The Linear Interaction Energy (LIE) method was used to estimate the free-energy of ATP binding to wild-type and mutant Kir6.2 channels. Comparisons of the theoretical binding free energies for ATP with those determined from mutational experiments enabled the identification of the most probable conformation of ATP bound to the Kir6.2 channel. A set of LIE parameters was defined that may enable prediction of the effects of additional Kir6.2 mutations within the ATP binding site on the affinity for ATP.

  19. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.

    PubMed Central

    Cooke, J P; Rossitch, E; Andon, N A; Loscalzo, J; Dzau, V J

    1991-01-01

    Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of 60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by L,N-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions of potassium, verapamil, calcium-free medium, and antagonists of the KCa channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses to other agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on micro-carrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator; the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the KCa channel) on the endothelial cell membrane that leads to the release of nitric oxide. Images PMID:1719029

  20. Oxidative Modulation of Voltage-Gated Potassium Channels

    PubMed Central

    Sahoo, Nirakar; Hoshi, Toshinori

    2014-01-01

    Abstract Significance: Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as reactive oxygen species (ROS) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. Recent Advances: Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling provide a broader phenomenology and emerging mechanistic insights. Critical Issues: Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. Future Directions: High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs. Antioxid. Redox Signal. 21, 933–952. PMID:24040918

  1. hERG Potassium Channel Blockade by the HCN Channel Inhibitor Bradycardic Agent Ivabradine

    PubMed Central

    Melgari, Dario; Brack, Kieran E.; Zhang, Chuan; Zhang, Yihong; El Harchi, Aziza; Mitcheson, John S.; Dempsey, Christopher E.; Ng, G. André; Hancox, Jules C.

    2015-01-01

    Background Ivabradine is a specific bradycardic agent used in coronary artery disease and heart failure, lowering heart rate through inhibition of sinoatrial nodal HCN‐channels. This study investigated the propensity of ivabradine to interact with KCNH2‐encoded human Ether‐à‐go‐go–Related Gene (hERG) potassium channels, which strongly influence ventricular repolarization and susceptibility to torsades de pointes arrhythmia. Methods and Results Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. IhERG was inhibited with an IC50 of 2.07 μmol/L for the hERG 1a isoform and 3.31 μmol/L for coexpressed hERG 1a/1b. The voltage and time‐dependent characteristics of IhERG block were consistent with preferential gated‐state‐dependent channel block. Inhibition was partially attenuated by the N588K inactivation‐mutant and the S624A pore‐helix mutant and was strongly reduced by the Y652A and F656A S6 helix mutants. In docking simulations to a MthK‐based homology model of hERG, the 2 aromatic rings of the drug could form multiple π‐π interactions with the aromatic side chains of both Y652 and F656. In monophasic action potential (MAP) recordings from guinea‐pig Langendorff‐perfused hearts, ivabradine delayed ventricular repolarization and produced a steepening of the MAPD90 restitution curve. Conclusions Ivabradine prolongs ventricular repolarization and alters electrical restitution properties at concentrations relevant to the upper therapeutic range. In absolute terms ivabradine does not discriminate between hERG and HCN channels: it inhibits IhERG with similar potency to that reported for native If and HCN channels, with S6 binding determinants resembling those observed for HCN4. These findings may have important implications both clinically and for future bradycardic drug design. PMID:25911606

  2. Docetaxel modulates the delayed rectifier potassium current (IK) and ATP-sensitive potassium current (IKATP) in human breast cancer cells.

    PubMed

    Sun, Tao; Song, Zhi-Guo; Jiang, Da-Qing; Nie, Hong-Guang; Han, Dong-Yun

    2015-04-01

    Ion channel expression and activity may be affected during tumor development and cancer growth. Activation of potassium (K(+)) channels in human breast cancer cells is reported to be involved in cell cycle progression. In this study, we investigated the effects of docetaxel on the delayed rectifier potassium current (I K) and the ATP-sensitive potassium current (I KATP) in two human breast cancer cell lines, MCF-7 and MDA-MB-435S, using the whole-cell patch-clamp technique. Our results show that docetaxel inhibited the I K and I KATP in both cell lines in a dose-dependent manner. Compared with the control at a potential of +60 mV, treatment with docetaxel at doses of 0.1, 1, 5, and 10 µM significantly decreased the I K in MCF-7 cells by 16.1 ± 3.5, 30.2 ± 5.2, 42.5 ± 4.3, and 46.4 ± 9% (n = 5, P < 0.05), respectively and also decreased the I KATP at +50 mV. Similar results were observed in MDA-MB-435S cells. The G-V curves showed no significant changes after treatment of either MCF-7 or MDA-MB-435S cells with 10 μM docetaxel. The datas indicate that the possible mechanisms of I K and I KATP inhibition by docetaxel may be responsible for its effect on the proliferation of human breast cancer cells.

  3. Hemin inhibits the large conductance potassium channel in brain mitochondria: a putative novel mechanism of neurodegeneration.

    PubMed

    Augustynek, Bartłomiej; Kudin, Alexei P; Bednarczyk, Piotr; Szewczyk, Adam; Kunz, Wolfram S

    2014-07-01

    Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH. One of them is a massive release of hemin from the decomposed erythrocytes. It has been previously shown, that hemin can inhibit the large-conductance Ca(2+)-regulated potassium channel in the plasma membrane. However, it remained unclear whether this phenomenon applies also to the mitochondrial large-conductance Ca(2+)-regulated potassium channel. The aim of the present study was to determine the impact of hemin on the activity of the large conductance Ca(2+)-regulated potassium channel in the brain mitochondria (mitoBKCa). In order to do so, we have used a patch-clamp technique and shown that hemin inhibits mitoBKCa in human astrocytoma U-87 MG cell line mitochondria. Since opening of the mitochondrial potassium channels is known to be cytoprotective, we have elucidated whether hemin can attenuate some of the beneficiary effects of potassium channel opening. We have studied the effect of hemin on reactive oxygen species synthesis, and mild mitochondrial uncoupling in isolated rat brain mitochondria. Taken together, our data show that hemin inhibits mitoBKCa and partially abolishes some of the cytoprotective properties of potassium channel opening. Considering the role of the mitoBKCa in cytoprotection, it can be presumed that its inhibition by hemin may be a novel mechanism contributing to the severity of the ICH symptoms. However, the validity of the presented results shall be further verified in an experimental model of ICH.

  4. Sequence of a probable potassium channel component encoded at shaker locus of drosophila

    SciTech Connect

    Tempel, B.L.; Papazian, D.M.; Schwarz, T.L.; Jan, Y.N.; Jan, L.Y.

    1987-08-24

    Potassium currents are crucial for the repolarization of electrically excitable membranes, a role that makes potassium channels a target for physiological modifications that alter synaptic efficacy. The Shaker locus of Drosophila is thought to encode a K/sup +/ channel. The sequence of two complementary DNA clones from the Shaker locus is reported here. The sequence predicts an integral membrane protein of 70,200 daltons containing seven potential membrane-spanning sequences. In addition, the predicted protein is homologous to the vertebrate sodium channel in a region previously proposed to be involved in the voltage-dependent activation of the Na/sup +/ channel. These results support the hypothesis that Shaker encodes a structural component of a voltage-dependent K/sup +/ channel and suggest a conserved mechanism for voltage activation.

  5. Apical potassium channels in the rat connecting tubule.

    PubMed

    Frindt, Gustavo; Palmer, Lawrence G

    2004-11-01

    Apical membrane K channels in the rat connecting tubule (CNT) were studied using the patch-clamp technique. Tubules were isolated from the cortical labyrinth of the kidney and split open to provide access to the apical membrane. Cell-attached patches were formed on presumed principal and/or connecting tubule cells. The major channel type observed had a single-channel conductance of 52 pS, high open probability and kinetics that were only weakly dependent on voltage. These correspond closely to the "SK"-type channels in the cortical collecting duct, identified with the ROMK (Kir1.1) gene product. A second channel type, which was less frequently observed, mediated larger currents and was strongly activated by depolarization of the apical membrane voltage. These were identified as BK or maxi-K channels. The density of active SK channels revealed a high degree of clustering. Although heterogeneity of tubules or of cell types within a tubule could not be excluded, the major factor underlying the distribution appeared to be the presence of channel clusters on the membrane of individual cells. The overall density of channels was higher than that previously found in the cortical collecting tubule (CCT). In contrast to results in the CCT, we did not detect an increase in the overall density of SK channels in the apical membrane after feeding the animals a high-K diet. However, the activity of amiloride-sensitive Na channels was undetectable under control conditions but was increased after both 1 day (90 +/- 24 pA/cell) or 7 days (385 +/- 82 pA/cell) of K loading. Thus one important factor leading to an increased K secretion in the CNT in response to increased dietary K is an increased apical Na conductance, leading to depolarization of the apical membrane voltage and an increased driving force for K movement out into the tubular lumen. PMID:15280155

  6. Divalent ion trapping inside potassium channels of human T lymphocytes

    PubMed Central

    1989-01-01

    Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions approximately 25% of the channels were loaded with Ca2+, whereas under conditions with 10 microM internal Ca2+ the proportion of channels loaded with Ca2+ increased to approximately 50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation. PMID:2786551

  7. Potassium channels keep mobile cells on the go.

    PubMed

    Schwab, Albrecht; Hanley, Peter; Fabian, Anke; Stock, Christian

    2008-08-01

    Cell motility is a prerequisite for the creation of new life, and it is required for maintaining the integrity of an organism. Under pathological conditions, "too much" motility may cause premature death. Studies over the past few years have revealed that ion channels are essential for cell motility. This review highlights the importance of K+ channels in regulating cell motility. PMID:18697995

  8. Mechanisms of maurotoxin action on Shaker potassium channels.

    PubMed Central

    Avdonin, V; Nolan, B; Sabatier, J M; De Waard, M; Hoshi, T

    2000-01-01

    Maurotoxin (alpha-KTx6.2) is a toxin derived from the Tunisian chactoid scorpion Scorpio maurus palmatus, and it is a member of a new family of toxins that contain four disulfide bridges (, Eur. J. Biochem. 254:468-479). We investigated the mechanism of the maurotoxin action on voltage-gated K(+) channels expressed in Xenopus oocytes. Maurotoxin blocks the channels in a voltage-dependent manner, with its efficacy increasing with greater hyperpolarization. We show that an amino acid residue in the external mouth of the channel pore segment that is known to be involved in the actions of other peptide toxins is also involved in maurotoxin's interaction with the channel. We conclude that, despite the unusual disulfide bridge pattern, the mechanism of the maurotoxin action is similar to those of other K(+) channel toxins with only three disulfide bridges. PMID:10920011

  9. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    PubMed Central

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  10. Characterization of single potassium channels in mouse pancreatic acinar cells.

    PubMed Central

    Schmid, A; Schulz, I

    1995-01-01

    1. Single K(+)-selective channels with a conductance of about 48 pS (pipette, 145 mM KCl; bath, 140 mM NaCl + 4.7 mM KCl) were recorded in the patch-clamp whole-cell configuration in isolated mouse pancreatic acinar cells. 2. Neither application of the secretagogues acetylcholine (second messenger, inositol 1,4,5-trisphosphate) or secretin (second messenger, cAMP), nor addition of the catalytic subunit of protein kinase A to the pipette solution changed the activity of the 48 pS K+ channel. 3. Intracellular acidification with sodium propionate (20 mM) diminished activity of the 48 pS channel, whereas channel open probability was increased by cytosolic alkalization with 20 mM NH4Cl. 4. BaCl2 (5 mM), TEA (10 mM) or apamin (1 microM) added to the bath solution had no obvious effect on the kinetics of the 48 pS channel. Similarly, glibenclamide and diazoxide failed to influence the channel activity. 5. When extracellular NaCl was replaced by KCl, whole-cell recordings revealed an inwardly rectifying K+ current carried by a 17 pS K+ channel. 6. The inwardly rectifying K+ current was not pH dependent and could largely be blocked by Ba2+ but not by TEA. 7. Since the 48 pS K+ channel is neither Ca2+ nor cAMP regulated, we suggest that this channel could play a role in the maintenance of the negative cell resting potential. PMID:7623283

  11. Computational Studies of Venom Peptides Targeting Potassium Channels.

    PubMed

    Chen, Rong; Chung, Shin-Ho

    2015-12-01

    Small peptides isolated from the venom of animals are potential scaffolds for ion channel drug discovery. This review article mainly focuses on the computational studies that have advanced our understanding of how various toxins interfere with the function of K⁺ channels. We introduce the computational tools available for the study of toxin-channel interactions. We then discuss how these computational tools have been fruitfully applied to elucidate the mechanisms of action of a wide range of venom peptides from scorpions, spiders, and sea anemone. PMID:26633507

  12. Computational Studies of Venom Peptides Targeting Potassium Channels

    PubMed Central

    Chen, Rong; Chung, Shin-Ho

    2015-01-01

    Small peptides isolated from the venom of animals are potential scaffolds for ion channel drug discovery. This review article mainly focuses on the computational studies that have advanced our understanding of how various toxins interfere with the function of K+ channels. We introduce the computational tools available for the study of toxin-channel interactions. We then discuss how these computational tools have been fruitfully applied to elucidate the mechanisms of action of a wide range of venom peptides from scorpions, spiders, and sea anemone. PMID:26633507

  13. Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    PubMed Central

    Schaarschmidt, Grit; Wegner, Florian; Schwarz, Sigrid C.; Schmidt, Hartmut; Schwarz, Johannes

    2009-01-01

    Background Voltage-gated potassium (Kv) channels are among the earliest ion channels to appear during brain development, suggesting a functional requirement for progenitor cell proliferation and/or differentiation. We tested this hypothesis, using human neural progenitor cells (hNPCs) as a model system. Methodology/Principal Findings In proliferating hNPCs a broad spectrum of Kv channel subtypes was identified using quantitative real-time PCR with a predominant expression of the A-type channel Kv4.2. In whole-cell patch-clamp recordings Kv currents were separated into a large transient component characteristic for fast-inactivating A-type potassium channels (IA) and a small, sustained component produced by delayed-rectifying channels (IK). During differentiation the expression of IA as well as A-type channel transcripts dramatically decreased, while IK producing delayed-rectifiers were upregulated. Both Kv currents were differentially inhibited by selective neurotoxins like phrixotoxin-1 and α-dendrotoxin as well as by antagonists like 4-aminopyridine, ammoniumchloride, tetraethylammonium chloride and quinidine. In viability and proliferation assays chronic inhibition of the A-type currents severely disturbed the cell cycle and precluded proper hNPC proliferation, while the blockade of delayed-rectifiers by α-dendrotoxin increased proliferation. Conclusions/Significance These findings suggest that A-type potassium currents are essential for proper proliferation of immature multipotent hNPCs. PMID:19584922

  14. Adrenergic modulation of the delayed rectifier potassium channel in calf cardiac Purkinje fibers.

    PubMed Central

    Bennett, P; McKinney, L; Begenisich, T; Kass, R S

    1986-01-01

    We have investigated the modulation of the delayed rectifier potassium channel in calf cardiac Purkinje fibers by the neurohormone norepinephrine. We find that 0.5 microM norepinephrine increases this K channel current by a factor of 2.7. A maximal increase of about four was found for concentrations of 1 microM and above. Norepinephrine produced a small (less than 5 mV) and variable shift of the K channel reversal potential toward more negative values. The kinetics of the potassium channel are well described by a two-exponential process, both in the absence and presence of norepinephrine. However, norepinephrine substantially decreases the slower time constant with no significant effect on the fast time constant. Potassium channel activation curves in the presence of norepinephrine are very similar to control curves except at large positive potentials. A simple sequential three-state model for this channel can reproduce these data both with and without norepinephrine. The logarithms of the rate constants derived from this model are quadratic functions of voltage, suggesting the involvement of electric field-induced dipoles in the gating of this channel. Most of the kinetic effects of norepinephrine appear to be on a single rate constant. PMID:2424513

  15. Non-Michaelis-Menten kinetics model for conductance of low-conductance potassium ion channels.

    PubMed

    Tolokh, Igor S; Tolokh, Illya I; Cho, Hee Cheol; D'Avanzo, Nazzareno; Backx, Peter H; Goldman, Saul; Gray, C G

    2005-02-01

    A reduced kinetics model is proposed for ion permeation in low-conductance potassium ion channels with zero net electrical charge in the selectivity filter region. The selectivity filter is assumed to be the only conductance-determining part of the channel. Ion entry and exit rate constants depend on the occupancy of the filter due to ion-ion interactions. The corresponding rates are assumed slow relative to the rates of ion motion between binding sites inside the filter, allowing a reduction of the kinetics model of the filter by averaging the entry and exit rate constants over the states with a particular occupancy number. The reduced kinetics model for low-conductance channels is described by only three states and two sets of effective rate constants characterizing transitions between these states. An explicit expression for the channel conductance as a function of symmetrical external ion concentration is derived under the assumption that the average electrical mobility of ions in the selectivity filter region in a limited range of ion concentrations does not depend on these concentrations. The simplified conductance model is shown to provide a good description of the experimentally observed conductance-concentration curve for the low-conductance potassium channel Kir2.1, and also predicts the mean occupancy of the selectivity filter of this channel. We find that at physiological external ion concentrations this occupancy is much lower than the value of two ions observed for one of the high-conductance potassium channels, KcsA.

  16. ATP Sensitive Potassium Channels in the Skeletal Muscle Function: Involvement of the KCNJ11(Kir6.2) Gene in the Determination of Mechanical Warner Bratzer Shear Force

    PubMed Central

    Tricarico, Domenico; Selvaggi, Maria; Passantino, Giuseppe; De Palo, Pasquale; Dario, Cataldo; Centoducati, Pasquale; Tateo, Alessandra; Curci, Angela; Maqoud, Fatima; Mele, Antonietta; Camerino, Giulia M.; Liantonio, Antonella; Imbrici, Paola; Zizzo, Nicola

    2016-01-01

    The ATP-sensitive K+-channels (KATP) are distributed in the tissues coupling metabolism with K+ ions efflux. KATP subunits are encoded by KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1), and ABCC9 (SUR2) genes, alternative RNA splicing give rise to SUR variants that confer distinct physiological properties on the channel. An high expression/activity of the sarco-KATP channel is observed in various rat fast-twitch muscles, characterized by elevated muscle strength, while a low expression/activity is observed in the slow-twitch muscles characterized by reduced strength and frailty. Down-regulation of the KATP subunits of fast-twitch fibers is found in conditions characterized by weakness and frailty. KCNJ11 gene knockout mice have reduced glycogen, lean phenotype, lower body fat, and weakness. KATP channel is also a sensor of muscle atrophy. The KCNJ11 gene is located on BTA15, close to a QTL for meat tenderness, it has also a role in glycogen storage, a key mechanism of the postmortem transformation of muscle into meat. The role of KCNJ11 gene in muscle function may underlie an effect of KCNJ11 genotypes on meat tenderness, as recently reported. The fiber phenotype and genotype are important in livestock production science. Quantitative traits including meat production and quality are influenced both by environment and genes. Molecular markers can play an important role in the genetic improvement of animals through breeding strategies. Many factors influence the muscle Warner-Bratzler shear force including breed, age, feeding, the biochemical, and functional parameters. The role of KCNJ11gene and related genes on muscle tenderness will be discussed in the present review. PMID:27242541

  17. Combinatorial augmentation of voltage-gated KCNQ potassium channels by chemical openers

    PubMed Central

    Xiong, Qiaojie; Sun, Haiyan; Zhang, Yangming; Nan, Fajun; Li, Min

    2008-01-01

    Noninactivating potassium current formed by KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) subunits resembles neuronal M-currents which are activated by voltage and play a critical role in controlling membrane excitability. Activation of voltage-gated potassium channels by a chemical opener is uncommon. Therefore, the mechanisms of action are worthy further investigation. Retigabine and zinc pyrithione are two activators for KCNQ channels but their molecular interactions with KCNQ channel remain largely elusive. Here we report that retigabine and zinc pyrithione recognize two different sites of KCNQ2 channels. Their agonistic actions are noncompetitive and allow for simultaneous binding of two different activators on the same channel complex, hence giving rise to combinatorial potentiation with characteristic properties of both openers. Examining their effects on mutant channels, we showed zinc pyrithione is capable of opening nonconductive channels and coapplication of zinc pyrithione and retigabine could restore a disease mutant channel similar to wild type. Our results indicate two independent activator binding sites present in KCNQ channels. The resultant combinatorial potentiation by multiple synthetic chemical openers indicates that KCNQ channels are accessible to various types and combinations of pharmacological regulation. PMID:18272489

  18. Astemizole Derivatives as Fluorescent Probes for hERG Potassium Channel Imaging.

    PubMed

    Wang, Beilei; Liu, Zhenzhen; Ma, Zhao; Li, Minyong; Du, Lupei

    2016-03-10

    The detection and imaging of hERG potassium channels in living cells can provide useful information for hERG-correlation studies. Herein, three small-molecule fluorescent probes, based on the potent hERG channel inhibitor astemizole, for the imaging of hERG channels in hERG-transfected HEK293 cells (hERG-HEK293) and human colorectal cancer cells (HT-29), are described. These probes are expected to be applied in the physiological and pathological studies of hERG channels. PMID:26985309

  19. OsKAT2 is the prevailing functional inward rectifier potassium channels in rice guard cell.

    PubMed

    Hwang, Hyunsik; Yoon, Jin-Young; Cho, Hana; Kim, Beom-Gi

    2013-01-01

    AtKAT1 plays roles as a major channel to uptake K(+) in guard cell when stomata open in dicot model plant Arabidopsis. In a recent publication, we isolated 3 KAT-like potassium channels in rice. We expressed them in CHO cell to identify electrophysiological characteristics of the channels. OsKAT2 showed much bigger inwardly rectifying potassium channel activities among them. The histochemical X-glu staining of transgenic rice leaf blades expressing β-glucuronidase fused with OsKAT2 promoter showed that the OsKAT2 is dominantly expressed in rice guard cell. These findings indicate that OsKAT2 may be a functional ortholog of AtKAT1 in rice. Thus this gene will be the prime target for engineering the guard cell movement to improve drought tolerance in monocot plants, including most major crops.

  20. Regulation of Aldosterone Biosynthesis by the Kir3.4 (KCNJ5) Potassium Channel

    PubMed Central

    Velarde-Miranda, Carolina; Gomez-Sanchez, Elise P.; Gomez-Sanchez, Celso E.

    2013-01-01

    Summary The G-protein-activated inwardly rectifying potassium channel Kir3.4 is expressed in the zona glomerulosa cell membrane and transports potassium out of the cell. Angiotensin II stimulation of aldosterone secretion is mediated in part by suppression of the transcription of KCNJ5, the gene coding for Kir3.4, and blocking channel activity. This results in membrane depolarization, mobilization of intracellular calcium, activation of the calcium-calmodulin pathway, and increasing gene transcription of steroidogenic enzymes required for aldosterone secretion. In 40–60% of aldosterone-producing adenomas there is a somatic mutation in the region of the KCNJ5 gene that codes for the selectivity filter that decreases potassium selectivity, allowing sodium to leak into the cells, thus depolarizing the membrane and initiating events that result in increased aldosterone synthesis. The mechanism by which mutated KCNJ5 induces cell proliferation and adenoma formation remains unclear. PMID:23829355

  1. Characterization of apical potassium channels induced in rat distal colon during potassium adaptation.

    PubMed Central

    Butterfield, I; Warhurst, G; Jones, M N; Sandle, G I

    1997-01-01

    1. Chronic dietary K+ loading stimulates an active K+ secretory process in rat distal colon, which involves an increase in the macroscopic apical K+ conductance of surface epithelial cells. In the present study, the abundance and characteristics of K+ channels constituting this enhanced apical K+ conductance were evaluated using patch clamp recording techniques. 2. In isolated non-polarized surface cells, K+ channels were seen in 9 of 90 (10%) cell-attached patches in cells from control animals, and in 247 of 437 (57%) cell-attached patches in cells from K(+)-loaded animals, with a significant (P < 0.001) shift in distribution density. Similarly, recordings from cell-attached patches of the apical membrane of surface cells surrounding the openings of distal colonic crypts revealed identical K+ channels in 1 of 11 (9%) patches in control animals, and in 9 of 13 (69%) patches in K(+)-loaded animals. 3. In isolated surface cells and surface cells in situ, K+ channels had mean slope conductances of 209 +/- 6 and 233 +/- 14 pS, respectively, when inside-out patches were bathed symmetrically in K2SO4 solution. The channels were sensitive to 'cytosolic' Ca2+ concentration, were voltage sensitive at 'cytosolic' Ca2+ concentrations encountered in colonic epithelial cells, and were inhibited by 1 mM quinidine, 20 mM TEA or 5 mM Ba2+ ions. 4. The data show that dietary K+ loading increases the abundance of Ca(2+)- and voltage-sensitive large-conductance K+ channels in the apical membrane of surface cells in rat distal colon. These channels constitute the enhanced macroscopic apical K+ conductance previously identified in these cells, and are likely to play a critical role in the active K+ secretory process that typifies this model of colonic K+ adaptation. PMID:9218214

  2. Distinct potassium channels on pain-sensing neurons.

    PubMed

    Rasband, M N; Park, E W; Vanderah, T W; Lai, J; Porreca, F; Trimmer, J S

    2001-11-01

    Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K(+) (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 alpha subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in A delta and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na(+) channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 alpha subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells. PMID:11698689

  3. The effect of copper sulfate, potassium permanganate, and peracetic acid on Ichthyobodo necator in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ichthyobodo necator is a single celled biflagellate that can cause significant mortalities in fish, particularly young, tank-reared fish. Copper sulfate (CuSO4), potassium permanganate (KMnO4) and peracetic acid (PAA) were evaluated for effectiveness against Ichthybodosis in juvenile channel catfis...

  4. Modification of sodium and potassium channel gating kinetics by ether and halothane

    SciTech Connect

    Bean, B.P.; Shrager, P.; Goldstein, D.A.

    1981-03-01

    The effects of ether and halothane on the kinetics of sodium and potassium currents were investigated in the crayfish giant axon. Both general anesthetics produced a reversible, dose-dependent speeding up of sodium current inactivation at all membrane potentials, with no change in the rising phase of the currents. Double-pulse inactivation experiments with ether also showed faster inactivation, but the rate of recovery from inactivation at negative potentials was not affected. Ether shifted the midpoint of the steady-state fast inactivation curve in the hyperpolarizing direction and made the curve steeper. The activation of potassium currents was faster with ether present, with no change in the voltage dependence of steady-state potassium currents. Ether and halothane are known to perturb the structure of lipid bilayer membranes; the alterations in sodium and potassium channel gating kinetics are consistent with the hypothesis that the rats of the gating processes of the channels can be affected by the state of the lipids surrounding the channels, but a direct effect of ether and halothane on the protein part of the channels cannot be ruled out.

  5. Altered potassium ATP channel signaling in mesenteric arteries of old high salt-fed rats

    PubMed Central

    Whidden, Melissa A.; Basgut, Bilgen; Kirichenko, Nataliya; Erdos, Benedek; Tümer, Nihal

    2016-01-01

    [Purpose] Both aging and the consumption of a high salt diet are associated with clear changes in the vascular system that can lead to the development of cardiovascular disease; however the mechanisms are not clearly understood. Therefore, we examined whether aging and the consumption of excess salt alters the function of potassium ATP-dependent channel signaling in mesenteric arteries [Methods] Young (7 months) and old (29 months) Fischer 344 x Brown Norway rats were fed a control or a high salt diet (8% NaCl) for 12 days and mesenteric arteries were utilized for vascular reactivity measurements. [Results] Acetylcholine-induced endothelium relaxation was significantly reduced in old arteries (81 ± 4%) when compared with young arteries (92 ± 2%). Pretreatment with the potassium-ATP channel blocker glibenclamide reduced relaxation to acetylcholine in young arteries but did not alter dilation in old arteries. On a high salt diet, endothelium dilation to acetylcholine was significantly reduced in old salt arteries (60 ± 3%) when compared with old control arteries (81 ± 4%). Glibenclamide reduced acetylcholine-induced dilation in young salt arteries but had no effect on old salt arteries. Dilation to cromakalim, a potassium-ATP channel opener, was reduced in old salt arteries when compared with old control arteries. [Conclusion] These findings demonstrate that aging impairs endothelium-dependent relaxation in mesenteric arteries. Furthermore, a high salt diet alters the function of potassium-ATP-dependent channel signaling in old isolated mesenteric arteries and affects the mediation of relaxation stimuli. PMID:27508155

  6. Role of Calcium-activated Potassium Channels in Atrial Fibrillation Pathophysiology and Therapy.

    PubMed

    Diness, Jonas G; Bentzen, Bo H; Sørensen, Ulrik S; Grunnet, Morten

    2015-11-01

    Small-conductance Ca(2+)-activated potassium (SK) channels are relative newcomers within the field of cardiac electrophysiology. In recent years, an increased focus has been given to these channels because they might constitute a relatively atrial-selective target. This review will give a general introduction to SK channels followed by their proposed function in the heart under normal and pathophysiological conditions. It is revealed how antiarrhythmic effects can be obtained by SK channel inhibition in a number of species in situations of atrial fibrillation. On the contrary, the beneficial effects of SK channel inhibition in situations of heart failure are questionable and still needs investigation. The understanding of cardiac SK channels is rapidly increasing these years, and it is hoped that this will clarify whether SK channel inhibition has potential as a new anti-atrial fibrillation principle. PMID:25830485

  7. Role of Calcium-activated Potassium Channels in Atrial Fibrillation Pathophysiology and Therapy.

    PubMed

    Diness, Jonas G; Bentzen, Bo H; Sørensen, Ulrik S; Grunnet, Morten

    2015-11-01

    Small-conductance Ca(2+)-activated potassium (SK) channels are relative newcomers within the field of cardiac electrophysiology. In recent years, an increased focus has been given to these channels because they might constitute a relatively atrial-selective target. This review will give a general introduction to SK channels followed by their proposed function in the heart under normal and pathophysiological conditions. It is revealed how antiarrhythmic effects can be obtained by SK channel inhibition in a number of species in situations of atrial fibrillation. On the contrary, the beneficial effects of SK channel inhibition in situations of heart failure are questionable and still needs investigation. The understanding of cardiac SK channels is rapidly increasing these years, and it is hoped that this will clarify whether SK channel inhibition has potential as a new anti-atrial fibrillation principle.

  8. Ligand action on sodium, potassium, and calcium channels: role of permeant ions.

    PubMed

    Zhorov, Boris S; Tikhonov, Denis B

    2013-03-01

    Ion channels are targets for many naturally occurring toxins and small-molecule drugs. Despite great progress in the X-ray crystallography of ion channels, we still do not have a complete understanding of the atomistic mechanisms of channel modulation by ligands. In particular, the importance of the simultaneous interaction of permeant ions with the ligand and the channel protein has not been the focus of much attention. Considering these interactions often allows one to rationalize the highly diverse experimental data within the framework of relatively simple structural models. This has been illustrated in earlier studies on the action of local anesthetics, sodium channel activators, as well as blockers of potassium and calcium channels. Here, we discuss the available data with a view to understanding the use-, voltage-, and current carrying cation-dependence of the ligand action, paradoxes in structure--activity relationships, and effects of mutations in these ion channels.

  9. Renal outer medullary potassium channel knockout models reveal thick ascending limb function and dysfunction.

    PubMed

    Wang, Tong

    2012-02-01

    The renal outer medullary potassium channel (ROMK) is an adenosine triphosphate-sensitive inward-rectifier potassium channel (Kir1.1 or KCNJ1) highly expressed in the cortical and medullary thick ascending limbs (TAL), connecting segment (CNT) and cortical collecting duct (CCD) in the mammalian kidney, where it serves to recycle potassium (K(+)) across the apical membrane in TAL and to secrete K(+) in the CNT and CCD. ROMK channel mutations cause type II Bartter's syndrome with salt wasting and dehydration, and ROMK knockout mice display a similar phenotype of Bartter's syndrome in humans. Studies from ROMK null mice indicate that ROMK is required to form both the small-conductance (30pS, SK) K channels and the 70pS (IK) K channels in the TAL. The availability of ROMK(-/-) mice has made it possible to study electrolyte transport along the nephron in order to understand the TAL function under physiological conditions and the compensatory mechanisms of salt and water transport under the conditions of TAL dysfunction. This review summarizes previous progress in the study of K(+) channel activity in the TAL and CCD, ion transporter expression and activities along the nephron, and renal functions under physiological and pathophysiological conditions using ROMK(-/-) mice. PMID:22038261

  10. Effects of donepezil on hERG potassium channels.

    PubMed

    Chae, Yun Ju; Lee, Hong Joon; Jeon, Ji Hyun; Kim, In-Beom; Choi, Jin-Sung; Sung, Ki-Wug; Hahn, Sang June

    2015-02-01

    Donepezil is a potent, selective inhibitor of acetylcholinesterase, which is used for the treatment of Alzheimer's disease. Whole-cell patch-clamp technique and Western blot analyses were used to study the effects of donepezil on the human ether-a-go-go-related gene (hERG) channel. Donepezil inhibited the tail current of the hERG in a concentration-dependent manner with an IC50 of 1.3 μM. The metabolites of donepezil, 6-ODD and 5-ODD, inhibited the hERG currents in a similar concentration-dependent manner; the IC50 values were 1.0 and 1.5 μM, respectively. A fast drug perfusion system demonstrated that donepezil interacted with both the open and inactivated states of the hERG. A fast application of donepezil during the tail currents inhibited the open state of the hERG in a concentration-dependent manner with an IC50 of 2.7 μM. Kinetic analysis of donepezil in an open state of the hERG yielded blocking and unblocking rate constants of 0.54 µM(-1)s(-1) and 1.82 s(-1), respectively. The block of the hERG by donepezil was voltage-dependent with a steep increase across the voltage range of channel activation. Donepezil caused a reduction in the hERG channel protein trafficking to the plasma membrane at low concentration, but decreased the channel protein expression at higher concentrations. These results suggest that donepezil inhibited the hERG at a supratherapeutic concentration, and that it did so by preferentially binding to the activated (open and/or inactivated) states of the channels and by inhibiting the trafficking and expression of the hERG channel protein in the plasma membrane.

  11. Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain

    PubMed Central

    Knaus, Hans‐Günther; Schwarzer, Christoph

    2015-01-01

    ABSTRACT The sodium‐activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high‐conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093–2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26587966

  12. Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles

    PubMed Central

    Aimon, Sophie; Manzi, John; Schmidt, Daniel; Poveda Larrosa, Jose Antonio; Bassereau, Patricia; Toombes, Gilman E. S.

    2011-01-01

    Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability. PMID:21998666

  13. Effect of astragaloside IV against rat myocardial cell apoptosis induced by oxidative stress via mitochondrial ATP-sensitive potassium channels.

    PubMed

    Guan, Feng-Ying; Yang, Shi-Jie; Liu, Jinxiang; Yang, Si-Rui

    2015-07-01

    Astragaloside is one of the most common traditional Chinese medicines and is derived from Astragalus membranaceus. Astragaloside IV (AsIV) is a monomer located in an extract of astragaloside. The current study investigated the protective effects of AsIV against hydrogen peroxide (H2O2)-induced injury in cardiocytes and elucidated the mechanisms responsible for this protective effect. Cultured neonatal rat cardiocytes were divided into five experimental groups as follows: i) Dimethyl sulfoxide; ii) H2O2; iii) AsIV+H2O2; iv) AsIV+H2O2+5-hydroxydecanoate (5-HD); and v) nicorandil+H2O2. Cardiocyte survival was analyzed using an MTT assay. Lactate dehydrogenase (LDH) release was also assessed to evaluate the viability of the cells. Intracellular reactive oxygen species (ROS) were measured by 2,7-dichlorodihydrofluorescein diacetate staining. The apoptotic rate was measured by flow cytometry. Mitochondrial membrane potential (ΔΨm) and intracellular calcium were observed using a laser confocal microscopy system. The results indicated that AsIV promoted the survival of cardiocytes (P<0.05), attenuated LDH release (P<0.05), ROS production (P<0.01) and apoptosis (P<0.01), stabilized the ΔΨm and reduced intracellular calcium overload (P<0.01) compared with the H2O2 group. The mitochondrial adenosine triphosphate-sensitive potassium channel (mitoKATP) inhibitor 5-HD was observed to partially reverse the protective effect of AsIV. Following treatment with 5-HD, the survival of cardiocytes was reduced (P<0.05), LDH release (P<0.01) and ROS production (P<0.05) were stimulated, ΔΨm and intracellular calcium change were increased (P<0.01) and apoptosis was increased (P<0.01) compared with the AsIV+H2O2 group. Thus, AsIV has potential for use in the suppression of apoptosis resulting from H2O2 exposure, and mitoKATP activation may underlie this protective mechanism. PMID:25739067

  14. Atomic basis for therapeutic activation of neuronal potassium channels

    NASA Astrophysics Data System (ADS)

    Kim, Robin Y.; Yau, Michael C.; Galpin, Jason D.; Seebohm, Guiscard; Ahern, Christopher A.; Pless, Stephan A.; Kurata, Harley T.

    2015-09-01

    Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

  15. Atomic basis for therapeutic activation of neuronal potassium channels

    PubMed Central

    Kim, Robin Y.; Yau, Michael C.; Galpin, Jason D.; Seebohm, Guiscard; Ahern, Christopher A.; Pless, Stephan A.; Kurata, Harley T.

    2015-01-01

    Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2–5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators. PMID:26333338

  16. Magnesium modulates ROMK channel-mediated potassium secretion.

    PubMed

    Yang, Lei; Frindt, Gustavo; Palmer, Lawrence G

    2010-12-01

    The ability of intracellular and extracellular Mg(2+) to block secretory K(+) currents through ROMK channels under physiologic conditions is incompletely understood. We expressed ROMK2 channels in Xenopus oocytes and measured unitary currents in the inside-out and cell-attached modes of the patch-clamp technique. With 110 mM K(+) on both sides of the membrane, 0.2 to 5 mM Mg(2+) on the cytoplasmic side reduced outward currents, but not inward currents, at V(m) > 0. With 11 or 1.1 mM extracellular K(+) ([K(+)](o)), ≥0.2 mM Mg(2+) blocked outward currents in the physiologic V(m) range (0 to -60 mV). With decreasing [K(+)](o), the apparent dissociation constant of the blocker decreased, but the voltage dependence of block did not significantly change. Whole-cell recordings from principal cells of rat cortical collecting ducts revealed similar inhibitory effects of intracellular Mg(2+). Mg(2+) added to the extracellular solution also reduced single-channel currents with an affinity that increased as [K(+)](o) decreased. In conclusion, physiologic concentrations of intracellular and extracellular Mg(2+) can influence secretory K(+) currents through ROMK channels. These effects could play a role in the modulation of K(+) transport under conditions of K(+) and/or Mg(2+) depletion. PMID:21030597

  17. Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

    PubMed

    Peigneur, Steve; Yamaguchi, Yoko; Kawano, Chihiro; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki

    2016-05-31

    Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency. PMID:27159046

  18. K2P potassium channels, mysterious and paradoxically exciting.

    PubMed

    Goldstein, Steve A N

    2011-08-01

    New evidence reveals that the common electrolyte disorder hypokalemia can induce K2P1 channels that are normally selective for K+ to break the rules and conduct Na+. This defiant behavior leads to paradoxical depolarization of many cells in the heart, increasing the risk for lethal arrhythmia. The new research resolves a mystery uncovered 50 years ago and bestows an array of new riddles. Here, I discuss how K2P1 might achieve this alchemy--through stable residence of the K+ selectivity filter in a Na+-conductive state between its open and C-inactive configurations--and predict that other K+ channels and environmental stimuli will be discovered to produce the same excitatory misconduct. PMID:21868351

  19. Voltage-sensitive potassium channels in Limulus ventral photoreceptors

    PubMed Central

    1978-01-01

    The steady-state slope conductance of Limulus ventral photoreceptors increases markedly when the membrane is depolarized from rest. The ionic basis of this rectification has been examined with a voltage- clamp technique. Tail currents that occur when membrane potential is repolarized after having been depolarized have been identified. The tail currents reverse direction at a voltage that becomes more positive when Ko is increased. Rectification is reduced by extracellular 4- aminopyridine and by intracellular injection of tetra-ethyl-ammonium (TEA). These results indicate that the membrane rectification around resting potential is due primarily to voltage-sensitive K+ channels. The increase in gK caused by depolarization is not mediated by a voltage-dependent rise in in Cai++, since intracellular injection of Ca++ causes a decrease rather than an increase in slope conductance. TEA can be used to examine the functional role of the K+ channels because it blocks them without substantially affecting the light- activated Na+ conductance. The effect of TEA on response-intensity curves shows that the K+ channels serve to compress the voltage range of receptor potentials. PMID:621492

  20. A paradox concerning ion permeation of the delayed rectifier potassium ion channel in squid giant axons.

    PubMed Central

    Clay, J R

    1991-01-01

    1. The fully activated current-voltage relation (I-V) of the delayed rectifier potassium ion channel in squid giant axons has a non-linear dependence upon the driving force, V-EK, as I have previously demonstrated, where V is membrane potential and EK is the equilibrium potential for potassium ions. 2. The non-linearity of the I-V relation and its dependence upon external potassium ion concentration are both well described, phenomenologically, by the Goldman-Hodgkin-Katz (GHK) flux equation, as I have also previously demonstrated. As illustrated below, this result can be modelled using the Eyring rate theory of single-file diffusion of ions through a channel in the low-occupancy limit of the theory. 3. The GHK equation analysis and the low-occupancy limit of the Eyring rate theory are both consistent with the independence principle for movement of ions through the channel, which is at odds with tracer flux ratio results from the delayed rectifier, published elsewhere. Those results suggest that the channel is multiply occupied by two, or perhaps three, ions. 4. The resolution of this paradox is provided by a triple-binding site, multiple-occupancy model in which only one vacancy, at most, is allowed in the channel. This model predicts current-voltage relations which are consistent with the data (and with the phenomenological prediction of the GHK flux equation). The model is also consistent, approximately, with the tracer flux ratio results. PMID:1822560

  1. Voltage-gated potassium+ channel expression in coronary artery smooth muscle cells of SHR and WKY.

    PubMed

    Hu, Zhi; Ma, Aiqun; Zhang, Yushun; Xi, Yutao; Fan, Lihong; Wang, Tingzhong; Zhang, Tingting

    2014-12-01

    This study aims to compare the expression of genes and the molecular characteristic of voltage-gated K(+) channels, which make great effort in maintaining and controlling smooth muscle contraction, cellular membrane potential, and intracellular calcium ion currents in artery smooth muscle cells of SHR and WKY. Expression of potassium ions family in coronary artery was detected through reverse transcription polymerase chain reaction quantitatively. Significant levels of voltage-gated K(+) channels α1.2, α1.5, and β1.1 expression were all proved to be significantly higher in smooth muscles of SHR than WKY. Whole-cell voltage-gated K(+) channel currents were larger in SHR artery smooth muscles than the ones of WKY. Moreover, the voltage dependence of voltage-gated potassium channel activation was more negative in artery smooth muscle of SHR than that of WKY, while voltage dependence of availability was not different. The above diversity of voltage-gated potassium channel detected in gene expression and electrical character in coronary artery smooth muscle of SHR than that of WKY might be an underling mechanism associated with the membrane potential depolarization in artery smooth muscle of SHR.

  2. Analysis and functional implications of phosphorylation of neuronal voltage-gated potassium channels

    PubMed Central

    Cerda, Oscar; Trimmer, James S.

    2012-01-01

    Phosphorylation is the most common and abundant posttranslational modification to eukaryotic proteins, regulating a plethora of dynamic cellular processes. Here, we review and discuss recent advances in our knowledge of the breadth and importance of reversible phosphorylation in regulating the expression, localization and function of mammalian neuronal voltage-gated potassium (Kv) channels, key regulators of neuronal function. We highlight the role of modern mass spectrometric techniques and phosphospecific antibodies that reveal the extent and nature of phosphorylation at specific sites in Kv channels. We also emphasize the role of reversible phosphorylation in dynamically regulating diverse aspects of Kv channel biology. Finally, we discuss as important future directions the determination of the mechanistic basis for how altering phosphorylation state affects Kv channel expression, localization and function, the nature of macromolecular signaling complexes containing Kv channels and enzymes regulating their phosphorylation state, and the specific role of Kv channel phosphorylation in regulating neuronal function during physiological and pathophysiological events. PMID:20600597

  3. Nitric Oxide Regulates The Lymphatic Reactivity Following Hemorrhagic Shock Through Atp-Sensitive Potassium Channel.

    PubMed

    Zhang, Li-Min; Qin, Li-Peng; Zhang, Yu-Ping; Zhao, Zi-Gang; Niu, Chun-Yu

    2016-06-01

    Lymphatic reactivity has been shown to exhibit a biphasic change following hemorrhagic shock, and nitric oxide (NO) is involved in this process. However, the precise mechanism responsible for NO regulation of the lymphatic reactivity along with the progression of hemorrhagic shock is unclear. Therefore, the present study was to investigate how NO participates in regulating the shock-induced biphasic changes in lymphatic reactivity and its underlying mechanisms. First, the expressions or contents of inducible NO synthase, nitrite plus nitrate, and elements of cAMP-PKA-KATP and cGMP-PKG-KATP pathway in thoracic ducts tissue were assessed. The results revealed that levels of nitrite plus nitrate, cAMP, cyclic guanosine monophosphate (cGMP), p-PKA, and p-PKG were increased gradually along with the process of shock. Second, the roles of cAMP-PKA-KATP and cGMP-PKG-KATP in NO regulating lymphatic response to gradient substance P were evaluated with an isolated lymphatic perfusion system. The results showed that the NOS substrate (L-Arg), PKA donor (8-Br-cAMP) decreased the reactivity of shock 0.5 h-lymphatics, and that the PKA inhibitor (H-89) and KATP inhibitor (glibenclamide) restrained the effects of L-Arg while glibenclamide abolished the effects of 8-Br-cAMP. Meanwhile, NOS antagonist (L-NAME), protein kinase G (PKG) inhibitor (KT-5823), and soluble guanylate cyclase inhibitor (ODQ) increased the reactivity of shock 2 h-lymphatics, whereas KATP opener (pinacidil) inhibited these elevated effects induced by either L-NAME, ODQ, or KT-5823. Taken together, these results indicate that NO regulation of lymphatic reactivity during shock involves both cAMP-PKA-KATP and cGMP-PKG-KATP pathways. These findings have potential significance for the treatment of hemorrhagic shock through regulating lymphatic reactivity. PMID:26796572

  4. Electrical pumping of potassium ions against an external concentration gradient in a biological ion channel

    NASA Astrophysics Data System (ADS)

    Queralt-Martín, María; García-Giménez, Elena; Aguilella, Vicente M.; Ramirez, Patricio; Mafe, Salvador; Alcaraz, Antonio

    2013-07-01

    We show experimentally and theoretically that significant currents can be obtained with a biological ion channel, the OmpF porin of Escherichia coli, using zero-average potentials as driving forces. The channel rectifying properties can be used to pump potassium ions against an external concentration gradient under asymmetric pH conditions. The results are discussed in terms of the ionic selectivity and rectification ratio of the channel. The physical concepts involved may be applied to separation processes with synthetic nanopores and to bioelectrical phenomena.

  5. Fluorogenic Probe for the Human Ether-a-Go-Go-Related Gene Potassium Channel Imaging

    PubMed Central

    2016-01-01

    The first small-molecule fluorogenic probe A1 for imaging the human Ether-a-go-go-Related Gene (hERG) potassium channel based on the photoinduced electron transfer (PET) off–on mechanism was described herein. After careful biological evaluation, this probe had the potential of detecting and imaging the hERG channel at the molecular and cellular level. Moreover, the competitive binding mechanism of this probe would presumably minimize the effects on the electrophysiological properties of the hERG channel. Therefore, this probe may serve as a powerful toolkit to the hERG-associated study. PMID:25665091

  6. Two-pore domain potassium channels: potential therapeutic targets for the treatment of pain.

    PubMed

    Mathie, Alistair; Veale, Emma L

    2015-05-01

    Recent evidence points to a pivotal contribution of a variety of different potassium channels, including two-pore domain potassium (K2P) channels, in chronic pain processing. Expression of several different K2P channel subunits has been detected in nociceptive dorsal root ganglion neurons and trigeminal ganglion neurons, in particular, TREK1, TREK2, TRESK, TRAAK, TASK3 and TWIK1 channels. Of these, the strongest body of evidence from functional studies highlights the importance of TREK1, TRESK and, recently, TREK2 channels. For example, TREK1 knockout mice are more sensitive than wild-type mice to a number of painful stimuli but less sensitive to morphine-induced analgesia. TRESK knockdown mice show behavioural evidence of increased pain and increased sensitivity to painful pressure. Importantly, familial migraine with aura is associated with a dominant-negative mutation in human TRESK channels. Thus, the functional up-regulation of K2P channel activity may be a useful strategy in the development of new therapies for the treatment of pain. Whilst there are few currently available compounds that selectively and directly enhance the activity of TRESK and TREK2 channels, recent advances have been made in terms of identifying compounds that activate TREK1 channels and in understanding how they might act on the channel. Large-scale bio-informatic approaches and the further development of databases of putative ligands, channel structures and putative ligand binding sites on these structures may form the basis for future experimental strategies to detect novel molecules acting to enhance K2P channel activity that would be useful in the treatment of pain.

  7. Modeling the binding modes of Kv1.5 potassium channel and blockers.

    PubMed

    Yang, Qian; Du, Lupei; Wang, Xiaojian; Li, Minyong; You, Qidong

    2008-09-01

    The ultra-rapid delayed rectifier potassium current (I(Kur)), encoded by Kv1.5 gene, is the critical determinant of Phase I repolarization of action potential duration (APD). The evidences that Kv1.5 gene expresses more extensively in human atrial myocytes than in ventricle and the I(Kur) currents has not been recorded in the human ventricle, suggest Kv1.5 potassium channel as a selective target for the treatment of atrial fibrillation (AF). Recent mutagenesis studies have provided us some evidences that are useful in designing Kv1.5 blockers. In order to further evaluate these molecular biological information, the homology model of Kv1.5 potassium channel was established based on the Kv1.2 crystal structure (PDB entry: 2A79) using MODELLER 9v2 program. After the molecular dynamics refinement, the optimized homology model was assessed as a reliable structure by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and DOPE graph. The results of molecular docking studies on different Kv1.5 inhibitors are in agreement with the published mutagenesis data. Based on the docking conformations, a pharmacophore model was developed by HipHop algorithm in order to probe the common features of blockers. By analyzing the results, active site architecture, certain key residues and pharmacophore common-features that are responsible for substrate specificity were identified on the Kv1.5 potassium channel, which would be very helpful in understanding the blockade mechanism of Kv1.5 potassium channel and providing insights into rational design of novel Kv1.5 blockers. PMID:18485768

  8. Modeling the binding modes of Kv1.5 potassium channel and blockers.

    PubMed

    Yang, Qian; Du, Lupei; Wang, Xiaojian; Li, Minyong; You, Qidong

    2008-09-01

    The ultra-rapid delayed rectifier potassium current (I(Kur)), encoded by Kv1.5 gene, is the critical determinant of Phase I repolarization of action potential duration (APD). The evidences that Kv1.5 gene expresses more extensively in human atrial myocytes than in ventricle and the I(Kur) currents has not been recorded in the human ventricle, suggest Kv1.5 potassium channel as a selective target for the treatment of atrial fibrillation (AF). Recent mutagenesis studies have provided us some evidences that are useful in designing Kv1.5 blockers. In order to further evaluate these molecular biological information, the homology model of Kv1.5 potassium channel was established based on the Kv1.2 crystal structure (PDB entry: 2A79) using MODELLER 9v2 program. After the molecular dynamics refinement, the optimized homology model was assessed as a reliable structure by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and DOPE graph. The results of molecular docking studies on different Kv1.5 inhibitors are in agreement with the published mutagenesis data. Based on the docking conformations, a pharmacophore model was developed by HipHop algorithm in order to probe the common features of blockers. By analyzing the results, active site architecture, certain key residues and pharmacophore common-features that are responsible for substrate specificity were identified on the Kv1.5 potassium channel, which would be very helpful in understanding the blockade mechanism of Kv1.5 potassium channel and providing insights into rational design of novel Kv1.5 blockers.

  9. Trafficking of an endogenous potassium channel in adult ventricular myocytes

    PubMed Central

    Wang, Tiantian; Cheng, Yvonne; Dou, Ying; Goonesekara, Charitha; David, Jens-Peter; Steele, David F.; Huang, Chen

    2012-01-01

    The roles of several small GTPases in the expression of an endogenous potassium current, Ito,f, in adult rat ventricular myocytes have been investigated. The results indicate that forward trafficking of newly synthesized Kv4.2, which underlies Ito,f in these cells, requires both Rab1 and Sar1 function. Expression of a Rab1 dominant negative (DN) reduced Ito,f current density by roughly one-half relative to control, mCherry-transfected myocytes. Similarly, expression of a Sar1DN nearly halved Ito,f current density. Rab11 is not essential to trafficking of Kv4.2, as expression of a Rab11DN had no effect on Ito,f over the time frames investigated here. In a process dependent on intact endoplasmic reticulum (ER)-to-Golgi transport, however, overexpression of wild-type Rab11 resulted in a doubling of Ito,f density; block of ER-to-Golgi traffic by Brefeldin A completely abrogated the effect. Also implicated in the trafficking of Kv4.2 are Rab5 and Rab4. Rab5DN expression increased endogenous Ito,f by two- to threefold, nonadditively with inhibition of dynamin-dependent endocytosis. And, in a phenomenon similar to that previously reported for myoblast-expressed Kv1.5, Rab4DN expression roughly doubled endogenous peak transient currents. Colocalization experiments confirmed the involvement of Rab4 in postinternalization trafficking of Kv4.2. There was little role evident for the lysosome in the degradation of internalized Kv4.2, as overexpression of neither wild-type nor DN isoforms of Rab7 had any effect on Ito,f. Instead, degradation may depend largely on the proteasome; the proteasome inhibitor MG132 significantly increased Ito,f density. PMID:22914645

  10. [The role of adenosine Al receptors and mitochondrial K+ATP channels in the mechanism of increasing the resistance to acute hypoxia in the combined effects of hypoxia and hypercapnia].

    PubMed

    Tregub, P P; Kulikov, V P; Stepanova, L A; Zabrodina, A S; Nagibaeva, M E

    2014-01-01

    We studied the role of the role of mitoK+ATp channels and Al-adenosine receptor in the mechanism of increasing the resistance to acute hypoxia after hypoxic, hypercapnic and hypercapnic-hypoxic preconditioning. It is shown that mitochondrial ATP-sensitive potassium channels and Al-adenosine receptors, an important mechanism of preconditioning have a high value to increase the resistance to acute hypoxia/ischemia in the combined effect of hypoxia and hypercapnia. However, with regard to the adenosine receptor, this mechanism is realized without the participation hypercapnic component, which apparently starts neuroprotection without activation of the adenosine Al receptors. PMID:25980226

  11. Diversity of Potassium Channels in Human Umbilical Artery Smooth Muscle Cells

    PubMed Central

    Martín, Pedro; Rebolledo, Alejandro; Palomo, Ana Rocio Roldán; Moncada, Melisa; Piccinini, Luciano

    2014-01-01

    Through their control of cell membrane potential, potassium (K+) channels are among the best known regulators of vascular tone. This article discusses the expression and function of K+ channels in human umbilical artery smooth muscle cells (HUASMCs). We review the bibliographic reports and also present single-channel data recorded in freshly isolated cells. Electrophysiological properties of big conductance, voltage- and Ca2+-sensitive K+ channel and voltage-dependent K+ channels are clearly established in this vessel, where they are involved in contractile state regulation. Their role in the maintenance of membrane potential is an important control mechanism in the determination of the vessel diameter. Additionally, small conductance Ca2+-sensitive K+ channels, 2-pore domains K+ channels and inward rectifier K+ channels also appear to be present in HUASMCs, while intermediate conductance Ca2+-sensitive K+ channels and ATP-sensitive K+ channels could not be identified. In both cases, additional investigation is necessary to reach conclusive evidence of their expression and/or functional role in HUASMCs. Finally, we discuss the role of K+ channels in pregnancy-related pathologies like gestational diabetes and preeclampsia. PMID:24084522

  12. Chronic ethanol-induced changes in cardiac and neuronal ATP-sensitive potassium channels

    SciTech Connect

    Bangalore, R.; Hawthorn, M.; Triggle, D.J. )

    1992-02-26

    The present study was designed to investigate the effect of chronic ethanol consumption on cardiac and neuronal ATP-sensitive potassium channels. These channels have been shown to be regulated under diseased conditions such as congestive heart failure. Rats were chronically fed with a liquid diet containing ethanol or equicaloric amount of dextrin for the three weeks. This diet induced tolerance to ethanol as assessed by the longer time the ethanol fed rats could stay on a rotorod compared to control rats when challenged with an i.p. injection of ethanol, ATP-sensitive potassium channels were characterized using ({sup 3}H)glibenclamide binding to membrane preparations from heart, olfactory bulb, hippocampus, striatum, cerebellum, cortex, brain stem and spinal cord. Chronic ethanol consumption caused a significant increase in the K{sub D} value in the hippocampus and cerebellum, and a significant decrease in the K{sub D} value in the cortex. The K{sub D} value did not change in other brain areas and heart with chronic ethanol consumption. In contrast, chronic ethanol caused a significant decrease in the B{sub max} value in the heart, and a slight but significant increase in the B{sub max} value in the spinal cord. Chronic ethanol did not affect the B{sub max} value in other brain areas. ATP-sensitive potassium channels are differently regulated by ethanol in cardiac and neuronal preparations.

  13. Potassium channel-mediated relaxation to acetylcholine in rabbit arteries.

    PubMed

    Cowan, C L; Palacino, J J; Najibi, S; Cohen, R A

    1993-09-01

    Endothelium-dependent relaxation is associated with smooth muscle hyperpolarization in many arteries which may account for relaxation that persists in the presence of nitric oxide inhibitors such as NG-nitro-L-arginine methyl ester (L-NAME). Acetylcholine (ACh)-induced relaxations of the rabbit thoracic and abdominal aorta and iliac and carotid arteries were studied for the relative contribution of nitric oxide-dependent and -independent mechanisms in rings suspended for measurement of isometric tension. Although relaxation of the thoracic aorta to ACh (10(-6) M) was almost blocked completely by L-NAME (3 x 10(-5) M), the maximal relaxation in the abdominal aorta, carotid and iliac arteries was only reduced by 28, 26 and 62%, respectively. In rings of abdominal aorta, L-NAME blocked the ACh-stimulated (10(-6) M) rise in cyclic GMP verifying that relaxation which persists in L-NAME-treated rings is not mediated by nitric oxide. The L-NAME resistant response was nearly abolished by elevated external K+ in rings of abdominal aorta and carotid artery, suggesting this relaxation may be mediated by a membrane potential sensitive mechanism. Furthermore, tetraethylammonium (10(-3) M) partially and charybdotoxin (5 x 10(-8) M) completely inhibited the remaining L-NAME-resistant relaxation in both abdominal aorta and carotid artery, suggesting a role for Ca(++)-activated K(+)-channels. Blockers of ATP-sensitive K+ channels also inhibited the L-NAME resistant relaxation in the abdominal aorta only.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8396636

  14. Eucalyptol induces hyperexcitability and epileptiform activity in snail neurons by inhibiting potassium channels.

    PubMed

    Zeraatpisheh, Zahra; Vatanparast, Jafar

    2015-10-01

    The effects of eucalyptol (1,8-cineole) were studied on the activity of central neurons of land snail Caucasotachea atrolabiata. Eucalyptol (3 mM) depolarized the membrane potential and increased the frequency of spontaneous activity in a time dependent and reversible manner. These effects were associated with suppression of afterhyperpolarization and significant reduction of amplitude and slope of rising and falling phases of action potentials. While the eucalyptol-induced suppression of action potential amplitude and rising slope were essentially dependent on membrane depolarization, its actions on repolarization slope and afterhyperpolarization were not affected by resetting the membrane potential close to the control value. These findings suggest an inhibitory action on the potassium channels that underlie repolarization and afterhyperpolarization. Eucalyptol also increased the frequency of driven action potentials but suppressed the post stimulus inhibitory period, indicating an inhibitory action on calcium-activated potassium channels. A higher concentration of eucalyptol, 5mM, reversibly changed the pattern of activity to burst firing associated with paroxysmal depolarization shift (PDS). Low doses of eucalyptol and potassium channel blockers, tetraethylammonium and 4-aminopyridine, synergistically acted to induce burst firing. At high concentration (30 mM), tetraethylammonium was able to induce burst firing and PDS. The sodium currents and ion channel phosphorylation by protein kinases A and C were not required for the eucalyptol-induced epileptiform activity, but calcium currents were essential for this action. Our findings show the excitatory and epileptogenic action of eucalyptol, which is most likely mediated through direct inhibitory action on potassium channels.

  15. Molecular bases for the asynchronous activation of sodium and potassium channels required for nerve impulse generation.

    PubMed

    Lacroix, Jérôme J; Campos, Fabiana V; Frezza, Ludivine; Bezanilla, Francisco

    2013-08-21

    Most action potentials are produced by the sequential activation of voltage-gated sodium (Nav) and potassium (Kv) channels. This is mainly achieved by the rapid conformational rearrangement of voltage-sensor (VS) modules in Nav channels, with activation kinetics up to 6-fold faster than Shaker-type Kv channels. Here, using mutagenesis and gating current measurements, we show that a 3-fold acceleration of the VS kinetics in Nav versus Shaker Kv channels is produced by the hydrophilicity of two "speed-control" residues located in the S2 and S4 segments in Nav domains I-III. An additional 2-fold acceleration of the Nav VS kinetics is provided by the coexpression of the β1 subunit, ubiquitously found in mammal tissues. This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials.

  16. A bursting potassium channel in isolated cholinergic synaptosomes of Torpedo electric organ.

    PubMed Central

    Edry-Schiller, J; Ginsburg, S; Rahamimoff, R

    1991-01-01

    1. Pinched-off cholinergic nerve terminals (synaptosomes) prepared from the electric organ of Torpedo ocelata were fused into large structures (greater than 20 microns) using dimethyl sulphoxide and polyethylene glycol 1500, as previously described for synaptic vesicles from the same organ. 2. The giant fused synaptosomes were easily amenable to the patch clamp technique and 293 seals with a resistance greater than 4 G omega were obtained in the 'cell-attached' configuration. In a large fraction of the experiments, an 'inside-out' patch configuration was achieved. 3. Several types of unitary ionic currents were observed. This study describes the most frequently observed single-channel activity which was found in 247 out of the 293 membrane patches (84.3%). 4. The single-channel current-voltage relation was linear between -60 and 20 mV and showed a slope conductance of 23.8 +/- 1.3 pS when the pipette contained 350-390 mM-Na+ and the bath facing the inside of the synaptosomal membrane contained 390 mM-K+. 5. From extrapolated reversal potential measurements, it was concluded that this channel has a large selectivity for K+ over Na+ (70.4 +/- 11.5, mean +/- S.E.M.). Chloride ions are not transported significantly through this potassium channel. 6. This potassium channel has a low probability of opening. The probability of being in the open state increases upon depolarization and reaches about 1% when the inside of the patch is 20 mV positive compared to the pipette side. 7. The mean channel open time increases with depolarization; thus the product current x time (= charge) also increases upon depolarization, showing properties of an outward rectifier. 8. The potassium channel in the giant synaptosome membrane has a bursting behaviour. Open-time distribution, closed-time distribution and a Poisson analysis indicate that the minimal kinetic scheme requires one open state and three closed states. PMID:1654418

  17. Enhanced role of potassium channels in relaxations to acetylcholine in hypercholesterolemic rabbit carotid artery.

    PubMed

    Najibi, S; Cowan, C L; Palacino, J J; Cohen, R A

    1994-05-01

    The effect of hypercholesterolemia for 10 wk on endothelium-dependent relaxations to acetylcholine was studied in isolated rings of rabbit carotid artery and abdominal aorta contracted with phenylephrine or elevated potassium. In these arteries obtained from hypercholesterolemic rabbits, endothelium-dependent relaxations to acetylcholine were not significantly different from those of normal rabbits. In normal and hypercholesterolemic arteries, partial relaxation persisted in the presence of NG-nitro-L-arginine methyl ester (L-NAME), which blocked acetylcholine-induced increases in arterial guanosine 3',5'-cyclic monophosphate (cGMP). Combined treatment with L-NAME and the calcium-dependent potassium-channel inhibitor, charybdotoxin, blocked relaxations in both groups, suggesting that L-NAME-resistant relaxations are mediated by an endothelium-derived hyperpolarizing factor. Charybdotoxin alone or depolarizing potassium had no significant effect on normal carotid artery or normal and hypercholesterolemic abdominal aorta but significantly inhibited relaxations of the carotid artery from cholesterol-fed rabbits. The enhanced role of calcium-dependent potassium channels and the hyperpolarizing factor in relaxation of the hypercholesterolemic carotid artery suggested by these results was likely related to the fact that acetylcholine failed to stimulate cGMP only in that artery. These data suggest that endothelium-dependent relaxation in these rabbit arteries is mediated by nitric oxide-cGMP-dependent and -independent mechanisms. In hypercholesterolemia, the contribution of nitric oxide-cGMP in the carotid artery is reduced, but a hyperpolarizing factor and calcium-dependent potassium channels maintain normal acetylcholine-induced relaxation. PMID:7515589

  18. Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels

    PubMed Central

    Kuzmenkov, Alexey I.; Nekrasova, Oksana V.; Kudryashova, Kseniya S.; Peigneur, Steve; Tytgat, Jan; Stepanov, Alexey V.; Kirpichnikov, Mikhail P.; Grishin, Eugene V.; Feofanov, Alexey V.; Vassilevski, Alexander A.

    2016-01-01

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology. PMID:27650866

  19. Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels.

    PubMed

    Kuzmenkov, Alexey I; Nekrasova, Oksana V; Kudryashova, Kseniya S; Peigneur, Steve; Tytgat, Jan; Stepanov, Alexey V; Kirpichnikov, Mikhail P; Grishin, Eugene V; Feofanov, Alexey V; Vassilevski, Alexander A

    2016-01-01

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology. PMID:27650866

  20. VOLTAGE-GATED POTASSIUM CHANNELS AT THE CROSSROADS OF NEURONAL FUNCTION, ISCHEMIC TOLERANCE, AND NEURODEGENERATION

    PubMed Central

    Shah, Niyathi Hegde; Aizenman, Elias

    2013-01-01

    Voltage-gated potassium (Kv) channels are widely expressed in the central and peripheral nervous system, and are crucial mediators of neuronal excitability. Importantly, these channels also actively participate in cellular and molecular signaling pathways that regulate the life and death of neurons. Injury-mediated increased K+ efflux through Kv2.1 channels promotes neuronal apoptosis, contributing to widespread neuronal loss in neurodegenerative disorders such as Alzheimer’s disease and stroke. In contrast, some forms of neuronal activity can dramatically alter Kv2.1 channel phosphorylation levels and influence their localization. These changes are normally accompanied by modifications in channel voltage-dependence, which may be neuroprotective within the context of ischemic injury. Kv1 and Kv7 channel dysfunction leads to neuronal hyperexcitability that critically contributes to the pathophysiology of human clinical disorders such as episodic ataxia and epilepsy. This review summarizes the neurotoxic, neuroprotective, and neuroregulatory roles of Kv channels, and highlights the consequences of Kv channel dysfunction on neuronal physiology. The studies described in this review thus underscore the importance of normal Kv channel function in neurons, and emphasize the therapeutic potential of targeting Kv channels in the treatment of a wide range of neurological diseases. PMID:24323720

  1. Potassium channel activator attenuates salicylate-induced cochlear hearing loss potentially ameliorating tinnitus.

    PubMed

    Sun, Wei; Liu, Jun; Zhang, Chao; Zhou, Na; Manohar, Senthilvelan; Winchester, Wendy; Miranda, Jason A; Salvi, Richard J

    2015-01-01

    High dose sodium salicylate causes moderate, reversible hearing loss and tinnitus. Salicylate-induced hearing loss is believed to arise from a reduction in the electromotile response of outer hair cells (OHCs) and/or reduction of KCNQ4 potassium currents in OHCs, which decreases the driving force for the transduction current. Therefore, enhancing OHC potassium currents could potentially prevent salicylate-induced temporary hearing loss. In this study, we tested whether opening voltage-gated potassium channels using ICA-105665, a novel small molecule that opens KCNQ2/3 and KCNQ3/5 channels, can reduce salicylate-induced hearing loss. We found that systemic application of ICA-105665 at 10 mg/kg prevented the salicylate-induced amplitude reduction and threshold shift in the compound action potentials recorded at the round window of the cochlea. ICA-105665 also prevented the salicylate-induced reduction of distortion-product otoacoustic emission. These results suggest that ICA-105665 partially compensates for salicylate-induced cochlear hearing loss by enhancing KCNQ2/3 and KCNQ3/5 potassium currents and the motility of OHCs. PMID:25904892

  2. Cultured giant fiber lobe of squid expresses three distinct potassium channel activities in selective combinations.

    PubMed

    Griggs, W D; Hanyu, Y; Matsumoto, G

    1996-07-01

    Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days, indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally expressed on the giant axon membrane to the somatic membrane.

  3. Detailed Examination of a Single Conduction Event in a Potassium Channel

    PubMed Central

    2013-01-01

    Although extensively studied, it has proved difficult to describe in detail how potassium ion channels conduct cations and water. We present a computational study that, by using stratified umbrella sampling, examines nearly an entire conduction event of the Kv1.2/2.1 paddle chimera and thereby identifies the expected stable configurations of ions and waters in the selectivity filter of the channel. We describe in detail the motions of the ions and waters during a conduction event, focusing on how waters and ions enter the filter, the rotation of water molecules inside the filter, and how potassium ions are coordinated as they move from a water to a protein environment. Finally, we analyze the small conformational changes undergone by the protein, showing that the stable configurations are most similar to the experimental crystal structure. PMID:24143269

  4. Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification

    SciTech Connect

    Kamb, A.; Weir, M.; Rudy, B.; Varmus, H.; Kenyon, C. )

    1989-06-01

    The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. The authors have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, they have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, they have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, they have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.

  5. Encephalitis due to antibodies to voltage gated potassium channel (VGKC) with cerebellar involvement in a teenager.

    PubMed

    Langille, Megan M; Desai, Jay

    2015-01-01

    Encephalitis due to antibodies to voltage gated potassium channel (VGKC) typically presents with limbic encephalitis and medial temporal lobe involvement on neuroimaging. We describe a case of 13 year girl female with encephalitis due to antibodies to VGKC with signal changes in the cerebellar dentate nuclei bilaterally and clinical features that suggested predominant cerebellar involvement. These have never been reported previously in the literature. Our case expands the phenotypic spectrum of this rare condition.

  6. Targeting solid tumours with potassium channel activators. A return to fundamentals?

    PubMed

    Trechot, Philippe

    2014-01-01

    From a pharmacological point of view nicotinamide and minoxidil are potassium channel activators. Nicotinamide is used as a radiosensitizer in ARCON (accelerated radiotherapy combined with carbogen breathing and nicotinamide) therapeutic strategy with promising results but not confirmed so far. Minoxidil has never been considered by radiotherapists. Based from recent pathophysiological considerations we suggest a new perspective for the use of these two "old" molecules in order to target solid tumours. PMID:25371295

  7. Breaking the silence: functional expression of the two-pore-domain potassium channel THIK-2.

    PubMed

    Renigunta, Vijay; Zou, Xinle; Kling, Stefan; Schlichthörl, Günter; Daut, Jürgen

    2014-09-01

    THIK-2 belongs to the 'silent' channels of the two-pore-domain potassium channel family. It is highly expressed in many nuclei of the brain but has so far resisted all attempts at functional expression. THIK-2 has a highly conserved 19-amino-acid region in its N terminus (residues 6-24 in the rat orthologue) that is missing in the closely related channel THIK-1. After deletion of this region (THIK-2(Δ6-24) mutant), functional expression of the channel was observed in Xenopus oocytes and in mammalian cell lines. The resulting potassium current showed outward rectification under physiological conditions and slight inward rectification with symmetrical high-K(+) solutions and could be inhibited by application of halothane or quinidine. Another THIK-2 mutant, in which the putative retention/retrieval signal RRR at positions 14-16 was replaced by AAA, produced a similar potassium current. Both mutants showed a distinct localisation to the surface membrane when tagged with green fluorescent protein and expressed in a mammalian cell line, whereas wild-type THIK-2 was mainly localised to the endoplasmic reticulum. These findings suggest that deletion of the retention/retrieval signal RRR enabled transport of THIK-2 channels to the surface membrane. Combining the mutation THIK-2(Δ6-24) with a mutation in the pore cavity (rat THIK-2(I158G)) gave rise to a 12-fold increase in current amplitude, most likely due to an increase in open probability. In conclusion, the characteristics of THIK-2 channels can be analysed in heterologous expression systems by using trafficking and/or gating mutants. The possible mechanisms that enable THIK-2 expression at the surface membrane in vivo remain to be determined. PMID:24297522

  8. Disruption of the potassium channel regulatory subunit KCNE2 causes iron-deficient anemia.

    PubMed

    Salsbury, Grace; Cambridge, Emma L; McIntyre, Zoe; Arends, Mark J; Karp, Natasha A; Isherwood, Christopher; Shannon, Carl; Hooks, Yvette; Ramirez-Solis, Ramiro; Adams, David J; White, Jacqueline K; Speak, Anneliese O

    2014-12-01

    Iron homeostasis is a dynamic process that is tightly controlled to balance iron uptake, storage, and export. Reduction of dietary iron from the ferric to the ferrous form is required for uptake by solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (Slc11a2) into the enterocytes. Both processes are proton dependent and have led to the suggestion of the importance of acidic gastric pH for the absorption of dietary iron. Potassium voltage-gated channel subfamily E, member 2 (KCNE2), in combination with potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1), form a gastric potassium channel essential for gastric acidification. Deficiency of either Kcne2 or Kcnq1 results in achlorhydia, gastric hyperplasia, and neoplasia, but the impact on iron absorption has not, to our knowledge, been investigated. Here we report that Kcne2-deficient mice, in addition to the previously reported phenotypes, also present with iron-deficient anemia. Interestingly, impaired function of KCNQ1 results in iron-deficient anemia in Jervell and Lange-Nielsen syndrome patients. We speculate that impaired function of KCNE2 could result in the same clinical phenotype.

  9. Screening and cDNA Cloning of Kv1 Potassium Channel Toxins in Sea Anemones

    PubMed Central

    Yamaguchi, Yoshikazu; Hasegawa, Yuichi; Honma, Tomohiro; Nagashima, Yuji; Shiomi, Kazuo

    2010-01-01

    When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of 125I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3′RACE and 5′RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ1.3-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34–63% identities with the other type 1 potassium channel toxins. PMID:21339955

  10. Crystal structure of the PAS domain of the hEAG potassium channel

    PubMed Central

    Tang, Xue; Shao, Juan; Qin, Xiaohong

    2016-01-01

    KCNH voltage-gated potassium channels play critical roles in regulating cellular functions. The channel is composed of four subunits, each of which contains six transmembrane helices forming the central pore. The cytoplasmic parts of the subunits present a Per–Arnt–Sim (PAS) domain at the N-terminus and a cyclic nucleotide-binding homology domain at the C-terminus. PAS domains are conserved from prokaryotes to eukaryotes and are involved in sensing signals and cellular responses. To better understand the functional roles of PAS domains in KCNH channels, the structure of this domain from the human ether-à-go-go channel (hEAG channel) was determined. By comparing it with the structures of the Homo sapiens EAG-related gene (hERG) channel and the Drosophila EAG-like K+ (dELK) channel and analyzing the structural features of the hEAG channel, it was identified that a hydrophobic patch on the β-sheet may mediate interaction between the PAS domain and other regions of the channel to regulate its functions. PMID:27487920

  11. Vascular KCNQ (Kv7) potassium channels as common signaling intermediates and therapeutic targets in cerebral vasospasm.

    PubMed

    Mani, Bharath K; O'Dowd, James; Kumar, Lalit; Brueggemann, Lioubov I; Ross, Masey; Byron, Kenneth L

    2013-01-01

    Cerebral vasospasm after subarachnoid hemorrhage (SAH) is characterized by prolonged severe constriction of the basilar artery, which often leads to ischemic brain damage. Locally elevated concentrations of spasmogenic substances induce persistent depolarization of myocytes in the basilar artery, leading to continuous influx of calcium (Ca) through voltage-sensitive Ca channels and myocyte contraction. Potassium (K) channel openers may have therapeutic utility to oppose membrane depolarization, dilate the arteries, and reduce ischemia. Here, we examined the involvement of vascular Kv7 K channels in the pathogenesis of cerebral vasospasm and tested whether Kv7 channel openers are effective therapeutic agents in a rat model of SAH. Patch-clamp experiments revealed that 3 different spasmogens (serotonin, endothelin, and vasopressin) suppressed Kv7 currents and depolarized freshly isolated rat basilar artery myocytes. These effects were significantly reduced in the presence of a Kv7 channel opener, retigabine. Retigabine (10 μM) also significantly blocked L-type Ca channels, reducing peak inward currents by >50%. In the presence of a selective Kv7 channel blocker, XE991, the spasmogens did not produce additive constriction responses measured using pressure myography. Kv7 channel openers (retigabine or celecoxib) significantly attenuated basilar artery spasm in rats with experimentally induced SAH. In conclusion, we identify Kv7 channels as common targets of vasoconstrictor spasmogens and as candidates for therapeutic intervention for cerebral vasospasm.

  12. Vascular KCNQ (Kv7) potassium channels as common signaling intermediates and therapeutic targets in cerebral vasospasm

    PubMed Central

    Mani, Bharath K.; O'Dowd, James; Kumar, Lalit; Brueggemann, Lioubov I.; Ross, Masey; Byron, Kenneth L.

    2012-01-01

    Cerebral vasospasm following subarachnoid hemorrhage (SAH) is characterized by prolonged severe constriction of the basilar artery, which often leads to ischemic brain damage. Locally elevated concentrations of spasmogenic substances induce persistent depolarization of myocytes in the basilar artery, leading to continuous influx of calcium (Ca2+) through voltage-sensitive Ca2+ channels and myocyte contraction. Potassium (K+) channel openers may have therapeutic utility to oppose membrane depolarization, dilate the arteries, and reduce ischemia. Here, we examined the involvement of vascular Kv7 K+ channels in the pathogenesis of cerebral vasospasm and tested whether Kv7 channel openers are effective therapeutic agents in a rat model of SAH. Patch-clamp experiments revealed that three different spasmogens (serotonin, endothelin and vasopressin) suppressed Kv7 currents and depolarized freshly isolated rat basilar artery myocytes. These effects were significantly reduced in the presence of a Kv7 channel opener, retigabine. Retigabine (10 μmol/L) also significantly blocked L-type Ca2+ channels, reducing peak inward currents by >50%. In the presence of a selective Kv7 channel blocker, XE991, the spasmogens did not produce additive constriction responses measured using pressure myography. Kv7 channel openers (retigabine or celecoxib) significantly attenuated basilar artery spasm in rats with experimentally-induced SAH. In conclusion, we identify Kv7 channels as common targets of vasoconstrictor spasmogens and as candidates for therapeutic intervention for cerebral vasospasm. PMID:23107868

  13. Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning

    PubMed Central

    Rathour, Rahul Kumar; Malik, Ruchi; Narayanan, Rishikesh

    2016-01-01

    Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor. PMID:27094086

  14. Fear conditioning suppresses large-conductance calcium-activated potassium channels in lateral amygdala neurons.

    PubMed

    Sun, P; Zhang, Q; Zhang, Y; Wang, F; Wang, L; Yamamoto, R; Sugai, T; Kato, N

    2015-01-01

    It was previously shown that depression-like behavior is accompanied with suppression of the large-conductance calcium activated potassium (BK) channel in cingulate cortex pyramidal cells. To test whether BK channels are also involved in fear conditioning, we studied neuronal properties of amygdala principal cells in fear conditioned mice. After behavior, we made brain slices containing the amygdala, the structure critically relevant to fear memory. The resting membrane potential in lateral amygdala (LA) neurons obtained from fear conditioned mice (FC group) was more depolarized than in neurons from naïve controls. The frequencies of spikes evoked by current injections were higher in neurons from FC mice, demonstrating that excitability of LA neurons was elevated by fear conditioning. The depolarization in neurons from FC mice was shown to depend on BK channels by using the BK channel blocker charybdotoxin. Suppression of BK channels in LA neurons from the FC group was further confirmed on the basis of the spike width, since BK channels affect the descending phase of spikes. Spikes were broader in the FC group than those in the naïve control in a manner dependent on BK channels. Consistently, quantitative real-time PCR revealed a decreased expression of BK channel mRNA. The present findings suggest that emotional disorder manifested in the forms of fear conditioning is accompanied with BK channel suppression in the amygdala, the brain structure critical to this emotional disorder.

  15. Crystal structure of the PAS domain of the hEAG potassium channel.

    PubMed

    Tang, Xue; Shao, Juan; Qin, Xiaohong

    2016-08-01

    KCNH voltage-gated potassium channels play critical roles in regulating cellular functions. The channel is composed of four subunits, each of which contains six transmembrane helices forming the central pore. The cytoplasmic parts of the subunits present a Per-Arnt-Sim (PAS) domain at the N-terminus and a cyclic nucleotide-binding homology domain at the C-terminus. PAS domains are conserved from prokaryotes to eukaryotes and are involved in sensing signals and cellular responses. To better understand the functional roles of PAS domains in KCNH channels, the structure of this domain from the human ether-à-go-go channel (hEAG channel) was determined. By comparing it with the structures of the Homo sapiens EAG-related gene (hERG) channel and the Drosophila EAG-like K(+) (dELK) channel and analyzing the structural features of the hEAG channel, it was identified that a hydrophobic patch on the β-sheet may mediate interaction between the PAS domain and other regions of the channel to regulate its functions. PMID:27487920

  16. Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning.

    PubMed

    Rathour, Rahul Kumar; Malik, Ruchi; Narayanan, Rishikesh

    2016-01-01

    Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor.

  17. [Comparative assessment of nephroprotective properties of potassium and calcium channel modulators in experimental renal injury].

    PubMed

    Filipets, N D; Gozhenko, A I

    2014-01-01

    The experiments in white laboratory rats have shown that a single intragasrtric administration of the new fluorine-containing potassium channel opener flocalin in a dose of 5 mg/kg in the initial stage of sublimate nephropathy increased the glomerular filtration rate, reduced creatininemia, increased urinary creatinine excretion, and decreased proteinuria. Under similar conditions, the administration of the calcium channel blocker diltiazem in a dose of 5 mg/kg (intragasrtric) showed a less pronounced antiproteinuric effect as compared to that of flocalin. A comparative assessment of the influence of flocalin and diltiazem on the basic renal function markers demonstrated predominant nephroprotective effect of flocalin in the treatment of acute toxic nephropathy.

  18. Ion conduction in the KcsA potassium channel analyzed with a minimal kinetic model.

    PubMed

    Mafé, Salvador; Pellicer, Julio

    2005-02-01

    We use a model by Nelson to study the current-voltage and conductance-concentration curves of bacterial potassium channel KcsA without assuming rapid ion translocation. Ion association to the channel filter is rate controlling at low concentrations, but dissociation and transport in the filter can limit conduction at high concentration for ions other than K+. The absolute values of the effective rate constants are tentative but the relative changes in these constants needed to qualitatively explain the experiments should be of significance. PMID:15783362

  19. Ion conduction in the KcsA potassium channel analyzed with a minimal kinetic model.

    PubMed

    Mafé, Salvador; Pellicer, Julio

    2005-02-01

    We use a model by Nelson to study the current-voltage and conductance-concentration curves of bacterial potassium channel KcsA without assuming rapid ion translocation. Ion association to the channel filter is rate controlling at low concentrations, but dissociation and transport in the filter can limit conduction at high concentration for ions other than K+. The absolute values of the effective rate constants are tentative but the relative changes in these constants needed to qualitatively explain the experiments should be of significance.

  20. Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1.

    PubMed

    Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

    2016-01-01

    KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4(+) T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. PMID:27542194

  1. Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

    PubMed Central

    Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

    2016-01-01

    KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. DOI: http://dx.doi.org/10.7554/eLife.16093.001 PMID:27542194

  2. Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel.

    PubMed

    Li, Qingxin; Wong, Ying Lei; Ng, Hui Qi; Gayen, Shovanlal; Kang, CongBao

    2014-12-01

    The hERG (human ether-a-go-go related gene) potassium channel is a voltage-gated potassium channel containing an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2-S3 linker, S3, S4 and the S4-S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4-S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3-S4 and S4-S4-S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating.

  3. Potassium channel blockers from the venom of the Brazilian scorpion Tityus serrulatus ().

    PubMed

    Martin-Eauclaire, Marie-France; Pimenta, Adriano M C; Bougis, Pierre E; De Lima, Maria-Elena

    2016-09-01

    Potassium (K(+)) channels are trans-membrane proteins, which play a key role in cellular excitability and signal transduction pathways. Scorpion toxins blocking the ion-conducting pore from the external side have been invaluable probes to elucidate the structural, functional, and physio-pathological characteristics of these ion channels. This review will focus on the interaction between K(+) channels and their peptide blockers isolated from the venom of the scorpion Tityus serrulatus, which is considered as the most dangerous scorpion in Brazil, in particular in Minas-Gerais State, where many casualties are described each year. The primary mechanisms of action of these K(+) blockers will be discussed in correlation with their structure, very often non-canonical compared to those of other well known K(+) channels blockers purified from other scorpion venoms. Also, special attention will be brought to the most recent data obtained by proteomic and transcriptomic analyses on Tityus serrulatus venoms and venom glands. PMID:27349167

  4. Potassium channel blockers from the venom of the Brazilian scorpion Tityus serrulatus ().

    PubMed

    Martin-Eauclaire, Marie-France; Pimenta, Adriano M C; Bougis, Pierre E; De Lima, Maria-Elena

    2016-09-01

    Potassium (K(+)) channels are trans-membrane proteins, which play a key role in cellular excitability and signal transduction pathways. Scorpion toxins blocking the ion-conducting pore from the external side have been invaluable probes to elucidate the structural, functional, and physio-pathological characteristics of these ion channels. This review will focus on the interaction between K(+) channels and their peptide blockers isolated from the venom of the scorpion Tityus serrulatus, which is considered as the most dangerous scorpion in Brazil, in particular in Minas-Gerais State, where many casualties are described each year. The primary mechanisms of action of these K(+) blockers will be discussed in correlation with their structure, very often non-canonical compared to those of other well known K(+) channels blockers purified from other scorpion venoms. Also, special attention will be brought to the most recent data obtained by proteomic and transcriptomic analyses on Tityus serrulatus venoms and venom glands.

  5. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle.

    PubMed

    Bolotina, V M; Najibi, S; Palacino, J J; Pagano, P J; Cohen, R A

    1994-04-28

    Nitric oxide is the major endothelium-derived relaxing factor (EDRF), and it is thought to relax smooth muscle cells by stimulation of guanylate cyclase, accumulation of its product cyclic GMP, and cGMP-dependent modification of several intracellular processes, including activation of potassium channels through cGMP-dependent protein kinase. Here we present evidence that both exogenous nitric oxide and native EDRF can directly activate single Ca(2+)-dependent K+ channels (K+Ca) in cell-free membrane patches without requiring cGMP. Under conditions when guanylate cyclase was inhibited by methylene blue, considerable relaxation of rabbit aorta to nitric oxide persisted which was blocked by charybdotoxin, a specific inhibitor of K+Ca channels. These studies demonstrate a novel direct action of nitric oxide on K+Ca channels. PMID:7512692

  6. Role of potassium channels in the nitrergic nerve stimulation-induced vasodilatation in the guinea-pig isolated basilar artery

    PubMed Central

    Jiang, Fan; Li, Chun Guang; Rand, Michael J

    1998-01-01

    We studied the effects of various K+ channel blockers on the vasodilator responses of guinea-pig isolated basilar arteries to nitrergic nerve stimulation, the nitric oxide (NO) donor sodium nitroprusside (SNP), and the membrane permeable guanosine-3′, 5′-cyclic monophosphate (cyclic GMP) analogue 8-bromo-cyclic GMP (8-Br-cyclic GMP).In endothelium-denuded preparations which were contracted with prostaglandin F2α (1 μM), electrical field stimulation (EFS, 10 Hz for 30 s) produced a vasodilatation which was totally blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester L-NAME; 100 μM) (n=3) and by the selective NO-sensitive guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 1 μM) (n=4). The vasodilator response to SNP (100 nM) was not reduced by L-NAME but was abolished by ODQ (1 μM) (n=4).EFS-elicited vasodilatation was partly but significantly reduced by the non-selective K+ channel blockers tetraethylammonium (TEA, 1 and 3 mM) and 4-aminopyridine (4-AP, 3 mM), and by the large-conductance calcium-activated K+ channel (KCa channel) blockers charybdotoxin (ChTX, 150 nM) and iberiotoxin (IbTX, 30 and 100 nM). In contrast, the ATP-sensitive K+ channel (KATP channel) blocker glibenclamide (1–10 μM) and the small-conductance KCa channel blocker apamin (100–500 nM) did not affect EFS-induced vasodilatation.The vasodilator response elicited by SNP (10–100 nM) was significantly reduced by TEA (3 mM) and ChTX (150 nM) but not by apamin (500 nM) or glibenclamide (1 μM). The vasodilatation elicited by 8-Br-cyclic GMP (100 μM) was also reduced by TEA (3 mM) and ChTX (150 nM).The results indicate that the vasodilatations induced by nitrergic nerve stimulation and the NO donor SNP in endothelium-denuded guinea-pig basilar artery depend on the formation of intracellular cyclic GMP. The increased cyclic GMP level activates large-conductance KCa channels which

  7. The inwardly rectifying potassium channel Kir1.1: development of functional assays to identify and characterize channel inhibitors.

    PubMed

    Felix, John P; Priest, Birgit T; Solly, Kelli; Bailey, Timothy; Brochu, Richard M; Liu, Chou J; Kohler, Martin G; Kiss, Laszlo; Alonso-Galicia, Magdalena; Tang, Haifeng; Pasternak, Alexander; Kaczorowski, Gregory J; Garcia, Maria L

    2012-10-01

    The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartter's syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization. PMID:22881347

  8. hERG potassium channels and the structural basis of drug-induced arrhythmias.

    PubMed

    Mitcheson, John S

    2008-05-01

    hERG potassium channels have a critical role in the normal electrical activity of the heart. The block of hERG channels can cause the drug-induced form of long QT syndrome, a cardiac disorder that carries an increased risk of cardiac arrhythmias and sudden death. hERG channels are extraordinarily sensitive to block by large numbers of structurally diverse drugs. In previous years, the risk of compounds causing this cardiotoxic side effect has been a common reason for the failure of compounds in preclinical safety trials. Pharmaceutical companies have successfully utilized and developed higher throughput techniques for the early detection of compounds that block hERG, and this has helped reduce the number of compounds that fail in the late stages of development. Nevertheless, this screening-based approach is expensive, consumes chemistry resources, and bypasses the problem rather than shedding light on it. Crystal structures of potassium channels have facilitated studies into the structural basis for the gating and block of hERG channels. Most drugs bind within the inner cavity, and the individual amino acids that form the drug binding site have been identified by site-directed mutagenesis approaches. Gating processes have an important influence on the drug-binding site. Recent advances in our understanding of channel activation and inactivation are providing insight into why hERG channels are more susceptible to block than other K (+) channels. Knowledge of the structure of the drug-binding site and precise nature of interactions with drug molecules should assist efforts to develop drugs without the propensity to cause cardiac arrhythmias. PMID:18447395

  9. Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels.

    PubMed

    Yu, Haibo; Lin, Zhihong; Mattmann, Margrith E; Zou, Beiyan; Terrenoire, Cecile; Zhang, Hongkang; Wu, Meng; McManus, Owen B; Kass, Robert S; Lindsley, Craig W; Hopkins, Corey R; Li, Min

    2013-05-21

    Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels. PMID:23650380

  10. Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems

    PubMed Central

    Roy Chowdhury, Uttio; Bahler, Cindy K.; Holman, Bradley H.; Dosa, Peter I.; Fautsch, Michael P.

    2015-01-01

    Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm’s canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development. PMID:26535899

  11. Differential effects of ethanol on electrical properties of various potassium channels expressed in oocytes.

    PubMed

    Anantharam, V; Bayley, H; Wilson, A; Treistman, S N

    1992-09-01

    The effects of ethanol on a number of electrophysiological parameters were examined in 10 different voltage-gated potassium channels expressed in Xenopus oocytes. None of the channels examined was highly sensitive to ethanol, but there was significant variability among the channels tested at concentrations of ethanol of 200 mM and greater. The response to ethanol was not determined exclusively by membership in a genetic subfamily. In addition, the relative sensitivity among different channels could vary independently for different electrical parameters. For example, current amplitude in DRK1 was insensitive to ethanol, even at concentrations as high as 600 mM, whereas this was one of the more sensitive channels with respect to the kinetics of current inactivation. The opposite situation was true for ShA1. Therefore, ethanol at high concentrations may selectively perturb discrete regions of channel proteins. This is supported by the finding that removal of 318 amino acids from the cytoplasmic carboxyl terminus of DRK1 results in a channel whose current amplitude shows greater sensitivity to ethanol than does DRK1. Thus, the effects of ethanol on the channel may not be limited to interactions at the lipid-protein interface. PMID:1406600

  12. Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels

    SciTech Connect

    Xia Menghang; Shahane, Sampada A.; Huang, Ruili; Titus, Steven A.; Shum, Enoch; Zhao Yong; Southall, Noel; Zheng, Wei; Witt, Kristine L.; Tice, Raymond R.; Austin, Christopher P.

    2011-05-01

    The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K{sup +}) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC{sub 50} potencies ranging from 0.26 to 22 {mu}M. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC{sub 50} value of 260 nM in the thallium influx assay and 80 nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo.

  13. Subsets of ATP-sensitive potassium channel (KATP) inhibitors increase gap junctional intercellular communication in metastatic cancer cell lines independent of SUR expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gap junctional intercellular communication (GJIC) is a process whereby cells share molecules and nutrients with each other by physical contact through cell membrane pores. In tumor cells, GJIC is often altered, suggesting that this process may be important in the context of cancer. Certain ion chan...

  14. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

    PubMed Central

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M.; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-01-01

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596

  15. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

    PubMed

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-06-15

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.

  16. Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

    PubMed Central

    Chowdhury, Sandipan; Haehnel, Benjamin M.

    2014-01-01

    Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate. PMID:25311635

  17. Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels.

    PubMed

    Chowdhury, Sandipan; Haehnel, Benjamin M; Chanda, Baron

    2014-11-01

    Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4-S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3-5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.

  18. Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore

    PubMed Central

    Chokshi, Rikki H.; Larsen, Aaron T.; Bhayana, Brijesh

    2015-01-01

    Compounds PKTHPP (1-{1-[6-(biphenyl-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]-pyrimidin-4-yl]piperidin-4-yl}propan-1-one), A1899 (2ʹ′-[(4-methoxybenzoylamino)methyl]biphenyl-2-carboxylic acid 2,4-difluorobenzylamide), and doxapram inhibit TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore (K2P) potassium channel function and stimulate breathing. To better understand the molecular mechanism(s) of action of these drugs, we undertook studies to identify amino acid residues in the TASK-3 protein that mediate this inhibition. Guided by homology modeling and molecular docking, we hypothesized that PKTHPP and A1899 bind in the TASK-3 intracellular pore. To test our hypothesis, we mutated each residue in or near the predicted PKTHPP and A1899 binding site (residues 118–128 and 228–248), individually, to a negatively charged aspartate. We quantified each mutation's effect on TASK-3 potassium channel concentration response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in Fischer rat thyroid epithelial monolayers; channel function was measured in an Ussing chamber. TASK-3 pore mutations at residues 122 (L122D, E, or K) and 236 (G236D) caused the IC50 of PKTHPP to increase more than 1000-fold. TASK-3 mutants L122D, G236D, L239D, and V242D were resistant to block by PKTHPP, A1899, and doxapram. Our data are consistent with a model in which breathing stimulant compounds PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding at a common site within the channel intracellular pore region, although binding outside the channel pore cannot yet be excluded. PMID:26268529

  19. Expression and Stress-Dependent Induction of Potassium Channel Transcripts in the Common Ice Plant1

    PubMed Central

    Su, Hua; Golldack, Dortje; Katsuhara, Maki; Zhao, Chengsong; Bohnert, Hans J.

    2001-01-01

    We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mm NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant. PMID:11161018

  20. Kv1.3 potassium channel mediates macrophage migration in atherosclerosis by regulating ERK activity.

    PubMed

    Kan, Xiao-Hong; Gao, Hai-Qing; Ma, Zhi-Yong; Liu, Lin; Ling, Ming-Ying; Wang, Yuan-Yuan

    2016-02-01

    Ion channels expressed in macrophages have been tightly related to atherosclerosis by coupling cellular function. How the voltage-gated potassium channels (Kv) affect macrophage migration remain unknown. The aim of our study is to investigate whether Kv1.3-ERK signaling pathway plays an important role in the process. We explored the expression of Kv1.3 in coronary atherosclerotic heart disease and found Kv1.3 channel was increased in acute coronary syndrome patients. Treatment of RAW264.7 cells with Kv1.3 small interfering RNA, suppressed cell migration. The expression of phosphorylated ERK1/2 also decreased after knockdown of Kv1.3. On the other hand, overexpression of Kv1.3 channel promoted cell migration and ERK1/2 phosphorylation. U-0126, the mitogen-activated protein kinase inhibitors, could reverse macrophage migration induced by Kv1.3 channel overexpression. Downregulation of Kv1.3 channel by siRNA could not further inhibit cell migration when cells were treated with U-0126. It means that ERK is downstream signal of Kv1.3 channel. We concluded that Kv1.3 may stimulate macrophage migration through the activation of ERK.

  1. PKC and AMPK regulation of Kv1.5 potassium channels

    PubMed Central

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi; Petersen, Frederic; MacAulay, Nanna; Rasmussen, Hanne Borger; Jespersen, Thomas

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K+ current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems. PMID:26043299

  2. PKC and AMPK regulation of Kv1.5 potassium channels.

    PubMed

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi; Petersen, Frederic; MacAulay, Nanna; Rasmussen, Hanne Borger; Jespersen, Thomas

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K(+) current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4-2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems. PMID:26043299

  3. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

    PubMed Central

    Smith, Ryan C; McClure, Marisa C; Smith, Margaret A; Abel, Peter W; Bradley, Michael E

    2007-01-01

    Background Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium. Methods Functional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots. Results The Kv channel blocker 4-aminopyridine (4-AP) caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar) but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were found in isolated myometrium

  4. Effects of Resibufogenin and Cinobufagin on voltage-gated potassium channels in primary cultures of rat hippocampal neurons.

    PubMed

    Hao, Shuang; Bao, Yong-Ming; An, Li-Jia; Cheng, Wei; Zhao, Rong-Guo; Bi, Jing; Wang, He-Shuang; Sun, Chang-Sen; Liu, Ji-Wen; Jiang, Bo

    2011-12-01

    Outward delayed rectifier potassium channel and outward transient potassium channel have multiple important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG), on the outward delayed rectifier potassium current (IK) and outward transient potassium current (IA) in rat hippocampal neurons. RBG and CBG have similar structures and both were isolated from the venom gland of toad skin. RBG inhibited both IK and IA, whereas CBG inhibited IK without noticeable effect on IA. Moreover, at 1 μM concentration both RBG and CBG could alter some channel kinetics and gating properties of IK, such as steady-state activation and inactivation curves, open probability and time constants. These findings suggested that IK is probably a target of bufadienolides, which may explain the mechanisms of bufadienolides' pathological effects on central nervous system. PMID:21798339

  5. Ion channel profile of TRPM8 cold receptors reveals a role of TASK-3 potassium channels in thermosensation.

    PubMed

    Morenilla-Palao, Cruz; Luis, Enoch; Fernández-Peña, Carlos; Quintero, Eva; Weaver, Janelle L; Bayliss, Douglas A; Viana, Félix

    2014-09-11

    Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold-sensitive neurons, combining bacterial artificial chromosome (BAC) transgenesis with a molecular-profiling approach in fluorescence-activated cell sorting (FACS)-purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3, and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity. PMID:25199828

  6. Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

    PubMed

    Linta, Leonhard; Boeckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2013-07-01

    The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

  7. A hyperprostaglandin E syndrome mutation in Kir1.1 (renal outer medullary potassium) channels reveals a crucial residue for channel function in Kir1.3 channels.

    PubMed

    Derst, C; Wischmeyer, E; Preisig-Müller, R; Spauschus, A; Konrad, M; Hensen, P; Jeck, N; Seyberth, H W; Daut, J; Karschin, A

    1998-09-11

    Loss of function mutations in kidney Kir1.1 (renal outer medullary potassium channel, KCNJ1) inwardly rectifying potassium channels can be found in patients suffering from hyperprostaglandin E syndrome (HPS), the antenatal form of Bartter syndrome. A novel mutation found in a sporadic case substitutes an asparagine by a positively charged lysine residue at amino acid position 124 in the extracellular M1-H5 linker region. When heterologously expressed in Xenopus oocytes and mammalian cells, current amplitudes from mutant Kir1.1a[N124K] channels were reduced by a factor of approximately 12 as compared with wild type. A lysine at the equivalent position is present in only one of the known Kir subunits, the newly identified Kir1.3, which is also poorly expressed in the recombinant system. When the lysine residue in guinea pig Kir1.3 (gpKir1.3) isolated from a genomic library was changed to an asparagine (reverse HPS mutation), mutant channels yielded macroscopic currents with amplitudes increased 6-fold. From single channel analysis it became apparent that the decrease in mutant Kir1.1 channels and the increase in mutant gpKir1.3 macroscopic currents were mainly due to the number of expressed functional channels. Coexpression experiments revealed a dominant-negative effect of Kir1.1a[N124K] and gpKir1.3 on macroscopic current amplitudes when coexpressed with wild type Kir1.1a and gpKir[K110N], respectively. Thus we postulate that in Kir1.3 channels the extracellular positively charged lysine is of crucial functional importance. The HPS phenotype in man can be explained by the lower expression of functional channels by the Kir1. 1a[N124K] mutant. PMID:9727001

  8. Alteration in rectification of potassium channels in perinatal hypoxia ischemia brain damage.

    PubMed

    Chen, Penghui; Wang, Liyan; Deng, Qiyue; Ruan, Huaizhen; Cai, Wenqin

    2015-01-15

    Oligodendrocyte progenitor cells (OPCs) are susceptible to perinatal hypoxia ischemia brain damage (HIBD), which results in infant cerebral palsy due to the effects on myelination. The origin of OPC vulnerability in HIBD, however, remains controversial. In this study, we defined the HIBD punctate lesions by MRI diffuse excessive high signal intensity (DEHSI) in postnatal 7-day-old rats. The electrophysiological functional properties of OPCs in HIBD were recorded by patch-clamp in acute cerebral cortex slices. The slices were intracellularly injected with Lucifer yellow and immunohistochemically labeled with NG2 antibody to identify local OPCs. Passive membrane properties and K(+) channel functions in OPCs were analyzed to estimate the onset of vulnerability in HIBD. The resting membrane potential, membrane resistance, and membrane capacitance of OPCs were increased in both the gray and white matter of the cerebral cortex. OPCs in both the gray and white matter exhibited voltage-dependent K(+) currents, which consisted of the initiated rectified potassium currents (IA) and the sustained rectified currents (IK). The significant alternation in membrane resistance was influenced by the diversity of potassium channel kinetics. These findings suggest that the rectification of IA and IK channels may play a significant role in OPC vulnerability in HIBD.

  9. Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation

    PubMed Central

    Conti, Luca; Renhorn, Jakob; Gabrielsson, Anders; Turesson, Fredrik; Liin, Sara I; Lindahl, Erik; Elinder, Fredrik

    2016-01-01

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions – a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel. PMID:27278891

  10. Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization

    PubMed Central

    Auffenberg, Eva; Jurik, Angela; Mattusch, Corinna; Stoffel, Rainer; Genewsky, Andreas; Namendorf, Christian; Schmid, Roland M.; Rammes, Gerhard; Biel, Martin; Uhr, Manfred; Moosmang, Sven; Michalakis, Stylianos; Wotjak, Carsten T.; Thoeringer, Christoph K.

    2016-01-01

    Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research. PMID:26757616

  11. Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation

    NASA Astrophysics Data System (ADS)

    Conti, Luca; Renhorn, Jakob; Gabrielsson, Anders; Turesson, Fredrik; Liin, Sara I.; Lindahl, Erik; Elinder, Fredrik

    2016-06-01

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

  12. Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation

    NASA Astrophysics Data System (ADS)

    Conti, Luca; Renhorn, Jakob; Gabrielsson, Anders; Turesson, Fredrik; Liin, Sara I.; Lindahl, Erik; Elinder, Fredrik

    2016-06-01

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions – a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

  13. Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation.

    PubMed

    Conti, Luca; Renhorn, Jakob; Gabrielsson, Anders; Turesson, Fredrik; Liin, Sara I; Lindahl, Erik; Elinder, Fredrik

    2016-01-01

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd(2+) bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K(+) coordination, a hallmark for C-type inactivation. An engineered Cd(2+) bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel. PMID:27278891

  14. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

    NASA Astrophysics Data System (ADS)

    Lange, Adam; Giller, Karin; Hornig, Sönke; Martin-Eauclaire, Marie-France; Pongs, Olaf; Becker, Stefan; Baldus, Marc

    2006-04-01

    The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane-similar to the catalytic function of the active site of an enzyme-and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.

  15. Novel potassium channels encoded by the Shaker locus in Drosophila photoreceptors.

    PubMed

    Hardie, R C; Voss, D; Pongs, O; Laughlin, S B

    1991-03-01

    The Shaker gene, responsible for A-type potassium channels in Drosophila muscle, encodes a large family of transcripts capable of generating a variety of kinetically distinct A channels when expressed in oocytes. We describe a distinct class of A channel encoded by the Shaker gene in a novel preparation of dissociated Drosophila photoreceptors. Whole-cell recordings reveal a rapidly inactivating A current that is absent in Shaker mutants and that can be readily isolated in cell-attached patches. Although very similar to their muscle counterparts, the photoreceptor A channels show a striking 40-50 mV negative shift in their voltage-operating range. Two mutations (ShE62 and T(1;Y)W32), which exclude only certain classes of Shaker transcripts, were used to show that photoreceptor A channels are encoded by multiple transcripts distinct from those encoding muscle A channels, while PCR techniques identified four transcripts (ShA1, ShA2, ShG1, and ShG2) in mRNA from dissected retina. PMID:2001287

  16. The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel

    PubMed Central

    Yazdi, Samira; Stein, Matthias; Elinder, Fredrik; Andersson, Magnus; Lindahl, Erik

    2016-01-01

    Voltage-gated potassium (KV) channels are membrane proteins that respond to changes in membrane potential by enabling K+ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs) induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD), the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker KV channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA) and the Shaker KV channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins. PMID:26751683

  17. SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification.

    PubMed

    Alqadah, Amel; Hsieh, Yi-Wen; Schumacher, Jennifer A; Wang, Xiaohong; Merrill, Sean A; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M; Chuang, Chiou-Fen

    2016-01-01

    The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

  18. SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

    PubMed Central

    Schumacher, Jennifer A.; Wang, Xiaohong; Merrill, Sean A.; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M.; Chuang, Chiou-Fen

    2016-01-01

    The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons. PMID:26771544

  19. Single-channel basis for the slow activation of the repolarizing cardiac potassium current, I(Ks).

    PubMed

    Werry, Daniel; Eldstrom, Jodene; Wang, Zhuren; Fedida, David

    2013-03-12

    Coassembly of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) with potassium voltage-gated channel, Isk-related family, member 1 (KCNE1) the delayed rectifier potassium channel I(Ks). Its slow activation is critically important for membrane repolarization and for abbreviating the cardiac action potential, especially during sympathetic activation and at high heart rates. Mutations in either gene can cause long QT syndrome, which can lead to fatal arrhythmias. To understand better the elementary behavior of this slowly activating channel complex, we quantitatively analyzed direct measurements of single-channel I(Ks). Single-channel recordings from transiently transfected mouse ltk(-) cells confirm a channel that has long latency periods to opening (1.67 ± 0.073 s at +60 mV) but that flickers rapidly between multiple open and closed states in non-deactivating bursts at positive membrane potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall open probability of only ∼0.15 after 4 s under our recording conditions. The mean single-channel conductance was determined to be 3.2 pS, but unlike any other known wild-type human potassium channel, long-lived subconductance levels coupled to activation are a key feature of both the activation and deactivation time courses of the conducting channel complex. Up to five conducting levels ranging from 0.13 to 0.66 pA could be identified in single-channel recordings at 60 mV. Fast closings and overt subconductance behavior of the wild-type I(Ks) channel required modification of existing Markov models to include these features of channel behavior. PMID:23431135

  20. Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes*

    PubMed Central

    Jara-Oseguera, Andrés; Ishida, Itzel G.; Rangel-Yescas, Gisela E.; Espinosa-Jalapa, Noel; Pérez-Guzmán, José A.; Elías-Viñas, David; Le Lagadec, Ronan; Rosenbaum, Tamara; Islas, León D.

    2011-01-01

    The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling. PMID:21454671

  1. 18F-FDG PET/CT findings in voltage-gated potassium channel limbic encephalitis.

    PubMed

    Kamaleshwaran, Koramadai Karuppuswamy; Iyer, Rajesh Shankar; Antony, Joppy; Radhakrishnan, Edathuruthy Kalarickal; Shinto, Ajit

    2013-05-01

    Limbic encephalitis (LE) can be associated with cancer, viral infection, or be idiopathic. One such rare but treatable form is associated with voltage-gated potassium channel (VGKC) antibodies. Typical abnormalities are seen in FDG PET/CT. We report a 39-year-old female patient who presented with 3 months of progressive faciobrachial dystonic seizures and limbic encephalitis. Her serum and cerebrospinal fluid Lgi1 antibody titers were elevated. FDG PET/CT showed basal ganglial hypermetabolism and associated abnormalities. Serial MRI demonstrated atrophic changes predominantly involving the temporal lobes. She is on immunosuppressive therapy and shows clinical improvement with lowering of antibody titers.

  2. Large-conductance Ca²⁺-activated potassium channel in mitochondria of endothelial EA.hy926 cells.

    PubMed

    Bednarczyk, Piotr; Koziel, Agnieszka; Jarmuszkiewicz, Wieslawa; Szewczyk, Adam

    2013-06-01

    In the present study, we describe the existence of a large-conductance Ca²⁺-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca²⁺, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β₂-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

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

    PubMed Central

    1989-01-01

    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. PMID:2472462

  4. Regulation of voltage-gated potassium channels by PI(4,5)P2

    PubMed Central

    Kruse, Martin; Hammond, Gerald R.V.

    2012-01-01

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) regulates activities of numerous ion channels including inwardly rectifying potassium (Kir) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (KV) channels might be regulated by PI(4,5)P2. Wide expression of KV channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of KV channels by PI(4,5)P2, we have coexpressed several of them in tsA-201 cells with a G protein–coupled receptor (M1R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P2 with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P2 at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited KV7.1, KV7.2/7.3, and Kir2.1 channel current by 90–95%. Activation of M1R inhibited KV7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P2 regulation of activity of KV1.1/KVβ1.1, KV1.3, KV1.4, and KV1.5/KVβ1.3, KV2.1, KV3.4, KV4.2, KV4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for KV1.1/KVβ1.1 and KV3.4, resulting in up-regulation of current density upon activation of M1R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P2 at the plasma membrane by enzymes does not seem to influence activity of most tested KV channels, whereas it does strongly inhibit members of the KV7 and Kir families. PMID:22851677

  5. Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure.

    PubMed

    Zhou, Xiaoyan; Zhang, Zuo; Shin, Myung Kyun; Horwitz, Sarah Beth; Levorse, John M; Zhu, Lei; Sharif-Rodriguez, Wanda; Streltsov, Denis Y; Dajee, Maya; Hernandez, Melba; Pan, Yi; Urosevic-Price, Olga; Wang, Li; Forrest, Gail; Szeto, Daphne; Zhu, Yonghua; Cui, Yan; Michael, Bindhu; Balogh, Leslie Ann; Welling, Paul A; Wade, James B; Roy, Sophie; Sullivan, Kathleen A

    2013-08-01

    The renal outer medullary potassium channel (ROMK, KCNJ1) mediates potassium recycling and facilitates sodium reabsorption through the Na(+)/K(+)/2Cl(-) cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome. We have generated an ROMK knockout rat model in Dahl salt-sensitive background by using zinc finger nuclease technology and investigated the effects of knocking out ROMK on systemic and renal hemodynamics and kidney histology in the Dahl salt-sensitive rats. The ROMK(-/-) pups recapitulated features identified in the ROMK null mice. The ROMK(+/-) rats, when challenged with a 4% salt diet, exhibited a reduced blood pressure compared with their ROMK(+/+) littermates. More importantly, when challenged with an 8% salt diet, the Dahl salt-sensitive rats with 50% less ROMK expression showed increased protection from salt-induced blood pressure elevation and signs of protection from renal injury. Our findings in ROMK knockout Dahl salt-sensitive rats, together with the previous reports in humans and mice, underscore a critical role of ROMK in blood pressure regulation. PMID:23753405

  6. Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure.

    PubMed

    Zhou, Xiaoyan; Zhang, Zuo; Shin, Myung Kyun; Horwitz, Sarah Beth; Levorse, John M; Zhu, Lei; Sharif-Rodriguez, Wanda; Streltsov, Denis Y; Dajee, Maya; Hernandez, Melba; Pan, Yi; Urosevic-Price, Olga; Wang, Li; Forrest, Gail; Szeto, Daphne; Zhu, Yonghua; Cui, Yan; Michael, Bindhu; Balogh, Leslie Ann; Welling, Paul A; Wade, James B; Roy, Sophie; Sullivan, Kathleen A

    2013-08-01

    The renal outer medullary potassium channel (ROMK, KCNJ1) mediates potassium recycling and facilitates sodium reabsorption through the Na(+)/K(+)/2Cl(-) cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome. We have generated an ROMK knockout rat model in Dahl salt-sensitive background by using zinc finger nuclease technology and investigated the effects of knocking out ROMK on systemic and renal hemodynamics and kidney histology in the Dahl salt-sensitive rats. The ROMK(-/-) pups recapitulated features identified in the ROMK null mice. The ROMK(+/-) rats, when challenged with a 4% salt diet, exhibited a reduced blood pressure compared with their ROMK(+/+) littermates. More importantly, when challenged with an 8% salt diet, the Dahl salt-sensitive rats with 50% less ROMK expression showed increased protection from salt-induced blood pressure elevation and signs of protection from renal injury. Our findings in ROMK knockout Dahl salt-sensitive rats, together with the previous reports in humans and mice, underscore a critical role of ROMK in blood pressure regulation.

  7. Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel

    PubMed Central

    Ishida, Itzel G.; Rangel-Yescas, Gisela E.; Carrasco-Zanini, Julia

    2015-01-01

    Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13–14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker’s, accounting for the smaller Kv1.2 gating charge. PMID:25779871

  8. Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury

    PubMed Central

    Calvo, Margarita; Richards, Natalie; Schmid, Annina B; Barroso, Alejandro; Zhu, Lan; Ivulic, Dinka; Zhu, Ning; Anwandter, Philipp; Bhat, Manzoor A; Court, Felipe A; McMahon, Stephen B; Bennett, David LH

    2016-01-01

    Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. DOI: http://dx.doi.org/10.7554/eLife.12661.001 PMID:27033551

  9. Developmental expression of Kv1 voltage-gated potassium channels in the avian hypothalamus.

    PubMed

    Doczi, Megan A; Vitzthum, Carl M; Forehand, Cynthia J

    2016-03-11

    Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis.

  10. Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury.

    PubMed

    Calvo, Margarita; Richards, Natalie; Schmid, Annina B; Barroso, Alejandro; Zhu, Lan; Ivulic, Dinka; Zhu, Ning; Anwandter, Philipp; Bhat, Manzoor A; Court, Felipe A; McMahon, Stephen B; Bennett, David L H

    2016-04-19

    Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury.

  11. Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury.

    PubMed

    Calvo, Margarita; Richards, Natalie; Schmid, Annina B; Barroso, Alejandro; Zhu, Lan; Ivulic, Dinka; Zhu, Ning; Anwandter, Philipp; Bhat, Manzoor A; Court, Felipe A; McMahon, Stephen B; Bennett, David L H

    2016-01-01

    Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. PMID:27033551

  12. Imaging and quantification of recycled KATP channels.

    PubMed

    Cockcroft, Christopher J

    2013-01-01

    This chapter describes immunochemistry-based methods to investigate recycling of membrane proteins at the cell surface. Two methods are described, one qualitative and the other quantitative. Both methods consist of two rounds of extracellular antibody capture. Firstly, a primary antibody is captured by an extracellular epitope presented by the target membrane protein and is subsequently internalized. Secondly, the primary antibody-labelled protein is recycled back to the membrane where it is captured by a probe--conjugated secondary antibody. In the qualitative assay, the probe is a fluorophore, which can be imaged by fluorescence microscopy. In the quantitative assay, the probe is horse-radish peroxidase (HRP) and enzyme activity can be assayed by chemiluminescence. PMID:23529434

  13. Structural analysis of the S4-S5 linker of the human KCNQ1 potassium channel.

    PubMed

    Gayen, Shovanlal; Li, Qingxin; Kang, CongBao

    2015-01-01

    KCNQ1 plays important roles in the cardiac action potential and consists of an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. KCNQ1 is a voltage-gated potassium channel and its channel activity is regulated by membrane potentials. The linker between transmembrane helices 4 and 5 (S4-S5 linker) is important for transferring the conformational changes from the voltage-sensor domain to the pore domain. In this study, the structure of the S4-S5 linker of KCNQ1 was investigated by solution NMR, circular dichroism and fluorescence spectroscopic studies. The S4-S5 linker adopted a helical structure in detergent micelles. The W248 may interact with the cell membrane.

  14. Residues in a jellyfish shaker-like channel involved in modulation by external potassium.

    PubMed

    Grigoriev, N G; Spafford, J D; Spencer, A N

    1999-10-01

    The jellyfish gene, jShak2, coded for a potassium channel that showed increased conductance and a decreased inactivation rate as [K(+)](out) was increased. The relative modulatory effectiveness of K(+), Rb(+), Cs(+), and Na(+) indicated that a weak-field-strength site is present. Cysteine substituted mutants (L369C and F370C) of an N-terminal truncated construct, (jShak2Delta2-38) which only showed C-type inactivation, were used to establish the position and nature of this site(s). In comparison with jShak2Delta2-38 and F370C, L369C showed a greater relative increase in peak current when [K(+)](out) was increased from 1 to 100 mM because the affinity of this site was reduced at low [K(+)](out). Increasing [K(+)](out) had little effect on the rate of inactivation of L369C; however, the appearance of a second, hyperbolic component to the inactivation curve for F370C indicated that this mutation had increased the affinity of the low-affinity site by bringing the backbone oxygens closer together. Methanethiosulphonate reagents were used to form positively (MTSET), negatively (MTSES), and neutrally (MTSM) charged side groups on the cysteine-substituted residues at the purported K(+) binding site(s) in the channel mouth and conductance and inactivation kinetic measurements made. The reduced affinity of the site produced by the mutation L369C was probably due to the increased hydrophobicity of cysteine, which changed the relative positions of carbonyl oxygens since MTSES modification did not form a high-field-strength site as might be expected if the cysteine residues project into the pore. Addition of the side chain -CH(2)-S-S-CH(3), which is similar to the side chain of methionine, a conserved residue in many potassium channels, resulted in an increased peak current and reduced inactivation rate, hence a higher affinity binding site. Modification of cysteine substituted mutants occurred more readily from the inactivated state confirming that side chains probably rotate

  15. Different calcium influx characteristics upon Kv1.3 and IKCa1 potassium channel inhibition in T helper subsets.

    PubMed

    Orbán, Csaba; Bajnok, Anna; Vásárhelyi, Barna; Tulassay, Tivadar; Toldi, Gergely

    2014-07-01

    Functional imbalance between T helper subsets plays important role in the pathogenesis of autoimmune disorders. Transient increase of cytoplasmic calcium level, and sustention of negative membrane potential by voltage sensitive Kv1.3 and calcium-dependent IKCa1 potassium channels are essential for short-term lymphocyte activation, thus present possible target for selective immunomodulation. We aimed to investigate calcium influx sensitivity to the inhibition of potassium channels in the main T helper subsets. Peripheral blood from 11 healthy individuals was drawn and calcium influx kinetics following activation with phytohemagglutinin in Th1, Th2, Th17, and Treg cells were evaluated. Alteration of calcium influx induced by specific inhibitors of Kv1.3 and IKCa1 potassium channels, and the expression of Kv1.3 channels were also assessed. Highest cytoplasmic calcium concentration was observed in stimulated Th1 cells, while the lowest level was measured in Treg cells. In Th1 and Th17 cells, inhibition of both investigated potassium channels decreased calcium influx. In Th2 cells only the inhibitor of Kv1.3 channels, while in Treg cells none of the inhibitors had significant effect. Upon the inhibition of IKCa1 channels, short-term activation of proinflammatory cells was specifically decreased without affecting anti-inflammatory subsets, indicating that selective immunomodulation is possible in healthy individuals.

  16. Calcium-activated potassium channels mask vascular dysfunction associated with oxidized LDL exposure in rabbit aorta.

    PubMed

    Bocker, J M; Miller, F J; Oltman, C L; Chappell, D A; Gutterman, D D

    2001-05-01

    Endothelium-dependent vasodilation is impaired in atherosclerosis. Oxidized low density lipoprotein (ox-LDL) plays an important role, possibly through alterations in G-protein activation. We examined the effect of acute exposure to ox-LDL on the dilator responses of isolated rabbit aorta segments. We sought also to evaluate the specificity of this dysfunction for dilator stimuli that traditionally operate through a Gi-protein mechanism. Aortic segments were prepared for measurement of isometric tension. After contraction with prostaglandin F2alpha, relaxation to thrombin, adenosine diphosphate (ADP), or the endothelium-independent agonists, sodium nitroprusside (SNP) or papaverine was examined. Maximal relaxation to thrombin was impaired in the presence of ox-LDL (17.7+/-3.7% p<0.05) compared to control (no LDL) (52.6+/-4.0%). Ox-LDL did not affect maximal relaxation to ADP or SNP. However, in the presence of charybdotoxin (CHTX: calcium-activated potassium channel inhibitor) ox-LDL impaired relaxation to ADP (17.4+/-3.2%). CHTX did not affect control (no LDL) responses to ADP (69.6+/-5.0%) or relaxation to thrombin or papaverine. In conclusion, ox-LDL impairs relaxation to thrombin, but in the case of ADP, calcium-activated potassium channels compensate to maintain this relaxation. PMID:11605770

  17. Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.

    PubMed

    Waters, Christopher W; Varuzhanyan, Grigor; Talmadge, Robert J; Voss, Andrew A

    2013-05-28

    Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

  18. Genetic variants in potassium channels are associated with type 2 diabetes in Mongolian population

    PubMed Central

    Odgerel, Zagaa; Lee, Hee S; Erdenebileg, Narnygerel; Gandbold, Suren; Luvsanjamba, Munkhjargal; Sambuughin, Nyamkhishig; Sonomtseren, Sainbileg; Sharavdorj, Purevdulam; Jodov, Erdenezul; Altaisaikhan, Khasag; Goldfarb, Lev G

    2011-01-01

    Objectives Recent genome-wide association studies (GWAS) have identified more than 40 common sequence variants associated with type 2 diabetes (T2D). However, the results are not always the same in populations with differing genetic backgrounds. We evaluated a hypothesis that a North Asian population living in a geographic area with unusually harsh environmental conditions developed unique genetic risks. Methods We performed a population-based association study with 21 single-nucleotide polymorphisms (SNPs) in 9 genes selected according to the results of GWAS conducted in other populations. The study participants included 393 full-heritage Mongolian individuals, 177 diagnosed with T2D and 216 matched controls. Genotyping was performed by TaqMan methodology. Results The strongest association was detected with SNPs located within the potassium-channel coding KCNQ1 (highest OR=1.92; P=3.4×10−5) and ABCC8 (OR=1.79; P=5×10−4) genes. Genetic variants identified as strongly influencing the risk of T2D in other populations such as those in KCNJ11 or TCF7L2 genes did not show statistically significant association in Mongolia. Conclusions The strongest T2D risk-associated SNPs in Mongolians are located within 2 of 3 tested potassium-channel coding genes; accumulated variations in these genes may be related to environmental exposure to extreme cold. PMID:22151254

  19. Opening of the inward rectifier potassium channel alleviates maladaptive tissue repair following myocardial infarction.

    PubMed

    Liu, Chengfang; Liu, Enli; Luo, Tiane; Zhang, Weifang; He, Rongli

    2016-08-01

    Activation of the inward rectifier potassium current (IK1) channel has been reported to be associated with suppression of ventricular arrhythmias. In this study, we tested the hypothesis that opening of the IK1 channel with zacopride (ZAC) was involved in the modulation of tissue repair after myocardial infarction. Sprague-Dawley rats were subject to coronary artery ligation and ZAC was administered intraperitoneally (15 µg/kg/day) for 28 days. Compared with the ischemia group, treatment with ZAC significantly reduced the ratio of heart/body weight and the cross-sectional area of cardiomyocytes, suggesting less cardiac hypertrophy. ZAC reduced the accumulation of collagen types I and III, accompanied with decrease of collagen area, which were associated with a reduction of collagen deposition in the fibrotic myocardium. Echocardiography showed improved cardiac function, evidenced by the reduced left ventricular end-diastolic dimension and left ventricular end-systolic dimension, and the increased ejection fraction and fractional shortening in ZAC-treated animals (all P < 0.05 vs. ischemia group). In coincidence with these changes, ZAC up-regulated the protein level of the IK1 channel and down-regulated the phosphorylation of mammalian target of rapamycin (mTOR) and 70-kDa ribosomal protein S6 (p70S6) kinase. Administration of chloroquine alone, an IK1 channel antagonist, had no effect on all the parameters measured, but significantly blocked the beneficial effects of ZAC on cardiac repair. In conclusion, opening of the IK1 channel with ZAC inhibits maladaptive tissue repair and improves cardiac function, potentially mediated by the inhibition of ischemia-activated mTOR-p70S6 signaling pathway via the IK1 channel. So the development of pharmacological agents specifically targeting the activation of the IK1 channel may protect the heart against myocardial ischemia-induced cardiac dysfunction. PMID:27486024

  20. Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury

    PubMed Central

    Yilmaz, Tonguç Utku; Yazihan, Nuray; Dalgic, Aydın; Kaya, Ezgi Ermis; Salman, Bulent; Kocak, Mehtap; Akcil, Ethem

    2015-01-01

    Background & objectives: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury. One of the targets to protect against injury is ATP-dependent potassium (KATP) channels. These channels could be involved in EPO induced ischaemic preconditoning like a protective effect. We evaluated the cell cytoprotective effects of EPO in relation to KATP channel activation in the renal tubular cell culture model under hypoxic/normoxic conditions. Methods: Dose and time dependent effects of EPO, KATP channel blocker glibenclamide and KATP channel opener diazoxide on cellular proliferation were evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] under normoxic and hypoxic conditions in human renal proximal tubular cell line (CRL-2830). Evaluation of the dose and time dependent effects of EPO, glibenclamide and diazoxide on apoptosis was done by caspase-3 activity levels. Hypoxia inducible factor-1 alpha (HIF-1 α) mRNA levels were measured by semi-quantative reverse transcription polymerase chain reaction (RT)-PCR. Kir 6.1 protein expresion was evalutaed by Western blot. Results: Glibenclamide treatment decreased the number of living cells in a time and dose dependent manner, whereas EPO and diazoxide treatments increased. Glibenclamide (100 μM) treatment significantly blocked the anti-apoptotic effects of EPO (10 IU/ml) under both normoxic and hypoxic conditions. EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (P<0.01) whereas glibenclamide decreased (P<0.05) HIF-1 α mRNA expression. Glibenclamide significantly (P<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group. Interpretation & conclusions: Our results showed that the cell proliferative, cytoprotective and anti-apoptotic effects of EPO were associated with KATP channels in the renal tubular cell culture model under hypoxic

  1. Pharmacological evidence for the participation of NO-cGMP-KATP pathway in the gastric protective effect of curcumin against indomethacin-induced gastric injury in the rat.

    PubMed

    Díaz-Triste, Nadia Estela; González-García, Martha Patricia; Jiménez-Andrade, Juan Miguel; Castañeda-Hernández, Gilberto; Chávez-Piña, Aracely Evangelina

    2014-05-01

    Curcumin, main compound obtained from rizhoma of Curcuma longa, shows antitumoral, antioxidant, anticarcinogenic and gastric protective properties. Recently, it has been demonstrated that curcumin exerts its gastric protective action due to an increase in gastric nitric oxide (NO) levels. However, it is unknown whether these increased NO levels are associated with activation of intracellular signaling pathways. Thus, the purpose of this study was to investigate the role of NO-cGMP-KATP pathway in the gastric protective effect of curcumin during indomethacin-induced gastric injury in the rat. Adult female Wistar rats were gavaged with curcumin (3-300mg/kg, p.o.) or omeprazole (30mg/kg, p.o.) 30min before indomethacin insult (30mg/kg, p.o.). Other groups of rats were administered L-NAME (70mg/kg, i.p.; inhibitor of nitric oxide synthase), ODQ (10mg/kg, i.p.; inhibitor of soluble guanylate cyclase) or glibenclamide (1mg/kg, i.p.; blocker of ATP-sensitive potassium (KATP) channels) 30min before curcumin (30mg/kg, p.o.). 3h after indomethacin administration, rats were sacrificed and gastric injury was evaluated by determining total damaged area. A sample of gastric tissue was harvested and processed to quantify organic nitrite levels. Curcumin significantly protected against indomethacin-induced gastric injury and this effect was comparable to gastroprotective effect by omeprazole. L-NAME, ODQ and glibenclamide significantly prevented the curcumin-mediated gastric protective effect in the indomethacin-induced gastric injury model. Furthermore, curcumin administration induced a significant increase in gastric nitric oxide levels as compared to vehicle administration. Our results show for the first time that curcumin activates NO/cGMP/KATP pathway during its gastro protective action.

  2. Channeling your inner ear potassium: K(+) channels in vestibular hair cells.

    PubMed

    Meredith, Frances L; Rennie, Katherine J

    2016-08-01

    During development of vestibular hair cells, K(+) conductances are acquired in a specific pattern. Functionally mature vestibular hair cells express different complements of K(+) channels which uniquely shape the hair cell receptor potential and filtering properties. In amniote species, type I hair cells (HCI) have a large input conductance due to a ubiquitous low-voltage-activated K(+) current that activates with slow sigmoidal kinetics at voltages negative to the membrane resting potential. In contrast type II hair cells (HCII) from mammalian and non-mammalian species have voltage-dependent outward K(+) currents that activate rapidly at or above the resting membrane potential and show significant inactivation. A-type, delayed rectifier and calcium-activated K(+) channels contribute to the outward K(+) conductance and are present in varying proportions in HCII. In many species, K(+) currents in HCII in peripheral locations of vestibular epithelia inactivate more than HCII in more central locations. Two types of inward rectifier currents have been described in both HCI and HCII. A rapidly activating K(+)-selective inward rectifier current (IK1, mediated by Kir2.1 channels) predominates in HCII in peripheral zones, whereas a slower mixed cation inward rectifier current (Ih), shows greater expression in HCII in central zones of vestibular epithelia. The implications for sensory coding of vestibular signals by different types of hair cells are discussed. This article is part of a Special Issue entitled .

  3. The distribution of intermediate-conductance, calcium-activated, potassium (IK) channels in epithelial cells.

    PubMed

    Thompson-Vest, Nichola; Shimizu, Yasutake; Hunne, Billie; Furness, John B

    2006-02-01

    Intermediate-conductance, calcium-activated, potassium (IK) channels were first identified by their roles in cell volume regulation, and were later shown to be involved in control of proliferation of lymphocytes and to provide a K+ current for epithelial secretory activity. Until now, there has been no systematic investigation of IK channel localization within different epithelia. IK channel immunoreactivity was present in most epithelia, where it occurred in surface membranes of epithelial cells. It was found in all stratified epithelia, including skin, cornea, oral mucosa, vaginal mucosa, urothelium and the oesophageal lining. It occurred in the ducts of fluid-secreting glands, the salivary glands, lacrimal glands and pancreas, and in the respiratory epithelium. A low level of expression was seen in serous acinar cells. It was also found in other epithelia with fluid-exchange properties, the choroid plexus epithelium, the ependyma, visceral pleura and peritoneum, bile ducts and intestinal lining epithelium. However, there was little or no expression in vascular endothelial cells, kidney tubules or collecting ducts, lung alveoli, or in sebaceous glands. It is concluded that the channel is present in surface epithelia (e.g. skin) where it has a cell-protective role against osmotic challenge, and in epithelia where there is anion secretion that is facilitated by a K+ current-dependent hyperpolarization. It was also in some epithelial cells where its roles are as yet unknown. PMID:16441566

  4. Probing the energy landscape of activation gating of the bacterial potassium channel KcsA.

    PubMed

    Linder, Tobias; de Groot, Bert L; Stary-Weinzinger, Anna

    2013-01-01

    The bacterial potassium channel KcsA, which has been crystallized in several conformations, offers an ideal model to investigate activation gating of ion channels. In this study, essential dynamics simulations are applied to obtain insights into the transition pathways and the energy profile of KcsA pore gating. In agreement with previous hypotheses, our simulations reveal a two phasic activation gating process. In the first phase, local structural rearrangements in TM2 are observed leading to an intermediate channel conformation, followed by large structural rearrangements leading to full opening of KcsA. Conformational changes of a highly conserved phenylalanine, F114, at the bundle crossing region are crucial for the transition from a closed to an intermediate state. 3.9 µs umbrella sampling calculations reveal that there are two well-defined energy barriers dividing closed, intermediate, and open channel states. In agreement with mutational studies, the closed state was found to be energetically more favorable compared to the open state. Further, the simulations provide new insights into the dynamical coupling effects of F103 between the activation gate and the selectivity filter. Investigations on individual subunits support cooperativity of subunits during activation gating.

  5. Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel.

    PubMed

    Schow, Eric V; Freites, J Alfredo; Nizkorodov, Alex; White, Stephen H; Tobias, Douglas J

    2012-07-01

    Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.

  6. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    PubMed

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

  7. Roles of calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation of the rabbit middle cerebral artery

    PubMed Central

    Dong, Hui; Waldron, Gareth J; Cole, William C; Triggle, Christopher R

    1998-01-01

    The cellular mechanism(s) of action of endothelium-derived vasodilator substances in the rabbit middle cerebral artery (RMCA) were investigated. Specifically, the subtypes of potassium channels involved in the effects of endothelium-derived relaxing factors (EDRFs) in acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in this vessel were systematically compared. In the endothelium-intact RMCA precontracted with histamine (3 μM), ACh induced a concentration-dependent vasorelaxation, which was sensitive to indomethacin (10 μM) or NG-nitro-L-arginine (L-NOARG; 100 μM); pD2 values 8.36 vs 7.40 and 6.38, P<0.01 for both, n=6 and abolished by a combination of both agents. ACh caused relaxation in the presence of high K+ PSS (40 mM KCl), which was not affected by indomethacin, but abolished by L-NOARG and a combination of indomethacin and L-NOARG. In the presence of indomethacin, relaxation to ACh in the endothelium-intact RMCA precontracted with histamine was unaffected by either glibenclamide (10 μM), an ATP-sensitive K+ channel (KATP) blocker, 4-aminopyridine (4-AP, 1 mM) or dendrotoxin (DTX, 0.1 μM), delayed rectifier K+ channel (KV) blockers. However, relaxation responses to ACh were significantly inhibited by either LY83583 (10 μM) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 μM), guanylyl cyclase inhibitors, or charybdotoxin (CTX; 0.1 μM), iberiotoxin (ITX, 0.1 μM) and apamin (APA, 0.1 μM), large conductance Ca2+ -activated K+ channels (BKCa) blocker and small conductance Ca2+ -activated K+ channel (SKCa) blocker, respectively. In the presence of L-NOARG, relaxation to ACh was unaffected by glibenclamide or the cytochrome P450 mono-oxygenase inhibitor, clotrimazole (1 μM), but was significantly inhibited by either 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ  22,536, 10 μM) and 2′,3′-dideoxyadenosine (2′,3′-DDA, 30 μM), adenylyl cyclase inhibitors, or 4-AP, DTX, CTX, ITX and APA. In

  8. Solute inaccessible aqueous volume changes during opening of the potassium channel of the squid giant axon.

    PubMed Central

    Zimmerberg, J; Bezanilla, F; Parsegian, V A

    1990-01-01

    We have applied solutions with varying osmotic pressures symmetrically to the inside and outside of perfused, TTX-treated, giant axons. The potassium conductance G decreased with increasing osmotic stress, but there was no effect on either the shape or the position of the voltage-current curve. One must distinguish three possible actions of the osmotic agent: osmotic stress, channel blocking, and lowered solution conductivity. To do so, we compared results obtained working with pairs of internal and external solutions of either (a) equal osmotic stress, (b) equal conductivity, or (c) the same blocking agent. There was the same change in G irrespective of the type of stressing species (sorbitol or sucrose); this provides some evidence against a blocking mechanism. The conductivity of the external solution had a small effect on K currents; internal solution conductivity had none. A change in series resistance of the Schwann cell layer could account for the small effect of external solution conductivity. The primary cause of G depression appears, then, to be the applied osmotic stress. Using this result, we have developed models in which the channel has a transition between closed states under voltage control but osmotically insensitive and a closed/open step that is voltage-independent but osmotically sensitive. We have assumed that the conductance of this open state does not change with osmotic stress. In this way, we estimate that an additional 1,350 +/- 200 A3 or 40-50 molecules of solute-inaccessible water appear to associate with the average delayed rectifier potassium channel of the squid axon when it opens. PMID:2340341

  9. Involvement of a membrane potassium channel in heparan sulphate-induced activation of macrophages.

    PubMed

    Ren, Jian-Dong; Fan, Li; Tian, Fu-Zhou; Fan, Kai-Hua; Yu, Bo-Tao; Jin, Wei-Hua; Tan, Yong-Hong; Cheng, Long

    2014-03-01

    Increasing evidence has demonstrated that Toll-like receptor 4 (TLR4) -mediated systemic inflammatory response syndrome accompanied by multiple organ failure, is one of the most common causes of death in patients with severe acute pancreatitis. Recent reports have revealed that heparan sulphate (HS) proteoglycan, a component of extracellular matrices, potentiates the activation of intracellular pro-inflammatory responses via TLR4, contributing to the aggravation of acute pancreatitis. However, little is known about the participants in the HS/TLR4-mediated inflammatory cascades. Our previous work provided a clue that a membrane potassium channel (MaxiK) is responsible for HS-induced production of inflammatory cytokines. Therefore, in this report we attempted to reveal the roles of MaxiK in the activation of macrophages stimulated by HS. Our results showed that incubation of RAW264.7 cells with HS up-regulated MaxiK and TLR4 expression levels. HS could also activate MaxiK channels to promote the efflux of potassium ions from cells, as measured by the elevated activity of caspase-1, whereas this was significantly abolished by treatment with paxilline, a specific blocker of the MaxiK channel. Moreover, it was found that paxilline substantially inhibited HS-induced activation of several different transcription factors in macrophages, including nuclear factor-κB, p38 and interferon regulatory factor-3, followed by decreased production of tumour necrosis factor-α and interferon-β. Taken together, our investigation provides evidence that the HS/TLR4-mediated intracellular inflammatory cascade depends on the activation of MaxiK, which may offer an important opportunity for a new approach in therapeutic strategies of severe acute pancreatitis.

  10. Distribution of High-Conductance Calcium-Activated Potassium Channels in Rat Vestibular Epithelia

    PubMed Central

    Schweizer, Felix E.; Savin, David; Luu, Cindy; Sultemeier, David R.; Hoffman, Larry F.

    2011-01-01

    Voltage- and calcium-activated potassium channels (BK) are important regulators of neuronal excitability. BK channels seem to be crucial for frequency tuning in nonmammalian vestibular and auditory hair cells. However, there are a paucity of data concerning BK expression in mammalian vestibular hair cells. We therefore investigated the localization of BK channels in mammalian vestibular hair cells, specifically in rat vestibular neuroepithelia. We find that only a subset of hair cells in the utricle and the crista ampullaris express BK channels. BK-positive hair cells are located mainly in the medial striolar region of the utricle, where they constitute at most 12% of hair cells, and in the central zone of the horizontal crista. A majority of BK-positive hair cells are encapsulated by a calretinin-positive calyx defining them as type I cells. The remainder are either type I cells encapsulated by a calretinin-negative calyx or type II hair cells. Surprisingly, the number of BK-positive hair cells in the utricle peaks in juvenile rats and declines in early adulthood. BK channels were not found in vestibular afferent dendrites or somata. Our data indicate that BK channel expression in the mammalian vestibular system differs from the expression pattern in the mammalian auditory and the nonmammalian vestibular system. The molecular diversity of vestibular hair cells indicates a functional diversity that has not yet been fully characterized. The predominance of BK-positive hair cells within the medial striola of juvenile animals suggests that they contribute to a scheme of highly lateralized coding of linear head movements during late development. PMID:19731297

  11. Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.

    PubMed

    Cornfield, D N; Reeve, H L; Tolarova, S; Weir, E K; Archer, S

    1996-07-23

    At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation. PMID:8755608

  12. Potassium channels cloned from neuroblastoma cells display slowly inactivating outward currents in Xenopus oocytes.

    PubMed

    Ito, Y; Yokoyama, S; Higashida, H

    1992-05-22

    Messenger RNAs (mRNAs) specific for NGK1 and NGK2 potassium channels were synthesized from complementary DNAs (cDNAs) that had been cloned from mouse neuroblastoma x rat glioma hybrid NG108-15 cells. Outward pottasium currents were evoked by 5 s depolarizing voltage commands in Xenopus oocytes injected with NGK1- or NGK2-specific mRNAs. The NGK1 or NGK2 currents showed different activation and inactivation kinetics, and different pharmacological sensitivities. The threshold potential for activation of the NGK2 current (-14 mV) was more positive than that for the NGK1 (-36 mV). The NGK2 current showed faster inactivation during a 5 s depolarizing pulse than did the NGK1 current. Inactivation was best fit by time constants of 0.37, 1.5 and 19 s for the NGK2 current and 4.4 and 19 s for NGK1. Extracellularly applied tetraethylammonium chloride (TEA) was 1000 times more potent on the NGK2 current than the NGK1 current. Furthermore we examined outward current following co-injection of an equal amount of mRNAs for NGK1 and NGK2. The timecourse of inactivation differed from either alone or from a simple sum of the two individual currents. TEA sensitivity could not be explained by summation of the two homomultimeric channels. These findings suggest that both NGK1 and NGK2 proteins assemble to form heteromultimeric K+ channels in addition to homomultimeric K+ channels. NGK2 channels and the heteromultimeric channels may be responsible for the native transient outward current with slow inactivation in NG108-15 hybrid cells.

  13. New inhibitors of the Kvβ2 subunit from mammalian Kv1 potassium channels.

    PubMed

    Alka, Kumari; Dolly, J Oliver; Ryan, Barry J; Henehan, Gary T M

    2014-10-01

    The role of the redox state of Kvβ subunits in the modulation of Kv1 potassium channels has been well documented over the past few years. It has been suggested that a molecule that binds to or inhibits the aldo-keto reductase activity of Kvβ might affect the modulation of channel properties. Previous studies of possible modulators of channel activity have shown that cortisone and some related compounds are able to physically dissociate the channel components by binding to a site at the interface between α and β subunits. Herein, we describe some new inhibitors of rat brain Kvβ2, identified using an assay based on multiple substrate turnover. This approach allows one to focus on molecules that specifically block NADPH oxidation. These studies showed that, at 0.5mM, 3,4-dihydroxphenylacetic acid (DOPAC) was an inhibitor of Kvβ2 turnover yielding a ∼ 40-50% reduction in the aldehyde reductase activity of this subunit. Other significant inhibitors include the bioflavinoid, rutin and the polyphenol resveratrol; some of the known cardioprotective effects of these molecules may be attributable to Kv1 channel modulation. Cortisone or catechol caused moderate inhibition of Kvβ2 turnover, and the aldo-keto reductases inhibitor valproate had an even smaller effect. Despite the importance of the Kv1 channels in a number of disease states, there have been few Kvβ2 inhibitors reported. While the ones identified in this study are only effective at high concentrations, they could serve as tools to decipher the role of Kvβ2 in vivo and, eventually, inform the development of novel therapeutics.

  14. Modeling the concentration-dependent permeation modes of the KcsA potassium ion channel

    NASA Astrophysics Data System (ADS)

    Nelson, Peter Hugo

    2003-12-01

    The potassium channel from Streptomyces lividans (KcsA) is an integral membrane protein with sequence similarity to all known potassium channels, particularly in the selectivity filter region. A recently proposed model for ion channels containing either n or (n-1) single-file ions in their selectivity filters [P. H. Nelson, J. Chem. Phys. 177, 11396 (2002)] is applied to published KcsA channel K+ permeation data that exhibit a high-affinity process at low concentrations and a low-affinity process at high concentrations [M. LeMasurier et al., J. Gen. Physiol. 118, 303 (2001)]. The kinetic model is shown to provide a reasonable first-order explanation for both the high- and low-concentration permeation modes observed experimentally. The low-concentration mode ([K+]<200 mM) has a 200-mV dissociation constant of 56 mM and a conductance of 88 pS. The high-concentration mode ([K+]>200 mM) has a 200-mV dissociation constant of 1100 mM and a conductance of 500 pS. Based on the permeation model, and x-ray analysis [J. H. Morais-Cabral et al., Nature (London) 414, 37 (2001)], it is suggested that the experimentally observed K+ permeation modes correspond to an n=3 mechanism at high concentrations and an n=2 mechanism at low concentrations. The ratio of the electrical dissociation distances for the high- and low-concentration modes is 3:2, also consistent with the proposed n=3 and n=2 modes. Model predictions for K+ channels that exhibit asymmetric current-voltage (I-V) curves are presented, and further validation of the kinetic model via molecular simulation and experiment is discussed. The qualitatively distinct I-V characteristics exhibited experimentally by Tl+, NH+4, and Rb+ ions at 100 mM concentration can also be explained using the model, but more extensive experimental tests are required for quantitative validation of the model predictions.

  15. Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells

    PubMed Central

    Panyi, G.; Vámosi, G.; Bacsó, Z.; Bagdány, M.; Bodnár, A.; Varga, Z.; Gáspár, R.; Mátyus, L.; Damjanovich, S.

    2004-01-01

    Membrane proteins of cytotoxic T cells specifically reorganize to form an immunological synapse (IS) on interaction with their specific target. In this paper, we investigated the redistribution of Kv1.3 channels, which are the dominant voltage-gated potassium channels, in the plasma membrane of allogen-activated human cytotoxic T lymphocytes (CTLs) on interacting with their specific target cells. Kv1.3 channels bearing a FLAG epitope were expressed in the CTLs and the cell-surface distribution of fluorescently labeled ion channels was determined from confocal laser-scanning microscopy images. FLAG epitope-tagged Kv1.3 channels showed a patchy distribution in CTLs not engaged with target cells, whereas the channels were accumulated in the IS formed between CTLs and specific target lymphocytes. Localization of Kv1.3 channels in the IS might open an unrevealed possibility in the regulation of ion channel activity by signaling molecules accumulated in the IS. PMID:14745040

  16. Angiotensin II inhibits the ROMK-like small conductance K channel in renal cortical collecting duct during dietary potassium restriction.

    PubMed

    Wei, Yuan; Zavilowitz, Beth; Satlin, Lisa M; Wang, Wen-Hui

    2007-03-01

    Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

  17. Effects of common antitussive drugs on the hERG potassium channel current.

    PubMed

    Deisemann, Heike; Ahrens, Nadine; Schlobohm, Irene; Kirchhoff, Christian; Netzer, Rainer; Möller, Clemens

    2008-12-01

    A common over-the-counter (OTC) non-opioid antitussive drug, clobutinol, was recently withdrawn from the market due to its potential to induce cardiac arrhythmias by a blockade of the potassium channel coded by the human ether-à-go-go-related gene (hERG). In this study, we investigated the effects of a number of antitussive compounds on the hERG ion channel current using patch-clamp electrophysiology, and compared the effects to that of clobutinol. The compounds clobutinol, pentoxyverine, dextromethorphan, and codeine inhibited the outward current in hERG transfected cells with half-maximal inhibition concentrations (IC50) of 1.9 microM, 3.0 microM, 5.1 microM, and 97 microM, respectively. For theobromine, no significant effect on the hERG current at a concentration up to 100 microM was detected. Safety margins between the effects of the drugs on the hERG ion channel current and their calculated maximal free therapeutic plasma concentration were calculated. These results were compared to assess potential risks of the compounds to induce torsade de pointes-type arrhythmias.

  18. Effects of common antitussive drugs on the hERG potassium channel current.

    PubMed

    Deisemann, Heike; Ahrens, Nadine; Schlobohm, Irene; Kirchhoff, Christian; Netzer, Rainer; Möller, Clemens

    2008-12-01

    A common over-the-counter (OTC) non-opioid antitussive drug, clobutinol, was recently withdrawn from the market due to its potential to induce cardiac arrhythmias by a blockade of the potassium channel coded by the human ether-à-go-go-related gene (hERG). In this study, we investigated the effects of a number of antitussive compounds on the hERG ion channel current using patch-clamp electrophysiology, and compared the effects to that of clobutinol. The compounds clobutinol, pentoxyverine, dextromethorphan, and codeine inhibited the outward current in hERG transfected cells with half-maximal inhibition concentrations (IC50) of 1.9 microM, 3.0 microM, 5.1 microM, and 97 microM, respectively. For theobromine, no significant effect on the hERG current at a concentration up to 100 microM was detected. Safety margins between the effects of the drugs on the hERG ion channel current and their calculated maximal free therapeutic plasma concentration were calculated. These results were compared to assess potential risks of the compounds to induce torsade de pointes-type arrhythmias. PMID:19034038

  19. Assignment of the human hippocampal inward rectifier potassium channel (HIR) gene to 22q13.1

    SciTech Connect

    Budarf, M.L.; Barnoski, B.L.; Bell, C.J.

    1995-04-10

    The HIR gene encodes a small-conductance inward rectifier potassium channel (HIR or K{sub IR}2.3) that is found in heart and brain. Inward rectifiers are a specialized class of potassium channels that produce large inward currents at potentials negative to the potassium equilibrium potential and only small outward currents at more positive potentials. This asymmetry in K{sup +} conductance plays a key role in the excitability of muscle cells and neurons. Inward rectifier potassium channels are the major contributors to the basal potassium conductance in cardiac muscle, where their role is to modulate cell excitability and heart beat frequency, maintain the resting potential, and terminate the long-duration cardiac action potentials. In the central nervous system, inward rectifiers are involved in similar aspects of the modulation of cell excitability. The central role of inward rectifiers in cardiac and neuronal function suggest that they might be involved in the etiology of human cardiovascular and neurological diseases. 11 refs., 2 figs.

  20. Targeting Ion Channels: An Important Therapeutic Implication in Gastrointestinal Dysmotility in Patients With Spinal Cord Injury

    PubMed Central

    Radulovic, Miroslav; Anand, Preeti; Korsten, Mark A; Gong, Bing

    2015-01-01

    Gastrointestinal (GI) dysmotility is a severe, and common complication in patients with spinal cord injury (SCI). Current therapeutic methods using acetylcholine analogs or laxative agents have unwanted side effects, besides often fail to have desired effect. Various ion channels such as ATP-sensitive potassium (KATP) channel, calcium ions (Ca2+)-activated potassium ions (K+) channels, voltage-sensitive Ca2+ channels and chloride ion (Cl−) channels are abundantly expressed in GI tissues, and play an important role in regulating GI motility. The release of neurotransmitters from the enteric nerve terminal, innervating GI interstitial cells of Cajal (ICC), and smooth muscle cells (SMC), causes inactivation of K+ and Cl− channels, increasing Ca2+ influx into cytoplasm, resulting in membrane depolarization and smooth muscle contraction. Thus, agents directly regulating ion channels activity either in ICC or in SMC may affect GI peristalsis and would be potential therapeutic target for the treatment of GI dysmotility with SCI. PMID:26424038

  1. Presence of voltage-gated potassium channel complex antibody in a case of genetic prion disease

    PubMed Central

    Jammoul, Adham; Lederman, Richard J; Tavee, Jinny; Li, Yuebing

    2014-01-01

    Voltage-gated potassium channel (VGKC) complex antibody-mediated encephalitis is a recently recognised entity which has been reported to mimic the clinical presentation of Creutzfeldt-Jakob disease (CJD). Testing for the presence of this neuronal surface autoantibody in patients presenting with subacute encephalopathy is therefore crucial as it may both revoke the bleak diagnosis of prion disease and allow institution of potentially life-saving immunotherapy. Tempering this optimistic view is the rare instance when a positive VGKC complex antibody titre occurs in a definite case of prion disease. We present a pathologically and genetically confirmed case of CJD with elevated serum VGKC complex antibody titres. This case highlights the importance of interpreting the result of a positive VGKC complex antibody with caution and in the context of the overall clinical manifestation. PMID:24903967

  2. High grade glioma mimicking voltage gated potassium channel complex associated antibody limbic encephalitis.

    PubMed

    Athauda, Dilan; Delamont, R S; Pablo-Fernandez, E De

    2014-01-01

    Though raised titres of voltage gated potassium channel (VGKC) complex antibodies have been occasionally associated with extracranial tumours, mainly presenting as Morvan's Syndrome or neuromyotonia, they have not yet been reported to be associated with an intracranial malignancy. This is especially important as misdiagnosis of these conditions and delay of the appropriate treatment can have important prognostic implications. We describe a patient with a high grade glioma presenting with clinical, radiological, and serological features consistent with the diagnosis of VGKC antibody associated limbic encephalitis (LE). This is the first association between a primary brain tumour and high titre of VGKC complex antibodies. Clinicoradiological progression despite effective immunosuppressive treatment should prompt clinicians to look for alternative diagnoses. Further studies to elucidate a possible association between VGKC complex and other surface antigen antibodies with primary brain tumours should be carried out.

  3. Presence of voltage-gated potassium channel complex antibody in a case of genetic prion disease.

    PubMed

    Jammoul, Adham; Lederman, Richard J; Tavee, Jinny; Li, Yuebing

    2014-06-05

    Voltage-gated potassium channel (VGKC) complex antibody-mediated encephalitis is a recently recognised entity which has been reported to mimic the clinical presentation of Creutzfeldt-Jakob disease (CJD). Testing for the presence of this neuronal surface autoantibody in patients presenting with subacute encephalopathy is therefore crucial as it may both revoke the bleak diagnosis of prion disease and allow institution of potentially life-saving immunotherapy. Tempering this optimistic view is the rare instance when a positive VGKC complex antibody titre occurs in a definite case of prion disease. We present a pathologically and genetically confirmed case of CJD with elevated serum VGKC complex antibody titres. This case highlights the importance of interpreting the result of a positive VGKC complex antibody with caution and in the context of the overall clinical manifestation.

  4. Localization of two potassium channel {beta} subunit genes, KCNA1B and KCNA2B

    SciTech Connect

    Schultz, D.; Smith, L.; Thayer, M.

    1996-02-01

    The gating properties and current amplitudes of mammalian voltage-activated Shaker potassium channels are modulated by at least two associated {beta} subunits (Kv{beta}1.1 and Kv{beta}1.2). The human Kv{beta}1.1 gene (KCNA1B) resides on chromosome 3, as indicated by somatic cell hybrid mapping. More precise localization of KCNA1B to 3q26.1 was obtained with fluorescence in situ hybridization (FISH) and was corroborated by PCR screening of the CEPH YAC library. The human Kv{beta}1.2 gene (KCNA2B) resides on chromosome 1, as indicated by somatic cell hybrid mapping, and has been localized by FISH to 1p36.3. 20 refs., 2 figs.

  5. Presence of voltage-gated potassium channel complex antibody in a case of genetic prion disease.

    PubMed

    Jammoul, Adham; Lederman, Richard J; Tavee, Jinny; Li, Yuebing

    2014-01-01

    Voltage-gated potassium channel (VGKC) complex antibody-mediated encephalitis is a recently recognised entity which has been reported to mimic the clinical presentation of Creutzfeldt-Jakob disease (CJD). Testing for the presence of this neuronal surface autoantibody in patients presenting with subacute encephalopathy is therefore crucial as it may both revoke the bleak diagnosis of prion disease and allow institution of potentially life-saving immunotherapy. Tempering this optimistic view is the rare instance when a positive VGKC complex antibody titre occurs in a definite case of prion disease. We present a pathologically and genetically confirmed case of CJD with elevated serum VGKC complex antibody titres. This case highlights the importance of interpreting the result of a positive VGKC complex antibody with caution and in the context of the overall clinical manifestation. PMID:24903967

  6. Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to potassium channels.

    PubMed

    Martins, Carina C; Bassetto, Carlos A Zanutto; Santos, Jandyson M; Eberlin, Marcos N; Magalhães, Alvicler; Varanda, Wamberto; Gonzalez, Eduardo R Perez

    2016-02-01

    Herein, we report the synthesis and mass spectrometry studies of several N-alkylbenzenesulfonamides structurally related to sulfanilic acid. The compounds were synthesized using a modified Schotten-Baumann reaction coupled with Meisenheimer arylation. Sequential mass spectrometry by negative mode electrospray ionization (ESI(-)-MS/MS) showed the formation of sulfoxylate anion (m/z 65) observed in the mass spectrum of p-chloro-N-alkylbenzenesulfonamides. Investigation of the unexpected loss of two water molecules, as observed by electron ionization mass spectrometry (EI-MS) analysis of p-(N-alkyl)lactam sulfonamides, led to the proposal of corresponding fragmentation pathways. These compounds showed loss of neutral iminosulfane dioxide molecule (M-79) with formation of ions observed at m/z 344 and 377. These ions were formed by rearrangement on ESI(+)-MS/MS analysis. Some of the molecules showed antagonistic activity against Kv3.1 voltage-gated potassium channels.

  7. The BTB domains of the potassium channel tetramerization domain proteins prevalently assume pentameric states.

    PubMed

    Smaldone, Giovanni; Pirone, Luciano; Pedone, Emilia; Marlovits, Thomas; Vitagliano, Luigi; Ciccarelli, Luciano

    2016-06-01

    Potassium channel tetramerization domain-containing (KCTD) proteins are involved in fundamental physio-pathological processes. Here, we report an analysis of the oligomeric state of the Bric-à-brack, Tram-track, Broad complex (BTB) domains of seven distinct KCTDs belonging to five major clades of the family evolution tree. Despite their functional and sequence variability, present electron microscopy data highlight the occurrence of well-defined pentameric states for all domains. Our data also show that these states coexist with alternative forms which include open pentamers. Thermal denaturation analyses conducted using KCTD1 as a model suggest that, in these proteins, different domains cooperate to their overall stability. Finally, negative-stain electron micrographs of KCTD6(BTB) in complex with Cullin3 show the presence of assemblies with a five-pointed pinwheel shape. PMID:27152988

  8. Regulation of voltage-gated potassium channels by PI(4,5)P2.

    PubMed

    Kruse, Martin; Hammond, Gerald R V; Hille, Bertil

    2012-08-01

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) regulates activities of numerous ion channels including inwardly rectifying potassium (K(ir)) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K(V)) channels might be regulated by PI(4,5)P(2). Wide expression of K(V) channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K(V) channels by PI(4,5)P(2), we have coexpressed several of them in tsA-201 cells with a G protein-coupled receptor (M(1)R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P(2) with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P(2) at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K(V)7.1, K(V)7.2/7.3, and K(ir)2.1 channel current by 90-95%. Activation of M(1)R inhibited K(V)7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P(2) regulation of activity of K(V)1.1/K(V)β1.1, K(V)1.3, K(V)1.4, and K(V)1.5/K(V)β1.3, K(V)2.1, K(V)3.4, K(V)4.2, K(V)4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K(V)1.1/K(V)β1.1 and K(V)3.4, resulting in up-regulation of current density upon activation of M(1)R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P(2) at the plasma membrane by enzymes does not seem to influence activity of most tested K(V) channels, whereas it does strongly inhibit members of the K(V)7 and K(ir) families. PMID

  9. Recessive Mutations in KCNJ13, Encoding an Inwardly Rectifying Potassium Channel Subunit, Cause Leber Congenital Amaurosis

    PubMed Central

    Sergouniotis, Panagiotis I.; Davidson, Alice E.; Mackay, Donna S.; Li, Zheng; Yang, Xu; Plagnol, Vincent; Moore, Anthony T.; Webster, Andrew R.

    2011-01-01

    Inherited retinal degenerations, including retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), comprise a group of disorders showing high genetic and allelic heterogeneity. The determination of a full catalog of genes that can, when mutated, cause human retinal disease is a powerful means to understand the molecular physiology and pathology of the human retina. As more genes are found, remaining ones are likely to be rarer and/or unexpected candidates. Here, we identify a family in which all known RP/LCA-related genes are unlikely to be associated with their disorder. A combination of homozygosity mapping and exome sequencing identifies a homozygous nonsense mutation, c.496C>T (p.Arg166X), in a gene, KCNJ13, encoding a potassium channel subunit Kir7.1. A screen of a further 333 unrelated individuals with recessive retinal degeneration identified an additional proband, homozygous for a missense mutation, c.722T>C (p.Leu241Pro), in the same gene. The three affected members of the two families have been diagnosed with LCA. All have a distinct and unusual retinal appearance and a similar early onset of visual loss, suggesting both impaired retinal development and progressive retinal degeneration, involving both rod and cone pathways. Examination of heterozygotes revealed no ocular disease. This finding implicates Kir7.1 as having an important role in human retinal development and maintenance. This disorder adds to a small diverse group of diseases consequent upon loss or reduced function of inwardly rectifying potassium channels affecting various organs. The distinct retinal phenotype that results from biallelic mutations in KCNJ13 should facilitate the molecular diagnosis in further families. PMID:21763485

  10. Encephalitis and antibodies to DPPX, a subunit of Kv4.2 potassium channels

    PubMed Central

    Boronat, Anna; Gelfand, Jeffrey M.; Gresa-Arribas, Nuria; Jeong, Hyo-Young; Walsh, Michael; Roberts, Kirk; Martinez-Hernandez, Eugenia; Rosenfeld, Myrna R.; Balice-Gordon, Rita; Graus, Francesc; Rudy, Bernardo; Dalmau, Josep

    2012-01-01

    Objective To report a novel cell-surface autoantigen of encephalitis that is a critical regulatory subunit of the Kv4.2 potassium channels. Methods Four patients with encephalitis of unclear etiology and antibodies with a similar pattern of neuropil brain immunostaining were selected for autoantigen characterization. Techniques included immunoprecipitation, mass spectrometry, cell-base experiments with Kv4.2 and several dipeptidyl-peptidase-like protein-6 (DPPX) plasmid constructs, and comparative brain immunostaining of wild-type and DPPX-null mice. Results Immunoprecipitation studies identified DPPX as the target autoantigen. A cell based assay confirmed that all 4 patients, but not 210 controls, had DPPX antibodies. Symptoms included agitation, confusion, myoclonus, tremor, and seizures (one case with prominent startle response). All patients had pleocytosis, and three had severe prodromal diarrhea of unknown etiology. Given that DPPX “tunes up” the Kv4.2 potassium channels (involved in somatodendritic signal integration and attenuation of dendritic backpropagation of action potentials), we determined the epitope distribution in DPPX, DPP10 (a protein homologous to DPPX) and Kv4.2. Patients’ antibodies were found specific for DPPX, without reacting with DPP10 or Kv4.2. The unexplained diarrhea led to demonstrate a robust expression of DPPX in the myenteric plexus, which strongly reacted with patients’ antibodies. The course of neuropsychiatric symptoms was prolonged and often associated with relapses while decreasing immunotherapy. Long-term follow-up showed substantial improvement in 3 patients (1 is lost to follow-up). Interpretation Antibodies to DPPX associate with a protracted encephalitis characterized by CNS hyperexcitability (agitation, myoclonus, tremor, seizures), pleocytosis, and frequent diarrhea at symptom onset. The disorder is potentially treatable with immunotherapy. PMID:23225603

  11. Voltage-gated potassium channel-complex autoimmunity and associated clinical syndromes.

    PubMed

    Irani, Sarosh R; Vincent, Angela

    2016-01-01

    Voltage-gated potassium channel (VGKC)-complex antibodies are defined by the radioimmunoprecipitation of Kv1 potassium channel subunits from brain tissue extracts and were initially discovered in patients with peripheral nerve hyperexcitability (PNH). Subsequently, they were found in patients with PNH plus psychosis, insomnia, and dysautonomia, collectively termed Morvan's syndrome (MoS), and in a limbic encephalopathy (LE) with prominent amnesia and frequent seizures. Most recently, they have been described in patients with pure epilepsies, especially in patients with the novel and distinctive semiology termed faciobrachial dystonic seizures (FBDS). In each of these conditions, there is a close correlation between clinical measures and antibody levels. The VGKC-complex is a group of proteins that are strongly associated in situ and after extraction in mild detergent. Two major targets of the autoantibodies are leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2). The patients with PNH or MoS are most likely to have CASPR2 antibodies, whereas LGI1 antibodies are found characteristically in patients with FBDS and LE. Crucially, each of these conditions has a good response to immunotherapies, often corticosteroids and plasma exchange, although optimal regimes require further study. VGKC-complex antibodies have also been described in neuropathic pain syndromes, chronic epilepsies, a polyradiculopathy in porcine abattoir workers, and some children with status epilepticus. Increasingly, however, the antigenic targets in these patients are not defined and in some cases the antibodies may be secondary rather than the primary cause. Future serologic studies should define all the antigenic components of the VGKC-complex, and further inform mechanisms of antibody pathogenicity and related inflammation.

  12. High-throughput screening for small-molecule modulators of inward rectifier potassium channels.

    PubMed

    Raphemot, Rene; Weaver, C David; Denton, Jerod S

    2013-01-01

    Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels. The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS). Here, we describe how to develop a fluorescence-based thallium (Tl(+)) flux assay of Kir channel function for high-throughput compound screening. The assay is based on the permeability of the K(+) channel pore to the K

  13. Students' Understanding of External Representations of the Potassium Ion Channel Protein Part II: Structure-Function Relationships and Fragmented Knowledge

    ERIC Educational Resources Information Center

    Harle, Marissa; Towns, Marcy H.

    2012-01-01

    Research that has focused on external representations in biochemistry has uncovered student difficulties in comprehending and interpreting external representations. This study focuses on students' understanding of three external representations (ribbon diagram, wireframe, and hydrophobic/hydrophilic) of the potassium ion channel protein. Analysis…

  14. Evaluation of potassium permanganate against an experimental subacute infection of Flavobacterium columnare in channel catfish, Icatlurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficacy of potassium permanganate (KMnO4) as a prophylactic and therapeutic treatment for subacute infection of Flavobacterium columnare was demonstrated in experimentally infected channel catfish, Ictalurus punctatus. Catfish experimentally infected with F. columnare to mimic a subacute infec...

  15. Evaluation of potassium permanganate against an experimental subacute infection of Flavobacterium columnare in channel catfish, Icatlurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An experiment was performed to evaluate the efficacy of potassium permanganate (KMnO4) as a prophylactic and therapeutic treatment of an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus. Fish were cutaneously abraded and divided into five treatment...

  16. Comparative effects of copper sulfate or potassium permanganate on channel catfish concurrently infected with Flavobacterium columnare and Ichthyobodo necator

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An opportunistic study was conducted to determine the effects of two chemical therapeutants on channel catfish (CCF) Ictalurus punctatus concurrently infected Flavobacterium columnare and Ichthyobodo necator. Copper sulfate (CuSO4) and potassium permanganate (KMnO4) were investigated for their abil...

  17. Genetic Variation in Genes Encoding Airway Epithelial Potassium Channels Is Associated with Chronic Rhinosinusitis in a Pediatric Population

    PubMed Central

    Mentch, Frank; Grant, Struan F. A.; Desrosiers, Martin; Hakonarson, Hakon; Toskala, Elina

    2014-01-01

    Background Apical potassium channels regulate ion transport in airway epithelial cells and influence air surface liquid (ASL) hydration and mucociliary clearance (MCC). We sought to identify whether genetic variation within genes encoding airway potassium channels is associated with chronic rhinosinusitis (CRS). Methods Single nucleotide polymorphism (SNP) genotypes for selected potassium channels were derived from data generated on the Illumnia HumanHap550 BeadChip or Illumina Human610-Quad BeadChip for 828 unrelated individuals diagnosed with CRS and 5,083 unrelated healthy controls from the Children's Hospital of Philadelphia (CHOP). Statistical analysis was performed with set-based tests using PLINK, and corrected for multiple testing. Results Set-based case control analysis revealed the gene KCNMA1 was associated with CRS in our Caucasian subset of the cohort (598 CRS cases and 3,489 controls; p = 0.022, based on 10,000 permutations). In addition there was borderline evidence that the gene KCNQ5 (p = 0.0704) was associated with the trait in our African American subset of the cohort (230 CRS cases and 1,594 controls). In addition to the top significant SNPs rs2917454 and rs6907229, imputation analysis uncovered additional genetic variants in KCNMA1 and in KCNQ5 that were associated with CRS. Conclusions We have implicated two airway epithelial potassium channels as novel susceptibility loci in contributing to the pathogenesis of CRS. PMID:24595210

  18. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    PubMed

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation. PMID:25825713

  19. Molecular basis of hERG potassium channel blockade by the class Ic antiarrhythmic flecainide

    PubMed Central

    Melgari, Dario; Zhang, Yihong; El Harchi, Aziza; Dempsey, Christopher E.; Hancox, Jules C.

    2015-01-01

    The class Ic antiarrhythmic drug flecainide inhibits KCNH2-encoded “hERG” potassium channels at clinically relevant concentrations. The aim of this study was to elucidate the underlying molecular basis of this action. Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37 °C. Wild-type (WT) IhERG was inhibited with an IC50 of 1.49 μM and this was not significantly altered by reversing the direction of K+ flux or raising external [K+]. The use of charged and uncharged flecainide analogues showed that the charged form of the drug accesses the channel from the cell interior to produce block. Promotion of WT IhERG inactivation slowed recovery from inhibition, whilst the N588K and S631A attenuated-inactivation mutants exhibited IC50 values 4–5 fold that of WT IhERG. The use of pore-helix/selectivity filter (T623A, S624A V625A) and S6 helix (G648A, Y652A, F656A) mutations showed < 10-fold shifts in IC50 for all but V625A and F656A, which respectively exhibited IC50s 27-fold and 142-fold their WT controls. Docking simulations using a MthK-based homology model suggested an allosteric effect of V625A, since in low energy conformations flecainide lay too low in the pore to interact directly with that residue. On the other hand, the molecule could readily form π–π stacking interactions with aromatic residues and particularly with F656. We conclude that flecainide accesses the hERG channel from the cell interior on channel gating, binding low in the inner cavity, with the S6 F656 residue acting as a principal binding determinant. PMID:26159617

  20. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

    PubMed Central

    Castillo, Karen; Contreras, Gustavo F.; Pupo, Amaury; Torres, Yolima P.; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-01-01

    Being activated by depolarizing voltages and increases in cytoplasmic Ca2+, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation. PMID:25825713

  1. Kcnq1-5 (Kv7.1-5) potassium channel expression in the adult zebrafish

    PubMed Central

    2014-01-01

    Background KCNQx genes encode slowly activating-inactivating K+ channels, are linked to physiological signal transduction pathways, and mutations in them underlie diseases such as long QT syndrome (KCNQ1), epilepsy in adults (KCNQ2/3), benign familial neonatal convulsions in children (KCNQ3), and hearing loss or tinnitus in humans (KCNQ4, but not KCNQ5). Identification of kcnqx potassium channel transcripts in zebrafish (Danio rerio) remains to be fully characterized although some genes have been mapped to the genome. Using zebrafish genome resources as the source of putative kcnq sequences, we investigated the expression of kcnq1-5 in heart, brain and ear tissues. Results Overall expression of the kcnqx channel transcripts is similar to that found in mammals. We found that kcnq1 expression was highest in the heart, and also present in the ear and brain. kcnq2 was lowest in the heart, while kcnq3 was highly expressed in the brain, heart and ear. kcnq5 expression was highest in the ear. We analyzed zebrafish genomic clones containing putative kcnq4 sequences to identify transcripts and protein for this highly conserved member of the Kcnq channel family. The zebrafish appears to have two kcnq4 genes that produce distinct mRNA species in brain, ear, and heart tissues. Conclusions We conclude that the zebrafish is an attractive model for the study of the KCNQ (Kv7) superfamily of genes, and are important to processes involved in neuronal excitability, cardiac anomalies, epileptic seizures, and hearing loss or tinnitus. PMID:24555524

  2. Behavioral motor dysfunction in Kv3-type potassium channel-deficient mice.

    PubMed

    Joho, R H; Street, C; Matsushita, S; Knöpfel, T

    2006-08-01

    The voltage-gated potassium channels Kv3.1 and Kv3.3 are expressed in several distinct neuronal subpopulations in brain areas known to be involved in motor control such as cortex, basal ganglia and cerebellum. Depending on the lack of Kv3.1 or Kv3.3 channel subunits, mutant mice show different Kv3-null allele-dependent behavioral alterations that include constitutive hyperactivity, sleep loss, impaired motor performance and, in the case of the Kv3.1/Kv3.3 double mutant, also severe ataxia, tremor and myoclonus (Espinosa et al. 2001, J Neurosci 21, 6657-6665, Genes, Brain Behav 3, 90-100). The lack of Kv3.1 channel subunits is mainly responsible for the constitutively increased locomotor activity and for sleep loss, whereas the absence of Kv3.3 subunits affects cerebellar function, in particular Purkinje cell discharges and olivocerebellar system properties (McMahon et al. 2004, Eur J Neurosci 19, 3317-3327). Here, we describe two sensitive and non-invasive tests to reliably quantify normal and abnormal motor functions, and we apply these tests to characterize motor dysfunction in Kv3-mutant mice. In contrast to wildtype and Kv3.1-single mutants, Kv3.3-single mutants and Kv3 mutants lacking three and four Kv3 alleles display Kv3-null allele-dependent gait alterations. Although the Kv3-null allele-dependent gait changes correlate with reduced motor performance, they appear to not affect the training-induced improvement of motor performance. These findings suggest that altered cerebellar physiology in the absence of Kv3.3 channels is responsible for impaired motor task execution but not motor task learning.

  3. Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes.

    PubMed

    Andronic, Joseph; Bobak, Nicole; Bittner, Stefan; Ehling, Petra; Kleinschnitz, Christoph; Herrmann, Alexander M; Zimmermann, Heiko; Sauer, Markus; Wiendl, Heinz; Budde, Thomas; Meuth, Sven G; Sukhorukov, Vladimir L

    2013-02-01

    Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated K(V)1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K(2P)) channels in the RVD of human CD4(+) T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K(2P)5.1 and K(2P)18.1 as the most important K(2P) channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K(2P)5.1 and K(2P)18.1 on the RVD was comparable to that of K(V)1.3. K(2P)9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K(2P) channels. PMID:23041580

  4. Gambierol, a toxin produced by the dinoflagellate Gambierdiscus toxicus, is a potent blocker of voltage-gated potassium channels.

    PubMed

    Cuypers, Eva; Abdel-Mottaleb, Yousra; Kopljar, Ivan; Rainier, Jon D; Raes, Adam L; Snyders, Dirk J; Tytgat, Jan

    2008-05-01

    In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Na(v)1.1-Na(v)1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian K(v)1.1-K(v)1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 microM. In contrast, K(v)1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (>97%) and an half maximal inhibitory concentration (IC(50)) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments. PMID:18313714

  5. Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium channel TRESK

    PubMed Central

    Chae, Yun Jeong; Zhang, Jianan; Au, Paul; Sabbadini, Marta; Xie, Guo-Xi; Yost, C. Spencer

    2010-01-01

    Backgrcound We investigated the role of tandem pore domain potassium channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. Methods Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wildtype, knockout or heterozygote) was determined using the polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared to wildtype littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. Results TRESK knockout mice had normal development and behavior except for decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test). TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared to wildtype littermates; sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice following initial anesthesia testing was nearly threefold higher than that of wildtype littermates. Conclusions TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogenous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the idea that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations. PMID:21042202

  6. AMIGO is an auxiliary subunit of the Kv2.1 potassium channel

    PubMed Central

    Peltola, Marjaana A; Kuja-Panula, Juha; Lauri, Sari E; Taira, Tomi; Rauvala, Heikki

    2011-01-01

    Kv2.1 is a potassium channel α-subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (IK) in several neuronal types and a regulator of excitability during high-frequency firing. Here we identify AMIGO (amphoterin-induced gene and ORF), a neuronal adhesion protein with leucine-rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus-induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage-dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal IK at negative membrane voltages. In conclusion, our data indicate AMIGO as a function-modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability. PMID:22056818

  7. Biogenesis of the pore architecture of a voltage-gated potassium channel.

    PubMed

    Gajewski, Christine; Dagcan, Alper; Roux, Benoit; Deutsch, Carol

    2011-02-22

    The pore domain of voltage-gated potassium (Kv) channels consists of transmembrane helices S5 and S6, the turret, the pore helix, the selectivity filter, and the loop preceding S6, with a tertiary reentrant structure between S5 and S6. Using biogenic intermediates, mass tagging (pegylation), and a molecular tape measure, we explored the possibility that the first stages of pore formation occur prior to oligomerization of the transmembrane core. Pegylation of introduced cysteines shows that the pore helix, but not the turret, forms a compact secondary structure in the terminal 20 Å of the ribosomal tunnel. We assessed the tertiary fold of the pore loop in monomeric constructs by determining the relative accessibilities of select cysteines using the kinetics of pegylation. Turret residues are accessible at the extracellular surface. In contrast, pore helix residues are less accessible. All-atom molecular dynamics simulations of a single Kv monomer in a solvated lipid membrane indicate that secondary and tertiary folds are stable over 650 ns. These results are consistent with acquisition of a tertiary reentrant pore architecture at the monomer stage of Kv biogenesis and begin to define a plausible sequence of folding events in the formation of Kv channels.

  8. The ethylene bis-dithiocarbamate fungicide Mancozeb activates voltage-gated KCNQ2 potassium channel.

    PubMed

    Li, Ping; Zhu, Jin; Kong, Qingya; Jiang, Baifeng; Wan, Xia; Yue, Jinfeng; Li, Min; Jiang, Hualiang; Li, Jian; Gao, Zhaobing

    2013-06-01

    Mancozeb (manganese/zinc ethylene bis-dithiocarbamate) is an organometallic fungicide that has been associated with human neurotoxicity and neurodegeneration. In a high-throughput screen for modulators of KCNQ2 channel, a fundamental player modulating neuronal excitability, Mancozeb, was found to significantly potentiate KCNQ2 activity. Mancozeb was validated electrophysiologically as a KCNQ2 activator with an EC50 value of 0.92±0.23μM. Further examination showed that manganese but not zinc ethylene bis-dithiocarbamate is the active component for the positive modulation effects. In addition, the compounds are effective when the metal ions are substituted by iron but lack potentiation activity when the metal ions are substituted by sodium, signifying the importance of the metal ion. However, the iron (Fe(3+)) alone, organic ligands alone or the mixture of iron with the organic ligand did not show any potentiation effect, suggesting as the active ingredient is a specific complex rather than two separate additive or synergistic components. Our study suggests that potentiation on KCNQ2 potassium channels might be the possible mechanism of Mancozeb toxicity in the nervous system. PMID:23542819

  9. Dynamic memory of a single voltage-gated potassium ion channel: A stochastic nonequilibrium thermodynamic analysis

    SciTech Connect

    Banerjee, Kinshuk

    2015-05-14

    In this work, we have studied the stochastic response of a single voltage-gated potassium ion channel to a periodic external voltage that keeps the system out-of-equilibrium. The system exhibits memory, resulting from time-dependent driving, that is reflected in terms of dynamic hysteresis in the current-voltage characteristics. The hysteresis loop area has a maximum at some intermediate voltage frequency and disappears in the limits of low and high frequencies. However, the (average) dissipation at long-time limit increases and finally goes to saturation with rising frequency. This raises the question: how diminishing hysteresis can be associated with growing dissipation? To answer this, we have studied the nonequilibrium thermodynamics of the system and analyzed different thermodynamic functions which also exhibit hysteresis. Interestingly, by applying a temporal symmetry analysis in the high-frequency limit, we have analytically shown that hysteresis in some of the periodic responses of the system does not vanish. On the contrary, the rates of free energy and internal energy change of the system as well as the rate of dissipative work done on the system show growing hysteresis with frequency. Hence, although the current-voltage hysteresis disappears in the high-frequency limit, the memory of the ion channel is manifested through its specific nonequilibrium thermodynamic responses.

  10. Phosphatidylinositol 4,5-bisphosphate alters pharmacological selectivity for epilepsy-causing KCNQ potassium channels.

    PubMed

    Zhou, Pingzheng; Yu, Haibo; Gu, Min; Nan, Fa-jun; Gao, Zhaobing; Li, Min

    2013-05-21

    Pharmacological augmentation of neuronal KCNQ muscarinic (M) currents by drugs such as retigabine (RTG) represents a first-in-class therapeutic to treat certain hyperexcitatory diseases by dampening neuronal firing. Whereas all five potassium channel subtypes (KCNQ1-KCNQ5) are found in the nervous system, KCNQ2 and KCNQ3 are the primary players that mediate M currents. We investigated the plasticity of subtype selectivity by two M current effective drugs, retigabine and zinc pyrithione (ZnPy). Retigabine is more effective on KCNQ3 than KCNQ2, whereas ZnPy is more effective on KCNQ2 with no detectable effect on KCNQ3. In neurons, activation of muscarinic receptor signaling desensitizes effects by retigabine but not ZnPy. Importantly, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) causes KCNQ3 to become sensitive to ZnPy but lose sensitivity to retigabine. The dynamic shift of pharmacological selectivity caused by PIP2 may be induced orthogonally by voltage-sensitive phosphatase, or conversely, abolished by mutating a PIP2 site within the S4-S5 linker of KCNQ3. Therefore, whereas drug-channel binding is a prerequisite, the drug selectivity on M current is dynamic and may be regulated by receptor signaling pathways via PIP2. PMID:23650395

  11. Determinants of ligand selectivity in a cyclic nucleotide-regulated potassium channel.

    PubMed

    Pessoa, João; Fonseca, Fátima; Furini, Simone; Morais-Cabral, João H

    2014-07-01

    Cyclic nucleotide-binding (CNB) domains regulate the activity of channels, kinases, exchange factors, and transcription factors. These proteins are highly variable in their ligand selectivity; some are highly selective for either cAMP or cGMP, whereas others are not. Several molecular determinants of ligand selectivity in CNB domains have been defined, but these do not provide a complete view of the selectivity mechanism. We performed a thorough analysis of the ligand-binding properties of mutants of the CNB domain from the MlotiK1 potassium channel. In particular, we defined which residues specifically favor cGMP or cAMP. Inversion of ligand selectivity, from favoring cAMP to favoring cGMP, was only achieved through a combination of three mutations in the ligand-binding pocket. We determined the x-ray structure of the triple mutant bound to cGMP and performed molecular dynamics simulations and a biochemical analysis of the effect of the mutations. We concluded that the increase in cGMP affinity and selectivity does not result simply from direct interactions between the nucleotide base and the amino acids introduced in the ligand-binding pocket residues. Rather, tighter cGMP binding over cAMP results from the polar chemical character of the mutations, from greater accessibility of water molecules to the ligand in the bound state, and from an increase in the structural flexibility of the mutated binding pocket.

  12. Cloxyquin (5-chloroquinolin-8-ol) is an activator of the two-pore domain potassium channel TRESK.

    PubMed

    Wright, Paul D; Weir, Gregory; Cartland, Jamie; Tickle, David; Kettleborough, Catherine; Cader, M Zameel; Jerman, Jeff

    2013-11-15

    TRESK is a two-pore domain potassium channel. Loss of function mutations have been linked to typical migraine with aura and due to TRESK’s expression pattern and role in neuronal excitability it represents a promising therapeutic target. We developed a cell based assay using baculovirus transduced U20S cells to screen for activators of TRESK. Using a thallium flux system to measure TRESK channel activity we identified Cloxyquin as a novel activator. Cloxyquin was shown to have an EC50 of 3.8 μM in the thallium assay and displayed good selectivity against other potassium channels tested. Activity was confirmed using whole cell patch electrophysiology, with Cloxyquin causing a near two fold increase in outward current. The strategy presented here will be used to screen larger compound libraries with the aim of identifying novel chemical series which may be developed into new migraine prophylactics.

  13. Cloxyquin (5-Chloroquinolin-8-ol) is an activator of the two-pore domain potassium channel TRESK.

    PubMed

    Wright, Paul D; Weir, Gregory; Cartland, Jamie; Tickle, David; Kettleborough, Catherine; Cader, Zameel; Jerman, Jeff

    2013-10-25

    TRESK is a two-pore domain potassium channel. Loss of function mutations have been linked to typical migraine with aura and due to TRESK's expression pattern and role in neuronal excitability it represents a promising therapeutic target. We developed a cell based assay using baculovirus transduced U20S cells to screen for activators of TRESK. Using a thallium flux system to measure TRESK channel activity we identified Cloxyquin as a novel activator. Cloxyquin was shown to have an EC50 of 3.8μM in the thallium assay and displayed good selectivity against other potassium channels tested. Activity was confirmed using whole cell patch electrophysiology, with Cloxyquin causing a near two fold increase in outward current. The strategy presented here will be used to screen larger compound libraries with the aim of identifying novel chemical series which may be developed into new migraine prophylactics.

  14. Glucose sensitivity of mouse olfactory bulb neurons is conveyed by a voltage-gated potassium channel

    PubMed Central

    Tucker, Kristal; Cho, Sukhee; Thiebaud, Nicolas; Henderson, Michael X; Fadool, Debra Ann

    2013-01-01

    The olfactory bulb has recently been proposed to serve as a metabolic sensor of internal chemistry, particularly that modified by metabolism. Because the voltage-dependent potassium channel Kv1.3 regulates a large proportion of the outward current in olfactory bulb neurons and gene-targeted deletion of the protein produces a phenotype of resistance to diet-induced obesity in mice, we hypothesized that this channel may play a role in translating energy availability into a metabolic signal. Here we explored the ability of extracellular glucose concentration to modify evoked excitability of the mitral neurons that principally regulate olfactory coding and processing of olfactory information. Using voltage-clamp electrophysiology of heterologously expressed Kv1.3 channels in HEK 293 cells, we found that Kv1.3 macroscopic currents responded to metabolically active (d-) rather than inactive (l-) glucose with a response profile that followed a bell-shaped curve. Olfactory bulb slices stimulated with varying glucose concentrations showed glucose-dependent mitral cell excitability as evaluated by current-clamp electrophysiology. While glucose could be either excitatory or inhibitory, the majority of the sampled neurons displayed a decreased firing frequency in response to elevated glucose concentration that was linked to increased latency to first spike and decreased action potential cluster length. Unlike modulation attributed to phosphorylation, glucose modulation of mitral cells was rapid, less than one minute, and was reversible within the time course of a patch recording. Moreover, we report that modulation targets properties of spike firing rather than action potential shape, involves synaptic activity of glutamate or GABA signalling circuits, and is dependent upon Kv1.3 expression. Given the rising incidence of metabolic disorders attributed to weight gain, changes in neuronal excitability in brain regions regulating sensory perception of food are of consequence

  15. ATP-mediated vasodilatation occurs via activation of inwardly rectifying potassium channels in humans.

    PubMed

    Crecelius, Anne R; Kirby, Brett S; Luckasen, Gary J; Larson, Dennis G; Dinenno, Frank A

    2012-11-01

    Circulating ATP possesses unique vasomotor properties in humans and has been hypothesized to play a role in vascular control under a variety of physiological conditions. However, the primary downstream signalling mechanisms underlying ATP-mediated vasodilatation remain unclear. The purpose of the present experiment was to determine whether ATP-mediated vasodilatation is independent of nitric oxide (NO) and prostaglandin (PG) synthesis and occurs primarily via the activation of Na(+)/K(+)-ATPase and inwardly rectifying potassium (K(IR)) channels in humans. In all protocols, young healthy adults were studied and forearm vascular conductance (FVC) was calculated from forearm blood flow (measured via venous occlusion plethysmography) and intra-arterial blood pressure to quantify local vasodilatation. Vasodilator responses (%FVC) during intra-arterial ATP infusions were unchanged following combined inhibition of NO and PGs (n = 8; P > 0.05) whereas the responses to KCl were greater (P < 0.05). Combined infusion of ouabain (to inhibit Na(+)/K(+)-ATPase) and barium chloride (BaCl(2); to inhibit K(IR) channels) abolished KCl-mediated vasodilatation (n = 6; %FVC = 134 ± 13 vs. 4 ± 5%; P < 0.05), demonstrating effective blockade of direct vascular hyperpolarization. The vasodilator responses to three different doses of ATP were inhibited on average 56 ± 5% (n = 16) following combined ouabain plus BaCl(2) infusion. In follow-up studies, BaCl(2) alone inhibited the vasodilator responses to ATP on average 51 ± 3% (n = 6), which was not different than that observed for combined ouabain plus BaCl(2) administration. Our novel results indicate that the primary mechanism of ATP-mediated vasodilatation is vascular hyperpolarization via activation of K(IR) channels. These observations translate in vitro findings to humans in vivo and may help explain the unique vasomotor properties of intravascular ATP in the human circulation.

  16. PIST (GOPC) modulates the oncogenic voltage-gated potassium channel KV10.1

    PubMed Central

    Herrmann, Solveig; Ninkovic, Milena; Kohl, Tobias; Pardo, Luis A.

    2013-01-01

    Although crucial for their correct function, the mechanisms controlling surface expression of ion channels are poorly understood. In the case of the voltage-gated potassium channel KV10.1, this is determinant not only for its physiological function in brain, but also for its pathophysiology in tumors and possible use as a therapeutic target. The Golgi resident protein PIST binds several membrane proteins, thereby modulating their expression. Here we describe a PDZ domain-mediated interaction of KV10.1 and PIST, which enhances surface levels of KV10.1. The functional, but not the physical interaction of both proteins is dependent on the coiled-coil and PDZ domains of PIST; insertion of eight amino acids in the coiled-coil domain to render the neural form of PIST (nPIST) and the corresponding short isoform in an as-of-yet unknown form abolishes the effect. In addition, two new isoforms of PIST (sPIST and nsPIST) lacking nearly the complete PDZ domain were cloned and shown to be ubiquitously expressed. PIST and KV10.1 co-precipitate from native and expression systems. nPIST also showed interaction, but did not alter the functional expression of the channel. We could not document physical interaction between KV10.1 and sPIST, but it reduced KV10.1 functional expression in a dominant-negative manner. nsPIST showed weak physical interaction and no functional effect on KV10.1. We propose these isoforms to work as modulators of PIST function via regulating the binding on interaction partners. PMID:23966943

  17. Suppression of the Eag1 potassium channel sensitizes glioblastoma cells to injury caused by temozolomide

    PubMed Central

    Sales, Thais Torquato; Resende, Fernando Francisco Borges; Chaves, Natália Lemos; Titze-De-Almeida, Simoneide Souza; Báo, Sônia Nair; Brettas, Marcella Lemos; Titze-De-Almeida, Ricardo

    2016-01-01

    Glioblastoma multiforme (GBM) is the most aggressive type of human primary brain tumor. The standard treatment protocol includes radiotherapy in combination with temozolomide (TMZ). Despite advances in GBM treatment, the survival time of patients diagnosed with glioma is 14.5 months. Regarding tumor biology, various types of cancer cell overexpress the ether à go-go 1 (Eag1) potassium channel. Therefore, the present study examined the role of Eag1 in the cell damage caused by TMZ on the U87MG glioblastoma cell line. Eag1 was inhibited using a channel blocker (astemizole) or silenced by a short-hairpin RNA expression vector (pKv10.1-3). pKv10.1-3 (0.2 µg) improved the Eag1 silencing caused by 250 µM TMZ, as determined by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. Additionally, inhibiting Eag1 with the vector or astemizole (5 µM) reduced glioblastoma cell viability and sensitized cells to TMZ. Cell viability decreased by 63% for pKv10.1-3 + TMZ compared with 34% for TMZ alone, and by 77% for astemizole + TMZ compared with 46% for TMZ alone, as determined by MTT assay. In addition, both the vector and astemizole increased the apoptosis rate of glioblastoma cells triggered by TMZ, as determined by an Annexin V apoptosis assay. Collectively, the current data reveal that Eag1 has a role in the damage caused to glioblastoma by TMZ. Furthermore, suppression of this channel can improve the action of TMZ on U87MG glioblastoma cells. Thus, silencing Eag1 is a promising strategy to improve GBM treatment and merits additional studies in animal models of glioma. PMID:27698831

  18. Suppression of the Eag1 potassium channel sensitizes glioblastoma cells to injury caused by temozolomide

    PubMed Central

    Sales, Thais Torquato; Resende, Fernando Francisco Borges; Chaves, Natália Lemos; Titze-De-Almeida, Simoneide Souza; Báo, Sônia Nair; Brettas, Marcella Lemos; Titze-De-Almeida, Ricardo

    2016-01-01

    Glioblastoma multiforme (GBM) is the most aggressive type of human primary brain tumor. The standard treatment protocol includes radiotherapy in combination with temozolomide (TMZ). Despite advances in GBM treatment, the survival time of patients diagnosed with glioma is 14.5 months. Regarding tumor biology, various types of cancer cell overexpress the ether à go-go 1 (Eag1) potassium channel. Therefore, the present study examined the role of Eag1 in the cell damage caused by TMZ on the U87MG glioblastoma cell line. Eag1 was inhibited using a channel blocker (astemizole) or silenced by a short-hairpin RNA expression vector (pKv10.1-3). pKv10.1-3 (0.2 µg) improved the Eag1 silencing caused by 250 µM TMZ, as determined by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. Additionally, inhibiting Eag1 with the vector or astemizole (5 µM) reduced glioblastoma cell viability and sensitized cells to TMZ. Cell viability decreased by 63% for pKv10.1-3 + TMZ compared with 34% for TMZ alone, and by 77% for astemizole + TMZ compared with 46% for TMZ alone, as determined by MTT assay. In addition, both the vector and astemizole increased the apoptosis rate of glioblastoma cells triggered by TMZ, as determined by an Annexin V apoptosis assay. Collectively, the current data reveal that Eag1 has a role in the damage caused to glioblastoma by TMZ. Furthermore, suppression of this channel can improve the action of TMZ on U87MG glioblastoma cells. Thus, silencing Eag1 is a promising strategy to improve GBM treatment and merits additional studies in animal models of glioma.

  19. ATP-mediated vasodilatation occurs via activation of inwardly rectifying potassium channels in humans

    PubMed Central

    Crecelius, Anne R; Kirby, Brett S; Luckasen, Gary J; Larson, Dennis G; Dinenno, Frank A

    2012-01-01

    Circulating ATP possesses unique vasomotor properties in humans and has been hypothesized to play a role in vascular control under a variety of physiological conditions. However, the primary downstream signalling mechanisms underlying ATP-mediated vasodilatation remain unclear. The purpose of the present experiment was to determine whether ATP-mediated vasodilatation is independent of nitric oxide (NO) and prostaglandin (PG) synthesis and occurs primarily via the activation of Na+/K+-ATPase and inwardly rectifying potassium (KIR) channels in humans. In all protocols, young healthy adults were studied and forearm vascular conductance (FVC) was calculated from forearm blood flow (measured via venous occlusion plethysmography) and intra-arterial blood pressure to quantify local vasodilatation. Vasodilator responses (%ΔFVC) during intra-arterial ATP infusions were unchanged following combined inhibition of NO and PGs (n= 8; P > 0.05) whereas the responses to KCl were greater (P < 0.05). Combined infusion of ouabain (to inhibit Na+/K+-ATPase) and barium chloride (BaCl2; to inhibit KIR channels) abolished KCl-mediated vasodilatation (n= 6; %ΔFVC = 134 ± 13 vs. 4 ± 5%; P < 0.05), demonstrating effective blockade of direct vascular hyperpolarization. The vasodilator responses to three different doses of ATP were inhibited on average 56 ± 5% (n= 16) following combined ouabain plus BaCl2 infusion. In follow-up studies, BaCl2 alone inhibited the vasodilator responses to ATP on average 51 ± 3% (n= 6), which was not different than that observed for combined ouabain plus BaCl2 administration. Our novel results indicate that the primary mechanism of ATP-mediated vasodilatation is vascular hyperpolarization via activation of KIR channels. These observations translate in vitro findings to humans in vivo and may help explain the unique vasomotor properties of intravascular ATP in the human circulation. PMID:22777673

  20. Gold nanoparticle-spermidine complex blocks the inward rectifier potassium channel

    PubMed Central

    Chin, Chur

    2014-01-01

    A previous study showed that negatively charged gold nanoparticles block ion pores by binding to the sulfur group of the cysteine loop of the ion channel when small molecules like amine lead the nanoparticles inside the ion pore. Cells were voltage clamped at -100 mV. Subsequently a bath application of 30 μM Ach produced a current followed by the extracellular application of 100 mM spermidine and 50 nM of nanoparticle complex. Peak amplitude was then recorded. The addition of Ach (30 uM) reversed the effect, and we recorded inhibition of the peak amplitude. We also recorded electrocardiogram (EKG) and the atria effective refractory period (AERP) after treatment with the complex in the atrium of a rabbit heart in a Langendorff apparatus. Upon external application of the complex, the Ach-activated current was blocked by 48.8% ± 3.1% with 82.7% ± 3.1% reversal. In recording the EKG and the AERP after the addition of the complex including 30 mM spermidine with 50 nM nanoparticles, the complete resolution of atrial fibrillation at 50 s and the elongation of AERP from 46 to 52 was observed, which unveils a new class 3 anti arrythmic agent using gold nanoparticles with spermidine. Negatively charged gold nanoparticles (0.8 nm) block ion pores after penetrating the cell membrane with spermidine, thus entering the cells with a polyamine transporter and acting at the intracellular face of the channel via binding to the sulfur group of the human inward rectifying potassium channel- I(KAch). PMID:25006531

  1. Down-state model of the voltage-sensing domain of a potassium channel.

    PubMed

    Schow, Eric V; Freites, J Alfredo; Gogna, Karun; White, Stephen H; Tobias, Douglas J

    2010-06-16

    Voltage-sensing domains (VSDs) of voltage-gated potassium (Kv) channels undergo a series of conformational changes upon membrane depolarization, from a down state when the channel is at rest to an up state, all of which lead to the opening of the channel pore. The crystal structures reported to date reveal the pore in an open state and the VSDs in an up state. To gain insights into the structure of the down state, we used a set of experiment-based restraints to generate a model of the down state of the KvAP VSD using molecular-dynamics simulations of the VSD in a lipid bilayer in excess water. The equilibrated VSD configuration is consistent with the biotin-avidin accessibility and internal salt-bridge data used to generate it, and with additional biotin-avidin accessibility data. In the model, both the S3b and S4 segments are displaced approximately 10 A toward the intracellular side with respect to the up-state configuration, but they do not move as a rigid body. Arginine side chains that carry the majority of the gating charge also make large excursions between the up and down states. In both states, arginines interact with water and participate in salt bridges with acidic residues and lipid phosphate groups. An important feature that emerges from the down-state model is that the N-terminal half of the S4 segment adopts a 3(10)-helical conformation, which appears to be necessary to satisfy a complex salt-bridge network.

  2. The inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons.

    PubMed

    Zhang, Yan-Zhuo; Zhang, Rui; Zeng, Xian-Zhang; Song, Chun-Yu

    2016-03-11

    Excessive K(+) efflux via activated voltage-gated K(+) channels can deplete intracellular K(+) and lead to long-lasting membrane depolarization which will promote neuronal apoptosis during ischemia/hypoxia injury. The Kv2.1 potassium channel was the major component of delayed rectifier potassium current (Ik) in pyramidal neurons in cortex and hippocampus. The neuronal protective effect of propofol has been proved. Delayed rectifier potassium current (Ik) has been shown to have close relationship with neuronal damage. The study was designed to test the inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons. Whole-cell patch clamp recordings and Western blot analysis were used to investigate the electrophysiological function and protein expression of Kv2.1 in rat parietal cortical neurons after propofol treatment. We found that propofol concentration-dependently inhibited Ik in pyramidal neurons. Propofol also caused a downward shift of the I-V curve of Ik at 30μM concentration. Propofol significantly inhibited the expression of Kv2.1 protein level at 30μM, 50μM, 100μM concentration. In conclusion, our data showed that propofol could inhibit Ik, probably via depressing the expression of Kv2.1 protein in rat cerebral parietal cortical neurons.

  3. Inhibition by acrolein of light-induced stomatal opening through inhibition of inward-rectifying potassium channels in Arabidopsis thaliana.

    PubMed

    Islam, Md Moshiul; Ye, Wenxiu; Matsushima, Daiki; Khokon, Md Atiqur Rahman; Munemasa, Shintaro; Nakamura, Yoshimasa; Murata, Yoshiyuki

    2015-01-01

    Acrolein is a reactive α,β-unsaturated aldehyde derived from lipid peroxides, which are produced in plants under a variety of stress. We investigated effects of acrolein on light-induced stomatal opening using Arabidopsis thaliana. Acrolein inhibited light-induced stomatal opening in a dose-dependent manner. Acrolein at 100 μM inhibited plasma membrane inward-rectifying potassium (Kin) channels in guard cells. Acrolein at 100 μM inhibited Kin channel KAT1 expressed in a heterologous system using Xenopus leaves oocytes. These results suggest that acrolein inhibits light-induced stomatal opening through inhibition of Kin channels in guard cells.

  4. Two conserved arginine residues from the SK3 potassium channel outer vestibule control selectivity of recognition by scorpion toxins.

    PubMed

    Feng, Jing; Hu, Youtian; Yi, Hong; Yin, Shijin; Han, Song; Hu, Jun; Chen, Zongyun; Yang, Weishan; Cao, Zhijian; De Waard, Michel; Sabatier, Jean-Marc; Li, Wenxin; Wu, Yingliang

    2013-05-01

    Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca(2+)-activated K(+) (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the "new" blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile. PMID:23511633

  5. Correlation between potassium channel expression and sensitivity to drug-induced cell death in tumor cell lines.

    PubMed

    Leanza, Luigi; O'Reilly, Paul; Doyle, Anne; Venturini, Elisa; Zoratti, Mario; Szegezdi, Eva; Szabo, Ildiko

    2014-01-01

    Plasma membrane (PM) and mitochondrial (mt) ion channels - particularly potassium channels - became oncological targets soon after the discovery that they are involved both in the regulation of proliferation and apoptosis. Some members of the Kv Shaker family, namely Kv1.1, Kv1.3, Kv1.5 and Kv11.1 (Herg), and the intermediate-conductance calcium-activated potassium KCa3.1 (IK) channels have been shown to contribute to apoptosis in various cell lines. Kv1.3, Kv1.5 and IK are located in the plasma membrane but also in the mitochondrial inner membrane, where they participate in apoptotic signalling. Interestingly, an altered protein expression of some of the channels mentioned above has been reported in neoplastic cell lines/tissues, but a systematic quantification addressing the protein expression of the above potassium channels in tumor cell lines of different origin has not been carried out yet. In the present study we investigated whether expression of specific potassium channels, at the mRNA and protein level, can be correlated with cell sensitivity to various apoptotic stimuli, including chemotherapeutic drugs, in a panel of cancer cell lines. The results show correlation between the protein expression of the Kv1.1 and Kv1.3 channels and susceptibility to death upon treatment with staurosporine, C2-ceramide and cisplatin. Furthermore, we investigated the correlation between Kv channel expression and sensitivity to three distinct membrane-permeant Kv1.3 inhibitors, since these drugs have recently been shown to be able to induce apoptosis and also reduce tumor volume in an in vivo model. Higher protein expression of Kv1.3 significantly correlated with lower cell survival upon treatment with clofazimine, one of the Kv1.3 inhibitors. These results suggest that expression of Kv1.1 and Kv1.3 sensitizes tumour cells of various origins to cytotoxins. Data reported in this work regarding potassium channel protein expression in different cancer cell lines may be exploited

  6. Involvement of Potassium and Cation Channels in Hippocampal Abnormalities of Embryonic Ts65Dn and Tc1 Trisomic Mice.

    PubMed

    Stern, Shani; Segal, Menahem; Moses, Elisha

    2015-09-01

    Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca(2+)]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude. Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~ 90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~ 65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype. PMID:26501103

  7. Involvement of Potassium and Cation Channels in Hippocampal Abnormalities of Embryonic Ts65Dn and Tc1 Trisomic Mice

    PubMed Central

    Stern, Shani; Segal, Menahem; Moses, Elisha

    2015-01-01

    Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca2+]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude. Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~ 90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~ 65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype. PMID:26501103